Aoac.methods.1980
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This document summarizes an official methods of analysis publication from 1980. It has been incorporated by reference and is legally binding for US citizens. The publication contains standardized methods for analyzing various agricultural and food products developed by the Association of Official Analytical Chemists (AOAC) through collaborative studies. It provides methods for analyzing products like fertilizers, plants, disinfectants, hazardous substances, and pesticide formulations. The summarized document establishes the referenced publication as an authoritative source for analytical testing in the United States.
![In clinical chemistry, within-laboratory precision (repeatability)
is critical, and often is of greater interest to clinical laboratories
than absolute accuracy or agreement with the values of other
laboratories (reproducibility). In drug analysis, a high degree of
accuracy is required in the therapeutic range because the
analytical values determining the identity, strength, quality, and
purity of pharmaceutical preparations, as laid down in phar-
macopoeial specifications, are directly related to clinical value.
With polynuclear hydrocarbons, specificity is important, since
some of these compounds are carcinogenic while others are
not. In applying the famous Delaney clause of the United States
Federal Food, Drug, and Cosmetic Act, all attributes of the
analytical methods are secondary to the detection of extremely
small concentrations (detectability), or to exhibiting a high
degree of response for small changes in concentration (sensi-
tivity).
There is a very special case involving accuracy, where the
"true value" is determined by the method of analysis. Many
legal specifications and standards for food and agricultural
products define ill-defined components such as moisture, fat,
protein, and crude fiber in terms of reference methods. There-
fore, the precision of these methods becomes the limiting factor
for their performance. In fact, most analyses involved in com-
mercial transactions require primarily that the buyer and seller
agree on the same value (analytically and economically), re-
gardless of where it stands on an absolute scale.
The point of these examples is that although methods of
analysis are characterized by a number of attributes-accuracy,
precision, specificity, sensitivity, detectability, dependability,
and practicality-no method is so flawless that all these qualities
can be maximized simultaneously. For any particular analysis,
the analyst must determine, on the basis of the purpose of the
analysis, which attributes are essential and which may be
compromised.
Unfortunately, the literature is replete with examples indicat-
ing that an individual analyst, and especially the originator of
a method of analysis, is not an unbiased judge of the relative
merits of the methods of analysis which he develops and uses.
In our experience, the collaborative study provides impartial
data on the suitability of the method. The data, in many cases,
speak for themselves.
The collaborative study, or ring test or round robin test, as it
is called in other organizations, provides the basic information
on the performance of analytical methods. The extent of the
information will depend on the number of samples provided,
the number of analyses performed, and the number of labora-
tories participating. The data should be unbiased because the
composition of the samples is known only to the administrator
of the study. Some of the requirements of the study and their
relationship to the characteristics and attributes of the method
are as follows:
(1) Accuracy. Samples must be of defined composition (by
spiking, by formulation, or by analytical consensus).
(2) Specificity. Samples should contain related analytes.
(3) Sensitivity. Samples should differ from each other or from
negative samples by a known amount.
(4) Applicability. Samples should include the concentration
range and matrix components of interest.
(5) Blanks. Samples should include different matrices with
"none" of the component of interest.
(6) Precision. Instructions should request replicate analyses
by the same or different analysts in the same laboratory,
preferably on different days. By far a better procedure is to
include "blind" (unknown to the analyst) replicate samples in
the series.
(7) Practicality. Instructions should request information as to
xiii
the actual and elapsed time required for the analyses; the
availability of reagents, equipment, and standards; and any
necessary substitutions. When practice samples are included,
the number of analyses required to achieve the stated recovery
and repeatability, should be reported.
PROCEDURAL DETAILS OF COLLABORATIVE STUDY
As numerous beginners in this field have discovered, much
preliminary work must be done before sending out samples:
(1) The method must be chosen and demonstrated to apply
to the matrices and concentrations of interest.
(2) The critical variables in the method should have been
determined and the need for their control emphasized [a rug-
gedness test (5) is useful for this purpose].
(3) The method should be written in detail by the Associate
Referee and tested by an analyst not previously connected with
its development.
(4) Unusual standards, reagents, and equipment must be
available from usual commercial sources of supply, or sufficient
quantities must be prepared or obtained to furnish to the
participants.
(5) The samples must be identical and homogeneous so that
the analytical sample error is only a negligible fraction of the
expected analytical error.
(6) A sufficient number of samples must be prepared to cover
typical matrices and the concentration range of interest (toler-
ance, maximum or minimum specifications, likely levels of
occurrence, etc.).
(7) Samples must be stable and capable of surviving the
rigors of commercial transportation.
(8) Reserve samples should be prepared and preserved to
replace lost samples and to permit reanalysis of samples con-
sidered as outliers to attempt to discover the cause of abnormal
results.
(9) The instructions must be clear. They should be reviewed
by someone not connected with the study to uncover potential
misunderstandings and ambiguities.
(10) If the analyte is subject to change (e.g., bacterial levels,
nitroglycerin tablets), provision must be made for all participants
to begin the analysis at the same time.
(11) Practice samples of a known and declared composition
should be furnished with instructions not to analyze the un-
knowns until a specified degree of recovery and repeatability
(or other attribute) has been achieved.
(12) Provision should be made when necessary for submis-
sion of standard curves, tracings of recorder charts, or photo-
graphs of thin-layer plates in order to assist in determining
possible causes of error.
OTHER TYPES OF INTERLABORATORY STUDIES
This type of collaborative study, which is designed to deter-
mine the characteristics of a method, must be carefully distin-
guished from other types of interlaboratory studies which by
design or through ignorance provide other kinds of information.
The most important types of other studies are:
(1) Those studies which require the collaborators to investi-
gate the variability of parts of methods or applicability to
different types of samples. (An interlaboratory study is usually
an inefficient way of obtaining this type of information.)
(2) Those studies which permit an analyst to use any method
he desires. Such studies invariably produce such a wide scatter
of results that the data are of little value for evaluation of
methods. They may be useful in selecting a method from a
number of apparently equivalent methods, provided the purpose](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-15-320.jpg)
![(21) Unless otherwise indicated, temps are expressed as
degrees Centigrade.
Table 1. Nominal Dimensions of Standard Test Sieves (U.S.A.
Standard Series)
Sieve Designation
Nominal Nominal
International Sieve Wire
Standard' U.S.A. Opening, Diameter,
(ISO) Standard inches mm
12.5 mmb '/2 in.b 0.500 2.67
11.2 mm 7/'6 in. 0.438 2.45
9.5 mm 3/8 in. 0.375 2.27
8.0 mm 5/,6 in. 0.312 2.07
6.7 mm 0.265 in. 0.265 1.87
6.3 mmb
'/. in." 0.250 1.82
5.6 mm No. 3'/2 0.223 1.68
4.75 mm No.4 0.187 1.54
4.00 mm No.5 0.157 1.37
3.35 mm No.6 0.132 1.23
2.80 mm No.7 0.111 1.10
2.38 mm No.8 0.0937 1.00
2.00 mm No. 10 0.0787 0.900
1.70 mm No. 12 0.0661 0.810
1.40 mm No. 14 0.0555 0.725
1.18 mm No. 16 0.0469 0.650
1.00 mm No. 18 0.0394 0.580
850 "me No. 20 0.0331 0.510
710 "m No. 25 0.0278 0.450
600 "m No. 30 0.0234 0.390
500 "m No. 35 0.0197 0.340
425 "m No. 40 0.0165 0.290
355 "m No. 45 0.0139 0.247
300 "m No. 50 0.Q117 0.215
250 "m No. 60 0.0098 0.180
212 "m No. 70 0.0083 0.152
180 "m No. 80 0.0070 0.131
150 "m No. 100 0.0059 0.110
125 "m No. 120 0.0049 0.091
106 "m No. 140 0.0041 0.076
90 "m No. 170 0.0035 0.064
75 "m No. 200 0.0029 0.053
63 "m No. 230 0.0025 0.044
53 "m No. 270 0.0021 0.037
a These standard designations correspond to the values for test sieve
apertures recommended by the International Organization for Standard-
ization, Geneva, Switzerland.
b These sieves are not in the standard series but they have been
included because they are in common usage.
c 1000 I-'m = 1 mm.
Standard Operations
(22) Operations specified as "wash (rinse, ext. etc.) with two
(three, four, etc.) 10 mL (or other voL) portions H20 (or other
solv.)" mean that the operation is to be performed with indicated
vol. of solv. and repeated with same vol. of solv. until number
of portions required have been used.
(23) Definitions of terms used in methods involving spectro-
photometry are those given in JAOAC 37, 54(1954). Most
important principles and definitions are:
(a) More accurate instrument may be substituted for less
accurate instrument (e.g., spectrophtr may replace colorimeter)
where latter is specified in method. Wavelength specified in
method is understood to be that of max. absorbance (A). unless
no peak is present.
(b) Absorbance(s) (A).-Neg. logarithm to base 10 of ratio of
transmittance (T) of sample to that of ref. or std material. Other
xvi
names that have been used for quantity represented by this
term are optical density, extinction, and absorbancy.
(e) Absorptivity(ies) (a).-Absorbance per unit concn and cell
length. a = A/be, where b is in cm and c in gil, or a = (A/be)
x 1000, if c is in mg/L. Other names that have been used for
this or related quantities are extinction coefficient, specific
absorption, absorbance index, and £16';'".
(d) Transmittance(s) (T).-Ratio of radiant power transmitted
by sample to radiant power incident on sample, when both are
measured at same spectral position and with same slit width.
Beam is understood to be parallel radiation and incident at right
angles to plane parallel surface of sample. If sample is soln,
solute transmittance is quantity usually desired and is detd
directly as ratio of transmittance of soln in cell to transmittance
of solv. in an equal cell. Other names that have been used for
this quantity are transmittancy and transmission.
(e) Standardization.-Spectrophtr may be checked for accu-
racy of wavelength scale by ref. to Hg lines: 239.95, 248.3,
253.65, 265.3, 280.4, 302.25, 313.16, 334.15, 365.43, 404.66,
435.83, 546.07, 578.0, and 1014.0 nm. To check consistency of
absorbance scale, prep. soln of 0.0400 g K2CrO./L 0.05N KOH
and det. absorbance at following wavelengths in 1 cm cell: 230
nm, 0.171; 275,0.757; 313.2,0.043; 375, 0.991; 400, 0.396. See
"Standards for Checking the Calibration of Spectrophotome-
ters," Letter Circular LC-l017, reissued Jan 1967, NBS.
(24) Least square treatment of data and calculation of regres-
sion lines.-This technic finds the best fitting straight line for
set of data such as std curve. It calcs that straight line whose
sum of squares of vertical deviations (usually A) of observations
from the line is smaller than corresponding sum of squares of
deviations from any other line. Equation of straight line is:
Y = a + bX,
where a is intercept at Y axis (X = 0). and b is slope of line.
Least square estimates of constants are:
b = I (X, Y,) - [(IX,I Y,)/n]
IXf - (IX,)2/n
a=Y-bX,
where I = "sum of" the n individual values of indicated
operation, and X and Yare the averages of the X and Y points.
Example: To find "best" straight line relating A (Y) to concn
(X):
Observation Concn Absorbance
No. CI X, Y, X~ X,V,
1 80 1.270 6400 101.6
2 60 1.000 3600 60.0
3 40 0.700 1600 28.0
4 30 0.550 900 16.5
5 20 0.250 400 5.0
6 10 0.100 100 1.0
7 0 0.050 0 0.0
Totals:
n=7 IX, = 240 IY, = 3.92 IXl = 13000 I(X,Y,) = 212.1
x= IX,!n = 240/7 = 34.29
Y = IY,!n = 3.92/7 = 0.56
b = 212.1 - (240)(3.92)/7 77.7 = 0.0163
13000 - (240)2/7 4771
a = 0.56 - 0.0163(34.29) = 0.001
Best equation is then:
Y = 0.00 + 0.0163X
If for sample, A = 0.82, corresponding concn (X) would be:
X = (Y - 0.00)/0.0163 =0.82/0.0163 = 50.3.](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-18-320.jpg)
![4 1. AGRICULTURAL liMING MATERIALS AOAC METHODS (1980)
1.018 Oxides of Iron, Aluminum, Phosphorus,
and Titanium (9)-Official Final Action
(Alternatively, Fe, AI, Mn, P, and Ti may be detd
colorimetrically as in 1.025-1.040.)
To 125 mL aliquot Soln X from 1.017(a), add 10 mL HCI and
few drops Me red indicator; heat to gentle boil and add NH.OH
(1 +1) until ppt forms and indicator just changes to distinct
yellow. Boil ,,;2 min and filter rapidly. Wash ppt!H3 times with
hot 2% NH"NOa soln. Return ppt and filter to original beaker,
add 10 mL HCI, and macerate fi Iter with policeman. Dil. with
H,O, heat to dissolve ppt, dil. to ca 200 mL, and reppt as above.
Wash thoroly with the hot NH.NOa soln until CI-free. Combine
first and second filtrates and save for Ca and Mg detns.
Place ppt in Pt crucible and dry. Ignite gently to oxidize C,
heat to bright red ca 10 min, cool in desiccator, and weigh in
covered crucible as Fe,Oa + Al,Oa + P,Os + TiO,.
1.019 Calcium (9)-Official Final Action
Conc. combined filtrates and washings from 1.018 to ca 50
mL; make slightly alk. with NH.OH (1+1); while still hot, add
satd (NH.),C,O. soln dropwise as long as any ppt forms, and
then enough excess to convert Mg salts also to oxalate. Heat to
bp, let stand ",,3 hr, decant clear soln thru filter, pour 15-20 mL
hot H,O on ppt, and again decant clear soln thru filter. Dissolve
any ppt remaining on filter by washing with hot HCI (1 +9) into
original beaker, wash 6 times with hot H,O, and then reppt at
bp by adding NH.OH and a little satd (NH.),C,O. soln. Let stand
as before, filter thru same filter, and wash with hot H,O until CI-
free. Reserve filtrates and washings from both pptns for detn of
Mg,1.021.
Complete detn by one of following methods and report as
% CaO:
(a) Ignite ppt in crucible, either over S-free blast lamp, or in
elec. furnace at 950°, to const wt, cool in desiccator, and weigh
as CaO.
(b) Incinerate filter over low flame, mix ignited ppt with finely
pulverized and dried mixt. of equal parts of (NH.),SO.and NH.CI,
and drive off excess sulfate by carefully heating upper portion
of crucible. Complete ignition, cool in desiccator, and weigh as
CaSO•.
(c) Perforate apex of cone; wash CaC,O. ppt into beaker used
for pptn; then wash filter with hot H2S04 (1 +4), and titr. at 85-90°
with 0.1N KMnO•.
Magnesium (70)-Official Final Action
1.020 Reagent
Phosphate soln.-Dissolve 100 g (NH.),HP04 in hot H,O, dil.
to 1 L, and add 5 mL CHCla.
1.021 Determination
To combined filtrates and washings, 1.019, add 2 mL 1M citric
acid, 100 mL NH.OH, and 50 mL alcohol. Then add 25 mL of the
phosphate soIn, with const stirring, and let stand 12-24 hr.
Filter, wash twice with NH.OH (1 +9), and dissolve ppt in HNOa
(1 +4), washing soln into original beaker to vol. of 100-150 mL.
Add 1/10 vol. NH.OH and 2 drops of the phosphate soln. Stir
vigorously and let stand ""3 hr. Filter thru gooch, wash with
NH.OH (1 +9), moisten filter with satd soln of NH.NOa made
slightly ammoniacal, ignite, and weigh as Mg,P,07. Report as
% MgO. Correct wt Mg,P,07 for co-pptd Mn,P,07 by detg Mn as
in 33.127.
EDTA Titration Methods
Calcium and Magnesium (77)-Official Final Action
(Not applicable to samples with high
phosphate content or contg <2% Mg)
(Caution: See 51.050.)
1.022 Reagents
(a) Buffer soln.-pH 10. Dissolve 67.5 g NH.CI in 200 mL H,O,
add 570 mL NH40H, and dil. to 1 L.
(b) Potassium hydroxide-potassium cyanide soln.- Dissolve
280 g KOH and 66 g KCN in 1 L H,O.
(e) Potassium cyanide soln.-2%. Dissolve 2 g KCN in 100
mL H,O.
(d) Eriochrome black T indicator soln.-Dissolve 0.2 g indi-
cator (Eastman Kodak P6361, or equiv.) in 50 mL MeOH contg
2 g NH,OH.HCI. Store ,,;1 month.
(e) Magnesium std solns.-{).25 and 1.00 mg/mL. Dissolve
0.25 and 1.00 g Mg turnings in HCI (1 +10) and dil. each to 1 L
with double distd H,O.
(f) Calcium std soln.-1 mg/mL. Dissolve 2.4973 g CaCOa,
primary std grade, previously dried 2 hr at285°, in HCI (1 + 10).
Dil. to 1 L with double distd H,O.
(9) Calcein indicator.-Grind together 1 g indicator, 10 g
charcoal (Norite A is satisfactory), and 100 g KCI. (Indicator is
described in Anal. Chem. 28, 882 (1956), and is available from
G. Frederick Smith Chemical Co. and Eastman Kodak.)
(h) Disodium dihydrogen EDTA std solns.-(1) D.4%.-Dis-
solve 4 g Na,H,EDTA in 1 L H,O. Stdze against std Ca and Mg
solns. (2) D.1 %.-Prep. as in (1). using 1 g Na,H,EDTA, and stdze
against 0.25 mg/mL Mg std soln.
1.023 Standardization
(a) For calcium.-Pipet 10 mL std Ca soln into 300 mL erlen-
meyer and add 10 mL H,O. Add 10 mL KOH-KCN soln and ca 35
mg calcein indicator. Using mag. stirrer and artificial light, titr.
with 0.4% EDTA std soln to disappearance of all green. Titr. ""3
aliquots and use avo to calc. titer Ca soln = 10/mL EDTA soln.
(b) For magnesium.-Pipet 10 mL 0.25 and 1.00 mg/mL Mg
std solns into 300 mL erlenmeyers and add 100 mL H,O. Add 5
mL pH 10 buffer, 2 mL 2% KCN soln, and 10 drops eriochrome
black T indicator. Using mag. stirrer and artificial light, titr. with
0.1 and 0.4% EDTA std solns, resp., until color changes per-
manently from wine red to pure blue. Titr. ",,3 aliquots and use
avo to calc. titer Mg soln = 2.5/mL EDTA soln, or 10/mL EDTA
soln, resp.
1.024 Determination
Dry sample at 110° to canst wt and cool to room temp. Grind
to pass No. 60 or 80 sieve and mix thoroly. Accurately weigh ca
0.5 9 into 250 mL beaker, add 20 mL HCI (1+1), and evap. to
dryness on hot plate. Dissolve residue in 5 mL HCI (1 + 10), dil.
to ca 100 mL with H,O, and digest over low flame 1 hr. Cool,
transfer to 200 mL vol. flask, dil. to vol., mix, and let settle or
filter.
(a) Forcalcium.-Pipet 10 mL aliquot into 300 mL erlenmeyer
and titr. as in 1.023(a), observing end point thru soln and away
from light. % Ca = (Titer EDTA std soln for Cal x mL EDTA std
soln x 2/g sample.
(b) For magnesium.-(For agricultural limestones contg >4%
Mg.) For Ca + Mg, pipet 10 mL aliquot into 300 mL erlenmeyer
and titr. with 0.4% EDTA soln as in 1.023(b).
% Mg = (Titer EDTA std soln for Mg) x [(mL EDTA std soln
in Ca + Mg titrn) - (mL EDTA std soln in Ca titrn)] x 2/g sample.
(e) For magnesium.-(For agricultural limestones contg 2-4%](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-26-320.jpg)
![10 2. FERTILIZERS AOAC METHODS (1980)
(a) Materials containing small quantities of organic matter.-
Dissolve in 30 mL HN03 and 3-5 mL HCI, and boil until org.
matter is destroyed (30 min for liqs and suspensions).
*(b) Fertilizers containing much Fe or AI phosphate, and
basic slag. *-Dissolve in 15-30 mL HCI and 3-10 mL HN03•
(e) Organic material like cottonseed meal alone or in mix-
tures.-Evap. with 5 mL Mg(N03 )2 sol n, 2.019, ignite, and dissolve
in HCI.
*(d) Materials or mixtures containing large amounts of or-
ganic matter. *-Digestion with H2SO. and NaN03 or KN03• See
2.017(d), 11th ed.
(e) All fertilizers.-Boil gently 30-45 min with 20-30 mL HN03
in suitable flask (preferably Kjeldahl for samples contg large
amts of org. matter) to oxidize all easily oxidizable matter. Cool.
Add 10-20 mL 70-72% HCIO•. Boil very gently until soln is
colorless or nearly so and dense white fumes appear in flask.
Do not boil to dryness at any time (Danger!). (With samples
contg large amts of org. matter, raise temp. tI.. fuming point, ca
170°, over period of ;,1 hr.) Cool slightly, add 50 mL H20, and
boil few min.
Spectrophotometric Molybdovanadophosphate
Method (70)-Official Final Action
(Not applicable to materials yielding colored solns or solns
contg ions other than orthophosphate which form colored
complexes with molybdovanadate. Not recommended for
basic slag.)
2.021 Apparatus
Photometer.-Beckman Instruments, Inc. Model DU (current
models 24/25) spectrophtr with stray light filter and matched 1
cm cells. With other photometers analyst must det. suitability
for use and conditions for satisfactory performance. Means for
dispelling heat from light source is desirable.
2.022 Reagents
(a) Molybdovanadate reagent.-Dissolve 40 9 NH. molyb-
date.4H20 in 400 mL hot H20 and cool. Dissolve 2 9 NH.
metavanadate in 250 mL hot H20, cool, and add 450 mL 70%
HCIO•. (Caution: See 51.028(a) and (d).) Gradually add molybdate
soln to vanadate soln with stirring, and dil. to 2 L.
(b) Phosphate std soln.-Dry pure KH2PO. (52.15% P20 S) 2 hr
at 105°. Prep. solns contg 0.4-1.0 mg P20s/mL in 0.1 mg
increments by weighing 0.0767, 0.0959, 0.1151, 0.1342, 0.1534,
0.1726, and 0.1918 g KH2PO. and dilg each to 100 mL with H20.
Prep. fresh solns contg 0.4 and 0.7 mg P,Os/mL weekly.
2.023 Preparation ofStandardCurve
Pipet 5 mL aliquots of 7 std phosphate solns (2-5 mg P20 S/
aliquot) into 100 mL vol. flasks and add 45 mL H20. Then, within
5 min for entire series, add 20 mL molybdovanadate reagent by
buret or pipet, dil. to vol. and mix. Let stand 10 min.
Select 2 absorption cells (std and sample cells) and fill both
with 2 mg std. Set spectrophtr to 400 nm and adjust to zero A
with std cell. Sample cell must check zero A within 0.001 unit;
otherwise read A for sample cell and correct subsequent read-
ings. (Choose cell showing pos. A against other as sample cell
so that this pos. A is always subtracted.) Using sample cell, det.
A of other stds with instrument adjusted to zero A for 2 mg std.
After each detn empty and refill cell contg 2 mg std, and readjust
zero to avoid error that might arise from temp. changes. Plot A
against concn in mg P20s/mL std soln.
*Surplus metho~ee inside front cover.
2.024 Preparation ofSolution
Treat 1 g sample as in 2.020, preferably (e). when these acids
are suitable solv. (Soln should be free of N oxides and NOCI.)
(a) For P,Os content ";5%, dil. to 250 mL.
(b) For P20S content >5%, dil. to such vol. that 5 or 10 mL
aliquot contains 2-5 mg P20 S.
2.025 Determination
Pipet, into 100 mL vol. flasks, 5 mL aliquots of std phosphate
solns contg 2 and 3.5 mg P,Os/aliquot, resp., and develop color
as in 2.023. Adjust instrument to zero A for 2 mg std, and det.
A of 3.5 mg std. (It is essential that A of latter std be practically
identical with corresponding value on std curve.)
(a) Samples containing up to 5% P,Os.-Pipet, into 100 mL
vol. flask, 5 mL sample soln, 2.024(a). and 5 mL std phosphate
soln contg 2 mg P,Os. Develop color and det. A concurrently
with and in same manner as for std phosphate solns in preceding
par., with instrument adjusted to zero A for 2 mg std. Read P,Os
concn from std curve. With series of sample solns, empty and
refill cell contg 2 mg std after each detn.
% P,Os in sample = 100 x [(mg P,Os from std curve - 2)/20].
(b) Samples containing more than 5% P,Os.-Pipet 5 or 10
mL sample soln, 2.024(b). into 100 mL vol. flask. Without adding
std phosphate soln, proceed as in (a).
% P,Os in sample = 100 x (mg P20 Sfrom std
curve/mg sample in aliquot).
Gravimetric Quinolinium Molybdophosphate
Method (77)-Official Final Action
2.026 Reagents
(Store solns in polyethylene bottles.)
(a) Citric-molybdic acid reagent.-Dissolve 54 g 100% mo-
lybdic anhydride (Mo03) and 12 g NaOH with stirring in 400 mL
hot H,O, and cool. Dissolve 60 g citric acid in mixt. of 140 mL
Hel and 200 mL H20, and cool. Gradually add molybdic soln to
citric acid soln with stirring. Cool, filter, and dil. to 1 L. (Soln may
be green or blue; color deepens on exposure to light.) If
necessary, add 0.5% KBr03 soln dropwise until green color
pales. Store in dark.
(b) Quinoline soln.-Dissolve 50 mL synthetic quinoline, with
stirring, in mixt. of 60 mL HCI and 300 mL H20. Cool, dil. to 1 L,
and filter.
(e) Quimociac reagent.-Dissolve 70 g Na molybdate.2H,O in
150 mL H20. Dissolve 60 g citric acid in mixt. of 85 mL HN03 and
150 mL H20, and cool. Gradually add molybdate soln to citric
acid-HN03 mixt. with stirring. Dissolve 5 mLsynthetic quinoline
in mixt. of 35 mL HN03 and 100 mL H,O. Gradually add this soln
to molybdate-citric acid-HN03 soln, mix, and let stand 24 hr.
Filter, add 280 mL acetone, dil. to 1 L with H20, and mix.
2.027 Preparation of Solution
Treat 1 g sample as in 2.020, dilg to 200 mL.
2.028 Determination
Pipet, into 500 mL erlenmeyer, aliquot contg ,,;25 mg P20 S
and dil. to ca 100 mL with H20. Continue by one of the following
methods:
(a) Add 30 mL citric-molybdic acid reagent and boil gently 3
min. (Soln must be ppt-free at this time.) Remove from heat and
swirl carefully. Immediately add 10 mL quinoline soln from buret
with continuous swirling. (Add first 3-4 mL dropwise and
remainder in steady stream.) Or:](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-32-320.jpg)
![AOAC METHODS (1980) NITROGEN 15
2.057 Improved Kjeldahl Method for Nitrate-Free
Samples (20)-Official Final Action
(Caution: See 51.030 and 51.0S5.)
Place weighed sample (0.7-2.2 g) in digestion flask. Add 0.7
g HgO or 0.65 g metallic Hg, 15 g powd K2S04or anhyd. Na2S04,
and 25 mL H2S04, If sample >2.2 g is used, increase H2S04by 10
mL for each g sample. Place flask in inclined position and heat
gently until frothing ceases (if necessary, add small amt of
paraffin to reduce frothing); boil briskly until soln clears and
then ~30 min longer (2 hr for samples contg org. material).
Cool, add ca 200 mL H20, cool <25°, add 25 mL of the sulfide
or thiosulfate soln, and mix to ppt Hg. Add few Zn granules to
prevent bumping, tilt flask, and add layer of NaOH without
agitation. (For each 10 mL H2S04used, or its equiv. in dild H2S04,
add 15 g solid NaOH or enough soln to make contents strongly
alk.) (Thiosulfate or sulfide soln may be mixed with the NaOH
soln before addn to flask.) Immediately connect flask to distg
bulb on condenser, and, with tip of condenser immersed in std
acid and 5-7 drops indicator in receiver, rotate flask to mix
contents thoroly; then heat until all NH3 has distd (~150 mL
distillate). Remove receiver, wash tip of condenser, and titr.
excess std acid in distillate with std NaOH soln. Correct for blank
detn on reagents.
% N = [(mL std acid x normality acid) - (mL std NaOH
x normality NaOH)] x 1.4007/g sample
2.058 Improved Kjeldahl Method for Nitrate-Containing
Samples-Official Final Action
(Not applicable to liqs or to materials with high
CI:N03 ratio. Caution: See 51.030 and 51.0S5.)
Place weighed sample (0.7-2.2 g) in digestion flask. Add 40
mL H2S04contg 2 g salicylic acid. Shake until thoroly mixed and
let stand, with occasional shaking, ~30 min; then add (1) 5 g
Na2S2 0 3.5H20 or (2) 2 g Zn dust (as impalpable powder, not
granulated Zn or filings). Shake and let stand 5 min; then heat
over low flame until frothing ceases. Turn off heat, add 0.7 9
HgO (or 0.65 g metallic Hg) and 15 g powd K2S04 (or anhyd.
Na2S04), and boil briskly until soln clears, then ~30 min longer
(2 hr for samples contg org. material).
Proceed as in second par. of 2.057.
2.059
Comprehensive Nitrogen Method (21)
Official Final Action
(Applicable to all fertilizer samples.
Caution: See 51.030 and 51.079.)
Reagents
(a) Chromium metal.-100 mesh, low N (Fisher Scientific Co.
No. C-318 or Sargent-Welch Scientific Co. No. SC11432 is satis-
factory).
(b) Alundum.-Boiling stones. 8-14 mesh (Arthur H. Thomas
Co. No. 1590-D18, or equiv.).
(e) Dilute sulfuric acid.-Slowly add 625 mL H2S04to 300 mL
H20. Oil. to ca 1 L and mix. After cooling, dil. to 1 L with H20 and
mix. Avoid absorption of NH3 from air during prepn, particularly
if stream of air is used for mixing.
(d) Sodium thiosulfate or potassium sulfide soln.-160 g
Na2S20 3.5H20/L or 80 g K2S/L.
For other reagents, see 2.055.
2.0S0 Determination
Place 0.2-2.0 9 sample contg ~60 mg nitrate N in 500-800 mL
Kjeldahl flask and add 1.2 g Cr powder. Add 35 mL H20 or, with
liqs, amt to make total vol. 35 ml. Let stand 10 min with
occasional gentle swirling to dissolve all nitrate salts. Add 7 mL
HCI and let stand ~30 sec but ~10 min.
Place flask on preheated burner with heat input set at 7.0-7.5
min boil test, 2.05S(a). After heating 3.5 min, remove from heat
and let cool.
Add 22 g K2S04, 1.0 g HgO, and few granules Alundum. Add
40 mL dil. H2S04, (e). (If adequate ventilation is available, 25 mL
H2S04may be added instead of dil. H2S04, If org. matter which
consumes large amt of acid exceeds 1.0 g, add addnl 1.0 mL
H2S04for each 0.1 g org. matter in excess of 1.0 g.)
Place flask on burners set at 5 min boil test. (Pre-heated
burners reduce foaming with most samples. Reduce heat input
if foam fills ~% of bulb of flask. Use variable heat input until
this phase is past.) Heat at 5 min boil test until dense white
fumes of H2S04clear bulb of flask. Digestion is now complete for
samples contg ammoniacal, nitrate, and urea N. For other
samples, swirl flask gently and continue digestion 60 min more.
Proceed as in 2.057, second par., substituting 2.059(d) for
2.055(e).
2.0S1
Modified Comprehensive Nitrogen Method (22)
Official First Action
(Applicable to all fertilizer samples)
Reagents
See 2.055(a), (e), (f), (i), (j), (k), 2.059(a), (b), and in addn:
Copper sulfate pentahydrate (or anhydrous copper sulfate).-
Reagent grade, N-free.
2.0S2 Determination
(Caution: See 51.019 and 51.030.)
Proceed as in 2.0S0, par. 1 and 2, using 0.2-1.6 g sample. For
samples contg orgs other than urea or urea-form, use ~0.5 g
sample.
Add 15 g K2S04 or 12 g anhyd. Na2S04, 0.4 g anhyd. CUS04 or
0.6 g CuS04.5H20, and ca 0.8 g Alundum granules. Add 37 mL
H2S04 (1 +1). (If adequate ventilation is available, 20 mL H2S04
may be added instead of H2S04 (1 +1). If org. matter other than
urea exceeds 1.0 g, add addnl 1.0 mL H2 S04for each 0.1 g fat or
0.2 g other org. matter in excess of 1.0 g.)
Proceed as in 2.0S0, par. 4, substituting 75 min for 60 min in
last sentence.
Cool flask until it can be handled without gloves, and add ca
250 mL H2 0. Swirl to dissolve contents, and cool <25°. Add ca
0.8 g Alundum granules to minimize bumping, tilt flask, and
add layer of NaOH without agitation. (For each 10 ml H2S04used,
or its equiv. in H2S04(1 +1), add 15 g solid NaOH or enough soln
to make contents strongly alk.) Proceed as in 2.057, par. 2,
beginning "Immediately connect flask to distg bulb ..."
Raney Powder Method (21)
Official Final Action
(Applicable to all fertilizer samples except "nitric phosphates"
contg nonsulfate S. Caution: See 51.030 and 51.079.)
2.063 Reagents
(a) Raney catalyst powder No. 2813.-50% Ni, 50% AI (w. R.
Grace & Co., Davison Chemical Division, 10 E Baltimore St,
Baltimore, MD 21203). Caution: Raney catalyst powders react
slowly in H20 or moist air to form alumina; avoid prolonged
contact with air or moisture during storage or use.
(b) Sulfuric acid-potassium sulfate soln.-Slowly add 200 mL
H2S04 to 625 mL H20 and mix. Without cooling, add 106.7 9](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-37-320.jpg)
![AOAC METHODS (1980) OTHER ELEMENTS 21
with varying requirements of light source, burner flow rate, and
detector sensitivity, only general outline of operating parameters
is given in Table 2:04. Operator must become familiar with
settings and procedures adapted to his own app. and use table
only as guide to concn ranges and flame conditions.
(b) Disodium EDTA soln.-2.5%. Dissolve 25 g Na2H2EDTA in
L H20 and adjust to pH 7.0 with 5N NaOH, using pH meter.
Table 2:04 Operating Parameters
Wave- Range
Element length, A Flame /Lg/mL Remarks
Ca 4227 Rich Air-C,H, 2-20 1% La, 1% HCI
4227 Rich N,O-C,H, 2-20 Requires special
burner
Cu 3247 Air-C,H, 2-20
Fe 2483 Rich Air-C,H, 2-20
Mg 2852 Rich Air-C,H, 0.2-2 May need La
Mn 2795 Air-C,H, 2-20
Zn 2138 Air-C,H, 0.5-5
2.110 Standard Solutions
(Do not use <2 mL pipets or <25 mL vol. flasks. Automatic diln
app. may be used. Prep. std solns in 0-20/Lg range fresh daily.)
(a) Calcium solns.-(1) Stock soln.-25 /1-g Ca/mL. Dissolve
1.249 9 CaCOa in min. amt 3N HCI. Oil. to 1 L. Dil. 50 mL to 1 L.
(2) Working std solns.-{J, 5,10,15, and 20 /1-g Ca/mL contg 1%
La. To 25 mL vol. flasks add 0, 5, 10, 15, and 20 mL Ca stock
soln. Add 5 mL La stock soln and dil. to 25 mL.
(b) Copperstock soln.-l000 /1-g Cu/mL. Dissolve 1.000 9 pure
Cu metal in min. amt HNOa and add 5 mL HCI. Evap. almost to
dryness and dil. to 1 L with O.lN HCI.
(c) Iron stock soln.-l000 /1-g Fe/mL. Dissolve 1.000 9 pure Fe
wire in ca 30 mL 6N HCI with boiling. Dil. to 1 L.
(d) Lanthanum stock soln.-50 9 La/L. Dissolve 58.65 9 La20 a
(99.99%, Ventran Corp., Alfa Products, 8 Congress St, Beverly,
MA 01915, or equiv.) in 250 mL HCI, adding acid slowly. Dil. to
1 L.
(e) Magnesium stock soln.-l000 /1-g Mg/mL. Place 1.000 9
pure Mg metal in 50 mL H20 and slowly add 10 mL HCI. Dil. to
1 L.
(f) Manganese stock so/n.-1000 /1-g Mn/mL. Dissolve 1.582
9 Mn02 in ca 30 mL 6N HCI. Boil to remove CI and dil. to 1 L.
(g) Zinc stock soln.-l000 /1-g Zn/mL. Dissolve 1.000 9 pure
Zn metal in ca 10 mL 6N HCI. Dil. to 1 L.
(h) Other std solns.-Dil. aliquots of solns (b). (c), (e), (f), and
(g) with 0.5N HCI to make ;;.4 std solns of each element within
range of detn.
2.111 Preparation of Sample Solutions
(Caution: See 51.019, 51.025, and 51.028.)
(a) Inorganic materials and mixed fertilizers.-Dissolve 1.00
9 well ground sample in 10 mL HCI in 150 mL beaker. Boil and
evap. soln nearly to dryness on hot plate. Do not bake residue.
Redissolve residue in 20 mL 2N HCI, boiling gently if necessary.
Filter thru fast paper into 100 mL vol. flask, washing paper and
residue thoroly with H2 0. Measure absorption of soln directly,
or dil. with 0.5N HCI to obtain solns within ranges of instrument.
If Ca is to be detd, add enough La stock soln to make final diln
1% La (i.e., 5 mL La to 25 mL flask, 20 mL to 100 mL flask, etc.).
(b) Fertilizers containing organic matter (tankage, corncobs,
cottonseed hulls, etc. ).-Place 1.00 g sample in 150 mL beaker
(Pyrex, or equiv.). Char on hot plate and ignite 1 hr at 5000
with
muffle door propped open to allow free access of air. Break up
cake with stirring rod and dissolve in 10 mL HCI as in (a).
(c) Fertilizers containing fritted trace elements.- Dissolve
";;;1.00 g well ground sample in 5 mL HCIO. and 5 mL HF. Boil
and evap. to dense HCIO. fumes. Dil. carefully with H20, filter,
and proceed as in (a). Alternatively, dissolve sample in 10 mL
HC!. 5 mL HF, and 10 mL MeOH. Evap. to dryness. Add 5 mL HCI
and evap. Repeat HCI addn and evapn. Dissolve residue as in
(a). (Normally Pt ware should be used; Pyrex or other glassware
may be used if Na, K, Ca, and Fe are not to be detd.)
(d) For manganese.-(1) Acid-soluble, for both Mn+2 and
Mn+4
.-5ee (a). (b). and (c), and 2.139(b). (2) Acid-soluble, for
Mn+2 only.-5ee 2.139(a), 2.140, and 2.141*. (3) Water-soluble,
for Mn+2
only.-5ee 2.142.
(e) For iron and zinc.-(1) Aqueous extraction.-Place 1.00 9
sample in 250 mL beaker, add 75 mL H20, and boil 30 min. Filter
into 100 mL vol. flask, washing paper with H20. Dil. to vol. and
redil. if necessary. (2) Chelation extraction.-Place 1.00 g sample
in 250 mL beaker, and add 5 cm (2") mag. stirrer bar and 100 mL
2.5% EDTA soln. Stir exactly 5 min, and filter thru Whatman No.
41 paper, or equiv. If filtrate is cloudy, refilter immediately thru
fine paper (Whatman No.5, or equiv.). Redil., if necessary, with
0.5N HCI.
2.112 Determination
(P interferes in Ca and may interfere in Mg detn with air-C2H2
burners. Eliminate interference by adding La stock soln to std
and sample solns so that final dilns contain 1% La. P does not
interfere with Ca detn when N20-C2H2 flame is used.)
Set up instrument as in Table 2:04, or previously established
optimum settings for app. to be used. Less sensitive secondary
lines (Gatehouse and Willis, Spectrochim. Acta 17, 710(1961))
may be used to reduce necessary diln, if desired. Read ;;.4 std
solns within anal. range before and after each group of 6-12
samples. Flush burner with H20 between samples, and re-
establish 0 absorption point each time. Prep. calibration curve
from avo of each std before and after sample group. Read concn
of samples from plot of absorption against /1-g/mL.
2.113 Calculations
% Element = (/1-g/mL) x (F/sample wt) x 10-4
,
2.114
F = mL original diln x mL final diln/mL aliquot, if
original 100 mL vol. is dild.
Acid-Soluble Boron (40)-Official Final Action
Apparatus
Use high sensitivity glass electrode pH meter for titrn. Use
assembly with burets, electrodes, and mech. stirrer, arranged
for convenient use with 250 mL beaker. Use ordinary 50 mL
burets for the 0.025N NaOH and 0.02N HCI.
2.115 Reagents
(a) Boric acid std soln.-Dissolve 1 g HaBOa in H20 and dil. to
1 L. 1 mL = 0.1748 mg B.
(b) Sodium hydroxide std soln.-C02-free, ca 0.025N. Stdze as
follows: Pipet 25 mL std HaBOa soln into 250 mL beaker, add 3.0
g NaCI, acidify to Me red, dil. to 150 mL, boil to expel CO2, cool,
and titr. potentiometrically as in 2.116. Det. blank by repeating
titrn, substituting 25 mL H20 for HaB03 soln. Calc. B equivalence
as follows:
mg B/mL = 4.369/[(mL NaOH soln) - (mL blank)].
Protect from atm. CO2 by soda-lime tubes or other suitable
means.
(c) Methyl red indicator.--Dissolve 0.1 9 Me red in 50 mL
alcohol, dil. to 100 mL with H20, and filter if necessary.](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-43-320.jpg)
![AOAC METHODS (1980) OTHER ELEMENTS 25
2.145 Determination
Place 2 g sample in 300 mL erlenmeyer and add 10 mL HN03
and 5 mL H2S04• Digest on hot plate to white fumes. If soln
darkens, owing to org. matter, cool slightly, add little more
HN03, and digest again to white fumes, repeating operation if
necessary until org. matter appears to be destroyed. Cool, add
50 mL H20, boil ca 1 min, and cool to room temp.
Add bromocresol green, then NH40H until indicator changes
to light green (pH 4). Cool again to room temp., and if indicator
changes back to more acid color, add NH4 0H dropwise until
indicator becomes light green again, avoiding excess. Add 2 g
NH4HF2 (Toxic. Caution: See 51.084), mix well, and let stand ca
5 min. Add 8--10 g KI, mix well, and titr. with std Na2S20 3 soln to
light yellow. Add ca 1 mL starch soln and continue titrg slowly
until color is nearly same as just before addn of the KI and
becomes no darker on standing 20 sec. Report as % Cu.
2.146 Atomic Absorption Method
See 2.109-2.113.
Sodium-Official First Action
Flame Photometric Method (53)-Official First Action
2.147 Reagents
See 2.091(aHc), and in addn:
Sodium chloride.-Dry 2 hr at 105°.
2.148 Preparation ofSolution
Prep. soln as in 2.093(a), using 2.5 g sample «4% Na) or 1.25
g (4-20% Na).
2.149 Preparation ofStandard Curve
(a) Samples containing 1% or more sodium.-Proceed as in
2.094, using 1.2716 g NaCI, range of diln 0-40 ppm Na, intervals
~5 ppm, and full scale for 40 ppm Na.
(b) Samples containing less than 1% sodium.-Proceed as in
2.094, using 1.2716 g NaCI, range of diln 0-10 ppm Na, intervals
2 ppm, and full scale for 10 ppm Na.
2.150 Determination
(Caution: See 51.007.)
Transfer 25 mL «4% Na) or 10 mL (4-20% Na) sample soln
to 250 mL vol. flask, dil. to vol. with H20, and mix (if internal std
instrument is used, add required amt LiN03 before dilg to voL).
Atomize portions of sample several times to obtain reliable avo
readings for each soln. Det. ppm Na from std curve (a) or (b).
Calc. % Na as follows:
0-4%: ppm Na!10 = % Na
4-20%: ppm Na!2 = % Na
Zinc-Official Final Action
2.151 * Gravimetric Method (54) *
(For samples contg ;;.0.1 % Znj
Digestion with HN03 and H2S04, sepn of interfering sulfides
from acid soln with H2S, pptn ofZnS at pH 3 with H2S, and ignition
to ZnO. See 2.138, 12th ed.
2.152 * Colorimetric Method (55) *
(For samples contg <4% Zn)
Digestion with HN03 and H2S04, and detn with dithizone. See
2.139, 12th ed.
Zincon Ion Exchange Method (56)
(Clean all glassware with hot chromic acid or HN03 (1 + 1). Rinse
thoroly with H20. Caution: See 51.023, 51.026, and 51.030.)
2.153 Reagents
(a) Anion exchange resin.-100-200 mesh, strong base, poly-
styrene alkyl quaternary amine, 7% cross linkage, CGA-540 (J.
T. Baker Chemical Co., No. 4602, or equiv.).
(b) Zincon indicator.-Dissolve 0.12 g zincon (0-[[0:-[(2-
hydroxy-5-sulfophenyl)azo] benzyl idene] hydrazino] benzoic acid,
Na salt) (J. T. Baker Chemical Co., No. X690) in 5 mL 0.3N NaOH
and dil. to 100 mL with H2 0. Prep. fresh weekly.
(c) Hydrochloric acid solns.-(T) O.5N.-Dil. 20 mL HCI to 500
mL with H20. (2) O.25N.-Dil. 2 mL HCI to 100 mL with H20. (3)
O.005N.-Dil. 2.5 mL HCI to 6 L with H20.
(d) Borate buffer soln.-pH 9.8. Dissolve 4 g H3B03 in 140 mL
H20. Add 5 mL NH40H by pipet and then dropwise to pH 9.8.
Check daily.
(e) Ammonium thiocyanate.-1M. Dissolve 0.76 g NH4 CNS in
10 mL H2 0.
(f) Zinc std solns.-(T) Stock soln.-1000 ppm. Dissolve 1.000
g pure Zn metal in small amt HCI-HN03 (1+1). Evap. to small
vol., add 3 mL HCI, and heat. Oil. to 1 L with H20. (2) Working
soln.-10 ppm. Oil. 10 mL stock soln to 1 L with H20.
(g) Sodium hydroxide soln.-{).3N. Dissolve ca 1.25 g NaOH
in 100 mL H20.
2.154 Preparation of Resin Column
Wash 12 g new resin in 250 mL beaker with H20 until washings
are neut. Introduce resin as slurry into 25 x 2.2 cm chromatgc
tube with fritted glass disk and stopcock at bottom. Mark vol.
levels on column at 10,40, and 50 mL above packed resin and
on 250 mL separator at 90 mL. (Keep resin wet and store under
liq. when not in use.) Connect separator to top of column thru
stopper. Attach inverted U-shaped glass dispensing tube to 250
mL vol. flask thru vented stopper or cork and connect with Zn-
free plastic tubing to stopcock of column with stopcock grease.
See Fig. 2:08.
Mount reservoir (aspirator bottle or carboy) contg ;;. 1 L 0.005N
HCI high enough to effect backwashing. Attach Zn-free tubing
and pinch clamp.
2.155 Flow Calibration
Use sweep sec hand of watch or stopwatch to establish flow
rates. Det. number drops!mL leaving dispensing tube. Remove
separator and vol. flask; drain and then backwash resin (see
2.157). Remove reservoir tubing, open stopcock, elute 40 drops
from dispensing tube, and measure vol. Use this factor to
convert 0.5 mL/min (required in Zn elution, 2.157) to drops/sec.
2.156 Preparation of Sample
Remove separator and elution tubing from column. Activate
resin by draining column and adding 50 mL 0.5N HCI. Drain
column to 40 mL mark.
(a) Samples containing 0.14% or more zinc. -Dissolve 1.000
g well-ground sample in 10 mL HCI and 5 mL HN03 in 250 mL
beaker. Evap. to near dryness on hot plate. (Caution: Do not
bake.) Redissolve residue in ca 40 mL 0.5N HCI, boiling gently
if necessary. Filterthru Whatman No. 41 paper into 100 mL vol.
flask. Thoroly wash residue and dil. filtrate to vol. with 0.5N HCI.
Drain column to 10 mL mark. Tap column to pack resin. Pipet
aliquot contg 0.7-0.8 mg Zn onto column. Elute sample soln at
ca 5 sec/drop.
* Surplus method-5ee inside front cover.](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-47-320.jpg)
![28 2. FERTILIZERS AOAC METHODS (1980)
than those in H20. Observed pH in Cael2 soln is virtually
independent of initial amt salt present in soil, whereas pH
readings in H20 can be modified by salts such as fertilizer
materiaL)
2.173 Ash (61)-Official Final Action
Place uncovered (retain cover for weighing) Vycor or porcelain
dish contg dried sample from moisture detn in furnace. Gradually
bring to 5500
and hold until completely ashed. Cover with
retained AI foil cover, cool, and weigh.
% Ash (report to nearest 0.1%) = g ash x 100/g as-received
sample taken for moisture detn. (If moisture Method II was used,
g as-received sample = 10.0.)
Sand (60)---Procedure
2.174 Preparation ofSample
Air-dryas in 2.167.
2.175 Determination
(Caution: See 51.056.)
Place 25 g air-dried, ground sample into 125 mL tall-form
beaker, or equiv. Nearly fill beaker with CHCI3, stir briefly, and
let settle ca 1 min. With spoon, discard most floating org.
material, decant remaining org. material and CHCI3, taking care
not to disturb settled portion (sand), and air-dry to remove
residual CHCI3• (Stirring aids drying.)
When dry, weigh settled portion and calc. as % sand (includes
other minerals present such as limestone, etc.).
% Sand = (g air-dried settled residue x 100)/g air-dried sample
2.176 Organic Matter (67)-Official Final Action
% Org. matter = 100.0 - (% moisture + % ash).
2.177 Total Nitrogen (60)---Procedure
Det. N as in 2.057, using well mixed, air-dried, ground sample
equiv. to 10.0 g sample on as-received basis.
Det. g air-dried sample equiv. to 10.0 g as-received sample as
in 2.167.
Water Capacity and Volumes (62)
Official Final Action
2.178 Apparatus
Dispensing apparatus.-2 dispensing burets, 250 mL in 1 mL
subdivisions, ±2 mL tolerance, pinchcock type; 1-hole No.6
rubber stopper; straight polyethylene drying tube with serrated
rubber tubing fittings, 15 cm long, %" od, %" id (Cenco Instru-
ment Corp. No. 14782-2); and stainless steel screen circle, ca
16 mesh and 28.7 mm diam.
Assemble dispensing app. as follows: Discard serrated rubber
tubing fittings from polyethylene drying tube and use tube only.
Center stainless steel screen on one end of tube and seal.
(Soldering iron is usefuL) Adjust length of tube to match con-
venient graduation of buret; then scallop end without screen to
allow for H20 drainage, and insert into dispensing buret with
screen side up.
2.179 Preparation ofSample
See 2.165.
2.180 Determination
Det. moisture content on sep. sample by 2.166 or 2.167.
Weigh buret fitted with plastic tube and screen. Working
rapidly to prevent moisture losses, mix sample thoroly, place
on top of NO.4 screen, and shake until sieving is complete. Use
only portion that has passed thru sieve for detn. Firmly pack
buret with 25 cm (10") of 4 mesh sample as follows: Attach
rubber stopper to delivery end of buret. Add ca 20 mL portions,
firmly tapping 3 times vertically from ht of 15 cm (6") on rubber
stopper, for final ht of 25 cm. (This will ensure that ht of final
wet vol. is 19--25 cm.) Remove stopper; weigh buret to nearest
g. Position buret to use sink as drain. Place H20 source (19 L
(5 gaL) bottle) equipped with siphon device above level of buret.
Connect clamped rubber tubing of siphon device to buret with
glass tubing (ca 13 cm (5") long, constricted at one end) inserted
into one-hole rubber stopper fitting tightly into top of buret.
Attach rubber tubing with pinch clamp to delivery end of buret.
Open both clamps and pass H20 thru sample ~24 hr, maintaining
water reservoir over sample at all times. (Moss-type samples
may float but gradually settle as sample becomes wet.) After
initial soaking, regulate H20 flow thru column by adjusting screw
clamp at delivery end of buret. On-flow of H20 should be ca
equal to out-flow; flow of ca 1 drop/sec is suitable.) When
sample is supersatd, close both clamps and let sample settle in
H20 ca 5 min. Top surface of sample should be as level as
possible.
Raise buret and replace rubber tubing on delivery end of buret
with 250 mL dispensing buret filled with H20, using rubber
stopper for connection. Connect two burets tightly, with no air
leaks. Remove siphon device and open outlet clamps of both
burets to empty. (Suction created is equiv. to ca 38 cm (15") H20.
Check for air leaks to ensure that std suction is exerted on
sample. It is important to remove excess H2 0 as described.)
Measure ht of wet peat. Ht should be 19--25 cm. Record vol. in
mL and weigh buret, plastic tube with screen, and wet peat to
nearest g.
Wet sample again as above ~ 1 hr, drain by suction, record
vol., and weigh. Repeat until consistent results are obtained.
2.181 Calculations
(a) Saturated Volume Weights, g/mL
As-recd = g as-recd sample/mL wet vol.
Oven-dried = g dried sample/mL wet vol., where g dried
sample = g as-recd sample x [(100 - % moisture)/100j.
Wet = g wet sample/mL wet vol.
(b) Water-Holding Capacity, %
(1) Weight basis:
As-recd = [(g wet sample - g as-recd sample) x 100]/g
as-recd sample
Oven-dried = [(g wet sample - g dried sample) x 100]/g
dried sample
(2) Volume basis:
Water vol. = [(g wet sample - g dried sample) x 100j/(mL
wet vol. x 1.0)
(e) Dry Peat Volume, %
Dry peat vol. = (g dried sample x 100)/(mL wet vol. x 1.5)
(d) Air Volume, %
Air vol. = 100 - (% water vol. + % dry peat voL)
2.182
Alternative Methods (60)
Volume---Procedure
Principle
Method consists of dividing particles of peat from original
container by passing them thru 12.7 mm (0.5") sieve and allowing
them to fall into voL-measuring container.](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-50-320.jpg)
![48 3. PLANTS AOAC METHODS (1980)
min at time. Repeat grinding frequently during 30 min; then
without delay, transfer quant. to 100 mL vol. flask with H20. Add
3 mL 5% uranyl acetate soln to ppt protein, dil. to vol. with H20,
mix well, and centrf. portion of mixt. Pipet 10 mL clear supernate
into 25 x 150 mm test tube. Add ca 100 mg Celite, 5 mL 20%
NaCI soln, and 2 mL I-KI reagent, and mix well. Let stand
overnight, centrf., and decant.
Wash starch-I ppt by suspending it in 5 mL alc. NaCI soln,
centrf., and decant. Add 2 mL alc. NaOH soln to packed ppt.
Gently shake and tap tube until ppt is no longer blue. (Do not
use stirring rod; allow ample time for complex to decompose.)
Wash walls of tube with 5 mL alc. NaCI soln, centrf. liberated
starch, and wash with 5 mL alc. NaCI soln as before. Add 2 mL
0.7N HCI to ppt. Stopper tube loosely with size 00 crucible, and
heat 2.5 hr in boiling H20 bath. (Bath should have cover with
holes to accommodate tubes; holes not occupied by tubes must
be covered.) Cool, and transfer quant. to 25 mL vol. flask. Add
drop phenol red, 50.008, and neutze with IN NaOH. Discharge
color with O. TN oxalic acid, dil. to vol., and mix well. Transfer
5 mL aliquot to 25 x 200 mL Pyrex test tube, add exactly 5 mL
Somogyi reagent, and stopper tube with size 00 crucible. Heat
together with several blanks contg 5 mL H20 and 5 mL Somogyi
reagent in vigorously boiling H20 bath exactly 15 min. Remove
tube from bath and cool to 25-30°. Add 1 mL 2.5% KI soln down
wall of tube without agitation and then add 3 mL 1.5N H2S04
rapidly with agitation. After all Cu20 dissolves, titr. soln with
0.005N Na2S203' adding starch indicator, (h)' just before end
point is reached. Treat blank solns similarly.
% Starch = [50(mL blank - mL sample)
x 0.90/mg sample] x (N/0.005) x G x 100,
where 50 = diln factor, 0.90 = factor glucose to starch, N =
actual normality Na2S203 soln, and G = mg glucose equiv. to 1
mL 0.005N Na2S20 3•
Lignin (35)-Official Final Action
Direct Method
3.122 Preparation ofSample
Grind sample in mill to pass No. 80 sieve and dry at 105°. Ext
wei~hed sample (5-10 g) 30 hr in Soxhlet app. with alcohol-
benzene soln (32 parts alcohol and 68 parts benzene by wt). Dry
material in oven to free it from solvs and place in flask of
suitable size. Add 150 mL H20/g sample, and reflux 3 hr. Filter
mixt. while still hot, preferably thru weighed fritted glass cru-
cible, and transfer extd material to flask. Add 1% HCI (111 g
coned HCI + 3890 mL H20) in proportion of 150 mL acid soln/g
plant material, and reflux 3 hr. Filter mixt. while still hot thru
fritted glass crucible previously used, wash with H20 until acid-
free, dry at 105°, and weigh. Calc. % total loss due to successive
extn with alcohol-benzene soln, hot H20, and 1% HCI. (With
samples not especially rich in carbohydrates and proteins, extn
with hot H20 may be omitted.)
3.123 Apparatus
App., Fig. 3:06, consists of: (1) 1.5 L bottle, A, to which is
attached by 2-hole rubber stopper 250 mL dropping funnel, C,
having lower end of stem bent as illustrated and placed close
to bottom of A; (2) Drechsel gas-washing bottle, D; (3) 3 Pyrex
test tubes, 38 x 300 mm diam., G, G', G", connected in parallel
by device, 0, and immersed in wooden box, L, filled with
crushed ice, H; and (4) bottle contg H20 for absorption of excess
HCI, K. G, G', and G" are provided with 2-hole rubber stoppers;
glass tube with right angle bend extends thru 1 hole nearly to
bottom of test tube, and similar tube extending ca 10 mm into
test tube passes thru other hole. Rubber connections and
stopcocks for regulating flow of gas are provided as indicated
in diagram. A is filled with ca 500 mL H2S04 and C with HCI; HCI
flowing thru stoptock B into A generates HCI gas, which is dried
by H2S04 in D, and flows into G, G', and G" contg samples and
fuming HCI reagent.
3.124 Reagent
Fuming hydrochloric acid.-(Caution: See 51.031.) Density
1.212-1.223 at 15°. To 500 g NaCI in 1 L g-s Pyrex distg flask,
add cold soln of 250 mL H20 in 450 mL H2S04• Connect side tube
of distg flask to glass tube passing thru H2S04 wash bottle, and
connect outlet tube of H2S04 wash bottle to another glass tube,
immersed in flask contg 3 L HCI. Surround flask contg HCI with
crushed ice. Heat distg flask with small flame and pass HCI gas
into acid soln until it attains sp gr of 1.212-1.223 at 15°. Keep
"
FIG. 3:06--Apparatus for determining lignin](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-70-320.jpg)
![AOAC METHOOS (1980) TOBACCO 53
contg ~0.1 9 alkaloids; if spectrophtric, use ~2 9 sample.) (If
Griffith still is used, use 0.05--0.2 9 sample.) Place 25 mL HCI
(1 +4) in receiver (1 L vol. flask is desirable) and place receiver
so that condenser tube dips into acid. (With Griffith still, use 10
mL HCI (1 +4) in 250 mL vol. flask.) Add 50 mL alkali-salt soln
to distn flask so that sample is rinsed into bottom of flask. (With
Griffith still, use 5 mL alkali-salt soln.) If large vol. of liq. is
required for proper function of still, add more alkali-salt soln;
do not dil. Connect flask to app. immediately and steam distil
with as rapid current of steam as can be condensed efficiently.
Effluent condensate should not be above room temp. Apply
heat to distn flask from burner, mantle, or other heat source to
keep vol. in flask approx. const. Collect ca 900 mL condensate
(or distil addnl 100 mL after condensate shows no nicotine by
silicotungstic acid test). (With Griffith stili, collect 225 mL.) Dil.
distillate to vol.
3.155 Determination
(a) Spectrophotometric.-Dil. aliquots of distillate (if neces-
sary) with O.OSN HCI so that A at 259 nm is 0.5--0.8 and read A
at 236, 259, and 282 nm. Calc. correctedA'259 = 1.059 x [observed
A 259 - 'h (A236 + Am)] after correcting all observed A values to
original distillate vol. basis. Concn, c, of alkaloids as nicotine in
gjL is given by c = A'259 /(a x b), where a is absorptivity at 259
nm, and b is cell length in cm.
% alkaloid (as nicotine) = c x vol. distillate (L) x 100/g
sample.
(b) Gravimetric.-Det. alkaloids in distillate as in 6.176, but
double amt of silicotungstic acid specified, i.e., 2 mL/each 10
mg alkaloids expected.
Cundiff-Markunas Method (45)-OHicial Final Action
(Total alkaloids (as nicotine), tertiary alkaloids (as nicotine)' and
secondary alkaloids (as nornicotine))
3.156 Reagents
(a) Benzene-chloroform soln.-Mix equal parts by vol. of
benzene and CHCI3 and sat. with H20.
(b) Sodium hydroxide soln.-36%. Dissolve 500 9 NaOH in
H2 0 and dil. to 1 L.
(c) Dilute acetic acid.-5%. Dil. 50 mL HOAc to 1 L with H20.
(d) Crystal violet indicator.-Dissolve 0.5 9 crystal violet in
100 mL HOAc.
(e) Perchloric acid std soln.---O.025N. Add 4.7 mL 72% HCIO.
to freshly opened 51b bottle HOAc and mix. (Caution: See 51.022
and 51.028(a) and (d).) Stdze as follows: Accurately weigh 0.1 9
KH phthalate (NBS) into 125 mL erlenmeyer, add 50 mL HOAc,
and heat to dissolve. Cool, add 2 drops indicator, and titr. to
blue-green end point. Perform blank titrn on 50 mL HOAc and
2 drops indicator soln, and correct vol. of titrant.
N = wt KH phthalate x 4.896/mL HCIO. soln.
3.157 Determination
Accurately weigh 2.5 9 finely ground tobacco into 250 mL
erlenmeyer. Add 15 mL 5% HOAc and swirl until tobacco is
thoroly wetted. Pipet 100 mL benzene-CHCl3 soln into flask, and
then 10 mL 36% NaOH soln. Stopper flask tightly and shake 20
min, using wrist-action shaker. Add 4.5-5 9 (2 teaspoonfuls)
Filter-Cel, mix, and filter most of benzene layer thru Whatman
No.2 paper into second flask. If filtrate has any turbidity, add
2-2.5 9 (1 teaspoonful) addnl Filter-Cel and refilter thru Whatman
No.2 paper. Filtrate must be clear.
Pipet 25 mL aliquots of filtrate into each of two 125 mL
erlenmeyers. Pass stream of air over surface of soln in first flask
5 min, add 2 drops indicator, and titr. to green end point with
0.025N HCIO•. Add 1.0 mL Ac20 to second flask and let stand
~15 min. Add 25 mL HOAc and 2 drops indicator, and titr. to
blue-green end point with 0.02SN HCIO•. Take first appearance
of blue-green thruout soln as end point. For each series of
analyses perform blank titrns and correct respective vols of
titrant.
Calc. % alkaloids as follows: % total alkaloids (as nicotine) =
V, x N x 32.45/wt sample; % tertiary alkaloids (as nicotine) =
(2V2 - V,) x N x 32.45/wt sample; % secondary alkaloids (as
nornicotine) = 2(V, - V2 ) x N x 29.64/wt sample; where V, =
vol. titrant for nonacetylated aliquot; V2 = vol. titrant for acet-
ylated aliquot; and N = normality HCIO•.
3.158
Nicotine on Cambridge Filter Pads
Gas-Liquid Chromatographic Method (46)
Official First Action
Apparatus and Reagents
(a) Gas chromatograph.-With flame ionization detector,
heated injection port, and thermostated column oven. Following
conditions have been found satisfactory: Column, 1.8 m (6') x
'Is" stainless steel; packing, 2% KOH and 10% Carbowax 20M
(based on final packing wt) on 45-60 mesh calcined diat. earth
(such as Chromosorb W, or equiv.), resieved before use to mesh
range to remove fines and lumps; temps (0): column 165,
detector and injection port 200-250; carrier gas flow, ca 40
mL/min. Adjust H and air flows for max. sensitivity and stability.
Under these conditions, column should have ht equiv. to the-
oretical plate (HETP) <1 mm and resolution of >2, calcd with
nicotine and anethole.
(b) Measuring system.-Measure peak areas with electronic
integrator or other system with resolution of ~1 count/mv-sec.
(e) Mechanical shaker.-Capable of extg ~99% nicotine. Bur-
rell Wrist-Action shaker has been found satisfactory.
(d) Extracting soln.-2-Propanol contg 1 mg anethole/mL as
internal std for nicotine. If H20 is also to be detd, add 20 mg
::tOH/mL 2-propanol as addnl internal std.
(e) Nicotinestdsolns.-( 1) Stockso/n.-Weigh 2.500 9 nicotine,
3.153, or equiv. amt of nicotine salt. Transfer quant. into 100 mL
vol. flask, and dil. to vol. with extg soln. (2) Working std solns.-
Pipet 1,2,3,4, and 5 mL stock soln into five 100 mL vol. flasks,
and dil. to vol. with extg soln (0.25, 0.50, 0.75, 1.00, and 1.25 mg
nicotine/mL). (Caution: See precaution in 3.153.)
3.159 Extraction
Place Cambridge filter material in flask or serum bottle ac-
comodated by shaker used, add 10.00 mL extg soln, stopper,
and shake until ~99% of nicotine is extd (usually ca 15 min).
3.160 Standardization
Prime column with aliquots of 1.25 mgjmL std soln. Let
baseline stabilize, inject 1 ILL each std soln in succession, and
repeat sequence 3 times. Det. area ratio (nicotine:anethole) for
each injection, and calc. slope and intercept of response curve,
preferably by method of least squares (See Definition of Terms
and Explanatory Notes No. (24)). Correlation coefficient should
be ~0.99 and intercept ,,;;0.05 mgjmL.
3.161 Determination
Prime column with aliquots of ext, 3.159. Let baseline stabilize,
and inject 1 ILL of each sample soln. Calc. nicotine concn in soln
(C, mg/mL) = mx + b, where m = slope of stdzn curve, b
intercept, and x = area ratio of nicotine to anethole.
Nicotine yield/cigaret = (C x 10.00)/(No. cigarets/pad)](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-75-320.jpg)
![62 4. DISINFECTANTS AOAC METHODS (1980)
polysorbate 80, and 1.25 mL phosphate buffer, (e); dil. with H20
to 1 L and adjust to pH 7.2. Dispense in 100 mL portions and
autoclave 20 min at 121°.
(d) Neutralizer blanks.-For use with ",;200 ppm quaternary
NH, compd. Mix 100 mL neutralizer stock soln, (cl, 25 mL 0.25M
phosphate buffer stock soln, (e), and 1675 mL H20. Dispense 9
mL portions into 20 x 150 mm tubes. Autoclave 20 min at 121°.
(e) Phosphate buffer stock soln.-O.25M. Dissolve 34.0 g
KH2PO, in 500 mL H20, adjust to pH 7.2 with 1N NaOH, and dil.
to 1 L.
(f) Phosphate buffer dilution water.-Add 1.25 mL 0.25M
phosphate buffer stock soln, (e), to 1 L H20 and dispense in 99
mL portions. Autoclave 20 min at 121°.
(g) Test organisms.-Use Escherichia coli ATCC No. 11229 or
Staphylococcus aureus ATCC 6538. Incubate 24 and 48 hr, resp.
Maintain stock cultures on nutrient agar (AOAC), (a)(3), at
refrigerator temp.
4.024 Resistance to Phenol of Test Cultures
Det. resistance to phenol at least every 3 months by
4.001-4.005. Resistance of E. coli should be equiv. to that
specified for S. typhi in 4.004 and that for Staph. aureus equiv.
to that specified for this organism in 4.005.
4.025 Apparatus
(a) Glassware.-250 mL wide-mouth erlenmeyers; 100 mL
graduate; Mohr, serological, and/ or bacteriological (APHA spec-
ification) pipets; 20 x 150 mm test tubes. Sterilize at 180° in hot
air oven ;,,2 hr.
(b) Petri dishes.-Sterile.
(c) French square bottles.-175 mL, borosilicate. Use of other
containers will give variable results.
(d) Water bath.-Controlled at 25°.
4.026 Preparation of Culture Suspension
From stock culture inoculate tube of nutrient agar A,
4.023(a)( 1), and make ;,,3 consecutive daily transfers (",;30),
incubating transfers 20-24 hr at 35-37°. Do not use transfers
>30 days. If only 1 daily transfer has been missed, no special
procedures are required; if 2 daily transfers are missed, repeat
with 3 daily transfers.
Prep. 175 mL French square culture bottles contg 20 mL
nutrient agar B, 4.023(a)(2), autoclave 20 min at 121°, and let
solidify with bottle in horizontal position. Inoculate culture
bottles by washing growth from slant with 5 mL phosphate
buffer diln H20, 4.023(f)' into 99 mL phosphate buffer diln H20,
and adding 2 mL of this suspension to each culture bottle, tilting
back and forth to distribute suspension; then drain excess liq.
Incubate 18-24 hr at 35-3]D, agar side down. Remove culture
from agar surface of 4 or more bottles, using 3 mL phosphate
buffer diln H20 and glass beads in each bottle to suspend
growth. Filter suspension thru Whatman No.2 paper prewet
with 1 mL sterile phosphate buffer, and collect in sterile tube.
(To hasten filtration, rub paper gently with sterile policeman.)
Stdze suspension to give avo of 10 x 109
organisms/mL by diln
with sterile phosphate buffer diln H20, 4.023(f).
If Lumetron colorimeter is used, dil. suspension in sterile
Lumetron tube to give % T according to Table 4:02.
If McFarland nephelometer and BaSO, stds are used, select
7 tubes of same id as that contg test culture suspension. Place
10 mL of each suspension of BaSO" prepd as indicated in Table
4:03, in each tube and seal tube. Stdze suspension to correspond
to No.4 std.
Table 4:03 Preparation of BaSO, Suspensions Corresponding
to Bacterial Concentrations
mL 2% BaCl, mL 1% H2SO4 Av. Bacterial
Std No. Soln (v/v) Soln Count/mL
1 4.0 96.0 5.0 x 10'
2 5.0 95.0 7.5
3 6.0 94.0 8.5
4 7.0 93.0 10.0
5 8.0 92.0 12.0
6 10.0 90.0 13.5
7 12.0 88.0 15.0
4.027 Synthetic Hard Water
Prep. Soln 1 by dissolving 31.74 g MgCI2 (or equiv. of hydrates)
and 73.99 g CaCI2in boiled distd H20 and dilg to 1 L. Prep. Soln
2 by dissolving 56.03 9 NaHCOa in boiled distd H20 and dilg to
1 L. Soln 1 may be heat sterilized; Soln 2 must be sterilized by
filtration. Place required amt Soln 1 in sterile 1 L flask and add
;,,600 mL sterile distd H20; then add 4 mL Soln 2 and dil. to 1
L with sterile distd H20. Each mL Soln 1 will give a water equiv.
to ca 100 ppm of hardness caicd as CaC03 by formula:
Total hardness as ppm Cae03
= 2.495 x ppm Ca + 4.115 x ppm Mg.
pH of all test waters ",;2000 ppm hardness should be 7.6-8.0.
Check prepd synthetic waters chemically for hardness at time
of tests, using following method or other methods described in
14th Ed. of Standard Methods for the Examination of Water,
Sewage, and Industrial Wastes.
4.028 Hardness Method
(a) EDTA std soln. -Dissolve 4.0 9 Na2H2EDTA.2H20 and 0.10
g MgCI2.6H20 in 800 mL H20 and adjust by subsequent diln so
that 1 mL of soln is equiv. to 1 mg CaC03 when titrd as in (e).
Check EDTA soln after prepn or, if com. purchased, against
CaC03 std at least every 2 months.
(b) Calcium std soln.-1 mL = 1 mg CaC03 • Weigh 1.00 9
CaCOa, dried overnight or longer at 105°, into 500 mL erlenmeyer
and add dil. HCI thru funnel until CaeOa is dissolved. Add 200
mL H20, boil to expel CO2, and cool. Add few drops Me red
indicator and adjust color to intermediate orange with dil. NH,OH
or Hel as required. Transfer quant. to 1 L vol. flask and dil. to
vol.
Table 4:02 Per Cent Light Transmission at Various Wavelengths Corresponding to Bacterial Concentrations
% Light Transmission with Filters. nm
Av. Bacterial
370 420 490 530 550 580 650 Count/mL
7.0 4.0 6.0 6.0 6.0 7.0 8.0 13.0 X 10'
8.0 5.0 7.0 7.0 7.0 8.0 9.0 11.5
9.0 6.0 8.0 8.0 8.0 9.0 10.0 10.2
10.0 7.0 9.0 9.0 9.0 11.0 11.0 8.6
11.0 8.0 10.0 10.0 10.0 12.0 13.0 7.7
13.0 9.0 12.0 12.0 12.0 13.0 15.0 6.7](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-84-320.jpg)
![5. Hazardous Substances
PAINT
lead (1)-Official Final Action
5.001 Reagents and Apparatus
(a) Lead std salns.-(1) Stock saln.-1 mg Pb/mL 1% HN03 •
Dissolve 159.9 mg Pb(N03)2 in HN03 (1 +99) and dil. to 100 mL
with HN03 (1 +99). (2) Intermediatesaln.-300 p,g/mL dil. HN03 ·
Oil. 15 mL stock soln to 50 mL with 0.5 mL HN03 and H20. (3)
Working salns.-To each of seven 100 mL vol. flasks contg 1 mL
HN03, add resp. 0, 1, 2, 3, 4, 5, and 6 mL intermediate soln and
dil. to vol. with H20 (0,3,6,9,12,15, and 18 p,g Pb/mL).
(b) Atomic absorption spectrophatameter.-With Pb hollow
cathode lamp and 4" single slot or 3 slot Boling burner head,
capable of detecting 0.5p,g Pb/mL, such as Perkin-Elmer Model
403. Operating conditions: 283.3 nm, 0.7 nm band width slit,
recorder response (if used) 0.2~1 sec time constant, air-C2H2
flame, with gas flows adjusted according to directions of man-
ufacturer.
(e) Heater for digestion.-Drill 7.5 cm AI block to hold ;;.16
test tubes, 16 x 150 mm. Place on hot plate capable of main-
taining medium at 160-170° (Corning PC 35, or equiv.). Sand
bath may be used instead of AI block.
(d) Boiling chips.-Unglazed boiling chips, 1.5 mm diam., Pb-
free.
5.002 Determination of Solids
Thoroly mix samples manually for 10 min or mech. for 5 min.
Accurately weigh 0.3-0.4 g into weighed AI dish, 63 mm diam.
Add 3-5 mL hexane or pet ether to oil-based paints or H20 to
latex paints and swirl to disperse. Warm on hot plate while
swirling until solv. has evapd and film is formed. Heat in oven
4 hr at 105°, cool, and weigh.
% Solids = g dried sample x 100/g sample.
5.003 Determination of Lead
Introduce ca 0.6 g (0.3 mL) thoroly mixed sample near bottom
of 16 x 150 mm test tube with syringe and weigh accurately.
Add 5±0.2 mL HN03 and 2 boiling chips to each, including
blanks. Place in block or bath at 90-100° so that liq. surface is
slightly above heated surface. (Use hood.) After initial fuming
has subsided, increase temp. until vapors are condensing in
top 1-2 cm of tube (bath temp., 160-170°) and maintain at this
temp. 3 hr. Cool to 50-60°, transfer to 25 mL vol. flask, including
chips and any ppt, and rinse with four 4 mL portions H20,
transferring as much residue as possible. Oil. to vol. with H20
and let settle 0.~1 hr. Floating residue may be removed by
aspiration thru disposable pipet.
Aspirate solns and stds into AA spectrophotometer, avoiding
introduction of ppt. If A of sample is greater than highest std,
dil. sample and re-aspirate. Det. p,g Pb/mL from std curve.
% Pb in paint solids = (p,g Pb/mL) x F x 10-2/
(g sample x % solids in sample)
F (diln factor) = 1/[{1/25) (b/c) (die) ...J,
where 25 = vol. original sample digest, b = aliquot of original
25 mL dild to c mL; d = aliquot of c (mL) dild to e mL; etc. For
dry paint films, % solids in sample = 100.
69
PREPARATIONS CONTAINING FLUORIDES
Fluoride (2)-Official Final Action
5.004 Apparatus
(a) pH meter.-With expanded mv scale (digital Model 110,
Corning Scientific Instruments, Medfield, MA 02052, or equiv.).
fluoride ion-selective electrode (Model 94-09, Orion Research
Inc., 11 Blackstone St, Cambridge, MA 02139, or equiv.), and
single junction ref. electrode, plastic sleeve-type (Model 90-01,
Orion Research Inc., or equiv.).
(b) Magnetic stirrer.-With Teflon-coated stirring bar. Use
asbestos or foam mat to insulate sample from motor heat.
(c) Beakers.-4.5 oz (135 mL). polypropylene, or equiv.
(d) Graph paper.-Linear or semi-antilog, vol. corrected No.
90-00-90 Gran's plot paper (Orion Research Inc., or equiv.).
5.005 Reagents
(a) Buffer soln.-pH 6.0. Add 77.0 g NH40Ac and 0.452 g NH4
citrate to 1 L H20. Adjust to pH 6.0 with HOAc.
(b) Fluoride std soln.-1 mg F/mL. Prep. 2.2108 g NaF
(reagent grade, dried 4 hr at 105°)/L buffer soln. (Caution: See
51.084.) Store in leakproof plastic bottles. Compare with 1 mg
F/mL soln prepd from USP Ref. Std; equiv. reading of ±1 mv
is satisfactory.
5.006 Determination
(Stir all solns constantly at same rate thruout titrns. Let electrodes
equilibrate ;;.2 min before addn of F std soln and 30 sec after
each addn of F std soln.)
(a) Blank.-Record mv values (E') of 100 mL buffer soln after
addn of 4 mL std F soln from 10 mL buret and after each addnl
mL up to 10 mL. (Preliminary mv values will not fall on linear
range of response curve.) Vol. std soln added = V'.
(b) Samples.-Est. molarity of samples from direct reading.
Oil. samples, if necessary, to ca 0.001M F. Transfer 50 mL sample
soln to beaker and add 50 mL buffer soln. Record initial mv
reading, using expanded scale (Eo). If initial reading is <-50
mv, soln is too concd. Oil. sample to avoid asymptotic slope.
Record mv values (E) after each mL F std soln is added up to 10
mL. Rinse electrodes with H20 between samples. Vol. std soln
added = V.
5.007 Calculations
(a) Linear graph paper.-For each addn of F std soln and
corresponding E value, calc. for blank:
Z' = antilog [log(Vo + V') - 0.017(E')],
where Va is original vol. soln to which F std soln was added
(100mL) and E' is treated algebraically (+ or - as read). PlotZ'
against mL (mg) F std soln added and extrapolate to intersection
of mL (mg) Faxis to obtain mL (mg) F in blank, Ve'. In graph,
assign horizontal axis to mL (mg) F, with 0 at center and mL
(mg) F increasing in both directions to left and right. Assign Z
values to vertical axis. Plot actual readings of mL (mg) F on
right portion of horizontal axis so that extrapolation will fall on
left portion of axis.](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-91-320.jpg)
![70 5. HAZARDOUS SUBSTANCES AOAC METHODS (1980)
Similarly, for original readings and each addn of F std soln
and corresponding E value, calc. for sample:
Z = antilog [log(Vo + V) - 0.017(E}j,
where Vo is original vol. soln to which F std soln was added (100
mL). Plot Z against mL (mg) F std soln on same graph as blank
and extrapolate to intersection of mL (mg) Faxis to obtain mL
(mg) F in sample, Ve'
(b) Semi-antilog paper.-Plot E directly for both blank and
sample, descending 5 mv for each major line crossing vertical
axis. At top of vertical axis place most neg. E reading which still
allows extrapolation of Ve on left portion of mL (mg) side of
horizontal axis. Obtain Ve and V,' by extrapolation to left side
of 0 mL (mg) F.
% F = (Ve - V.') x (8 x 100)/[W x C x 1000 (mg/g)].
where 8 = vol. of diln, W = mL or g sample, and C = aliquot
(50 mL max.) buffered to 100 mL.
PREPARATIONS CONTAINING METHANOL
Methanol (3)-Official Final Action
(Applicable in presence of acetone, BuOAc, EtOH, isopropanol,
hexane, MeEt ketone, CH2CI2, Me Cellosolve, paraffin, toluene,
and H20. This includes many paint removers, fuels, liq. sanders,
antifreezes, and paint products.)
5.008 Apparatus and Reagents
(a) Gas chromatograph.-With flame ionization detector and
oven capable oftemp. changes ~5°/min near 160° or preferably
temp. programmer. Column.-1.8 m (6') x 4 mm id packed with
120-150 mesh Porapak R (Waters Associates, Inc., PO Box 246,
Milford, MA 01757); condition 2 hr at 235°. Conditions: Temps
(0): injection ca 200, column ca 160, detector ca 210; N flow ca
25 mL/min; set electrometer so that 8 ILL std soln provides at
least half scale peak. Adjust column temp. and N flow so that
MeOH retention time is ca 5-7 min.
(b) Methanol std soln.--O.4% (v/v). Oil. 4.00 mL MeOH to 100
mL with dioxane; dil. 10.0 mL of this soln to 100 mL with
dioxane. Rinse pipet into flask before dilg to vol. with dioxane.
Prep. fresh daily.
5.009 Preparation of Sample
(a) For asphalt-base tar compounds and viscous adhesives.-
Refrigerate unopened sample container ~3 hr (longer for larger
containers) at 1-10°, open container, and mix well; close con-
tainer and refrigerate 30 min more. Transfer 1.5-3 g sample to
tared, 250 mL, wide-mouth g-s erlenmeyer (tared with stopper
in place). Let sample reach room temp. in stoppered erlenmeyer
and weigh. Refrigerate 30 min and quickly add 100.0 mL dioxane.
Stopper and shake mech. 1 hr. Refrigerate 30 min and filter thru
rapid paper (S&S sharkskin, or equiv.). Filter as quickly as
possible, covering funnel with watch glass and placing funnel
against neck of narrow-mouth g-s receiver. Proceed as in 5.010,
dilg with dioxane, if necessary.
(b) For other less viscous products.-Prep. soln with pipets
and vol. flasks to contain ca 0.4% (v/v) MeOH, dilg with dioxane.
Avoid excessive shaking of semiviscous products and do not fill
pipet above mark. (Use safety pipet filler to draw liq. to mark
and hold until transfer.) Wash pipet with dioxane and add
washings to soln.
If MeOH concn is unknown, prep. 2% soln. Prep. addnl dilns
as needed.
5.010 Determination
Inject portion std soln with 10 ILL syringe. Note vol. At RMA
(retention time relative to MeOH) ca 0.5, inject portion sample
soln. Note vol. At RMA ca 2 (from second injection), repeat
injection of std soln. At RMA ca 0.5 (from third injection), repeat
injection of sample soln. After MeOH from fourth injection
elutes, increase column temp. to 235° as rapidly as possible for
time ca 4 x R';'A until all dioxane (RMA ca 5) is removed from
column. Cool column to 160° and repeat sequence for subse-
quent sample. Modify injection time if necessary to sep. MeOH
from other peaks. (Note: Injection sequence is used only to save
time; it need not be used if desired.)
5.011 Calculation
Oet. retention areas for each MeOH peak by mUltiplying peak
ht by retention distance. Average retention areas for sample
(RA) and for std (RA 'I. Presence of solv. in column changes
retention times, requiring use of retention areas in calcn.
% MeOH (w/v) in sample soln = F x (RA/RA')
x (V'/V) x C x 0.79,
where F = diln factor, C = % (v/v) std soln, V and V' = vol.
sample and std soln injected, resp., and 0.79 = density of MeOH.
Volatile Denaturants in Alcoholic Products (4)
Official First Action
5.012 Apparatus and Reagents
(a) Gas chromatograph and integrator.-See 19.001(a) and
(b).
(b) Std solns.-fJ% (v/v). Oil. 6.00 mL of each denaturant of
interest to 100 mL with anhyd. alcohol in sep. vol. flasks. Approx.
slopes and retention times relative to n-PrOH are given in Table
5:01.
Table 5:01 Approximate Slopes and Retention Times Relative
to n-Propyl Alcohol (RT) for Denaturants
Compound
Acetone
Benzene
n-Butyl alcohol
sec-Butyl alcohol
Chloroform
Ethyl acetate
Ethylene glycol monoethyl ether
Ethylene glycol monomethyl ether
Isopropanol
Methanol
Methyl isobutyl ketone
Toluene
Slope
0.207
0.464
0.269
0.246
0.058
0.192
0.187
0.151
0.210
0.130
0.275
0.454
RT
0.694
2.309
2.283
1.621
1.543
1.640
3.868
2.071
0.727
0.266
5.436
5.302
5.013 Determination
Pipet 25 mL of each expected denaturant std soln into sep.
flasks and add 1.00 mL n-PrOH as internal std. Proceed as in
19.002, starting with "Cap immediately ...", except inject 0.3
ILL portions. Oet. peak areas and calc. slope for each compd as:
Sx = (PA x/PA i )/6.00,
where PAx and PA i = peak areas of compd X in std soln and of
n-PrOH internal std, resp., and 6.00 = % compd X in std soln.
Slopes and retention times should approximate those of Table
5:01.
% Compd X in sample = (PA/PA i ) = (1/Sx),
where PA = peak area of compd X in sample.](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-92-320.jpg)
![AOAC METHODS (1980) FUMIGANTS 85
ANT POISONS AND RODENTICIDES
6.139 * Alpha-Naphthylthiourea (40) *
Official First Action
(Caution: See 51.039, 51.041, and 51.046.)
N detn. See 4.132, 10th ed.
6.140 Thallous Sulfate (41)--Official Final Action
(Caution: See 51.019, 51.026, 51.031, and 51.041.)
Weigh sample contg 0.1-0.15 g TI2SO. (usually 10 g). transfer
to 800 mL Kjeldahl flask, and add 25 mL H2SO. followed by 5-10
mL HNOa. After first violent reaction ceases, heat until white
fumes of H2SO. appear. Add few drops fuming HNOa and
continue heating and adding HNOa until org. matter is destroyed,
as shown by colorless or light yellow soln. Cool, add 10-15 mL
H20, again cool, and wash contents of flask into 400 mL beaker,
continuing washing until vol. is 60-70 mL. Boil several min to
remove all HNOa, cool, and filter into 400 mL beaker. Wash with
hot H20 until vol. in beaker is 175 mL, neutze with NH.OH, and
then slightly acidify with H2SO. (1 +4). Add 1 g NaHSOa to ensure
reduction of thallic to thallous state. Heat to bp, add 50 mL 10%
K/ so/n, stir, and let stand overnight. Filter thru tight gooch contg
2 disks S&S 589 white ribbon paper covered by medium pad of
asbestos. Wash 4 or 5 times with 10 mL portions 1% KI soln,
and finally with absolute alcohol. Dry to canst wt at 1050
(1-1.5
hr), and weigh as TIL
% TI2S04 = (g Til x 0.7619 x 100)/g sample.
Warfarin (3-(a-Acetonylbenzyl)-4-hydroxycoumarin) (42)
Official Final Action
(Applicable to baits contg ca 0.025% and to concs contg ;,.0.5%
warfarin. Not applicable to pelleted baits or baits consisting of
cracked corn treated with alc. warfarin soln and aq. sugar soln,
and then dried.)
6.141 Reagents
(a) Sodium pyrophosphate soln.-1%. Dissolve 5 g Na,-
P20 7.10H2 0 in 500 mL H20.
(b) Petroleum ether, purified.-Ext 200 mL pet ether with
three 20 mL portions 1% Na,P20 7 soln.
(c) Warfarin std soln.-10 J-tg/mL. Dissolve 100 mg pure
warfarin (Wisconsin Alumni Research Foundation, P.O. Box
7365, Madison, WI 53707) in 100 mL 1% Na,P20 7 soln. Oil. 10
mL to 100 mL with 1% Na.P20 7 soln, and dil. 10 mL of second
soln to 100 mL with 1% Na,P20 7 soln.
6.142 Determination
Weigh 10 g sample (0.025%), 0.600 g (0.5%). or equiv. wt of
higher concn, into 125 mL g-s flask or 100 mL centrf. tube and
add 50 mL Et ether from pipet. Stopper tightly and shake mech.
ca 30 min. Transfer 5 or 10 mL to centrf. tube (or centrf. directly),
stopper, and centrf. 5 min at high speed or until clear. Take
precautions to avoid evapn of ether.
Pipet 10 mL 1% Na,P20 7soln into g-s 16 x 150 mm test tube
and add 2 mL centrfd ether ext from pipet. Stopper and shake
vigorously 2 min. Centrf. at high speed until aq. layer is clear.
Draw off ether layer, including any emulsion that remains, using
fine-tip glass tube attached to aspirator. Add ca 2 mL Et ether,
shake vigorously, centrf., and completely draw off ether layer.
Repeat ether extn, and then ext twice with purified pet ether in
same manner.
Prep. blank soln similarly, using 2 mL ether instead of 2 mL
ether ext.
Det. A of aq. soln in 1 cm silica cell at 308 nm against blank
soln in Beckman spectrophtr, model DU (replaced by models
24/25). or equiv. Det. A' (ca 0.46) of the std warfarin soln against
1% Na'P207 soln.
% Warfarin = (A/A') x (10-5 g std/mL)
x [100/(g sample x (2/50)(1/10))]
= (A/A') x (0.250/g sample).
FUMIGANTS
Fumigant Mixtures (43~fficial First Action
(Applicable to org. components of CS2, CCI., (CH2)2CI2, and
(CH2)2Br2 mixts. Precautions: Handle with care in hood or well-
ventilated area. Mixts are volatile, poisonous, and sometimes
flammable and may be fatal if inhaled or swallowed. They cause
skin and eye irritation. In case of contact, immediately remove
contaminated clothing and flush affected area with copious
amts of H20. Do not reuse clothing until free of contamination.
Do not use containers or equipment of AI, Mg, or their alloys.)
6.143 Principle
Components are detd by GLC. Peak area of each component
is measured and compared to stds of same fumigant mixt.
Precision of method is ±0.6% for each component.
6.144 Sampling
Obtain representative 1 L sample from container. Sample bulk
containers by means of weighted bottle, lowered toward bottom
and raised at such rate that it is % full when withdrawn. Sample
drums or small containers with thief or thru tap or valve located
so that sample comes from well below surface. Prevent contam-
ination of product or sample.
Place sample in clean, dry, and solv. vapor-tight glass bottle
of such size that it is nearly filled (not above shoulder) by
sample. Vapor-tight g-s bottles or screw-cap bottles with Sn foil
lined caps are satisfactory. Store samples at low temp.; cool to
<180
before opening for analysis.
6.145 Apparatus
(a) Gas chromatograph.-With flame ionization or thermal
conductivity detector. Typical operating conditions: Column
temp., 110°; injection port temp., 200°; flow rate, 80 mL He/ min.
(b) Recorder.-O.05-1.05 mv, full scale response. Integrator
may be used.
(c) Syringe.-Hamilton Co. 10 J-tL No. 701 N, or equiv.
(d) Column.-l.2 m (4') stainless steel, W' od, 0.194" id,
packed with reagent 6.146(a). Max. temp. is 160°. Other columns
can be used but chromatgc conditions and sample size must be
adjusted in accordance with column requirements. One such
column is: 3 m (10') stainless, a/,6" od, 0.12" id, packed with 20%
by wt N,N-bis-(2-cyanoethyl) formamide on 80-100 mesh Chro-
mosorb W, acid-washed. Columns are available from com.
suppliers. Criterion for use is emergence of each component of
mixt. of CS2, CCI" (CH2I.CI2, and (CH2J.Br2 as sep. peak.
6.146 Reagents
(a) Column packing.-30% by wt tricresyl phosphate on Chro-
mosorb P, 30--60 mesh.
(b) Carbon disulfide std.-ACS.
(c) Carbon tetrachloride std.-ACS.
(d) Ethylene dichloride std.-Purified 1,2-dichloroethane,
available from laboratory supply houses, or use center cut of
fractionation of com. product.
(e) Ethylene dibromide (1,2-dibromoethane) std.- Purified or
distd as in (d).](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-107-320.jpg)
![AOAC METHODS (1980) ALDRIN, DIELDRIN, ENDRIN 91
present. Stir thoroly and let stand overnight at room temp.
Before filtering, stir ppt to see that it settles quickly and is in
cryst. form. Filter on either ashless paper or gooch and wash
with HCI (1 +1000) at room temp. Continue washing for 2 or 3
fillings of filter after no more opalescence appears when few
mL fresh filtrate is tested with few drops nicotine distillate. With
paper, transfer paper and ppt to weighed Pt crucible, dry
carefully, and ignite until all C is destroyed. Finally heat over
Meker burner ,,;;10 min. Wt residue x 0.1141 = wt nicotine in
aliquot. With gooch, dry in oven 3 hr at 105° and weigh. Wt
residue x 0.1012 = wt nicotine in aliquot.
6.177
HALOGENATED PESTICIDES
Aldrin, Dieldrin, and Endrin-Offieial Final Action
* Total Chlorine by Sodium Biphenyl *
Reduction Method (53)
Principle
Org. halogen compds are decomposed by Na biphenyl and
liberated halide ion is titrd by Volhard method or potentiomet-
rically after extn with H20 from reaction medium. Applicable to
detn of aldrin, dieldrin, or endrin in dusts, granules, wettable
powders, emulsifiable concs, and solns in absence of other org.
CI-contg compds. More than trace amts of H20 and appreciable
amts of org. compds contg labile H cause excessive consumption
of Na biphenyl. Interference of S is avoided, when present, by
special treatment.
6.178 Reagents
(a) Dilute nitric acid.........fJ% by wt. Add 60 mL HN03 to 945 mL
H20.
(b) Sodium biphenyl reagent.-30% w/w. (Caution: See
51.034 and 51.038.) Place 300 mL dry toluene and 58 g Na in dry
2 L 3-neck flask equipped with adjustable speed sealed stirrer,
inlet for N, and reflux condenser. With stirrer off, and with slow
stream of N passing thru flask, warm until refluxing begins and
Na is entirely melted. Agitate vigorously until Na is finely
dispersed; then cool to <10°. Remove reflux condenser and add
1.25 L anhyd. ethylene glycol dimethyl ether. Add 390 g biphenyl
with moderate stirring and with slow stream of N passing thru
flask. Reaction should begin within few min, indicated by' blue
or green color which gradually darkens to black. Maintain temp.
at <30° with oil bath or other cooling medium not involving
hazard should flask contg Na break. Reaction should be complete
in 1 hr. Reagent protected from moisture and air has useful life
of 1-2 months at 25°.
(Premixed reagent, packed in'15 mL vials, each enough for 1
detn, is available from Southwestern Analytical Chemicals, Inc.,
PO Box 485, Austin, TX 78767.)
(e) Toluene.-Nitration grade, CI-free.
6.179 Preparation of Sample
(Caution: See 51.041 and 51.084.)
(a) Technical products.-Accurately weigh ca 0.1 g sample
into 125 mL separator contg 25-30 mL toluene. Cautiously add
10-14 g Na biphenyl reagent, mix by swirling, and let stand 5
min. If soln is not dark green, add addnl 10-14 g reagent.
(Dieldrin and endrin require 15 min reaction time afterfinal addn
of reagent.)
Destroy excess reagent by dropwise addn of H2 0, shaking
frequently between addns, until green color is completely re-
moved. Then slowly add 25 mL dil. HN03, with intermittent
swirling. Stopper separator, and mix with gentle rocking motion,
venting occasionally. Avoid vigorous shaking during this first
extn.
Let sep., rinse stopper and walls of separator with H20, and
drain aq. phase into 250 mL g-s erlenmeyer. Re-ext reaction
mixt. with two 25 mL portions dil. HN03, shaking vigorously.
Add aq. exts to erlenmeyer and det. CI.
(b) Emulsifiable concentrates and oil spray solns.- Mix tho-
roly and weigh sample contg 0.05-0.08 g CI into 250 mL
separator contg 25-30 mL toluene. Proceed as in (a). beginning
"Cautiously add ..."
(e) Dusts, granules, and wettable powders.-Weigh sample
contg 0.1-0.15 g active ingredient in paper Soxhlet extn thimble,
place in extn app., and ext with ca 150 mL acetone in 300 mL
flask 3 hr. Evap. ext to dryness on steam bath, dissolve residue
in few mL toluene, and quant. transfer to 250 mL separator,
using 25-30 mL toluene. Continue as in (a), beginning "Cau-
tiously add ..."
If S is brought into soln by decomposition of emulsifiers or
other compds such as org. thiophosphates, remove as follows:
Add 30% NaOH soln to acid soln in erlenmeyer until alk. to
phthln, and add 1 mL excess. Add 5 mL 30% H20 2, heat to bp
on hot plate, and boil ca 10 min. Let cool slightly, cautiously add
5 mL more 30% H20" and boil again ca 10 min. Cool, and add
small flake (ca 0.05 g) hydrazine sulfate to remove last traces of
H20 2• Neutze with dil. HN03 to phthln and add 2-3 mL excess.
6.180 Determination
(a) Colorless solns.-To acid aq. soln add 30 mL H,O, 10 mL
nitrobenzene, 3 mL ferric indicator, 6.019(e), and, from buret,
0.4-0.6 mL 0.05N KSCN. Swirling constantly, titr. with O.lN
AgN03 until red is discharged, and add 2-5 mL excess. Stopper
flask tightly and shake vigorously 15 sec. Without refilling buret,
titr. slowly with the 0.05N KSCN until end point approaches.
Stopper flask, shake vigorously 20-30 sec, and continue titrn
until 1 drop produces distinct reddish color which does not fade
on swirling or vigorous shaking.
(b) Colored solns or chloride in presence of bromide and/or
iodide.-To acid aq. soln add 30 mL H,O, transfer to 400 mL
beaker, adjust vol. to 200-250 mL, and add 0.5 g Ba(N03)2' Titr.
with O.lN AgN03 potentiometrically, with stirring, using cell
system of either glass ref. electrode and Ag indicating electrode
or Ag-AgCI electrode system, electronic voltmeter, and 10 mL
buret.
(e) Blank determination.-Det. blank on all reagents by adding
10-14 g Na biphenyl reagent to 25 mL toluene and continuing
as in 6.179(a).
6.181 Calculations
% Aldrin, dieldrin, or endrin = (net mL AgN03 - mL blank)
x normality x 35.45 x F/(10 x g sample). where F is 1.61 for
aldrin, 1.81 for dieldrin, and 1.74 for endrin. Net mL AgN03 =
[mL 0.1N AgN03 - (mL 0.05N KSCN/2)].
(For most accurate results, det. factor F for specific batch of
tech. pesticide used in formulation. Toxicant content is stenciled
on drum. Calc. F = PIC, where P = % purity (toxicant content
as stenciled on drum). and C = % CI by wt.)
Infrared Method (53)
6.182 Principle
Dieldrin and endrin in dusts, granules, wettable powders,
emulsifiable concs, and solns are purified on adsorbent columns.
Hexachloro-epoxy-octahydro-endo,exo-dimethanonaphthalene
*Surplus method-see inside front cover.](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-113-320.jpg)
![AOAC METHODS (1980) CHLORDANE 97
<2%. Without changing conditions, inject supernate from sam-
ple. If sample ratio differs by > ± 10% from std ratio, reweigh
sample. For detn, inject std, sample in duplicate, and std.
Measure HEOD and captan peak hts. When solv. peak tailing
extends into region of HEOD and captan peaks, draw curved
extension as baseline. Det. peak ht ratio captan:HEOD and
average the 2 values (Rs) for std injections just before and after
sample injections. Calc. and average peak ht ratio of the 2
samples (Rx).
% Captan = (Rx/Wx) x (Ws x P/Rs),
where Ws = g std, Wx = g sample, and P = % purity of captan
std.
Technical Chlordane
Total Chlorine Method (61}-Offlcial Final Action
(Caution: See 51.011, 51.018, 51.038, 51.039, 51.040, 51.041,
and 51.045.)
6.220 Standardization of Standard Solutions
(a) Sodium chloride std soln.-0.1N. Dissolve 5.845 g NaCI,
previously dried 2 hr at 105°, in H2 0, and dil. to 1 L in vol. flask.
(b) Silver nitrate std soln.-0.1N. Prep. as in 50.027. To 250
mL g-s erlenmeyer add 15.00 mL O.lN NaCI, (a), 50 mL H20, 10
mL HN03 (1 +1), boiled to expel oxides of N, and 25.00 mL of
the AgNOj soln. Add 3 mL nitrobenzene, stopper, and shake
vigorously 15 sec. Add 5 mL ferric indicator, 6.019(e), and back-
titr. with O.lN KSCN, (c), to reddish-brown end point. (Potenti-
ometric titrn using Ag indicator electrode and Ag-AgCI or glass
ref. electrode may be substituted for indicator method, but must
be used in both stdzn and detn.)
(c) Potassium thiocyanate std soln.-0.1N. Prep. and titr.
against AgN03 soln, (b), as in 50.030(b). Calc. F = mL AgN03
soln/mL KSCN soln.
Normality AgN03 soln = mL NaCI soln x 0.1000/(mL AgN03
soln --; mL KSCN soln x F).
6.221 Preparation of Sample
(a) Emulsifiable concentrate formulations.-Accurately
weigh sample contg 0.5±0.05 g tech. chlordane into 50 mL vol.
flask, dissolve, and dil. to vol. with toluene. Transfer 5 mL aliquot
to 125 mL separator, add 15 mL or g Na biphenyl reagent,
6.178(b), and swirl. If soln is not dark green, add more reagent.
Let stand 3 min and add 3-5 mL H20 dropwise. With stopper
removed, swirl soln gently to decompose excess reagent. Add
25 mL H20, stopper, and mix with gentle rocking motion. (Do
not shake vigorously.) Let layers sep. and drain lower aq. layer
into 250 mL erlenmeyer. Re-ext solv. layer with two 25 mL
portions 3N HN03 and combine aq. solns in erlenmeyer.
(b) Dusts, granular impregnates, and wettable powders.-
Accurately weigh sample contg 0.5±0.05 g tech. chlordane into
Soxhlet extn thimble. Ext with 80 mL benzene in Soxhlet app.
1 hr. Transfer to 100 mL vol. flask, washing with several 3 mL
portions benzene. Dil. to vol. with benzene and transfer 10 mL
aliquot to 125 mL separator. Proceed as in (a).
6.222 Determination
Add 15.00 mL O.lN AgN03 and 3 mL nitrobenzene to erlen-
meyer, stopper, and shake vigorously 15 sec. Rinse stopper, add
5 mL ferric indicator, 6.019(e), and back-titr. with O.lN KSCN to
reddish-brown end point. (Designate mL KSCN as D.)
Det. blank on reagents by pipetting 5 mL toluene into 125 mL
separator, add 15 mL or g Na biphenyl reagent, and proceed as
in 6.221(a), thru combining aq. solns in erlenmeyer. Add 15.00
mL O.lN NaCI, 25.00 mL O.lN AgN03, and 3 mL nitrobenzene,
and proceed as above. Calc. blank correction factor, C = mL
KSCN used in stdzn of AgN03 - mL KSCN used in blank detn.
% Chlorine = [15 - (C + D) x F] x normality AgN03 x 3.545/g
sample.
% Tech. chlordane = % CI x 1.56.
Colorimetric Method (62}-Offlcial Final Action
(Method is empirical; all conditions must be reproduced exactly
to attain good precision. Temp., reaction time, and vol. of
reagents affect color intensity.)
6.223 Apparatus
(a) Constant temperature bath.-Capable of maintaining
100±1° and holding twelve 20 x 150 mni test tubes.
(b) Cuvets.-l0 or 2 mm light path (available from Pyrocell
Mfg. Co., 91 Carver Ave, PO Box 176, Westwood, NJ 07675).
(c) Spectrophotometer.-Capable of accepting cuvets, (b).
6.224 Reagents
(a) Methanol.-90% (by voL).
(b) Methanol-benzene.-Mix 7 vols MeOH with 3 vols ben-
zene.
(c) Diethanolamine.-Purify by vac. distn at ca 20 mm Hg and
take middle fraction. (Caution: See 51.011 and 51.015.)
(d) Diethanolamine-KOH soln (Davidow reagent). -Mix 1 vol.-
reagent (c) with 2 vols 1.0N KOH in MeOH.
(e) Chlordane std solns.-l.5, 2.5, and 3.5 mg/mL. Ref. grade
(available from Velsicol Chemical Corp.). Dissolve tech. chlor-
dane in reagent (b). Discard stds after 2 weeks.
6.225 Preparation of Sample
(a) Liquid formulations.-Transfer weighed sample contg
200-300 mg tech. chlordane to 100 mL graduate and dil. to 100
mL with MeOH-benzene.
(b) High concentration solid formulations (10% chlordane or
more).-Treat as in (a) and shake vigorously several min. Let
settle 1 hr.
(c) Low concentration solid formulations (less than 10% chlor-
dane).-Transfer weighed sample contg 200-300 mg tech. chlor-
dane to Soxhlet and ext 1 hr with pentane. (Caution: See 51.039
and 51.074.) Evap. pentane on steam bath and transfer ext to
100 mL g-s graduate. Dil. to 100 mL with MeOH-benzene.
6.226 Determination
Pipet 2 mL aliquot prepd sample to 20 x 150 mm test tube.
Add No.8 grit SiC boiling chip and 2 mL Davidow reagent, and
place in 100° const temp. bath. Remove after exactly 45 min
and cool immediately in beaker of cold H20. Transfer to 10 mL
vol. flask and dil. to vol. with 90% MeOH. Transfer aliquot of
soln to 2 mm cell and read A at 550 nm within 15 min with 90%
MeOH as ref. (Comparable results are obtained by dilg soln to
50 mL and using 1 cm cello)
Treat 2 mL each std soln with each set of samples. (Read 1 std
soln before samples, 1 after half the samples are read, and 1
after last sample is read.)
6.227 Calculations
Calc. absorptivity (a) for each of 3 stds, and use avo in
subsequent calcns (expected a is ca 0.25): a =A '/W, where
A' = A std soln and W = mg tech. chlordane (2 x concn std soln
in mg/mL).
% Tech. chlordane in sample = A x 5000/(a x mg sample).](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-119-320.jpg)
![AOAC METHODS (1980) MALATHION 115
Add 35--40 mL cyclohexane to reagent blank followed by 2
mL Cu reagent and immediately dil. to vol. with cyclohexane.
Stopper flask and mix. Use this soln to zero spectrophtr, using
1 cm cells at max. A, ca 420 nm. Proceed as above for sample.
Det. A against reagent blank exactly 2 min after adding Cu
reagent. Slight haze may appear on initial diln with cyclohexane.
If it does not clear after adding Cu reagent, check for presence
of excessive H2 0 in analysis.
Water can be introduced by EtOAc, acetone, CH3CN, alcohol,
tetrahydrofuran, or contaminated glassware. Correct this con-
dition before proceeding with analysis.
6.369 Calculations
(a) Absorptivity of samples (a).-
(1) Tech. malathion and emulsifiable concs
a = (A x 50 x 50 x 50)/(mg sample x 5 x 5)
= (A x 5000)/mg sample
(2) Wettable powders and dusts
a = (A x 100 x 50 x 50)/(mg sample x 10 x 5)
= (A x 5000)/mg sample
(b) Absorptivity of stds (a').-
(1) Malathion: Use formula in (a)( 1).
(2) Potassium O,O-dimethyl phosphorodithioate
a' =[(A x 50 x 50 x 50)/(mg sample x 5 x 5)] x 0.5939
= (A x 2969)/mg sample,
where 0.5939 is factor to convert K salt to equiv. wt malathion.
(In general, a of stds are in range 17.2-17.6, corrected for quality
of std, at concn 1 mg/mL in 1 cm cell.)
(3) % Malathion = (a x % purity of std)/a'
6.370 Preparation of Standard Curve (Optional)
Transfer 3, 4,5, and 6 mL aliquots centrfgd EtOAc soln of std,
6.368, par. 3, into sep. 50 mL vol. flasks. Proceed with color
development, and plot A against wt malathion in aliquot. Use
std curve with considerable caution, since fade rate may vary
from day to day, depending on solvs used for analysis. Std
curve is not appropriate where fluctuations in temp. occur in
laboratory environment.
6.371 Interferences
Test for free O,O-dimethyl phosphorodithioic acid or other
interfering components in sample ext as follows: Transfer 2
drops ext to test tube, dil. with ca 10 mL cyclohexane, add 8
drops Cu reagent, and mix. Development of discernible yellow
in soln indicates necessity of obtaining sample-blank correction.
Obtain sample-blank correction as follows: Transfer 5 or 10
mL aliquot sample to 50 mL vol. flask and dil. to vol. with EtOAc.
(Do not add alc. NaOH soln.) For color development, transfer 5
mL dild sample soln and continue as in 6.368. Correct A for
base-treated sample and proceed with calcn.
Gas Chromatographic Method (96)-Official First Action
6.372 Apparatus
(a) Gas chromatograph.-With glass column, on-column in-
jection system, flame ionization detector, and electrometer with
sensitivity of ~1O-11 amp driving 1 mv recorder. Drift should be
<1%/hr. Totally solid state amplifier with FET input is recom-
mended. Electronic digital integrator or computer calcd area
measurements must be used. Integrator should have independ-
ent controls for selection of up and down slope sensitivities so
that start and stop integration points can be selected. Automated
sample injection system contributes significantly to precision.
Hewlett-Packard Model 7600 is suitable when equipped as
described. Equiv. instrumentation may be used but may require
modification of operating conditions to obtain good peak shape,
adequate resolution, and appropriate retention times.
Typical conditions for Hewlett-Packard Model 7600 (instru-
ment may have to be adjusted to give complete resolution of
well shaped peaks): Cycle timers (min): analysis and stop
integrate, 16; range, 103; temps (0): oven 180, injection port 200,
flame detector 300; gas flow rotameters (mL/min): H 35, air
425, He carrier gas 30; integrator settings (adjusted so that
deflections on slope meter do not exceed ±50% before injec-
tion): noise suppression max., slope sensitivity up and down
0.1, BL reset delay 0.15, area threshold 1000; retention times
(min): malathion 10, internal std 6, min. time between malathion
and internal std 3.5.
(b) Column.-Borosilicate glass tube 1.22 m (6') x 4 mm id,
6 mm od, bent to fit chromatograph and packed with 5% SP-
2401 or OV-210 on Gas-Chrom Q or Supelcoport (100/120 mesh).
Can be purchased as prepd packing from Supelco, Inc., (specify
"Pesticide Grade"); Alltech Associates, 202 Campus Dr, Arling-
ton Heights, IL 60006; and Applied Science Laboratories, Inc.
Use exclusively for malathion analysis.
(e) Glass wool.-Silane treated. (No. 14502, Applied Science
Laboratories, Inc.)
(d) Syringes.-10 ILL, Series 700, Hamilton Co.
(e) HI-EFF Fluidizer.-Applied Science Laboratories, Inc.
6.373 Reagents
(a) Internal std soln.-1.2% m-Diphenoxybenzene in CHCI3 •
Must not contain any impurities which elute at or near malathion
peak. Bring soln to consistent temp. above ambient (e.g. 25°)
before taking aliquots.
(b) Malathion std solns.-Accurately weigh ca 170, 200, and
230 mg malathion std (anal. grade, available from American
Cyanamid Co.) into sep. preweighed 25 mL vol. flasks. Add by
pipet 5 mL internal std soln and dil. to vol. with CHCI3 • Label
A, B, and C. Soln B is working std soln for detn; solns A
and C are used for linearity check and to guard against weighing
error in prepn of working std soln. Solns are stable ca 4 weeks
if kept tightly sealed in refrigerator. Warm to room temp. before
use. Soln B can be prepd independently of solns A and C, if
conditions of linearity check are met.
6.374 Preparation and Conditioning of Column
Weigh 6.25 g of trifluoropropylsilicone (SP-2401 or' OV-210)
in 250 mL beaker and dissolve in 125 mL EtOAc. Stir to obtain
vortex and add 25 g solid support (Gas-Chrom Q or Supelcoport,
100/120 mesh) with continued agitation. Filter slurry thru What-
man No.1 paper, or equiv., on buchner, using gentle vac. to
minimize evapn of soIv. Continue filtration until drop rate is ca
1/sec. Transfer packing to HI-EFF Fluidizer, connect source of N
thru pressure reducer to base, and place fluidizer on controlled
temp. hot plate set for 75°. Continue gas flow until soIv. vapors
can no longer be detected by odor, taking care that packing is
not blown out top of fluidizer.
To pack column, attach 75 mm funnel to exit end of prebent
glass tube. Tap tube with pencil or small wooden rod, and add
prepd packing in small amts until exit end is filled to ca 15 mm
from end. Move funnel to entrance end of column. Insert pledget
of silane-treated glass wool in exit end and attach source of
moderate vac. to this end. Continue to add packing slowly with
tapping until tube is filled to ca 20 mm from entrance end. Insert
pledget of silane-treated glass wool in entrance end, compress-
ing it only enough to hold it in place.
Condition column with He carrier gas flowing at 30 mL/min](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-137-320.jpg)
![AOAC METHODS (1980) NITROGEN 127
7.020 Calculations
(a) Record observed N vol., Vo = R2 - R" where Vo = observed
N vol. (ttL). R, = initial counter reading, and R2 = final counter
reading.
(b) Det. corrected N vol. (in ttL), Vc = Vo - (Vb + Vt), where
Vb = vol. blank (ttL), Vt = vol. correction for temp. (ttL) = C,(t2
- t,). Cf is obtained from Table 7:01 (based on final counter
reading); t2 and t, are in OK.
(c) Det. corrected barometric pressure, Pc = Po - (Pb + Pv).
where Po = observed barometric pressure (mm Hg), Pb =
barometric temp. correction (from Table 7:02), and Pv = pressure
correction for vapor pressure of KOH soln (from Table 7:03).
(Note: Empirical approximation of (Pb + Pv ) = 11.0 will be
satisfactorily accurate for Po between 740 and 780 mm Hg and
syringe temp. between 298 and 305°K.)
(d) Calc. % N = (Pc x Vc x 0.0449)/(T x W). where T = final
syringe temp. in oK and W = sample wt in mg.
Example:
Po = 750.1 mm Hg at 25°C; W = 148.91 mg
Start Finish
Counter readings, blank 500 ILL 524 ILL
Counter readings, sample 524 6955
t, = 302.rK, t2 = 303.00
K, Vo = 6955 - 524 = 6431 ILL
Vc = 6431 - [24 + C,(t2- t,)] = 6431 - (24 + 35 x 0.3) = 6396
ttL
Pc = 750.1 - (3.1 x 9.6) = 737.4
% N = (737.4 x 6396 x 0.04493)/(303.0 x 148.91) = 4.69%
(e) Calc. % protein = % N x 6.25, or % N x 5.70 in case of
wheat grains.
Table 7:03 Pressure correction (Pv) for vapor pressure of
KOH (for practical purposes, temp. of KOH is same as syringe)
7.021
Temperature, OK Pv (mm Hg)
288
293
298
303
308
313
4.1
5.7
7.4
9.6
12.5
16.5
Automated Method (tt)-Official Final Action
Principle
Automation of macro Kjeldahl method is in 6 steps: sample
and reagent addn, initial and final digestion, cooling and diln,
NaOH addn, steam distn and titrn, and automatic pumping of
flask contents to waste. Chemistry is carried out in macro
Kjeldahl flasks equipped with side arms which are rotated at 3
min intervals thru each successive step.
7.022 Apparatus
(a) Kjeldahl (protein/nitrogen) analyzer. -Kjel-Foss Auto-
matic, Model 16210 (Foss America, Inc., PO Box 504, Rt 82,
Fishkill, NY 12524). or equiv.
(b) Weighing papers.-120 x 120 mm N-free tissues, Foss
America, Inc., or equiv.
7.023 Reagents
(a) Kjel-tabs.-Contg 5 g K2S04and 0.25 g HgO (Foss America,
Inc.).
(b) Kjeldahl (protein/nitrogen) analyzer reagents.-Prep. fol-
lowing according to manufacturer's instructions: (1) Sulfuric
acid.-96--98%. (2) Hydrogen peroxide.-30-35%. (3) Ammo-
nium sulfate std solns.-(a) Std soln I.-Dissolve 30.000±0.030
g (NH4)2S04 in H20 and dil. to 1 L with H20. (b) Std soln 11.-
Dissolve 0.750±0.001 g (NH4)2S04 in H20 and dil. to 1 L with
H20. (4) Mixed indicator soln.-Dissolve 1.000 g Me red and
0.250 g methylene blue in alcohol and dil. to 1 L with alcohol.
Dil. 10 mL this soln to 1 L with H20. (5) Sodium hydroxide-
sodium thiosulfate soln.-40% NaOH-8% Na2S20 3.5H20. (6) Di-
lute sulfuric acid soln.--fJ.6%. Dil. 30 mL 96--98% H2S04to 5 L
with H20.
7.024 Determination
(Caution: See 51.019, 51.030, 51.065, and 51.070.)
Place 3 Kjel-tabs in special flask (500 mL of design compatible
to Foss instrument) in position 1. Shift dispenser arm over flask
and depress H2S04 lever, initiating simultaneous addn of 10 mL
30-35% H20 2 and 12-15 mL 96--98% H2S04 (depending on fat
content of sample). To flask, add accurately weighed sample
(ca 1.0 g if <45% protein, and ca 0.5 g if >45% protein) wrapped
in weighing paper and close lid. Flask automatically rotates to
position 2 where sample digests 3 min, and then to position 3
for 3 min addnl digestion. In position 4, flask is cooled by
centrifugal blower, lid opens automatically, and 140 mL H20 is
added automatically. Flask rotates to position 5, where NaOH-
Na2S20 3 soln is automatically introduced in excess. Released
NH3 is steam distd Quant. into 200 mL tall-form titrn beaker
contg 50 mL mixed indicator soln, and is simultaneously titrd
automatically with dil. H2S04soln delivered by photometrically
regulated syringe. Final position of syringe is measured by
potentiometer, output of which feeds electronic circuitry for
conversion to visual display and/or printout in % Nor % protein
with appropriate conversion factors. In position 6, flask is
emptied. Calibrate instrument initially each day with aliquots of
(NH4)2S04 std solns and check periodically as stated in operating
manual.
Semiautomated Method (12)-Official Final Action
7.025 Principle
Samples are digested in 250 mL calibrated tubes, using block
digestor. A of NH3-salicylate complex is read in flowcell at 660
nm, or NH3, is distd into std acid and back-titrd with std alkali.
7.026 Apparatus
(a) Block digestor.-Model BD-20 (Technicon Instruments
Corp.) or Model DS-20 (Tecator, Inc., 1898 S Flatiron Ct, Boulder,
CO 80301). Capable of maintaining 410° and digesting 20 samples
at a time in 250 mL calibrated volumetric tubes constricted at
top. Block must be equipped with removable shields to enclose
exposed area of tubes comfJ:etely at or above ht of constriction.
(b) Automatic analyzer.-AutoAnalyzer with following mod-
ules (Technicon Instruments Corp.), or equiv.: Sampler II or IV
with 40/hr (2: 1) cam (higher ratio cams result in carry-over and
poorer peak sepn); proportioning pump III; NH3 anal. cartridge
No. 116-D531-01 (or construct equiv. manifold from flow dia-
gram); AAII single channel colorimeter with 15 x 1.5-2.0 mm
id tubular flowcell, matched 660 nm interference filters, and
voltage stabilizer; and recorder of appropriate span. (See Fig.
7:01.)
7.027 Reagents
(a) Phosphate-tartrate buffer soln.-pH 14.0. Dissolve 50 g
NaK tartrate and 26.8 g Na2HP04.7H20 in 600 mL H20. Add
54 g NaOH and dissolve. Add 1 mL Brij-35 (Technicon Instru-
ments Corp.), dil. to 1 L with H20, and mix.](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-149-320.jpg)
![AOAC METHODS (1980) NITROGEN 129
Table 7:04 Sample Weight
Protein, %
6-24
25-40
41-50
51-60
61-90
>90
Sample, 9
1.5±0.1
1.0±0.1
0.B±0.1
0.HO.1
0.5±0.01
Weigh sample equiv.
to 50 mg N
Place stds in tray in increasing order of concn, followed by
group of samples. Analyze lowest concn std in duplicate, dis-
carding first peak. Precede and follow each group of samples
with std ref. curve to correct for possible drift. Analyze stds and
samples at rate of 40!hr, 2!1 sample-to-wash ratio. Prep. std
curve by averaging peak hts of first and second set of stds. Plot
avo peak ht stds against N concn contained in each 250 mL tube.
7.032
% Protein = [(mg N!250 mL from graph)
x 6.25 x 100l/mg sample
Titrimetric Determination
Digest as in 7.031. Cool 5 min and add only enough H20 to
dissolve salts (70-75 mL). Cool and attach digestion tube to
distn head according to manufacturer's directions. Place receiver
flask contg 25 mL std acid, 2.055(j}, and 5--7 drops Me red
indicator on platform. Condenser tip must be below surface of
std acid soln. Add 50 mL NaOH-K2S soln to tube and steam
distil vigorously until 125 mL distillate collects. Titr. excess acid
with std 0.1N NaOH soln, 2.055(k). Correct for reagent blank.
% N = [(mL std acid x normality acid) - (mL std NaOH
x normality NaOH)] x 1.4007!g sample
% crude protein = % N x 6.25
7.033
Urea and Ammoniacal Nitrogen (13)
Official Final Action
Reagents
(a) Defoaming so/n.-Dow Corning Corp. Antifoam B Emul-
sion.
(b) Urease so/n.-Prep. fresh soln by dissolving stdzd urease
in H20 so that each 10 mL neutzd soln will convert N of ~0.1 g
pure urea.
Standardization.-To det. alky of com. urease prepn dissolve
0.1 gin 50 mL H20 and titr. with 0.1N HCI, using Me red, 2.055(0.
Add same vol. 0.1N HCI to each 0.1 g urease in prepg urease
soln. To det. enzyme activity, prep. ca 50 mL neutzd 1% soln.
Add different amts of soln to 0.1 g samples pure urea and follow
with enzyme digestion and distn as in detn. Calc. activity of
urease prepn from amt of this urease soln that completely
converted urea, as detd by complete recovery of N by distn.
(c) Calcium chloride so/n.-Dissolve 25 g CaCI2 in 100 mL
H20.
7.034 Determination
Place 2 g sample in Kjeldahl flask with ca 250 mL H20. Add
10 mL urease soln, stopper tightly, and let stand 1 hr at room
temp. or 20 min at 40°. Cool to room temp. if necessary. Use
addnl urease soln if feed contains >5% urea (ca 12% protein
equiv.). Rinse stopper and neck with few mL H20. Add ~2 g
MgO (heavy type). 5 mL CaCI2 soln, and 3 mL defoaming soln,
and connect flask with condenser by Kjeldahl connecting bulb.
Distil 100 mL into measured vol. std acid, 2.055(j), and titr. with
std alkali, 2.055(k). using Me red, 2.055(0.
*Surplus method---see inside front cover.
Urea (14)--Official Final Action
(Applicable to animal feeds and their ingredients)
7.035 Apparatus
Spectrophotometer.-Instrument with max. band width 2.4
nm at 420 nm, with 1 em cells.
7.036 Reagents
(a) p-Dimethy/aminobenza/dehyde (DMAB) so/n.-Dissolve
16.00 g (Eastman Kodak Co. No. 95 only) in 1 L alcohol and add
100 mL HCI. Stable 1 month. Prep. new std curve with each new
batch of reagent.
(b) Zinc acetate soln.-Dissolve 22.0 g Zn(OAc)2.2H,O in H20,
add 3 mL HOAc, and dil. to 100 mL.
(c) Potassium ferrocyanide so/n.-Dissolve 10.6 g K4 Fe(CN)6
.3H20 in H20 and dil. to 100 mL.
(d) Vegetable charcoa/.-Darco G-60.
(e) Phosphate buffer so/n.-pH 7.0. Dissolve 3.403 g anhyd.
KH2P04 and 4.355 g anhyd. K2HP04 sep. in ca 100 mL portions
freshly distd H20. Combine solns and dil. to 1 L with H20.
(f) Urea std so/ns.-(1) Stock so/n.-5 mg!mL. Dissolve
5.000±0.001 g reagent grade urea in H20 and dil. to 1 L with
H20. (2) Working so/ns.--0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8,
and 2.0 mg urea!5 mL. Pipet 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20
mL stock soln into 250 mL vol. flasks and dil. to vol. with
phosphate buffer. (3) Reference so/n.-Use std soln contg 1.0
mg urea!5 mL as ref. std. Store at <24°. Stable 1 week.
7.037 Preparation of Standard Curve
Pipet 5 mL aliquots of working std solns into 20 x 150 mm
(25 mL) test tubes and add 5 mL DMAB soln to each. Prep.
reagent blank of 5 mL buffer soln and 5 mL DMAB soln. Shake
tubes thoroly and let stand 10 min in H2 0 bath at 25°. Read A
in 1 em cell at 420 nm with reagent blank at zero A. Plot A
against concn urea. Plot should be straight line; if not, repeat,
using new lot of DMAB.
7.038 Determination
Weigh 1.00 g ground sample into 500 mL vol. flask. Add 1 g
charcoal, ca 250 mL H20, 5 mL Zn(OAc)2 soln, and 5 mL K4 Fe(CN)6
soln. Shake mech. 30 min and dil. to vol. with H20. Let stand
until ppt settles. Decant thru Whatman No. 40 paper and collect
clear filtrate. Pipet 5 mL filtrate into test tube, add 5 mL DMAB
soln, and shake thoroly. Include reference std (5 mL soln (f)(3)
and 5 mL DMAB soln) and reagent blank with each 'group of
samples. Let stand 10 min in H20 bath at 250
• Read A at 420 nm
against reagent blank.
% Urea = (1.0 x AsamPle x 100)!(Astd X mg sample in aliquot).
7.039 * Albuminoid Nitrogen-Official Final Action *
Pptn with Cu(OH)2 and N detn. See 22.020-22.021, 10th ed.
7.040 * Amido Nitrogen-Official Final Action *
% total N - % albuminoid N = % amido N.
Nitrate and Nitrite Nitrogen (15}-Official Final Action
7.041 Princ;ple
Nitrate and nitrite are extd with Cd and Ba chloride soln. Bulk
of sol. proteins are pptd in alk. soln and clarified soln is passed
thru metallic Cd column, reducing nitrate to nitrite. Nitrite is
measured colorimetrically. (Caution: Cd salts are toxic. See
51.084.)](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-151-320.jpg)
![164 10. BEVERAGES: MALT BEVERAGES AND BREWING MATERIALS AOAC METHODS (1980)
ext directly from 52.009. If 100 mL beer was taken, correct as
follows:
Ext found x sp gr of dealcoholized beer
/sp gr of beer = g ext/l00 g beer.
10.022 Extract of Original Wort-Official Final Action
Calc. from following formula and report to first decimal place:
0= [(P x 2.0665) + E] x 100/[100 + (P x 1.0665)],
where 0 = ext of original wort; P = % alcohol by wt (g/100 g
beer); and E = % real ext, 10.021(a) or (b).
10.023 Alcohol by Volume-Official Final Action
See 11.005, but use pycnometers described in 10.106(i) and
10.107(b) or (c).
Alcohol by Weight
10.024 Specific Gravity Method-Official Final Action
See 9.021, but use pycnometers described in 10.106(i) and
10.107(b) or (c).
10.025
Refractometer Method (4)
Official Final Action
Apparatus
(a) Immersion refractometer.-Bausch & Lomb, Carl Zeiss,
Inc., 444 Fifth Ave, New York, NY 10018, or equiv., with prisms
covering range 1.32-1.37 n.
(b) Water bath.-5ee 10.106(k).
(c) Pycnometer.-5ee 10.106(i) and 10.107(b) or (c).
10.026 Calibration
Adjust refractometer light to give max. contrast between light
and dark fields. Adjust color compensator and focus for sharp,
color-free dividing line. H20, double-distd from glass, should
read ca 14.50 at 20°. Read H20 before each sample series. Rinse
prism with H20 after each sample and dry with soft tissue.
Prep. calibration curve to convert refractometer readings and
sp gr detns to % alcohol by wt by analyzing beers covering
alcohol range of interest by 10.024 and 10.027. Plot results,
using ordinates (R - N), where R = refractometer reading (Rbee,
- R wate,), N = 1000 x (sp gr - 1.00000), and abscissa = %
alcohol by wt, 10.024. Fit least squares line to adequate number
of points to get accuracy desired. Det. equation of line and
slope. (See Definitions of Terms and Explanatory Notes, Item
(24).) Calc. % alcohol by wt by formula or read from calibration
curve. % alcohol by wt = F x (R - N) + C, where F = slope of
calibration line, and C = const of calibration curve equation.
10.027 Determination
Det. sp gr of decarbonated beer by 10.016. Place refractometer
cuvet contg distd H20 and clear decarbonated beer samples in
20° H20 bath. Place prism of refractometer in H20 cuvet and
check temp. after 15 min. Make 5 readings to nearest 0.1 scale
division and average results (Rwate,). Transfer dry prism to beer
sample cuvet, wait ~1 min, make 5 readings to nearest 0.1 scale
division, and average results (Rbee,). Calc. % alcohol by wt by
formula or read from calibration curve.
10.028 Real Degree of Fermentation or Real
Attenuation-Official Final Action
Calc. as follows and report to first decimal place:
(orig. ext - real ext) x 100/orig. ext.
10.029 Apparent Degree of Fermentation or
Apparent Attenuation-Official Final Action
Calc. as follows and report to first decimal place:
(orig. ext - apparent ext) x 100/orig. ext.
10.030 End Fermentation (Yeast Fermentable Extract)
(Fermentable Sugars)-Official Final Action
Det. real ext, 10.021, or apparent ext, 10.020. Ferment 250 mL
beer with 1 g active compressed brewers yeast 24-48 hr at
15-25°, or until fermentation is complete, providing fermentation
flask with H20 or Hg seal. Filter; det. real ext, 10.021, or apparent
ext, 10.020. Fermentable sugars = difference in real ext before
and after fermentation; or fermentable sugars = 0.82 x differ-
ence in apparent ext before and after fermentation.
10.031 Caloric Content-Official First Action
Det. sp gr (= density), 10.016, % alcohol by wt, 10.024 or
10.027, real ext, 10.021, and ash, 10.047, of beer sample.
Calories/l00 g = (g real ext - % ash) x 4
+ (% EtOH by wt x 6.9);
Calories in vol. = (Calories/l00g) x (beer vol.20o x density)/100,
where vol. is expressed in units required by test.
10.032 * Glycerol-Official Final Action *
Dichromate oxdn method. See 11.010(b), 12th ed.
Total Acidity (5)-Official Final Action
10.033 Indicator Titration Method
Bring 250 mL H20 to bp and continue boiling 2 min. From
fast-flowing pipet add 25 mL beer previously decarbonated by
shaking and filtering, 10.001. After emptying pipet, continue
heating 60 sec, regulating heat so that soln resumes boiling
during final 30 sec. Remove from heat, stir 5 sec, and cool
rapidly to room temp.
Add 0.5 mL 0.5% phthln. Titr. with O.lN NaOH against white
background. Make frequent color comparisons with sample of
equal vol. and diln to which has been added approx. anticipated
amt of alkali but no indicator. Titr. to first appearance of faint
pink. Read buret. Add 0.2 mL more alkali; color should then be
permanent, definite pinkish red, indicative of overtitrn. Take first
buret reading as end point.
Observe strictly all details of method. However, 100 mL H20,
10 mL beer, and 0.2 mL indicator may be used in place of amts
specified above. (Use potentiometric titrn method, 10.034, for
beers of dark color which, even when dild, may not permit
judging phthln end point with necessary precision.)
Report results: (a) as lactic acid, to nearest 0.01% (1 mL O.lN
alkali = 0.0090 g lactic acid); or (b) as mL IN alkali, to nearest
0.1 mL, necessary for neutzn of 100 g beer.
10.034 Potentiometric Titration Method
Use glass-calomel electrode system. Decarbonate beer com-
pletely by shaking, 10.001. Using 50 mL undild sample (or such
amt as best suits titrn assembly)' titr. potentiometrically with
O.lN NaOH to pH 8.2. Add alkali in 1.5 mL portions to ca pH 7.6,
and in 0.15 mL portions from there to pH 8.2. Make sure that
complete equilibrium and good convergence are attained before
reading buret at exactly pH 8.2. Report results as in 10.033.
*Surplus method-see inside front cover.](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-186-320.jpg)
![AOAC METHODS (1980)
Precautions: Observe all details of good potentiometric tech-
nic, including following: Stdze potentiometer against fresh
0.05M K acid phthalate, 50.007(c). before and after any set of
titrns; read potentiometer to nearest 0.02 unit; use flexible
shielding around electrode leads and motor cords; ground
motor and motor cords, preferably to H20 pipes; avoid contact
between electrodes and glass beaker; use proper stirring speed
to ensure quick mixing but to avoid foaming which may tem-
porarily trap some of alkali added; stop titrn at ,,;pH 8.6 to
minimize alkali contamination of glass electrode; check batteries
frequently. Follow manufacturer's instructions for potentiometer
used.
Hydrogen-Ion Activity (pH)
Official Final Action
10.035 Potentiometric Method
Det. pH of undild sample, 10.001, using glass-calomel elec-
trode system. Follow manufacturer's instructions for potentiom-
eter used. Check pH meter before and after use against std K
acid phthalate buffer, 50.007(c). Observe precautions in 10.034.
Report results to nearest 0.05 pH.
10.036 Volatile Acids-Official Final Action
Using 100 mL beer, proceed as in 11.040. Express result as
HOAc, g/100 mL. 1 mL 0.1N alkali = 0.0060 g HOAc.
10.037 Carbohydrate Content-Official First Action
Det. sp gr (=density). 10.016, real ext, 10.021, ash, 10.047, and
protein, 10.045, of beer sample.
g Carbohydrate/100 g
= (g real ext/100g) - (% protein + % ash)
g Carbohydrate in vol.
= (g carbohydrate/100 g) x (beer vol.20o x density)/100,
where vol. is expressed in units required by test.
10.038 Reducing Sugars-Official Final Action
Dil. 25 mL prepd sample, 10.001, measured at 20°, to 100 mL
with H20 at same temp. Det. reducing sugars in 25 mL of this
soln by Munson-Walker method, 31.060, or dil. 50 mL beer with
H20 to 100 mL and use Lane-Eynon method, 31.059. Express
result as g maltose/100 mL beer. For conversion to % by wt,
divide results by sp gr of beer.
10.039 Dextrin-Official Final Action
To 25 mL prepd sample, 10.001, measured at 20° in 500 mL
boiling flask, add 15 mL HCI (sp gr 1.125) and dil. to 200 mL.
Attach flask to reflux condenser, and keep in boiling H20 bath
2 hr. Cool. nearly neutze with NaOH soln (1 + 1), dil. to 250 mL
in vol. flask, filter, and det. glucose as in 31.051.
g Dextrin/100 mL beer = [glucose (g/100 mL)
- (1.053 x maltose, 10.038)] x 0.9.
10.040
Diacetyl (6)-Official Final Action
ASBC Colorimetric Method
Apparatus
(a) Compressed carbon dioxide gas.-In steel cylinder with
reducing valve connected thru 1-hole rubber stopper into 1 neck
of boiling flask, (b), ending high up in flask.
(b) Glass distillation apparatus.-With following parts: boiling
flask, 2-neck, 500 mL; distg tube, mounted vertically on boiling
flask; condenser, H20-cooled, connected by 75° adapter, if
necessary, to distg tube so that condenser slopes downward;
BEER 165
curved, tapered tube adapter, connected to condenser delivery
tip to dip below liq. level in receiver; and receiver, such as 50
mL beaker, marked at 15 and 35 mL.
(c) Heating mantle.-For boiling flask, (b). or burner.
(d) Spectrophotometer or colorimeter.-Capable of measur-
ing A at 520 nm; with matched 1 cm cells.
10.041 Reagents
(a) Dimethylglyoxime std soln.-O.0674 mg/mL. Dissolve
0.1348 g dimethylglyoxime in 3-5 mL alcohol, dil. to 2 L with
H20, and mix well.
(b) Dipotassium hydrogen phosphate in aqueous acetone
soln.-O.827M. Dissolve 14.4 g K2HP04 (or 19.9 g K2HP04.3H20)
in H20, add 20 mL acetone, dil. to 100 mL with H20, and mix
well. Store in refrigerator.
(c) Ferrous sulfate soln.-5%. Dissolve 5 g FeS04.7H20 in 100
mL H2S04 (1 +99). and mix well. Discard when soln turns yellow
due to oxidn of Fe.
(d) Hydroxylamine hydrochloride soln.-6% aq. soln of
NH20H.HCI.
(e) Potassium dihydrogen phosphate alkaline soln.-0.30M.
Dissolve 1.0 g KH2P04 in 25 mL 0.1N NaOH, and mix well.
(f) Potassium sodium tartrate, saturated soln.-Shake 90 g K
Na tartrate well with 50 mL H20, and let settle.
(g) Silicone antifoam.-Dow Corning Antifoam A, or equiv.
10.042 Preparation of Standard Curve
In 20 mL vol. flasks, prep. series of dimethylglyoxime std
solns contg, in increments of 0.025 mg, 0.025--0.200 mg diacetyl
by mixing appropriate vols dimethylglyoxime std soln, (a) (0.741
x mg dimethylglyoxime/mL = mg diacetyl/mL). with 0.75 mL
6% NH20H.HCI soln and sufficient H20 to yield 15 mL. Heat each
soln 15 min in ca 80° H20 bath, cool to room temp., add 1.0 mL
0.827M K2HP04, and let stand 5 min. Successively add 0.6 mL
NH4 0H, 2.5 mL satd K Na tartrate soln, and 0.2 mL 5% FeS04
soln. Dil. to 20 mL with H20, and mix by inversion.
Prep. blank contg all reagents, except dimethylglyoxime, in
sufficient H20 to yield 20 mL.
Set spectrophtr to read 0 A at 520 nm with blank in cell. Obtain
A readings at 520 nm for dimethylglyoxime std solns contg
0.025--0.200 mg diacetyl equiv. Plot A at 520 nm against mg
diacetyl/20 mL. Std curve should be straight line passing thru
origin.
10.043 Determination
Place 25 mL 0.30M KH2P04 in 500 mL boiling flask. Add 250
mL cold beer, not decarbonated, and 1 drop antifoam. Fit 1 neck
of boiling flask to lower joint of vertical distg tube of distn app.
Place tip of adapter into 0.75 mL 6% NH20H.HCI soln and 2-3
mL H20 to seal adapter. Cool receiver during distn. If sample is
expected to have high diacetyl concn, e.g., if diacetyl can be
smelled or tasted readily, use 1.50 mL NH20H.HCI soln and
divide distillate into 2 equal portions. Use only 1 portion for
distn, and multiply result by 2.
Connect CO2line to feed into boiling flask well above liq. level,
and adjust CO2gas flow to maintain CO2 atm. in entire system.
Heat, and distil at ca 1 drop/sec. Collect 30 mL distillate.
Place receiver in ca 80° H2 0 bath 15 min. Raise temp. of H20
bath to 100° or transfer receiver to boiling H20 bath. Let distillate
evap. to 15 mL. Cool to room temp., and transfer quant. to 20
mL vol. flask.
Add 1.0 mL 0.827M K2HPO. and let stand 5 min. Successively
add 0.6 mL NH4 0H, 2.5 mL satd K Na tartrate soln, and 0.2 mL
FeS04 soln. Dil. to 20 mL with H20 and mix. Within 20 min, read
A of colored soln at 520 nm against blank.](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-187-320.jpg)
![AOAC METHODS (1980)
10.066 Determination
To cleaned 50 mL centrf. tube, add 25 mL cold sample,
measured in graduate, 3 mL H,S04 (1 +3), and 1 mL 30% H,O,.
If foam interferes with sample measurement, add 1 drop hexyl
alcohol. Mix, and place tube in boiling H20 bath 0.5 hr. If
excessive foaming occurs, add 1 drop hexyl alcohol. Remove
tube and cool to 25°. Add 5 or 10 mL, accurately measured,
lDBT soln, depending upon size of photometer cell, and stopper
tube. Ext at 25° by shaking vigorously 60 times. Re-ext again 4
times, giving 60 snapping shakes each time to obtain fine
emulsion, allowing partial sepn between extns. Digested sample
must be shaken vigorously with the lDBT soln; thoro and
complete emulsification must be obtained during each series of
extns or results may be low.
Centrf. tube 2-3 min and draw off clear, colored layer to
photometer cell of same size used in calibration, and det. A. If
droplets of aq. layer are carried into pipet, remove by flowing
solv. from pipet down wall of clean, dry test tube. H20 droplets
will adhere to test tube and clear solv. can be poured off into
cell.
Prep. reagent blank by extg, in clean 50 mL centrf. tube, 25
mL Cu-free H20 at 25° and 3 mL H2S04 (1 +3) with 5 (or 10) mL
lDBT soln and det. A,. To correct for A of color extd by solv.,
perform entire detn, omitting lDBT soln, but shaking with
toluene (or CCI4) and det. A 2• Do not give tubes used for this
solv.-extractable beer color blank preliminary cleaning with
lDBT soln, since carryover of lDBT may give high readings.
ppm Cu = [A -(A, + A 2)] x F,
where F is factor for converting A to ppm Cu.
10.067
Cuprethol Method {H)-Official Final Action
Apparatus
(a) Photometer.-Any com. instrument with blue-green or
green filter, or spectrophtr set at 445 nm and with 40-50 mm
cells.
(b) Copper-free glassware.-Clean all glassware with O.lN
HN03 and rinse thoroly with Cu-free distd H20.
10.068 Reagents
(a) Diethanolamine soln.-Dissolve 4.0 mL (HOCH2CH2)2NH
(Eastman Kodak Co., No. 1598) in 200 mL MeOH.
(b) Carbon disulfide soln.-Add 1.0 mL CS2 (free of pptd S)
to 200 mL MeOH.
(c) Cuprethol soln.-Mix 3 vols soln (a) and 1 vol. soln (b).
Prep. fresh daily. Also mix equal vols soln (a) and MeOH for
blank.
(d) Copper std solns.-Prep. as in 10.062(b).
(e) Buffer soln.-pH 4.4. Dissolve 63.3 g anhyd. NaOAc in ca
800 mL H20 contg 65 mL HOAc. Oil. to 1 L with H20.
(f) Copper-free distilled water.-Use distd H20 redistd from
all-glass app. thruout method.
10.069 Preparation of Standard Curve
Into series of g-s 100 mL vol. flasks add 0.0, 1.0, 2.0, 4.0, 8.0,
and 12.0 mL Cu working std soln contg 0.0, 0.4, 0.8, 1.6,3.2, and
4.8 ppm Cu, resp. Add H20 to 12 mL in each flask. Oil. to vol.
with degassed low-Cu beer, 10.065, mix, and proceed as in
10.070. Use 0.0 sample to zero instrument, and obtain A or scale
readings for 0.1,0.2,0.4,0.8, and 1.2 ppm added Cu. A over this
range follow Beer's law. Calc. avo factor, F, converting A or scale
readings to ppm Cu. If instrument response is not linear, draw
and use smooth curve for calcg ppm Cu.
BEER 169
10.070 Determination
Slowly pour 50 mL cold beer into 50 mL graduate; avoid
foaming. Transfer to 125 mL flask, add 25 mL buffer soln, (e),
and mix. Measure two 30 mL aliquots in 50 mL graduate and
transfer to sep. 50 mL flasks. Add 3 mL cuprethol soln, (c), to
one flask and 3 mL blank soln to other. Mix each and let stand
10 min. lero instrument with blank. Det. A in same size cell and
at same wavelength used in calibration. Calc. ppm Cu by
mUltiplying A or scale reading by F, or use curve.
Iron (12)-Official Final Action
10.071 Apparatus
Photometer.-Spectrophtr set at ca 505 nm or photometer
with filter in blue-green region, 500-550 nm, or preferably,
505-520 nm.
10.072 Reagents
(a) Color reagent: (1) 2,2'-Bipyridine.--O.2%. Dissolve 1 g
2,2'-bipyridine in 20 mL HOAc (1 +2) and dil. to 500 mL with
H2 0; or-
(2) o-Phenanthroline.--O.3%. Dissolve 1.5 g o-phenanthroline
in 500 mL H20 at 70°.
(b) Iron std soln.-O.l mg/mL. (1) From iron wire.-Dissolve
0.500 g reagent grade Fe wire, wiped free of oxide, in 5 mL HCI
(1 +4) and 1 mL HN03• Cover with watch glass, heat, and evap.
to dryness; add H20 and evap. to dryness again. Dissolve residue
in 3-5 mL HCI, cool, and transfer quant. to 500 mL vol. flask.
Add 2 drops satd Br-H20, dil. to vol. with H20, and mix. Transfer
50 mL of this soln to 500 mL vol. flask, add 2 drops Br-H2 0, dil.
to vol. with H20, and mix.
(2) From ferrous ammonium su/fate.-Dissolve 3.512 g
Fe(NH4b(S04)2.6H20 in H20, add 5 mL Hel, transfer quant. to 500
mL vol. flask, dil. to vol. with H20, and mix. Transfer 50 mL of
this soln to 500 mL vol. flask, dil. to vol. with H20, and mix.
(c) Ascorbic acid.-USP, ground to fine powder.
10.073 Preparation of Standard Curve
Prep. series of beer stds contg 0.0, 0.25, 0.50, 1.00, 2.00, and
3.00 ppm Fe as follows: Pipet 0.0, 0.25, 0.50,1.00,2.00, and 3.00
mL Fe std soln to series of 100 mL vol. flasks, add, from pipet,
enough H20 to total 3.00 mL, and dil. to vol. with decarbonated
beer, 10.074. Depending upon size cell to be used, develop color
in 25 or 50 mL aliquots of each of the beer stds as in 10.075(a)
or (b).
If T values are obtained, convert to A = -log T, and plot A
against ppm Fe. If straight line results, calc. factor, m, for
converting A to ppm Fe, y, by use of equation y = mA + b (b
= 0 if line passes thru origin). If instrument response is such
that curve is obtained, use this curve to calc. results.
10.074 Preparation of Sample
Adjust temp. of beer to 20-25°. Decarbonate by transferring
sample to large erlenmeyer and shaking, first gently and then
vigorously, until all gas is released. Do not filter unless necessary.
If filtration is required, make sure filter paper is Fe-free by
spotting sample of paper with drop of reagent prepd by dis-
solving 25 mg ascorbic acid in 2 mL color reagent. 10.072(a).
If beer sample is suspected of high Fe content, degas by
shaking only, and permit foam to subside before sampling.
10.075 Determination
Pipet 2 aliquots of degassed beer (25 or 50 mL as used in
prepn of std curve) irito 50 mL or 125 mL erlenmeyers; add 25](https://image.slidesharecdn.com/aoac-140402153033-phpapp01/85/Aoac-methods-1980-191-320.jpg)
Aoac.methods.1980
- 1. By Authority Of THE UNITED STATES OF AMERICA Legally Binding Document By the Authority Vested By Part 5 of the United States Code § 552(a) and Part 1 of the Code of Regulations § 51 the attached document has been duly INCORPORATED BY REFERENCE and shall be considered legally binding upon all citizens and residents of the United States of America. HEED THIS NOTICE: Criminal penalties may apply for noncompliance. Official Incorporator: THE EXECUTIVE DIRECTOR OFFICE OF THE FEDERAL REGISTER WASHINGTON, D.C. Document Name: CFR Section(s): Standards Body: e AOAC: Official Methods of Analysis, 1980 21 CFR 131.150(c) AOAC International
- 3. OFFICIAL METHODS OF ANALYSIS OF THE ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS WILLIAM HORWITZ, Editor THIRTEENTH EDITION, 1980 PUBLISHED BY THE ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS PO Box 540, BENJAMIN FRANKLIN STATION WASHINGTON, DC 20044
- 4. Direct inquiries related to the scientific content of Official Methods of Analysis to: Editor, Official Methods of Analysis Association of Official Analytical Chemists Box 540, Benjamin Franklin Station Washington, DC 20044 USA Direct inquiries related to the procurement of Official Methods of Analysis, supplements (Changes in Methods). Journal of the AOAC, or other AOAC publications to: Assistant Business Manager, Publications Association of Official Analytical Chemists 1111 N 19th Street (Suite 210) Arlington, VA 22209 USA (Telephone: 703-522-3032) COPYRIGHT 1920,1925, 1931, 1936, 1940, 1945, 1950, 1955, 1960, 1965, BYTHE ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS AND 1970, 1975, 1980 BY THE ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS The methods of the Association were also copyrighted in 1916, when they were published in the Journal of the Association of Official Agricultural Chemists Library of Congress Catalog Card Number: 20-21343 ISBN 0-935584-14-5 A copy of the 13th edition of this publication is on file with the Office of the Federal Register. U. S. Government Agencies may apply to the Director of the Office of the Federal Register for approval to incorporate this edition by reference in their regulations. The procedures that Federal agencies must follow in applying for the Director's approval are in Title 1, Part 51 of the Code of Federal Regulations. Composed by Monotype Composition Company, Inc. Baltimore, Maryland Printed and bound by George Banta Company, Inc. Menasha, Wisconsin ii
- 5. Preface to Thirteenth Edition The most noticeable physical change in this thirteenth edition of Official Methods of Analysis of the Association of Official Analytical Chemists ("The Book of Methods") is its size. A survey of users of the Book of Methods revealed an overwhelming desire to maintain the compendium as a single volume. The easiest way to do this was to increase the size of the page. Users also expressed a desire for a system that will keep the same reference number for a given method from edition to edition. A practical system that will maintain the continuity of the numbering system and the organizational structure of the methods within a chapter has not yet been devised. Approximately 175 new methods have been added during the current five year period (1974-1978); 83 methods have been deleted, replaced, or surplused. The approval of an average of only 35 new methods per year represents a marked decline from the 70 per year of the four year period of the previous edition (1970-1973) and the 50 per year of the five year period of the tenth edition (1965-1969). The decline is undoubtedly the result of a number of factors. Chief among them are the greater complexity of modern methods, requiring a large investment in resources that is not readily mobilized to fit an Associate Referee's schedule; and the fact that government agencies are attempting to obtain compliance, especially of the newer stat- utes, by promulgation of regulations and by auditing rather than by laboratory examinations. The greatest activity, as measured by approval of new meth- ods, is in the field of pesticide formulations, partly as a result of the active implementation of the cooperative agreement with the Collaborative International Pesticide Analytical Council (CIPAC). Other active areas include extraneous materials, vita- mins and other nutrients, dairy products, and microbiological methods. In fact, examination for and by biological constituents (filth, microbiological assays, and examination for food-borne pathogens) comprises approximately 17% of the new methods adopted. A comparison of the types of methods adopted between this and the previous edition reveals that gas-liquid chromatography has overtaken spectrophotometry in its various forms (visible, ultraviolet, and fluorescent) as the most predominant quanti- tative technique in Official Methods. High pressure (or perform- ance) liquid chromatography has spurted from none to 6% of the adopted methods within the relatively short period of five years. Electrometric methods (potentiometric, polarographic, and ion-selective) are now sufficiently numerous to deserve a separate category. Infrared methods are no longer a major factor in quantitation; they are now mainly used for identification and confirmation. A comparison of the present and previous editions is presented in Table 1. The figures given are only rough approximations because of the arbitrariness often re- quired in classifying a method and in deciding when a new method or revision is sufficiently independent to warrant con- tributing to the statistics. iii Table 1. Classification of new methods approved by the AOAC in the thirteenth and twelfth editions Method classification Gas-liquid chromatography Spectrophotometry Titrimetric Filth, isolation Automated Biological and microbiological High pressure liquid chromatography Atomic absorption Electrometric Chromatography (thin layer, etc.) Gravimetric Miscellaneous (physical, qualitative, etc.) 13th edition (1980) 18% 16 10 8 7 9 6 4 5 3 2 11 12th edition (1975) 14% 20 7 14 5 8 9 3 6 13 The most interesting new collaboratively studied method is the mass spectrometric method for the detection of adulteration of honey with high fructose corn sirup. Corn sirups (from a monocotyledonous plant) have a distinctly different 13C/12 C ratio than sirups from most dicotyledonous plants, which are the source of most honeys. The isotope ratio mass spectrometer required forthis determination is a highly specialized instrument, even in the field of mass spectrometry. Despite the rarity of the instrumentation, sufficient laboratories participated in the col- laborative study to establish the reproducibility of the method. A problem that has arisen is how to handle the numerous individual instruments of diverse design and manufacture that have been developed to automate a particular determination. Even if this problem is solved by incorporating all the available instruments into the initial collaborative study, the problem returns with the first "new and improved" modification. Different instrument designs and their subsequent modifications have been handled in the infrared determination of milk constituents by providing performance specifications which must be met by the basic instrument in general, when compared to a reference method or reference sample. In addition, the user must satisfy himself that his particular instrument also meets the perform- ance specifications by frequent comparisons with the reference method or sample. This requirement eliminates the need for repeated collaborative studies every time a manufacturer rede- signs or modifies his basic equipment, and in addition provides a continuous quality control technique on the performance of the instrument, method, and laboratory. It should not be overlooked that automation is not confined to physical and chemical determinations. The microbiological chapter contains three collaboratively studied instrumental methods for somatic cell count and an instrumental method for distributing a liquid sample for plate counting. Biochemical diagnostic kits have also been evaluated collaboratively for their
- 6. effectiveness in identifying Salmonella and other enteric isolates. A criticism that is often leveled at the AOAC validation mechanism is that it is too slow to keep up with the pace of requirements for methods by regulatory agencies and the regulated industry. This objection overlooks the point that the speed with which a method is validated is almost completely in the hands of those needing the validated method, rather than in the hands of the AOAC. The method must be tested for ruggedness, and the directions tested for clarity; samples must be prepared and distributed; laboratories must analyze the samples and report the results. Dubious results must be inves- tigated and occasionally samples must be reanalyzed. The data must be tabulated and analyzed, and a report written. There is no way to short-cut the process of obtaining intra- and interlab- oratory variability except to conduct the necessary experimental work and perform the necessary statistical calculations. Only then is the recommendation subject to the time restrictions of the AOAC mechanism. To ensure that the AOAC mechanism of annual approval is not holding up use of needed methods, a new temporary class of methods has been introduced entitled "Interim First Action." These are methods which, together with their supporting studies, have been received between annual meetings and have been sent through the customary reviewing procedure. To be designated Interim First Action, a method must have been approved by the appropriate Referee and Subcommittee, and by the Chairman of the Committee on Official Methods. The method only lacks the formal vote by those AOAC members having regulatory authority over the commodity involved. Such formal approval is usually provided at the next annual meeting, at which time the method becomes "First Action." Approximately five interim methods are approved annually. Several such methods appear in this volume, depend- ing upon the time of their submission and review in relation to the editorial status of the chapter to which they are assigned. The current status of these Interim First Action methods, as well as the status of any method, can be found by consulting the latest cumulative index to Changes in Methods, which appears as the final pages of each March issue of the Journal. For those users of AOAC methods who may not be familiar with the procedures by which a method is included in this volume, we are reprinting a paper prepared for the Joint International Symposium, "The Harmonization of Collaborative Studies," held in London, England 9-10 March 1978. The document was drafted by the AOAC Committee on Collaborative Studies: Elwyn D. Schall, Chairman; Charles W. Gehrke, William Horwitz, Anthony J. Malonski, James P. Minyard, Jr., Forrest W. Quackenbush, and Ernest S. Windham. This paper appeared in Analytical Chemistry (March 1978), 50, 337A-340A. The publication of this "Book of Methods" is possible only because of the extensive cooperation of thousands of analysts who have volunteered to direct, participate in, and review the numerous collaborative studies that form the basis for inclusion of the several thousand methods that appear herein. Special recognition is due to Mrs. Betty Johnson who has prepared an entirely new index for this edition. 28 September, 1979 William Horwitz, Ph.D. Editor Abstract from Preface to First Edition "In presenting this revision of the official and tentative methods of analysis of the Association of Official Agricultural Chemists, it is appropriate to give a brief statement of the organization of the Association, its purpose, and the procedure by which the methods are adopted. "Membership in the Association is institutional and includes the State Departments of Agriculture, the State Agricultural Colleges and Experiment Stations, the Federal Department of Agriculture, and the Federal, State, and City offices charged with the enforcement of food, feed, drug, fertilizer, insecticide and fungicide control laws. "The Association was founded at Philadelphia, Pa., September 9, 1884, by the following representative agricultural chemists of that time, the organization being the result of a series of informal meetings held the immediately preceding years: "Prof. H. W. Wiley, Chemist of the Department of Agriculture, Washington, D.C. Mr. Clifford Richardson, Assistant Chemist of the Department of Agriculture, Washington, D.C. Mr. Philip E. Chazal, State Chemist of South Carolina. Dr. Chas. W. Dabney, Jr., State Chemist of North Carolina. Dr. W. J. Gascoyne, State Chemist of Virginia. Dr. E. H. Jenkins, Connecticut Experiment Station. Prof. John A. Meyers, State Chemist of Mississippi. Prof. H. C. White, State Chemist of Georgia. Mr. C. DeGhequier, Secretary National Fertilizer Association. Dr. Schumann, Dr. Lehmann, Mr. Gaines and others." iv
- 7. Contents Preface to Thirteenth Edition ......................... . Preface to First Edition .............................. . List of Illustrations .................................. . Collaborative Study Procedures of the Association of Official Analytical Chemists ........................ . Definitions of Terms and Explanatory Notes .......... . CHAPTER 1. Agricultural Liming Materials .................... . Calcium Silicate Slags ........................ . Elemental Analysis ........................... . 2. Fertilizers ....................................... . Free Water ................................... . Phosphorus .................................. . Nitrogen ..................................... . Potassium ................................... . Other Elements ............................... . Peat ......................................... . 3. Plants .......................................... . Metals ....................................... . Nonmetals ................................... . Other Constituents ........................... . Pigments .................................... . Tobacco ..................................... . 4. Disinfectants .................................... . Phenol Coefficient ............................ . Use-Dilution Method .......................... . Available Chlorine Germicidal Equivalent Concentration .............................. . Sporicidal Test ............................... . Fungicidal Test ............................... . Germicidal and Detergent Sanitizers ........... . Germicidal Spray Products .................... . Water Disinfectants for Swimming Pools ....... . Tuberculocidal Activity ........................ . Bacteriostatic Activity of Laundry Additives .... . 5. Hazardous Substances .......................... . Paint ......................................... . Preparations Containing Fluorides ............. . Preparations Containing Methanol ............. . Preparations Containing Phenol ............... . Soda Lye .................................... . Earthenware ................................. . 6. Pesticide Formulations .......................... . General Methods ............................. . Arsenic .................................... . Lead ....................................... . Copper .................................... . Zinc ....................................... . Fluorine .................................... . PAGE iii iv ix xii xv 2 3 7 9 9 14 18 20 27 31 31 38 46 49 51 56 56 58 59 60 61 61 63 64 ' 65 67 69 69 69 70 71 71 71 72 72 72 74 74 75 75 v Contamination by Organochlorine Pesticides .. Volatility of Ester Forms of Hormone-Type Herbicides ............................... . Inorganic and Organometallic Pesticides and Adjuvants ................................ . Paris Green ................................ . Lead Arsenate .............................. . PAGE 77 77 78 78 78 Calcium Arsenate. . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Zinc Arsenite. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . 79 Copper Carbonate .. . . . . . . . . . . . . . . . . . . . . . . . . . 79 Copper Naphthenate . . . . . . . . . . . . . . . . . . . . . . . . . 79 Bordeaux Mixtures .......................... 80 Potassium Cyanate. . . . . . . . . . . . . . . . . . . . . . . . . . 81 Cyanides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Lime Sulfur Preparations. . . . . . . . . . . . . . . . . . . . . 81 Hypochlorites ............................... 82 Chloramine T ............................... 83 Mineral Oils. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Soap....................................... 83 Mineral Oil-Soap Emulsions. . . . . . . . . . . . . . .. . . 83 Mercurial Seed Disinfectants. . . . . . . . . . . . . .. . . 84 Ant Poisons and Rodenticides . . . . . . . . . . . . . . . . 85 Fumigants .................................. 85 Pesticides Related to Natural Products and Their Synergists ................................ 86 Allethrin .................................... 86 Derris and Cube Powder. . . . . . . . . . . . . . . . . .. . . 87 Pyrethrins . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . 89 Tobacco and Tobacco Products .............. 90 Halogenated Pesticides ........................ 91 Aldrin, Dieldrin, and Endrin .................. 91 Amiben ..................................... 93 BHC ........................................ 93 Balan or Trifluralin .......................... 95 Captan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Chlordane. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Chlorotoluron, Chloroxuron, or Metoxuron . . . . 99 Dichlobenil .. . . .. . ... . . . .. .. . .. . . ... . . . .. ... . 100 DDT.. . . . ... . . ..... . .. . . . .. . ... . . . . .. . .... . . 100 Dacthal ..................................... 102 Dicamba .................................... 102 2,4-D ....................................... 103 Dalapon .................................... 104 Dicofol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Fluometuron ................................ 105 Folpet ...................................... 105 Heptachlor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Picloram-2,4-D .............................. 106 Sodium Trichloroacetate ......... " .. . . .... .. 107 Nonhalogenated Pesticides .................... 107 Aldicarb .................................... 107 Amitrole .................................... 107 Carbaryl .................................... 108 DDVP ................... '" ..... " . . ... . .. . . 108 Diquat and Paraquat ............. " . . . . . . . . . . 109
- 8. PAGE Dithiocarbamates . . . . . . . . . . . . . . . . . . . . . .. . . . . . 110 Thiram ............................... '" . ... 111 Dodine...................................... 111 Formaldehyde. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Ethion ...................................... 112 Formothion ................................. 113 Malathion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Parathion ................................... 116 Phorate ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Sulfoxide ................................... 120 Tetraethylpyrophosphate .................... 120 Organic Thiocyanates . . . . . . . . . . . . . . . . . . . . . . . . 120 Thiocarbamates ............................. 121 Triazines, Diazinon, and Dichlorobenzilates . . . . 121 Triphenyltin Compounds. . . . . . .. . . . . . . .. . . . . . 122 Quaternary Ammonium Compounds ........... 123 7. Animal Feed..................................... 125 Nitrogen ...................................... 125 Other Constituents ............................ 132 Preservatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 8. Baking Powders and Baking Chemicals ........... . 9. Beverages: Distilled Liquors ..................... . Spirits ....................................... . Cordials and Liqueurs ........................ . 10. Beverages: Malt Beverages and Brewing Materials Beer ......................................... . Malt ......................................... . Cereal Adjuncts .............................. . Hops ........................................ . Brewing Sugars and Sirups ................... . Wort ......................................... . Yeast ........................................ . Brewers' Grains 11. Beverages: Wines ............................... . Preservatives ................................. . Flavors ...................................... . 12. Beverages: Nonalcoholic and Concentrates ....... . 13. Cacao Bean and Its Products .............. " ..... . Shell ......................................... . Cacao Products Processed with Alkalies ....... . Chocolate Liquor ............................. . Fat .......................................... . Dairy Ingredient Constituents ................. . Saccharine Ingredients Other Than Lactose .... . Starch ....................................... . 143 147 147 159 162 162 171 177 178 180 181 182 183 185 192 193 194 199 200 205 205 205 206 207 208 Chocolate Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 Theobromine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 14. Cereal Foods. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 211 Wheat Flour. . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 211 Wheat, Rye, Oats, Corn, Buckwheat, Rice, and Barley and Their Products Except Cereal Adjuncts .................................... 220 Soybean Flour ................................ 223 Bread....... . . . .... . ..... ..... ... . . . . . .... . ... 223 Baked Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 PAGE Baked Products Other Than Bread (Not Containing Fruit) ............................ 227 Fig Bars and Raisin-Filled Crackers ............. 227 Macaroni, Egg Noodles, and Similar Products. . . 228 15. Coffee and Tea ....................... " . . . . . .. . . . 233 Green Coffee. . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . 233 Roasted Coffee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 Tea........................................... 235 16. Dairy Products. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 238 Sampling ..................................... 238 Milk.......................................... 240 Automated Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 Cryoscopic Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Penicillins in Milk. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 255 Cream........................................ 256 Evaporated Milk. . . . . . . . . . . . . . . . . . . .. . . . . . .. . . . 258 Sweetened Condensed Milk ........ " . . . . . .. . . . 259 Dried Milk, Nonfat Dry Milk, and Malted Milk ... 259 Butter. .... ... .... . .. . . . . .... . ... . ....... . ..... 260 Cheese....................................... 264 Ice Cream and Frozen Desserts. . . . . . . . . . . . . . . . . 272 17. Eggs and Egg Products. . . . . . . .. . . . . . . . . . . . . . . . . . . 275 18. Fish and Other Marine Products ....... " . . . .. ..... 285 19. Flavors .......................................... 306 Alcohol ....................................... 306 Vanilla Extract and Its Substitutes .............. 306 Lemon, Orange, and Lime Extracts and Flavors. 313 Lemon and Orange Oils. . . . .... . ..... ... . ... . . . 316 Almond Extract. . .... . ... . . .. ... . ....... . ..... . 318 Cassia, Cinnamon, and Clove Extracts. ... . .... . 319 Ginger Extract. . ..... ..... . .... . . .... . . . . .... .. 319 Peppermint, Spearmint, and Wintergreen Extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 Anise and Nutmeg Extracts .................... 319 Other Extracts and Toilet Preparations. . . . . . . . . . 319 20. Food Additives: Direct. . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 Acidulants .................................... 323 Antioxidants .................................. 323 Chemical Preservatives ........................ 325 Emulsifying Agents. . . . . . . . . . . . . . . .. . . . . . . .. . . . 343 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 Miscellaneous. . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . 345 Nonnutritive Sweeteners. . . . . . . . . . . . . . . . . . . . . . . 347 Nutrients. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 Solvents ...................................... 352 21. Food Additives: Indirect. . .. .... . .... .. . . . ... ..... 354 22. Fruits and Fruit Products ......................... 359 Moisture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 Solids........................................ 362 Potassium .................................... 363 Sodium....................................... 364 Phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 Betaine ....................................... 365 Fruit Acids .................................... 366 Sugars........................................ 369 Anthocyanins ................................. 370 vi
- 9. PAGE PAGE Lemon Juice .................................. 372 Carbaryl .................................... 483 Sulphenone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485 23. Gelatin, Dessert Preparations, and Mixes . . . . . . . . . . 374 Perthane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485 DDT........................................ 485 24. Meat and Meat Products ......................... 376 Dichlone ................. . . . . . . . . . . . . . . . . . . . 487 Meat.......................................... 376 Guthion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488 Meat Extracts and Similar Products. . . . . . . . . . . . . 383 Dodine...................................... 488 Ethylenethiourea ............................ 488 25. Metals and Other Elements as Residues in Foods .. 385 Glyodin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489 Antimony ..................................... 385 Hexachlorobenzene and Mirex ............... 490 Arsenic ....................................... 385 Malathion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490 Cadmium ..................................... 388 Maleic Hydrazide .................. . . . . . . . . . . 490 Copper....................................... 391 Methoxychlor ............................... 491 Fluorine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 Mirex . . . . . . . .. . .. . . ... . . . . . . .. . . . . ... . .. . . . . 492 Lead.......................................... 397 Monofluoroacetic Acid. . . . . . . . . . . . . . . . . . . . . . . 492 Manganese ................................... 405 Naphthaleneacetic Acid. . . . . . . . . . . . . .. . . . . . . . 493 Mercury ...................................... 405 Nicotine .................................... 494 Nickel. . .. . ... ... . .... . .. . . .. . ... . ... . . . .. . ... . 409 Parathion ................................... 495 Selenium ..................................... 409 Piperonyl Butoxide .......................... 495 Tin........................................... 410 Thiram...................................... 495 Titanium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 Zinc.......................................... 411 30. Spices and Other Condiments .................... 497 Spices........................................ 497 26. Natural Poisons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414 Mycotoxins ................................... 414 Aflatoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 Prepared Mustard ............................. 500 Dressings for Foods ........................... 501 Vinegars. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 503 Minicolumn Detection Methods .............. 417 Quantitative Methods. . . . . . . . . . . . . . . . . . . . .. . . 418 Aflatoxin M, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427 Ochratoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428 Patulin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430 Sterigmatocystin ............................ 431 Zearalenone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432 Marine Toxins. . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . 433 Phytotoxins ................................... 433 31. Sugars and Sugar Products. . . . . . . . . . . . . . . . . . . . . . . 506 Sugars and Sirups. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506 Molasses and Molasses Products. .. . . . .. . .. . .. . 516 Confectionery. ... .. . . . .. . . .. . .. . .. . . . .. . . . .. . . 519 Honey........................................ 520 Maple Products ............................... 528 Sugar Beets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532 Starch Conversion Products. . . . . . . . . . . . . . . . . . . . 532 27. Nuts and Nut Products ........................... 435 32. Vegetable Products, Processed. . . . .. . . . .. .. . . .. .. . 537 Canned Products .............................. 537 Peanut Butter ................................. 435 Shredded Coconut. . .. . . . . . . . . . . . . . . . . . . . . . . . . . 436 Dried Vegetables .............................. 542 Frozen Vegetables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543 28. Oils and Fats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437 33. Waters; and Salt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547 .. ?hysical Constants ............................ 437 Water......................................... 547 Chemical Constants ........................... 438 Salt........................................... 565 Acid Constituents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441 Sterols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452 34. Color Additives .................................. 568 Tests for Specific Oils. . . . . . . . . . . . . . . . . . . . . . . . . . 456 Separation and Identification of Color Additives Chick Edema Factor ........................... 459 in Foods, Druq!'; and Cosmetics. . . . . . . . . .. . . . 568 Mono- and Diglycerides . . . . . . . . . . . . . . . . . . . . . . . . 461 Analysis Of r brcial Synthetic Organic Color t:'laxseed ...................................... 464 Additives .....:.............................. 570 29. Pesticide Residues ............................... 466 35. Cosmetics ....................................... 582 Multiresidue Methods ......................... 466 General Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582 General Method for Organochlorine and Deodorants and Anti-Perspirants ............... 583 Organophosphorus Pesticides. . . . . . . . . . . . . . 466 Dipilatories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587 Multiple Residue Methods for Face Powders ................................. 587 Organophosphorus Pesticides. . . . . . . . . . . . . . 476 Hair Preparations. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 587 Multiple Residue Methods for Fumigants ..... 480 Suntan Preparations. . . . . . . . . . . . . . . . . . . . . . . . . . . 589 Multiple Residue Method for Aryl N- Vanishing Cream. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 589 Methylcarbamate Insecticides .............. 480 Bioassay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590 Methods for Individual Residues ............... 481 Benzene Hexachloride ....................... 481 36. Drugs: General. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 591 Biphenyl .................................... 481 General Directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591 Aramite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483 Solvents ...................................... 591 Captan ...................................... 483 Halogenated Compounds. . . . . . . . . . . . . . . . . . . . . . 592 vii
- 10. PAGE Inorganic Drugs ............................... 594 Mydriatics and Myotics ........................ 604 Microscopic Tests ............................. 604 37. Drugs: Acidic .................... '" ........ '" .. 614 Acids......................................... 614 Antipyretic Drugs. .... . ... . ... ... . . ..... ..... .. 615 Hypnotic Drugs ................. " . .. . ... ..... . 619 Phenolic Drugs. . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . 625 Sulfonamide Drugs. .. . . . . . . . . . . . . . . . . . . . . . .. . . 629 Other Imide Drugs. . . . . . . . . . . . . . . . . .. . . . . . . .. . . 631 38. Drugs: Alkaloid and Related Bases ................ 636 Alkaloids, Opium. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 636 Alkaloids Other Than Opium ................... 639 Other Bases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649 39. Drugs: Neutral ................................... 667 Steroids and Related Hormones. . . . . . . . . . . . . . . . 667 Nonalkaloidal Vegetable Drugs and Their Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674 Miscellaneous. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678 40. Drugs: Illicit. .. .. ... . . ..... ... .... . . .... . . . ... .. . . 685 41. Drugs and Feed Additives in Animal Tissues. . . . . . . 688 ANOT......................................... 688 Arsenic ....................................... 688 Clopidol ...................................... 689 Decoquinate .................................. 690 Ethoxyquin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691 Melengestrol Acetate .......................... 691 Nalidixic Acid ................................. 693 Zoalene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694 42. Drugs in Feeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 695 Qualitative Tests .............................. 695 Arsenic ....................................... 695 Cyzine ........................................ 696 Aklomide ..................................... 697 p-Aminobenzoic Acid. . . . . . .. . . . . . . . . . . . . . . . . . . 697 Enheptin ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698 Amprolium ............................... " . . . 698 Arsanilic Acid ................................. 699 Bithionol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 699 Buquinolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 699 Cadmium Anthranilate. . . . . . . . . . . . . . . . . . . . . . 700 Carbadox ..................................... 700 Decoquinate .................................. 700 Dibutyltin Dilaurate ., ..................... " . . . 701 Diethylstilbestrol .............................. 702 Nitrophenide ....... . Nitarsone .......... . Phenothiazine ...... . Piperazine .......... . Pyrantel Tartrate ... . Racephenicol ....... . Reserpine ....... " .. Ronnel .............. , Roxarsone .......... , Sulfonamide Drugs .. Thiabendazole ....... , Zoalene ............. , Antibiotics .......... , 43. Vitamins and Other Nutri Chemical Methods ... Microbiological Metho Bioassay Methods ... Nutritionally Related C 44. Extraneous Materials: IsCl General ............ . Beverages and Bevera Dairy Products ..... . Nuts and Nut Product~ Grains and Their Prodl Baked Goods ....... . Breakfast Cereals ... . Eggs and Egg Product Poultry, Meat, and Fisl Products ......... . Fruits and Fruit Produ( Sugars and Sugar Prol Vegetables and Vegetc Spices and Other ConI Animal Excretions .. . Miscellaneous ...... . 45. Forensic Sciences ..... . Latent Fingerprints .. Glass Fragments .... Mineral Wool Insulatio Voice Print Identificati( 46. Microbiological Methods Eggs and Egg Product Frozen, Chilled, Precoe Commercial Sterility 0 Thermophilic Bacterial Clostridium perfringen Salmonella . ........ . Optical Somatic Cell C
- 11. FIGURE 1:01. 2:01. 2:02. 2:03. 2:04. 2:05. 2:06. 2:07. 2:08. 3:01. 3:02. 3:03. 3:04. 3:05. 3:06. 3:07. 4:01. 4:02. 6:01. 6:02. 6:03. 6:04. 6:05. 6:06. 6:07. 6:08. 6:09. 7:01. 7:02. 7:03. Illustrations Apparatus for Automatic Filtration and Measurement of Lime Solutions ........................................... . Sampling Pattern ................................................ " ............................................. . Sampling Cup ..... " ................................... , ........ " ............................................. . Missouri and Indiana Weighted Restricted-fill Fluid Fertilizer Sampling Bottles Designed to Fill While Being Lowered (and Raised) in Storage Tanks ................................................................................... . Sampling Apparatus for Ammoniacal Solutions, Including "Quick Coupler" for Attaching to Storage Tanks .......... . Apparatus for Control of Water Pressure ......................................................................... . Flow Diagram for Automated Analysis for Phosphorus ........................................................... . Flow Schematic for K20 in Fertilizers ............................................................................ . Apparatus for Elution of Resin Column .......................................................................... . Suction Device Used in Micro Method for Determining Calcium ................................................... . Schematic Drawing of Air Flow System Used in Semiautomated Analysis for Fluoride ............................. . Flow Diagram for Semiautomated Analysis for Fluoride .......................................................... . Schematic Drawing of Microdistillation Apparatus ................................................ , ............... . Microdistillation Column ........................................................................................ . Apparatus for Determining Lignin ............................................................................... . Distillation Apparatus ........................................................................................... . Transfer Loop and Manner of Using in Phenol Coefficient Technic ................................................ . Stainless Steel Spindle for Winding Test Fabric .................................................................. . Delivery Assembly for Sampling Pressurized Containers .......................................................... . Apparatus for Distilling Arsenious Chloride ...................................................................... . Apparatus for Determining Fluorine ............................................................................. . Partition Column and Solvent Evaporator ........................................................................ . Melting Point Apparatus ........................................................................................ . Distillation Apparatus ........................................................................................... . Carbon Disulfide Evolution Apparatus .................................................................... ..... . Absorption System for Thiram ............................................................................. : .... . Filtration Apparatus ............................................................................................ . Flow Diagram for Semiautomated Analysis for Crude Protein ..................................................... . Agitator ........................................................................................................ . Oklahoma State Filter Screen ................................................................................... . PAGE 2 7 7 7 8 9 12 20 26 32 41 42 43 43 48 54 57 67 72 73 76 94 94 99 110 111 122 128 131 133 7:04. Modified California State Buchner Funnel... .... . ... . ... . .. . . . ... .... . .... . ... .... . . . .... ... . . ... ... .. . .... ... ... . 133 7:05. Continuous Heater for Distilled Water, 1.25% Alkali, and 1.25% Acid ............................................... 133 8:01. Chittick Apparatus for Gasometric Determination of Carbon Dioxide ............................... :-................ 143 9:01. 100 mL and 50 mL Pycnometers ................................................................................. 148 9:02. Williams Tube. . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 9:03. Steam Distillation Flask. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 9:04. Apparatus for Determining Hydrogen Cyanide in Distilled Spirits................................................... 158 10:01. Absorption Buret................................................................................................ 166 11 :01. Distillation Apparatus for Chemical Determination of Alcohol in Wine .............................................. 186 11 :02. Volatile Acid Still (Cash Still) .......................... " ............ '" ..................................... '" . . 188 11 :03. Carbon Dioxide Apparatus; Manometric Method ...................................... '" ............... '" . ...... 190 11 :04. Carbon Dioxide Apparatus; Volumetric Method ........ '" ................ '" . '" . . . . ...... . . ...... ... .... ... . .. . . 192 12:01. Continuous Extraction Apparatus .......... " . '" ............................ ,. . . ..... ... . . ... . .. . ... ... . . ... ... .. 195 13:01. Spiral Vessel Sections. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 13:02. Stone Cells ..................................................................................................... 204 13:03. Graph Used in Correcting Cuprous Oxide for Effect of Sucrose ........................................ '" . . .. . ... .. 207 16:01. Apparatus for Drying Pentabromacetone by Aspiration ............................................................ 241 16:02. Liquid Extractor ...................................... '" . " ....................................... , ... . . .. .... .. 242 16:03. Flow Diagram for Determination of Fat in Milk ......... '" ......... , ..... '" . .... . . .... ... . .... ... ... . .. . . ... . .. . . 247 16:04. Modified Jones Reductor ........................................................................................ 266 16:05. Modified Plastic Desiccator ...................................................................................... 273 18:01. Extraction Apparatus............................................................................................ 291 18:02. Steam Distillation Assembly ..................................................................................... 292 ix
- 12. FIGURE PAGE 18:03. Muscle Bath .................................................................................................... 294 18:04. Electrophoresis Cabinet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 300 18:05. Disc Electrophoresis Apparatus .................................................................................. 300 18:06. Key to Identification of Canned Salmon Species by Scale Characteristics ........................................... 302 18:07. Sockeye (Red) Salmon Scale (Oncorhynchus nerka) ............................................................... 303 18:08. Chinook (King) Salmon Scale (Oncorhynchus tshawytscha) ........................................................ 304 18:09. Coho (Silver) Salmon Scale (Oncorhynchus kisutch) . . . . .. . . . . . .. .. . . . ... . . .. . . ... . . . . ... . . .. . .. ... . .. . . . .. . . ... . . . 304 18:10. Chum Salmon Scale (Oncorhynchus ketal ........................................................................ 304 18:11. Pink Salmon Scale (Oncorhynchus gorbuscha) ........ ............................... .... ................... ...... 304 19:01. Wilson Flask.................................................................................................... 315 19:02. Oil Separator Trap. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . 320 20:01. Steam Distillation Apparatus. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 20:02. Placement of Slides on Template for Applying Silica Gel Coating .................................................. 327 20:03. Steam Distillation Apparatus. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . 338 20:04. Apparatus for Modified Monier-Williams Method for Sulfur Dioxide. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 339 20:05. Alternative S02 Absorber. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . . .. . . . . 339 20:06. Gas Chromatograms of TBS, DBS, and TBS + DBS . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 20:07. Microsampling Die .............................................................................................. 348 20:08. GLC Effluent Collection Trap for Determination of Cyclohexylamine ................................................ 350 21 :01. Test Cell. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . 356 21:02. Oven Rack...................................................................................................... 357 22:01. Apparatus for Determining Volume of Frozen Fruits by Displacement............................................... 359 22:02. Capillary Viscometer ............................................................................................ 361 22:03. Electrical Circuit Diagram for Dried Fruit Moisture Tester.. . . . . ... . . .. . . ... . . .... . . .... .. .. . . ... . ... . ... . . . .. . . ... . 361 22:04. Connecting Tube Adapter for Direct Distillation ................................................................... 370 23:01. 0.5 Inch Bloom Gelometer Plunger ............................................................................... 374 23:02. 1.0 Inch Bloom Gelometer Plunger. . . . . .. .. . ... .. .. . . ... ... . . . ... . ... . . . .. . . . .. . . . . . ... ... . . . ... . .. . ... . . . .... ... 375 24:01. Helix Inlet Manifold. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 377 24:02. Phosphorus Analytical Manifold. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 378 24:03. Nitrogen Analytical Manifold. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . 379 25:01. Arsenic Apparatus. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . 386 25:02. Apparatus for Flameless Atomic Absorption Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406 25:03. Digestion Vessel ................................................................................................ 407 25:04. Special Digestion Apparatus for Mercury Residues. . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 408 26:01. Spotting and Scoring Patterns for 2-Dimensional TLC Plates. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 424 26:02. Plate Scraper for Removing Adsorbent From TLC Plates .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424 26:03. Schematic Showing Parts of Egg and Modes of Test Material Introduction. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . 426 28:01. Titer Stirring Assembly. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . 439 28:02. Liquid-Liquid Extractor. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . 442 28:03. Apparatus for Determining Reichert-Meissl and Polenske Values ................................................... 443 28:04. Constant Temperature Bath and Accessories. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . 444 28:05. Distribution Heads; Manifold. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445 28:06. Glass Micro Filter for Sterol Acetate Precipitates .................................................................. 452 28:07. Crystalline Forms of Free Sterols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 453 28:08. Apparatus for Determining Melting Point ......................................................................... 458 29:01. KCI Thermionic Detector Coil for In-series Dual Detection System .................................................. 468 29:02. KCI Thermionic Detector Coil for Parallel and In-series Split Dual Detection Systems ................................ 469 29:03. In-series Split Dual Detection System. . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . 469 29:04. Parallel Dual Detection System. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 469 29:05. In-series Dual Detection System. .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . 469 29:06. Sweep Co-distillation Apparatus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477 29:07. Evaporative Concentrator. . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . 484 29:08. Distillation Apparatus for Maleic Hydrazide Determination ......................................................... 491 30:01. Apparatus for Volatile Oil in Spices. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . 498 31 :01. Fractionator..................................................................................................... 506 31 :02. Carbon Combustion and Purification System. . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 527 31 :03. Distillation Apparatus for Determining Formaldehyde in Maple Sirup.. . . .... . ... .. . . . ... . ... . . ... . .. . . ... . ... . ... . . 530 33:01. Apparatus for Determination of Mercury by Flameless Atomic Absorption. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 560 33:02. Phosphorus Manifold. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563 34:01. Titanous Chloride Titration Apparatus ............................................................................ 574 34:02. Sulfiding Apparatus ............................................................................................. 579 36:01. Apparatus for Determining Arsenic in Iron-Arsenic Tablets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 594 36:02. Flow Diagrams for Semiautomated Analysis for Ferrous Sulfate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . 601 37:01. Bromine Apparatus.............................................................................................. 619 x
- 13. FIGURE PAGE 37:02. Flow Diagram for Automated Analysis for Acenocoumarol, Dicumarol, Phenprocoumon, Potassium Warfarin, and Sodium Warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629 38:01. Flow Diagram for Semiautomated Fluorometric Analysis for Reserpine............................................. 646 38:02. Flow Diagram for Phenylephrine Hydrochloride ................................................................... 661 38:03. Assembly of Debubbler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662 39:01. Flow Diagram for Semiautomated Analysis for Prednisolone or Prednisone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674 39:02. Flow Diagram for Automated Analysis for Digoxin ................................................................ 675 39:03. Scrubber Trap for Ammonia Distillation .......................................................................... 679 39:04. Flow Diagram for Automated Analysis of Methenamine and Methenamine Mandelate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683 43:01. Flow Diagram for Automated Analysis for Niacin and Niacinamide.. .... . .... ..... . . . ........ . .... .... ... . . ... . ... . 745 43:02. RRR-a-Tocopherol . . . . . . . .. . . . . . . .. . . . . . .. . . . . . . .. . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . 752 43:03. Line Test Chart. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771 43:04. Radii Sections Scored According to Line Test Chart ............................................................... 772 44:01. Mechanical Butter Stirrer ........................................................................................ 781 44:02. Wildman Trap Flask .......................................................................... , .... ........ . ... .. 781 44:03. Rot Fragment Counting Slide .................................................................................... 782 44:04. Sediment Filtering Apparatus, Unassembled ...................................................................... 786 44:05. Sediment Filtering Apparatus, Assembled ........................................................................ 786 44:06. Rot Fragments from Tomato Puree ...................... , . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . 800 44:07. Rot Fragments from Tomato Puree. . . . . . .. . . . . . . .. . . . . ... . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . 801 44:08. Mold Filaments in Tomato Products. . . . . .. . . . . . . .. . . . . . .. . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 802 44:09. Insect Penetration of Packaging .................................................................................. 817 46:01. Plastic Template Schematic for Microslide Assembly.............................................................. 842 46:02. Arrangement of Antisera and Homologous Reference Enterotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 843 46:03. Examples of 4 Possible Reactions in Bivalent Detection System .................................................... 844 46:04. Effect of Amount of Enterotoxin in Test Preparation on Development of Reference Line of Precipitation .............. 845 46:05. Appearance of Microslide Gel Diffusion Test as Monovalent System ............................................... 845 46:06. Precipitate Patterns in Microslide Gel Diffusion Test Demonstrating Nonspecific (Atypical) Lines of Precipitation. . . . . . 845 46:07. Optical Somatic Cell Counter Flow Diagram ....................................................... , . . . .. . . . . . . . . . . 846 46:08. Optical Somatic Cell Counter Flow Diagram. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 848 46:09. Optical Somatic Cell Counter Flow Diagram. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 849 47:01. Combustion Tube............................................................................................... 853 47:02. Carbon and Hydrogen Apparatus.... . . . .......... ... ......... ........... .... ..... . . . ... . .... .... ....... ....... . .. 855 47:03. Upper Section of Distilling Apparatus. . . . .. . . . . . . . . . . . . ... . . . . . . .. . . . . . .. . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . 857 47:04. Gravimetric Setup for Oxygen Determination ..................................................................... 859 47:05. Quartz Reaction Tube and Filling for Oxygen Determination ....................................................... 859 47:06. Details of Modified Clark Apparatus .............................................................................. 862 47:07. Modified Clark Apparatus. . . . . . . .. . . . . . . .. . . . . . . . . . . . . ... . . . . . . .. . . . . . .. . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . 863 48:01. Cross-section of Marinelli Beaker. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869 xi
- 14. Collaborative Study Procedures of the Association of Official Analytical Chemists The Association of Official Analytical Chemists (AOAC) is a unique, nonprofit scientific organization whose primary purpose is to serve the needs of government regulatory and research agencies for analytical methods. The goal of the Association is to provide methods which will perform with the necessary accuracy and precision under usual laboratory conditions (1). Since its formation in 1884 the AOAC has provided a mechanism to select methods of analysis from published literature or develop new methods, collaboratively test them through inter- laboratory studies, approve them, and publish the approved methods for a wide variety of materials relating to foods, drugs, cosmetics, agriculture, forensic science, and products affecting the public health and welfare. Its membership is composed of scientists from Federal, State, Provincial, and other regulatory bodies who work within the AOAC's established procedures as researchers, methods collaborators, and committee members. Although most of the members are from North America, many nations throughout the world are represented. The AOAC has almost a century of experience in utilizing the collaborative study as a means of determining the reliability of analytical methods for general purposes and, especially, for regulatory purposes. In fact, the AOAC's major contribution to analytical science has been to bring the collaborative study technique for the validation of analytical methods to a high degree of perfection. In such a study, laboratories analyze identical sample sets which cover the range of applicability of a method previously selected as being useful and practical. The purpose of the study is to establish the characteristics of the methods with respect to accuracy, precision, sensitivity, range, specificity, limit of detection, limit of reliable measurement, selectivity, practicality, and similar attributes, as required. ORGANIZATION AND PROCEDURES FOR AOAC COLLABORATIVE STUDIES The collaborative study is organized and directed by an analyst designated as the Associate Referee for the specific subject under investigation. Currently, some 600 Associate Referees appointed by the Association are responsible for as many topics. An Associate Referee is selected for his knowledge, interest, and experience in the subject matter field. He operates under the scientific guidance, support, and administrative supervision of a General Referee, who is in turn responsible for a product area. The Associate Referee reviews the literature and selects one or two of the better analytical methods available, modifying them as needed. Alternatively, he may develop or adapt a method used in his laboratory for the analyte and matrix under study, testing it thoroughly in his laboratory before designing a collaborative study. The General Referee is kept informed of such preliminary studies. The samples analyzed in a collaborative study are normally prepared and distributed to the participants by the Associate Referee. The Association follows the recommendations of You- den (2) that not fewer than five laboratories participate and that a minimum of six sample materials be sent to each. These are xii minima and, in practice, both are usually exceeded. In addition, a reference or practice sample is included, where possible. Laboratories with at least some experience in the general subject matter are selected as collaborators. Because the objec- tive of the study is to standardize the method, as contrasted to standardizing the analyst (3). all analysts are instructed to follow the method exactly as written even though they may not concur with the Associate Referee's selection among possible alterna- tives. The level ofthe analyte in the samples is usually unknown to the participants. All individual results obtained by the collaborators are re- ported to the Associate Referee, who compiles and evaluates them. Since statistical treatment of the data is considered essential in a rigorous evaluation of the method for accuracy, precision, sensitivity, and specificity, it is now required for all studies. The Association considers this of such importance that it provides statistical assistance in all cases where it is otherwise unavailable to the Associate Referee. A statistical manual (4) is also provided. The Associate Referee makes the initial judgment on the performance of the method. If he recommends approval, it passes to the General Referee and then to a committee of experts. If both recommend approval, the method is presented at the Association's annual business meeting for vote by the membership. Approved methods and supporting data are published in the Journal of the Association of Official Analytical Chemists. They are subject to scrutiny and general testing by other analysts for at least a year before final adoption. They may be modified and restudied collaboratively as needed, should feedback from gen- eral use reveal flaws in the method or in its written set of directions. Approved methods are included in the Association's "Official Methods of Analysis", a book of some 1000 pages which is updated every 4-5 years. The preceding summary of AOAC's modus operandi recog- nizes the need for healthy skepticism toward results obtained by analytical methods which have not undergone such rigorous scrutiny and interlaboratory testing of their accuracy, precision, dependability, specificity, and practicality. SELECTION OF METHODS FOR STUDY A certain degree of variability is associated with all measure- ments. Much of the research on analytical chemistry is an attempt to minimize that variability. But there are many different types of variability in analytical work. We often find that when we attempt to minimize one kind, we must necessarily permit expansion in another kind. In practical analytical chemistry, the problem often comes down to which variability is to be mini- mized. Some examples ofthis point may be helpful. In atomic weight determination, everything-especially practicality-is sacrificed for accuracy. A high degree of accuracy and practicality is required in the assay of precious metals, but the fire assay used is generally applicable to little else besides metals and minerals.
- 15. In clinical chemistry, within-laboratory precision (repeatability) is critical, and often is of greater interest to clinical laboratories than absolute accuracy or agreement with the values of other laboratories (reproducibility). In drug analysis, a high degree of accuracy is required in the therapeutic range because the analytical values determining the identity, strength, quality, and purity of pharmaceutical preparations, as laid down in phar- macopoeial specifications, are directly related to clinical value. With polynuclear hydrocarbons, specificity is important, since some of these compounds are carcinogenic while others are not. In applying the famous Delaney clause of the United States Federal Food, Drug, and Cosmetic Act, all attributes of the analytical methods are secondary to the detection of extremely small concentrations (detectability), or to exhibiting a high degree of response for small changes in concentration (sensi- tivity). There is a very special case involving accuracy, where the "true value" is determined by the method of analysis. Many legal specifications and standards for food and agricultural products define ill-defined components such as moisture, fat, protein, and crude fiber in terms of reference methods. There- fore, the precision of these methods becomes the limiting factor for their performance. In fact, most analyses involved in com- mercial transactions require primarily that the buyer and seller agree on the same value (analytically and economically), re- gardless of where it stands on an absolute scale. The point of these examples is that although methods of analysis are characterized by a number of attributes-accuracy, precision, specificity, sensitivity, detectability, dependability, and practicality-no method is so flawless that all these qualities can be maximized simultaneously. For any particular analysis, the analyst must determine, on the basis of the purpose of the analysis, which attributes are essential and which may be compromised. Unfortunately, the literature is replete with examples indicat- ing that an individual analyst, and especially the originator of a method of analysis, is not an unbiased judge of the relative merits of the methods of analysis which he develops and uses. In our experience, the collaborative study provides impartial data on the suitability of the method. The data, in many cases, speak for themselves. The collaborative study, or ring test or round robin test, as it is called in other organizations, provides the basic information on the performance of analytical methods. The extent of the information will depend on the number of samples provided, the number of analyses performed, and the number of labora- tories participating. The data should be unbiased because the composition of the samples is known only to the administrator of the study. Some of the requirements of the study and their relationship to the characteristics and attributes of the method are as follows: (1) Accuracy. Samples must be of defined composition (by spiking, by formulation, or by analytical consensus). (2) Specificity. Samples should contain related analytes. (3) Sensitivity. Samples should differ from each other or from negative samples by a known amount. (4) Applicability. Samples should include the concentration range and matrix components of interest. (5) Blanks. Samples should include different matrices with "none" of the component of interest. (6) Precision. Instructions should request replicate analyses by the same or different analysts in the same laboratory, preferably on different days. By far a better procedure is to include "blind" (unknown to the analyst) replicate samples in the series. (7) Practicality. Instructions should request information as to xiii the actual and elapsed time required for the analyses; the availability of reagents, equipment, and standards; and any necessary substitutions. When practice samples are included, the number of analyses required to achieve the stated recovery and repeatability, should be reported. PROCEDURAL DETAILS OF COLLABORATIVE STUDY As numerous beginners in this field have discovered, much preliminary work must be done before sending out samples: (1) The method must be chosen and demonstrated to apply to the matrices and concentrations of interest. (2) The critical variables in the method should have been determined and the need for their control emphasized [a rug- gedness test (5) is useful for this purpose]. (3) The method should be written in detail by the Associate Referee and tested by an analyst not previously connected with its development. (4) Unusual standards, reagents, and equipment must be available from usual commercial sources of supply, or sufficient quantities must be prepared or obtained to furnish to the participants. (5) The samples must be identical and homogeneous so that the analytical sample error is only a negligible fraction of the expected analytical error. (6) A sufficient number of samples must be prepared to cover typical matrices and the concentration range of interest (toler- ance, maximum or minimum specifications, likely levels of occurrence, etc.). (7) Samples must be stable and capable of surviving the rigors of commercial transportation. (8) Reserve samples should be prepared and preserved to replace lost samples and to permit reanalysis of samples con- sidered as outliers to attempt to discover the cause of abnormal results. (9) The instructions must be clear. They should be reviewed by someone not connected with the study to uncover potential misunderstandings and ambiguities. (10) If the analyte is subject to change (e.g., bacterial levels, nitroglycerin tablets), provision must be made for all participants to begin the analysis at the same time. (11) Practice samples of a known and declared composition should be furnished with instructions not to analyze the un- knowns until a specified degree of recovery and repeatability (or other attribute) has been achieved. (12) Provision should be made when necessary for submis- sion of standard curves, tracings of recorder charts, or photo- graphs of thin-layer plates in order to assist in determining possible causes of error. OTHER TYPES OF INTERLABORATORY STUDIES This type of collaborative study, which is designed to deter- mine the characteristics of a method, must be carefully distin- guished from other types of interlaboratory studies which by design or through ignorance provide other kinds of information. The most important types of other studies are: (1) Those studies which require the collaborators to investi- gate the variability of parts of methods or applicability to different types of samples. (An interlaboratory study is usually an inefficient way of obtaining this type of information.) (2) Those studies which permit an analyst to use any method he desires. Such studies invariably produce such a wide scatter of results that the data are of little value for evaluation of methods. They may be useful in selecting a method from a number of apparently equivalent methods, provided the purpose
- 16. is emphasized beforehand and the participants provide a de- scription of the method used in order to permit a correlation of the details of the methods with apparent biases and variabilities. (3) Those studies which are used for quality control purposes, whose participants are not permitted sufficient time to gain familiarity with the method, or who permit deviations to enter into the performance of the analyses on the grounds that the deviation is obviously an improvement which could not possibly affect the results of the analysis, or who claim to have a superior method. With this background information, it is now appropriate to introduce the following definitions which were agreed upon as part of the guidelines for collaboration between the AOAC and the Collaborative International Pesticide Analytical Council L~d. (CIPAC) (6). Collaborative study. An analytical study involving a number of laboratories analyzing the same sample(s) by the same method(s) for the purpose of validating the performance of the method(s). Preliminary interlaboratory study. An analytical study in which two or more laboratories evaluate a method to determine if it is ready for a collaborative study. Laboratory performance check. The analysis of very carefully prepared and homogeneous samples, normally of known active ingredient content, to establish or verify the performance of a laboratory or analyst. SUMMARY The collaborative study i's an experiment designed to evaluate the performance of a method of analysis through the analysis of a number of identical samples by a number of different laboratories. With proper design, it provides an unbiased eval- uation of the performance of a method in the hands of those analysts who will use it. A collaborative study must be distin- guished from those studies designed to choose a method or to determine laboratory or analyst performance. REFERENCES (1) AOAC, "Handbook of the AOAC", 4th ed., AOAC, Box 540, Benjamin Franklin Station, Washington, D.C. 20044, 1977. (2) W. J. Youden, "Accuracy of Analytical Procedures", J. Assoc. Off. Anal. Chern., 45, 169-73 (1962). (3) Harold Egan, "Methods of Analysis; An Analysis of Meth- ods", ibid., 60,260-7 (1977). (4) W. J. Youden and E. H. Steiner, "Statistical Manual of the AOAC: Statistical Techniques for Collaborative Tests. Plan- ning and Analysis of Results of Collaborative Tests", AOAC, Box 540, Benjamin Franklin Station, Washington, D.C. 20044, 1975. (5) W. J. Youden, "The Collaborative Test", J. Assoc. Off. Anal. Chern., 46, 55-62 (1963). (6) "Guidelines for Collaboration Between the Association of Official Analytical Chemists (AOAC) and the Collaborative International Pesticide Analytical Council Ltd. (CIPAC)", ibid., 57,447-9 (1974). BIBLIOGRAPHY Daniel Banes, "The Collaborative Study as a Scientific Concept", J. Assoc. Off. Anal. Chern., 52,203-06 (1969). William Horwitz, "Problems of Sampling and Analytical Meth- ods", ibid., 59, 1197-203 (1976). Reprinted with permission from: Analytical Chemistry (March 1978) 50, 337A-340A. Published 1978 American Chemical Society xiv
- 17. Definitions of Terms and Explanatory Notes Reagents (1) Term "H20" means distilled water, except where otherwise specified, and except where the water does not mix with the detn, as in "H20 bath." (2) Term "alcohol" means 95% ethanol by vol. Alcohol of strength x% may be prepd by dilg x mL 95% alcohol to 95 mL with H20. Absolute alcohol is 99.5% by vol. Formulae of specially denatured alcohols (SDA) used as reagents are as follows: SDA No. 100 parts alcohol plus 1 5 wood alcohol 2-B 3-A 12-A 13-A 23-A 30 0.5 5 5 10 10 10 benzene or rubber hydrocarbon solv. MeOH benzene ether acetone MeOH "Reagent" alcohol is 95 parts SDA 3-A plus 5 parts isopropanol. (3) Term "ether" means ethyl ether, peroxide-free by follow- ing test: To 420 mL ether in separator add 9.0 mL 1% NH4V03 in H2S04 (1+16). Shake 3 min and let sep. Drain lower layer into 25 mL g-s graduate, dil. to 10 mL with H2S04 (1 +16), and mix. Any orange color should not exceed that produced by 0.30 mg H20 2(1 mL of soln prepd by dilg 1 mL 30% H20 2to 100 mL with H20) and 9.0 mL 1% NH4V03in H2S04 (1 + 16). Peroxides may be eliminated by passing .;;700 mL ether thru 10 cm column of Woelm basic alumina in 22 mm id tube. (4) Reagents listed below, unless otherwise specified, have approx. strength stated and conform in purity with Recom- mended Specifications for Analytical Reagent Chemicals of American Chemical Society: Assay Sulfuric Acid . . . . . . . . . . . . . . . . . . . . . . . .. 95.0-98.0% H2S04 Hydrochloric acid .................... 36.5-38.0% HCI Nitric acid ........................... 69.0-71.0% HN03 Fuming nitric acid. . . . . . . . . . . .. . . . . . .. ;,,90% HN03 Acetic acid . . . . . . . . . . . . . . . . . . . . . . . . . .. ;,,99.7% HC2H30 2 Hydrobromic acid ......... " ..... . ... 47.0-49.0% HBr Ammonium hydroxide. . . . . . . . . . . . . . .. 28-30% NH3 Phosphoric acid. . . . . . . . . . . . . . . . . . . . .. ;,,85% H3P04 Where no indication of diln is given, reagent is of concn given above. (5) All other reagents and test solns, unless otherwise de- scribed in text, conform to requirements of American Chemical Society. Where such specifications have not been prepd, use highest grade reagent. When anhyd. salt is intended, it is so stated; otherwise the crystd product is meant. (6) Unless otherwise specified, phenolphthalein (phthln) used as indicator is 1% alc. soln; Me orange is 0.1 % aq. soln; Me red is 0.1 % alc. soln. (7) Directions for stdzg reagents are given in Chapter 50. (8) Unusual reagents not mentioned in reagent sections or cross referenced, other than common reagents normally found in laboratory, are italicized first time they occur in method. (9) Com. prepd reagentsolns must be checked for applicability to specific method. They may contain undeclared buffers, pre- servatives, chelating agents, etc. xv (10) In expressions (1 +2), (5+4), etc., used in connection with name of reagent, first numeral indicates vol. reagent used, and second numeral indicates vol. H20. For example, HCI (1 +2) means reagent prepd by mixing 1 vol. HCI with 2 vols H20. When one of reagents is solid, expression means parts by wt, first numeral representing solid reagent and second numeral H20. Solns for which the solv. is not specified are aq. solns. (11) In making up solns of definite percentage, it is understood thatx g substance is dissolved in H20 and dild to 100 mL. Altho not theoretically correct, this convention will not result in any appreciable error in any of methods given in this book. (12) Chromic acid cleaning soln is prepd by (1) adding 1 L H2SO. to ca 35 mL satd aq. Na2Cr207 soln; or (2) adding 2220 mL (9 Ib) H2S04 to ca 25 mL satd aq. Cr03 soln (170 g/100 mL). Reagents may be tech. grade. Use only after first cleaning by other means (e.g., detergent) and draining. Mixt. is expensive and hazardous. Use repeatedly until it is dild Of has a greenish tinge. Discard carefully with copious amts of H20. (13) All calcns are based on table of international atomic weights, 52.001. Apparatus (14) Burets, vol. flasks, and pipets conform to following Federal specifications (available from General Services Admin., Specification Activity 3F1, Washington Navy Yard, Bldg. 197, Washington, DC 20407): Buret NNN-B-00789a May 19, 1965 Flask, vol. NNN-F-00289d Feb 7, 1977 Pipet, vol. NNN-P-395c March 13, 1970 Pipet, measuring NNN-P-350c July 16,1973 See also NBS Circular 602, "Testing of Glass Volumetric Ap- paratus" (available as Com 73-10504 from NTIS, Springfield, VA 22151). (15) Standard taper ($) glass joints may be used instead of stoppers where the latter are specified or implied for connecting glass app. (16) Sieve designations, unless otherwise specified, are those described in Federal Specification RR-S-366e, Nov 9,1973 (avail- able from General Services Admin.). Designation" '100-mesh' (or other number) powder (material, etc.)" means material ground to pass thru std sieve No. 100 (or other number). Corresponding international std and US std sieves are given in Table 1. (17) Term "paper" means filter paper, unless otherwise spec- ified. (18) Term "high-speed blender" designates mixer with 4 canted, sharp-edge, stainless steel blades rotating at the bottom of 4-lobe jar at 10,000-12,000 rpm, or with equiv. shearing action. Suspended solids are reduced to fine pulp by action of blades and by lobular container, which swirls suspended solids into blades. Waring Blendor, or equiv., meets these require- ments. (19) "Flat-end rod" is glass rod with one end flattened by heating to softening in flame and pressing vertically on flat surface to form circular disk with flat bottom at end. (20) Designation and pore diam. range of fritted glassware are: extra coarse, 170-220 /-Lm; coarse, 40-60; medium, 10-15; fine, 4-5.5; Jena designations and pore diam. are: 1, 110 /-Lm; 2, 45; 3, 25; 4, 8.
- 18. (21) Unless otherwise indicated, temps are expressed as degrees Centigrade. Table 1. Nominal Dimensions of Standard Test Sieves (U.S.A. Standard Series) Sieve Designation Nominal Nominal International Sieve Wire Standard' U.S.A. Opening, Diameter, (ISO) Standard inches mm 12.5 mmb '/2 in.b 0.500 2.67 11.2 mm 7/'6 in. 0.438 2.45 9.5 mm 3/8 in. 0.375 2.27 8.0 mm 5/,6 in. 0.312 2.07 6.7 mm 0.265 in. 0.265 1.87 6.3 mmb '/. in." 0.250 1.82 5.6 mm No. 3'/2 0.223 1.68 4.75 mm No.4 0.187 1.54 4.00 mm No.5 0.157 1.37 3.35 mm No.6 0.132 1.23 2.80 mm No.7 0.111 1.10 2.38 mm No.8 0.0937 1.00 2.00 mm No. 10 0.0787 0.900 1.70 mm No. 12 0.0661 0.810 1.40 mm No. 14 0.0555 0.725 1.18 mm No. 16 0.0469 0.650 1.00 mm No. 18 0.0394 0.580 850 "me No. 20 0.0331 0.510 710 "m No. 25 0.0278 0.450 600 "m No. 30 0.0234 0.390 500 "m No. 35 0.0197 0.340 425 "m No. 40 0.0165 0.290 355 "m No. 45 0.0139 0.247 300 "m No. 50 0.Q117 0.215 250 "m No. 60 0.0098 0.180 212 "m No. 70 0.0083 0.152 180 "m No. 80 0.0070 0.131 150 "m No. 100 0.0059 0.110 125 "m No. 120 0.0049 0.091 106 "m No. 140 0.0041 0.076 90 "m No. 170 0.0035 0.064 75 "m No. 200 0.0029 0.053 63 "m No. 230 0.0025 0.044 53 "m No. 270 0.0021 0.037 a These standard designations correspond to the values for test sieve apertures recommended by the International Organization for Standard- ization, Geneva, Switzerland. b These sieves are not in the standard series but they have been included because they are in common usage. c 1000 I-'m = 1 mm. Standard Operations (22) Operations specified as "wash (rinse, ext. etc.) with two (three, four, etc.) 10 mL (or other voL) portions H20 (or other solv.)" mean that the operation is to be performed with indicated vol. of solv. and repeated with same vol. of solv. until number of portions required have been used. (23) Definitions of terms used in methods involving spectro- photometry are those given in JAOAC 37, 54(1954). Most important principles and definitions are: (a) More accurate instrument may be substituted for less accurate instrument (e.g., spectrophtr may replace colorimeter) where latter is specified in method. Wavelength specified in method is understood to be that of max. absorbance (A). unless no peak is present. (b) Absorbance(s) (A).-Neg. logarithm to base 10 of ratio of transmittance (T) of sample to that of ref. or std material. Other xvi names that have been used for quantity represented by this term are optical density, extinction, and absorbancy. (e) Absorptivity(ies) (a).-Absorbance per unit concn and cell length. a = A/be, where b is in cm and c in gil, or a = (A/be) x 1000, if c is in mg/L. Other names that have been used for this or related quantities are extinction coefficient, specific absorption, absorbance index, and £16';'". (d) Transmittance(s) (T).-Ratio of radiant power transmitted by sample to radiant power incident on sample, when both are measured at same spectral position and with same slit width. Beam is understood to be parallel radiation and incident at right angles to plane parallel surface of sample. If sample is soln, solute transmittance is quantity usually desired and is detd directly as ratio of transmittance of soln in cell to transmittance of solv. in an equal cell. Other names that have been used for this quantity are transmittancy and transmission. (e) Standardization.-Spectrophtr may be checked for accu- racy of wavelength scale by ref. to Hg lines: 239.95, 248.3, 253.65, 265.3, 280.4, 302.25, 313.16, 334.15, 365.43, 404.66, 435.83, 546.07, 578.0, and 1014.0 nm. To check consistency of absorbance scale, prep. soln of 0.0400 g K2CrO./L 0.05N KOH and det. absorbance at following wavelengths in 1 cm cell: 230 nm, 0.171; 275,0.757; 313.2,0.043; 375, 0.991; 400, 0.396. See "Standards for Checking the Calibration of Spectrophotome- ters," Letter Circular LC-l017, reissued Jan 1967, NBS. (24) Least square treatment of data and calculation of regres- sion lines.-This technic finds the best fitting straight line for set of data such as std curve. It calcs that straight line whose sum of squares of vertical deviations (usually A) of observations from the line is smaller than corresponding sum of squares of deviations from any other line. Equation of straight line is: Y = a + bX, where a is intercept at Y axis (X = 0). and b is slope of line. Least square estimates of constants are: b = I (X, Y,) - [(IX,I Y,)/n] IXf - (IX,)2/n a=Y-bX, where I = "sum of" the n individual values of indicated operation, and X and Yare the averages of the X and Y points. Example: To find "best" straight line relating A (Y) to concn (X): Observation Concn Absorbance No. CI X, Y, X~ X,V, 1 80 1.270 6400 101.6 2 60 1.000 3600 60.0 3 40 0.700 1600 28.0 4 30 0.550 900 16.5 5 20 0.250 400 5.0 6 10 0.100 100 1.0 7 0 0.050 0 0.0 Totals: n=7 IX, = 240 IY, = 3.92 IXl = 13000 I(X,Y,) = 212.1 x= IX,!n = 240/7 = 34.29 Y = IY,!n = 3.92/7 = 0.56 b = 212.1 - (240)(3.92)/7 77.7 = 0.0163 13000 - (240)2/7 4771 a = 0.56 - 0.0163(34.29) = 0.001 Best equation is then: Y = 0.00 + 0.0163X If for sample, A = 0.82, corresponding concn (X) would be: X = (Y - 0.00)/0.0163 =0.82/0.0163 = 50.3.
- 19. Many scientific and statistical calculators are preprogrammed to perform this calcn. (25) Common safety precautions are given in Chapter 51. Editorial Conventions (26) For sake of simplicity, abbreviations CI and I instead of CI2 and 12 are used for chlorine and iodine. Similar abbreviations have been used in other cases (0, N, H). The same abbreviation may also be used for the ion where no ambiguity will result. (27) Reagents and app. referenced with only a letter, e.g., (c). will be found in the reagent or apparatus section of that method. (28) To conserve space, most of the articles and some prep- ositions have been eliminated. (29) Names and addresses of manufacturers and suppliers, and trade names of frequently mentioned materials, are fur- nished below solely as a matter of identification and conven- ience, without implication of approval, endorsement, or certifi- cation. The same products available from other suppliers or other brands from other sources may serve equally well if proper tests indicate their use is satisfactory. These firms when mentioned in a method are given by name only (without addresses). Manufacturers and Suppliers Ace Glass, Inc., PO Box 688, 1430 N West Blvd, Vineland, NJ 08360 Aldrich Chemical Co., Inc., 940 W St. Paul Ave, Milwaukee, WI 53233 Allied Chemical Corp., Specialty Chemicals Div., PO Box 1087R, Morristown, NJ 07960 Aluminum Company of America, 1501 Alcoa Bldg, Pittsburgh, PA 15219 American Cyanamid Co., Agricultural Div., PO Box 400, Princeton, NJ 08540 American Instrument Co., Div. of Travenol Laboratories, Inc., 8030 Georgia Ave, Silver Spring, MD 20910 (ASBC) American Society of Brewing Chemists, 3340 Pilot Knob Rd, St. Paul, MN 55121 (ATCC) American Type Culture Collection, 12301 Parklawn Dr, Rockville, MD 20852 Analabs Inc., 80 Republic Dr, North Haven, CT 06473 Applied Science Laboratories, Inc. (Applied Science Division, Milton Roy Co.). PO Box 440, State College, PA 16801 Baird-Atomic, Inc., 125 Middlesex Tnpk, Bedford, MA 01730 J. T. Baker Chemical Co., 222 Red School Ln, Phillipsburg, NJ 08865 Barber-Colman Co., see Searle Analytic, Inc. Bausch & Lomb, Inc., Analytical Systems Div., 820 Linden Ave, Rochester, NY 14625 BBL, Div. of Bioquest, PO Box 243, Cockeysville, MD 21030 Beckman Instruments, Inc., 2500 Harbor Blvd, Fullerton, CA 92634 Becton, Dickinson, & Co., Rutherford, NJ 07070 Bio-Rad Laboratories, 32nd and Griffin Ave, Richmond, CA 94804 Brinkmann Instruments, Inc., Cantiague Rd, Westbury, NY 11590 Burdick & Jackson Laboratories, Inc., 1953 S Harvey St, Muske- gon, MI 49442 Burrell Corp., 2223 Fifth Ave, Pittsburgh, PA 15219 Calbiochem, 10933 N Torrey Pines Rd, La Jolla, CA 92037 Carborundum Co., PO Box 423, Niagara Falls, NY 14302 Cenco Inc., 2600 5 Kostner Ave, Chicago, IL 60623 Coleman Instruments Division, Perkin-Elmer Corp., 2000 York Rd, Oak Brook, IL 60521 Corning Glass Works, Laboratory Products Dept., Corning, NY 14830 xvii Curtin Matheson Scientific, Inc., PO Box 1546, Houston, TX 77001 Ddco Laboratories, PO Box 1058A, Detroit, MI 48232 Dohrmann Div. of Envirotech Corp., 3240 Scott Blvd, Santa Clara, CA 95050 Dow Chemical Co., Ag-Organics Dept., PO Box 1706, Midland, MI48640 Dow Corning Corp., Midland, MI 48640 E. I. du Pont de Nemours & Co., Wilmington, DE 19898 Eastman Kodak Co., Eastman Organic Chemicals, 343 State St, Rochester, NY 14650 Eaton-Dikeman Co., Mt. Holly Springs, PA 17065 Elanco Products Co., Div. of Eli Lilly Co., Elanco Analytical Laboratory, Dept. MC757, Indianapolis, IN 46206 Fisher & Porter Co., Lab Crest Scientific Div., County Line Rd, Warminster, PA 18974 Fisher Scientific Co., 711 Forbes Ave, Pittsburgh, PA 15219 Floridin Co., Berkeley Springs, WV 25411 Foss America Inc., PO Box 504, Route 82, Fishkill, NY 12524 GAF Corp., 140 W 51st St, New York, NY 10020 G.B. Fermentation Industries, Inc., 1 N Broadway, Des Plaines, IL 60016 Geigy Chemical Corp., Saw Mill River Rd, Ardsley, NY 10502 Hamilton Co., PO Box 17500, Reno NV 89510 Hess & Clark Laboratories, Div. of Rhodia, Inc., 7th and Orange Sts, Ashland, OH 44805 Hewlett-Packard Co., 1501 Page Mill Rd, Palo Alto, CA 94304 Hoffman-La Roche, Inc., Nutley, NJ 07110 ICI-America, Inc., Chemical Research Dept., Wilmington, DE 19899 ICN-K&K Laboratories, Inc., 121 Express St, Plainview, NY 11803 ICN Pharmaceuticals, Inc., Life Sciences Group, 26201 Miles Rd, Cleveland, OH 44128 johns-Manville Products Corp., Greenwood Plaza, Denver, CO 80217 Kimble Products, Owens-Illinois, PO Box 1035, Toledo, OH 43666 Kontes Glass Co., Spruce St, Vineland, NJ 08360 Labconco Corp., 8811 Prospect Ave, Kansas City, MO 64132 Eli Lilly & Co., 740 5 Alabama St, Indianapolis, IN 46206 Mallinckrodt Chemicals Works, Science Products Div., 2nd & Mallinckrodt Sts, St. Louis, MO 63147 MC/B Manufacturing Chemists, 2909 Highland Ave, Norwood, OH 45212 Matheson Scientific, Inc., see Curtin Matheson Scientific, Inc. Merck & Co., Inc., 126 E Lincoln Ave, Rahway, NJ 07065 Miles Laboratories, Inc., Elkhart, IN 46514 Monsanto Chemical Co., 800 N Lindberg Blvd, St. Louis, MO 63166 (NBS) National Bureau of Standards, Washington, DC 20234 (NF) National Formulary, see USP New York Laboratory Supply Co., 510 Hempstead Tnpk, West Hempstead, NY 11552 Orion Research Inc., 380 Putnam Ave, Cambridge, MA 02139 Perkin-Elmer Corp., 702-G Main Ave, Norwalk, CT 06856 Phillips Chemical Co., Division of Phillips Petroleum Co., Spe- cialty Chemicals, Drawer '0', Borger, TX 79007. Pierce Chemical Co., PO Box 117, Rockford, IL 61105 H. Reeve Angel & Co., Inc., 9 Bridewell PI, Clifton, NJ 07014 Rohm & Haas Co., Independence Mall West, Philadelphia, PA 19105 Salsbury Laboratories, Charles City, IA 50616 Sargent-Welch Scientific Co., 7300 N Linder Ave, Skokie, IL 60076 (S&S) Schleicher & Schuell, Inc., 543 Washington St, Keene, NH 03431
- 20. Schoeffellnstrument Corp., 24 Booker St, Westwood, NJ 07675 SGA Scientific, Inc., 735 Broad St, Bloomfield, NJ 07003 Scientific Products, Div. of American Hospital Supply Corp., 1430 Waukegan Rd, McGaw Park, IL 60085 Searle Analytic, Inc., 2000 Nuclear Dr, Des Plaines, IL 60018 Shell Oil Co., PO Box 2463, Houston, TX 77001 Sigma Chemical Co., PO Box 14508, Sl. Louis, MO 63178 G. Frederick Smith Chemical Co., PO Box 23344, Columbus, OH 43223 Sterwin Chemicals, Inc., 90 Park Ave, New York, NY 10016 Supelco, Bellefonte, PA 16823 Technicon Instruments Corp., 511 Benedict Ave, Tarrytown, NY 10591 Arthur H. Thomas Co., Vine St at 3rd, PO Box 779, Philadelphia, PA 19105 Ultra-Violet Products, Inc., 5100 Walnut Grove Ave, San Gabriel, CA 91778 Union Carbide Corp., Chemicals and Plastics, 270 Park Ave, New York, NY 10017 Union Carbide Corp., Agricultural Products and Services, PO Box 1906, Salinas, CA 93901 Uniroyal Chemical, Elm St, Naugatuck, CT 06770 The Upjohn Co., Kalamazoo, MI 49001 (USDA) U.S. Department of Agriculture, Office of Information, Washington, DC 20250 (USP) United States PharmacopeiaI Convention, Inc., 12601 Twinbrook Pkwy, Rockville, MD 20852 Varian Aerograph, 2700 Mitchell Dr, Walnut Creek, CA 94598 Varian Instrument Div., 611 Hansen Way, Palo Alto. CA 94303 Velsicol Chemical Corp., 341 E Ohio St, Chicago, IL 60611 VWR Scientific, PO Box 3200, San Francisco, CA 94119 Wallerstein Co., see G. B. Fermentation Industries, Inc. Waters Associates, Inc., Maple St, Milford, MA 01757 Winthrop Laboratories, Special Chemicals Dept., 90 Park Ave, New York, NY 10016 Trade Names Amberlite. Ion exchange resins. Rohm and Haas Co. Anakrom. Gas chromatography supports. Analabs, Inc. Celite. Diatomaceous products. Johns-Manville Products Corp. Chromosorb. Chromatographic supports and packings. Johns- Manville Products Corp. Dowex. Ion exchange resins. Dow Chemical Co. Florisil. Chromatographic adsorbents. Floridin Co. Gas-Chrom. Gas chromatography solid supports. Applied Sci- ence Laboratories, Inc. Hyf/o Super-Cel. Diatomaceous products. Johns-Manville Prod- ucts Corp. Skellysolve. Hydrocarbon solvents. Getty Refining and Market- ing Co., PO Box 1650, Tulsa, OK 74102 Tef/on. Chemically resistant polytetrafluoroethylene. E. I. du Pont de Nemours & Co. Tygon. Halogenated vinyl plastic. Norton Co., Plastics & Syn- thetics Div., 12 E Ave, Tallmadge, OH 44278 (30) The foillowing abbreviations, many of which conform with those of Chemical Abstracts, are used. In general, principle governing use of periods after abbreviations is that period is used where final letter of abbreviation is not the same as final letter of word it represents. Periods are not used with units, except inch(es) and gallon(s). xviii Abbreviation a A AA Ac ACS addn addnl alc. alk. alky amp amt anal. anhyd. AOCS app. approx. aq. ASTM atm. avo Be. bp Bu C ca calc. calcd calcg calcn Cat. No. centrf. centrfd centrfg Chap. chern. chromatgc chromatgd chromatgy Ci CI CIPAC cm compd com. conc. concd concg concn const contg cP cpm cryst. crystd crystg crystn cu in. dc del. detd Word absorptivity(ies) absorbance(s) thruout (not restricted to for- mulas); not absorption. A' is used for std; Ao for blank; 3 digit subscript numerals usually denote wavelengths in nm atomic absorption CH3CO- (acetyl, not acetate) American Chemical Society addition additional alcoholic (not alcohol) alkaline (not alkali) alkalinity ampere(s) amount analytical(ly) anhydrous American Oil Chemists' Society apparatus approximate(ly) aqueous American Society for Testing and Materials atmosphere, atmospheric average (except as verb) degree Baume boiling point butyl degrees Celsius (Centigrade) about, approximately calculate calculated calculating calculation Catalog Number centrifuge centrifuged centrifuging Chapter chemical(ly) chromatographic chromatographed chromatography curie(s) Color Index Collaborative International Pesticides Ana- lytical Council centimeter(s) compound commercial(ly) concentrate (as verb or noun) concentrated concentrating concentration constant containing centipoise counts per minute crystalline (not crystallize) crystallized crystallizing crystallization cubic inch(es) direct current determine determined
- 21. Abbreviation detg detn diam. diat. earth dil. dild dilg diln distd distg distn DMF DMSO EDTA e.g. elec. equiv. est. estd estg estn Et EtOH evap. evapd evapg evapn ext extd extg extn F FAO Fig. fl oz fp ft g g gal. GLC g-s HCHO HOAc HPLC hr ht id in. inorg. insol. IR ISO JAOAC kg L Ib liq. m Word determining determination diameter diatomaceous earth dilute diluted diluting dilution distilled distilling distillation N,N-dimethylformamide dimethyl sulfoxide ethylenedinitrilotetraacetic acid (or -tetra- acetate) for example electric(al) equivalent estimate estimated estimating estimation ethyl ethanol (the chemical entity C2H50H) evaporate evaporated evaporating evaporation extract extracted extracting extraction degrees Fahrenheit (DC = (5/9) x (OF - 32)) Food and Agriculture Organization Figure (illustration) fluid ounce(s) (29.57 mL) freezing point foot (30.48 cm) gram(s) gravity (in centrfg) gallon(s) (3.785 L) gas-liquid chromatography glass-stoppered formaldehyde acetic acid (not HAc) high pressure (or performance) liquid chro- matography hourIs) height inner diameter (or dimension) inch(es) (2.54 cm) inorganic insoluble infrared International Organization for Standardiza- tion Journal of the Association of Official Analyt- ical Chemists (after 1965) Journal of the Association of Official Agri- cultural Chemists (before 1966) kilogram(s) liter(s) pound(s) (453.6 g) liquid meter(s); milli-as prefix xix Abbreviation m M ma mag. max. mech. Me MeOH mg min min. mixt. mL mm mp mIL mv MW N N n NBS NCA neg. neut. neutze neutzd neutzg neutzn NF ng nm No. -OAc_ -OCN od org. oxidn oz p Pa par. pet ether phthln pos. powd ppb ppm ppt pptd pptg pptn Pr prep. prepd prepg prepn psi psig pt Word molal molar (as applied to concn). not molal milliampere (et amp) magnetic(ally) maximum mechanical(ly) methyl methyl alcohol milligram(s) minute(s) minimum mixture milliliter(s) millimeter(s) melting point millimicron (10-6 mm); use nanometer (nm) (10-9 m) millivolt molecular weight normal (as applied to concn); in equations. normality of titrating reagent Newton (105 dynes) refractive index National Bureau of Standards National Canners Association (now National Food Processors Associa.tion) negative neutral neutralize neutralized neutralizing neutralization National Formulary nanogram (10-9 g) nanometer (10-9 m); formerly mIL number acetate (cf Ac) cyanate outer diameter (or dimension) organic oxidation ounce(s) (28.35 g) pico (10-12 ) as prefix Pascal (1 Newton/m2 ; 9.87 x 10-6 atm.; 7.5 x 10-3 mm Hg (torr); 1.45 x 10--4 psi) paragraph(s) petroleum ether phenolphthalein positive powdered (as adjective) parts per billion (1/109 ) parts per million (1/106 ) preci pitate precipitated precipitating precipitation propyl prepare prepared preparing preparation pounds per square inch (absolute) pounds per square inch gage (atmospheric pressure = 0) pint(s) (473 mL)
- 22. Abbreviation GAC qt qual. quant. ® r-b ref. resp. rpm sat. satd satg satn -SCN SDF sec sep. sepd sepg sepn sol. soln solv. sp gr spectrophtr spectrophtric sq SRM std std dev. stdzd stdze stdzg stdzn T tech. temp. titr. titrd titrg titrn TLC USDA Word quaternary ammonium compound quart(s) (946 mL) qualitative(ly) quantitative(ly) Trademark name-(Registered) distance spot moved/distance soIv. moved (TLC) round-bottom (flask) reference respectively revolutions per minute saturate saturated saturating saturation thiocyanate special denatured formula (applied to alco- hol) second(s) separate(ly) separated separating separation soluble solution solvent specific gravity (apparent density) spectrophotometer spectrophotometric(ally) square Standard Reference Material of National Bu- reau of Standards standard standard deviation standardized standardize standardizing standardization transmittance technical temperature titrate titrated titrating itration thin layer chromatography United States Department of Agriculture xx Abbreviation USP UV v v/v vac. vol. w/w WHO wt JL I % > < Word United States Pharmacopeia ultraviolet volt(s) both components measured by vol. vacuum volume; also volumetric when used with flask both components measured by wt World Health Organization weight micron (0.001 mm); use micrometer (Iotm) (10-6 m) microgram(s) (10-6 g) microliter(s) (10-6 L) micrometer(s) (10-6 m); formerly lot difference (e.g., LlA = (A - A')) foot (feet) (1' = 30.48 cm) inch(es) (1" = 2.54 cm) per per cent (parts per 100); percentage more than; greater than; above; exceeds (use with numbers only) less than; under; below (use with numbers only) not more than; not greater than; equal to or less than not less than; equal to or greater than; equal to or more than; at least standard taper standard spherical joint (31) * This symbol indicates a method which is in or is being considered for "surplus" status. Such methods are satisfactory methods, having been subjected to collaborative studies and review. They are thought not to be in current use for various reasons: The purpose for which they were developed no longer exists; the product for which they were developed no longer is marketed; they have been replaced by other methods; etc. These methods retain their official status but are carried in this or next edition only by ref. Any laboratory who uses these methods and wishes the text retained or reprinted in next edition must so notify the AOAC. (32) Nos. appearing in titles of methods in bold face refer to the Selected References at the end of the Chap. These refs often contain the performance data supporting the adoption of the method.
- 23. 1. Agricultural liming Materials 1.001 Sampling (1)--Procedure (Caution: See 51.036.) Take sample representative of lot or shipment. Avoid dispro- portionate amt of surface or any modified or damaged zone. (a) Burnt or lump lime, in bulk.-Collect composite sample of ;;.10 shovelfuls/car, with proportionate amts from smaller lots, taking each shovelful from different part of lot or shipment. Immediately crush to pass 5 cm (2") diam. circular opening, mix thoroly and rapidly, reduce composite to ca 2 kg (5 Ib) sample by riffling or quartering, and place in labeled, dry, air-tight container. (b) Hydrated lime and ground burnt lime, in bags.-Select 10 bags from different parts of each lot or shipment of :%20 tons and 1 addnl bag for each addnl 5 tons. Use sampling tube to withdraw top to bottom core from each bag selected. Combine cores, mix thoroly and rapidly, reduce composite to ca 1 kg (2 Ib) by riffling or quartering, and place in dry, air-tight container. (c) Ground limestone and ground marl, in bags.-Proceed as in (b). (d) Ground limestone, ground burnt lime, ground marl, and slag, in bulk.-Use slotted sampling tube to withdraw samples to full sampler depth from 10 points in lot or shipment. Proceed as in (b), beginning "Combine cores, ..." 1.002 Mechanical Analysis (2)--Procedure (Caution: See 51.036.) If entire sample is not to be dried, obtain lesser portions by riffling or quartering. Dry at 110° to canst wt and cool to room temp. Obtain 90-150 g dry sample by riffling or quartering. Break any agglomerates formed during drying by rolling dry sample with hard rubber roller on hard rubber mat, wet sieving as in 2.011(a), or by equally effective means that does not result in crushing the limestone. (If wet sieving is used to break agglom- erates, do wet sieving on sieve having smallest opening to be used in final testing. After drying, transfer to sieves to be used in final testing. If only 1 sieve is to be used, do not transfer.) Quant. transfer weighed sample to 8" diam. std sieve or set of sieves (e.g., Nos. 10,20,40,60,80, and 100 or other appropriate combination). Sieve by lateral and vertical motion accompanied by jarring action. Continue ;;.5 min or until addnl 3 min of sieving time fails to change results of any sieve fraction by 0.5% of total sample wt. Do not overload any sieve when assaying closely sized materials. Det. wt of each sieve fraction and report as % of total sample wt. 1.003 Preparation of Sample (1)--Procedure Reduce dried sample, 1.002, to amt sufficient for analysis and grind ;;.225 g (0.5 Ib) reduced sample in mortar, ball mill, or other mech. app. to pass No. 60 sieve. Mix thoroly, and store in air-tight container. Neutralizing Value-Official Final Action (Uncorrected for sulfide content) 1.004 Reagents (a) Sodium hydroxide std soln.-O.25N. Prep. and stdze as in 50.032-50.036. (b) Hydrochloric acid std soln,-O.5N. Stdze against (a), using phthln. 1.005 Indicator Titration Method Place 0.5 g burnt or hydrated lime (1 g ground limestone or ground marl), prepd as in 1.003, in 250 mL erlenmeyer; add 50 mL HCI std soln and boil gently 5 min. Cool, and titr. excess acid with NaOH std soln, using phthln. For burnt and hydrated lime, report as % CaD; for limestone and marl. report as % CaC03 equivalence. % CaC03 equivalence of sample = 2.5 x (mL HCI - mL NaOH/2). % CaD equivalence = 2.8 x (mL HCI - mL NaOH/2). 1.006 Potentiometric Titration Method (3) (Applicable to liming materials contg large amt of Fe+2 or coloring matter, but not to silicate materials) Proceed as in 1.005 thru "Cool, ..." Transfer to 250 mL beaker and insert glass and calomel electrodes of pH meter, buret contg 0.25N NaOH, and mech. stirrer. Stir at moderate speed to avoid splash. Deliver NaOH rapidly to pH 5, then dropwise until soln attains pH 7 and remains canst 1 min while stirring. (If end point is passed, add, from 1 mL Mohr pipet, just enough 0.5N HCI to bring pH to <7, and back-titr. slowly to pH 7.) Add mL of excess acid, if used, to initial 50 mL in calcg. Report as % CaCOa or CaD equivalence as in 1.005. 1.007 Approximate Proportions of Calcium and Magnesium in Magnesic Limestone Slightly acidify titrd soln, 1.005 or 1.006, transfer to 250 mL vol. flask, and dil. to vol. Det. Ca in 50 mL aliquot as in 7.096, beginning " ... dil. to ca 100 mL ..." Subtract its CaCOa equivalence from total CaCOa equivalence, 1.005 or 1.006, and assign difference as CaCOa equivalence of the Mg content of the limestone. Caustic Value (4)-Official Final Action 1.008 Apparatus (Figure 1:01) Use 500 mL Pyrex erlenmeyer, A, and fritted glass filter (Corning Glass Works No. 39535, 30F), F. Connect filter to siphon tube B with thick-wall rubber tubing. Use receiving flasks M and N calibrated to deliver 50 and 100 mL, resp. S is suction flask. 1.009 Determination Transfer portion of sample, 1.003, to weighing bottle and det. wt bottle and contents in atm. of min. moisture and CO2 content. With polished, narrow-point spatula calibrated to hold ca 1.5 g, withdraw sample to be used and det. exact wt by difference. Insert sample directly into dry flask, A, fitted with tight rubber stopper. Prep. sucrose soln immediately before use by placing 25 g granulated sucrose in measuring flask calibrated to deliver 500 mL. Dissolve sucrose with cold CO2-free H20 and dil. to vol. Holding both erlenmeyer contg sample and flask contg sucrose soln in slightly inclined position, insert neck of sucrose soln flask short distance into erlenmeyer, and carefully transfer sucrose soln with synchronized rotary motion of both flasks to prevent granulation of lime. Stopper erlenmeyer securely, agitate, and
- 24. 2 1. AGRICULTURAL LIMING MATERIALS AOAC METHODS (1980) add, if desired, some clean dry beads. Completely dissolve uncoated caustic lime by six 1 min agitations at 2 or 3 min intervals. Invert flask to trap any sol id particles between stopper and neck and crush by carefully twisting stopper. Let stand 15 min and filter as follows: Connect filter cone F with siphon B and close stopcock D. Connect receiving flasks, apply suction, and quickly connect erlenmeyer A contg lime soln with stopper E. Open stopcock C and filter 25-50 mL soln. Close C and open D to release suction. Remove M and replace with similar dry flask. Close D, open C, and continue filtration until both M and N are filled at least to marks. To disconnect system, close stopcock C, and gently press down outlet of flask M and then outlet of flask N, to remove any excess liq. above marks. Let intermediate connection empty, open stopcock D, and remove M and N. Titr. first 50 mL, or pilot aliquot, of filtered soln with 0.5N HCI, using phthln. To covered 200 mL beaker add twice vol. 0.5N acid required for this titrn, add second (100 mL) aliquot of filtered soln to this acid and phthln, and complete titrn. Calc. caustic value of sample: X = 7V/W, where X = % active CaO; V = mL 0.5N acid used/l00 mL lime soln; W = g sample. Carbon Dioxide (5)-Official Final Action 1.010 Apparatus and Reagents Knorr alkalimeter with CO2 absorption train.-Fill guard tube of alkalimeter with Ascarite. Connect upper end of condenser to absorption train consisting of 5 or 6 U-shape, g-s drying tubes (or equiv.) joined in series. Fill first tube with H2S04 and second with Ag2S04-H 2S04 soln (10 g Ag2S04 in 100 mL H2S04) to remove acidic gases other than CO2, Fill third tube with Mg(CI04)2 to absorb H20. Fill inlet % of fourth and succeeding tubes with Ascarite to absorb CO2, and outlet Va of each tube with Mg(CI04),. Connect last tube in train with aspirating bottle or suction source. Condition app. daily before use, and also when freshly filled tube is placed in train, by aspirating air at rate of 2-3 bubbles/sec thru dry alkalimeter assembly and absorption train until CO2 absorption tubes attain const wt (usually 2Q--30 min). Tare against similarly packed tubes. Use std procedure for wiping tubes with dry, lint-free cloth before each weighing. 1.011 Determination Transfer 3 g burnt or hydrated lime or 0.5-1.0 g limestone or marl, prepd as in 1.003, to dry alkalimeter flask. Momentarily open stopcocks of first 2 CO2 absorption tubes to air to equalize pressure, weigh tubes sep., and place in position in train. With assembled alkalimeter connected to absorption train, adjust rate of aspiration of air thru system to ca 2 bubbles/sec. Close funnel stopcock, remove alkalimeter guard tube, fill funnel with 50 mL HCI (1 +4). and replace guard tube. Open funnel stopcock and let acid run slowly into flask, taking care that evolution of gas is so gradual as not to materially increase flow thru tubes. After all acid is added, agitate alkalimeter assembly to ensure com- plete dispersion of sample in acid soln. Continue aspiration, gradually heat contents of flask to bp, and boil 2-3 min after H20 begins to condense. Discontinue heating, and continue aspiration 15-20 min or until app. cools. Remove, equalize internal and external pressure, and reweigh absorption tubes. Increase in wt = wt CO2 , (Material increase in wt of second tube usually indicates exhaustion of first tube, but may result from too rapid evolution of CO2 in relation to aspiration rate.) Report % CaC03• CALCIUM SILICATE SLAGS 1.012 Neutralizing Value (6)-Official Final Action (Uncorrected for sulfide content) (a) Blast furnace slag.-Transfer 0.5 g sample, ground to pass No. 80 sieve, to 250 mL erlenmeyer. Wash down with small K FIG. 1:01-Apparatus for automatic filtration and measurement of lime solutions
- 25. AOAC METHODS (1980) ELEMENTAL ANALYSIS 3 portions H,O and add 35 mL 0.5N HCI while swirling. Heat to gentle boil over burner, agitating suspension continuously until bulk of sample dissolves. Boil 5 min and cool to room temp.; then dil. with CO,-free H,O to ca 150 mL and add 1 mL30% H,O, and 5 drops bromocresol green, 2.144(e). Back-titr. with O.5N NaOH, adding first 15 mL rapidly and titrg dropwise thereafter, vigorously agitating contents of stoppered flask after each addn, until indicator tint matches or slightly exceeds that of pH 5.2 phthalate buffer soln, 50.010, of like vol. and indicator concn, after 2-3 sec agitation. (b) Rock phosphate reduction furnace slag.-Transfer 0.5 g sample to 250 mL beaker. Wash down with small portions H,O and add, stirring continuously, 50 mL HOAc (1 +4). Heat to bp and boil 5 min, stirring frequently. Evap. to dryness on steam bath. Add 20 mL of the HOAc, dil. to 150 mL, and heat to bp; add NH.OH (1 +1) to distinct yellow of Me red. Digest ca 10 min on hot plate. Filter by gravity thru 9 cm paper, catching filtrate in 100 x 50 mm lipped Pyrex crystg dish; wash beaker 3 times and paper 5 addnl times with neut. O.5N NH.OAc. Evap. filtrate on hot plate. Adjust heat so bubbles breaking thru viscous surface film are released gently to avoid spattering. (To expedite dehydration, repeat treatments with 25 mL hot H,O and evapn 2 or 3 times.) Continue heating residue on hot plate until no HOAc odor remains. Heat addnl 10 min at full heat of hot plate; then ignite 10 min at 550°. Cool, wet residue with 15 mL H,O, place watch glass over dish, and add 25 mL 0.5N HCI thru lip of dish. Heat 5 min over burner at gentle simmer. Rinse watch glass, filter suspended matter on 9 cm paper, catching filtrate in 250 mL erlenmeyer, and wash dish and filter 3 times with hot H,O. Titr. excess acid with 0.5N NaOH to distinct yellow of Me red. Net acid used x 5 = neutzg value of slag in terms of % CaC03 equivalence. 1.013 Sulfide Sulfur (7)-Official Final Action (Note: CdSO. is toxic: see also 51.084.) (a) Zinc dust.-Low in Pb. Reagents (b) Absorbent soln.-Dissolve 20 g CdSO•.2%H,O in H,O and dil. to 1 L. Adjust to pH 5.6 potentiometrically or colorimetrically. If colorimetrically, match sep. 50 mL aliquot to buffer of same pH, SO.010. (e) Sodium hydroxide std soln.---D.1N. Prep. and stdze as in 50.032-50.036. (d) Std acid.---D.1N HCI. Stdze against std alkali, (c), using Me red. (e) Methyl red indicator.-Dissolve 0.2 g Me red in 100 mL alcohol. 1.014 Apparatus Fit 250 mL erlenmeyer with 2-hole No. 5.5 stopper. Insert thru stopper 60 mL separator with stem drawn out to 2 mm and bent upward at tip, adjusting separator so stem is 6 mm from bottom of flask. Also insert thru stopper 6 mm glass outlet tube. Connect with amber rubber tubing to inlet of 25 x 150 mm tube half filled with H,O and heated to near bp before and during detn. Connect in series 2 addnl tubes of same size, each contg 25 mL absorbent soln and held in 600 mL beaker filled with cold H,O. 1.015 Determination Fill absorbent tubes with absorbent soln and heat H20 tube to gentle boiling. Weigh 1 g slag, ground to pass No. 80 sieve, into evolution flask, add 1 g Zn dust, and wash down sides with 5-10 mL H20; mix with flat-end rod and connect flask to app. Add 50 mL HCI (1 +4) to separator and let acid flow into reaction flask while swirling contents. If necessary, apply pressure to transfer acid and close stopcock while a little of the acid is still above it. Heat to bp; then regulate to maintain active but not too vigorous boiling for 10 min. Swirl flask frequently after adding acid and for first 5 min of boiling. To disconnect, hold inlet in first absorbent tube firmly with one hand and quickly pull off rubber tubing with other hand without pinching. Filter CdS suspension by gravity thru 9 cm paper into 250 mL erlenmeyer and wash with H,O to vol. of 100 mL. Add 4 drops Me red indicator and agitate vigorously while titrg slowly with 0.1N NaOH to exact tint of ref. soln (50 mL absorbent soln dild to 100 mL, with identical indicator concn, in 250 mL erlenmeyer). If end point is passed so that Cd(OH), ppts, add 1-2 mL 0.1N HCI, let stand until ppt disappears, and complete titrn dropwise, agitating vigorously. % CaC03 equivalence of sulfide S in sample = net mL 0.1N NaOH!2 g Sulfide Sidetn = mL 0.1N NaOH x 0.0016 % Sulfide S = g sulfide S x 100 ELEMENTAL ANALYSIS Gravimetric Methods 1.016 Preparation of Sample Solution by Acid Digestion (S)-Official Final Action (Caution: See 51.019, 51.026, and 51.028.) Prep. samples as in 1.003, preferably in agate mortar. Grind silicates to pass No. 100 sieve, and dry all samples at 105°. Weigh 2 g limestone or 0.5 9 silicate. If sample contains org. matter, transfer to Pt crucible and place in cold furnace. Raise temp. gradually to 1000° and hold 15 min. Transfer sample to 400 mL beaker and, if ignited, moisten cautiously with H20. Add 10 mL HN03 and evap. on hot plate at low heat until mixt. becomes pasty. Cool, and add 10 mL H20 and 20 mL 60% HCIO•. Boil to heavy fumes of HCIO., cover, and fume slowly until soln is colorless or slightly yellow (5-10 min). Do not evap. to dryness. Cool to <100° and add 50 mL H20. Filter thru Whatman 41 H or finer paper into 250 mL vol. flask. Wash thoroly with hot H20 to remove all traces of HCID•. Reserve filtrate and washings for prepn of Sample Solns X and Y, 1.017(a) and (b). 1.017 Silica (S)-Official Final Action (See also 1.041-1.043.) (Caution: See 51.025 and 51.028.) Transfer paper with Si02 to uncovered Pt crucible and heat gently with low flame until paper chars without flame. Partially cover crucible and cautiously burn C. Finally cover completely and heat with blast lamp or in furnace at 1150-1200°. Cool in desiccator and weigh. Repeat to const wt (W). Treat with ca 1 mL H20, 2 drops H2SO. (1 + 1), and 10 mL HF. Cautiously evap. to dryness in hood. Heat 2 min at 1050-1100°, cool in desiccator, and weigh (8). W - 8 = g Si02 in sample. g SiO, x 0.4674 = g Si. (a) Sample Soln X.-(0.008 9 limestone or 0.002 g silicate/mL.) Fuse residue from Si detn with 0.5 g Na2C03 by heating covered crucible 10 min over Meker burner. Cool, fill crucible % full with H20, and add 2 mL 60% HCIO. dropwise, with stirring. Warm if necessary to dissolve melt. Add to filtrate and washings reserved for prepn of Sample Soln X in 1.016. Oil. to 250 mL with H20. (b) Sample Soln Y.-(0.00016 g limestone or 0.00004 g silicate!mL.) Oil. 10 mL Sample Soln X to 500 mL with H20.
- 26. 4 1. AGRICULTURAL liMING MATERIALS AOAC METHODS (1980) 1.018 Oxides of Iron, Aluminum, Phosphorus, and Titanium (9)-Official Final Action (Alternatively, Fe, AI, Mn, P, and Ti may be detd colorimetrically as in 1.025-1.040.) To 125 mL aliquot Soln X from 1.017(a), add 10 mL HCI and few drops Me red indicator; heat to gentle boil and add NH.OH (1 +1) until ppt forms and indicator just changes to distinct yellow. Boil ,,;2 min and filter rapidly. Wash ppt!H3 times with hot 2% NH"NOa soln. Return ppt and filter to original beaker, add 10 mL HCI, and macerate fi Iter with policeman. Dil. with H,O, heat to dissolve ppt, dil. to ca 200 mL, and reppt as above. Wash thoroly with the hot NH.NOa soln until CI-free. Combine first and second filtrates and save for Ca and Mg detns. Place ppt in Pt crucible and dry. Ignite gently to oxidize C, heat to bright red ca 10 min, cool in desiccator, and weigh in covered crucible as Fe,Oa + Al,Oa + P,Os + TiO,. 1.019 Calcium (9)-Official Final Action Conc. combined filtrates and washings from 1.018 to ca 50 mL; make slightly alk. with NH.OH (1+1); while still hot, add satd (NH.),C,O. soln dropwise as long as any ppt forms, and then enough excess to convert Mg salts also to oxalate. Heat to bp, let stand ",,3 hr, decant clear soln thru filter, pour 15-20 mL hot H,O on ppt, and again decant clear soln thru filter. Dissolve any ppt remaining on filter by washing with hot HCI (1 +9) into original beaker, wash 6 times with hot H,O, and then reppt at bp by adding NH.OH and a little satd (NH.),C,O. soln. Let stand as before, filter thru same filter, and wash with hot H,O until CI- free. Reserve filtrates and washings from both pptns for detn of Mg,1.021. Complete detn by one of following methods and report as % CaO: (a) Ignite ppt in crucible, either over S-free blast lamp, or in elec. furnace at 950°, to const wt, cool in desiccator, and weigh as CaO. (b) Incinerate filter over low flame, mix ignited ppt with finely pulverized and dried mixt. of equal parts of (NH.),SO.and NH.CI, and drive off excess sulfate by carefully heating upper portion of crucible. Complete ignition, cool in desiccator, and weigh as CaSO•. (c) Perforate apex of cone; wash CaC,O. ppt into beaker used for pptn; then wash filter with hot H2S04 (1 +4), and titr. at 85-90° with 0.1N KMnO•. Magnesium (70)-Official Final Action 1.020 Reagent Phosphate soln.-Dissolve 100 g (NH.),HP04 in hot H,O, dil. to 1 L, and add 5 mL CHCla. 1.021 Determination To combined filtrates and washings, 1.019, add 2 mL 1M citric acid, 100 mL NH.OH, and 50 mL alcohol. Then add 25 mL of the phosphate soIn, with const stirring, and let stand 12-24 hr. Filter, wash twice with NH.OH (1 +9), and dissolve ppt in HNOa (1 +4), washing soln into original beaker to vol. of 100-150 mL. Add 1/10 vol. NH.OH and 2 drops of the phosphate soln. Stir vigorously and let stand ""3 hr. Filter thru gooch, wash with NH.OH (1 +9), moisten filter with satd soln of NH.NOa made slightly ammoniacal, ignite, and weigh as Mg,P,07. Report as % MgO. Correct wt Mg,P,07 for co-pptd Mn,P,07 by detg Mn as in 33.127. EDTA Titration Methods Calcium and Magnesium (77)-Official Final Action (Not applicable to samples with high phosphate content or contg <2% Mg) (Caution: See 51.050.) 1.022 Reagents (a) Buffer soln.-pH 10. Dissolve 67.5 g NH.CI in 200 mL H,O, add 570 mL NH40H, and dil. to 1 L. (b) Potassium hydroxide-potassium cyanide soln.- Dissolve 280 g KOH and 66 g KCN in 1 L H,O. (e) Potassium cyanide soln.-2%. Dissolve 2 g KCN in 100 mL H,O. (d) Eriochrome black T indicator soln.-Dissolve 0.2 g indi- cator (Eastman Kodak P6361, or equiv.) in 50 mL MeOH contg 2 g NH,OH.HCI. Store ,,;1 month. (e) Magnesium std solns.-{).25 and 1.00 mg/mL. Dissolve 0.25 and 1.00 g Mg turnings in HCI (1 +10) and dil. each to 1 L with double distd H,O. (f) Calcium std soln.-1 mg/mL. Dissolve 2.4973 g CaCOa, primary std grade, previously dried 2 hr at285°, in HCI (1 + 10). Dil. to 1 L with double distd H,O. (9) Calcein indicator.-Grind together 1 g indicator, 10 g charcoal (Norite A is satisfactory), and 100 g KCI. (Indicator is described in Anal. Chem. 28, 882 (1956), and is available from G. Frederick Smith Chemical Co. and Eastman Kodak.) (h) Disodium dihydrogen EDTA std solns.-(1) D.4%.-Dis- solve 4 g Na,H,EDTA in 1 L H,O. Stdze against std Ca and Mg solns. (2) D.1 %.-Prep. as in (1). using 1 g Na,H,EDTA, and stdze against 0.25 mg/mL Mg std soln. 1.023 Standardization (a) For calcium.-Pipet 10 mL std Ca soln into 300 mL erlen- meyer and add 10 mL H,O. Add 10 mL KOH-KCN soln and ca 35 mg calcein indicator. Using mag. stirrer and artificial light, titr. with 0.4% EDTA std soln to disappearance of all green. Titr. ""3 aliquots and use avo to calc. titer Ca soln = 10/mL EDTA soln. (b) For magnesium.-Pipet 10 mL 0.25 and 1.00 mg/mL Mg std solns into 300 mL erlenmeyers and add 100 mL H,O. Add 5 mL pH 10 buffer, 2 mL 2% KCN soln, and 10 drops eriochrome black T indicator. Using mag. stirrer and artificial light, titr. with 0.1 and 0.4% EDTA std solns, resp., until color changes per- manently from wine red to pure blue. Titr. ",,3 aliquots and use avo to calc. titer Mg soln = 2.5/mL EDTA soln, or 10/mL EDTA soln, resp. 1.024 Determination Dry sample at 110° to canst wt and cool to room temp. Grind to pass No. 60 or 80 sieve and mix thoroly. Accurately weigh ca 0.5 9 into 250 mL beaker, add 20 mL HCI (1+1), and evap. to dryness on hot plate. Dissolve residue in 5 mL HCI (1 + 10), dil. to ca 100 mL with H,O, and digest over low flame 1 hr. Cool, transfer to 200 mL vol. flask, dil. to vol., mix, and let settle or filter. (a) Forcalcium.-Pipet 10 mL aliquot into 300 mL erlenmeyer and titr. as in 1.023(a), observing end point thru soln and away from light. % Ca = (Titer EDTA std soln for Cal x mL EDTA std soln x 2/g sample. (b) For magnesium.-(For agricultural limestones contg >4% Mg.) For Ca + Mg, pipet 10 mL aliquot into 300 mL erlenmeyer and titr. with 0.4% EDTA soln as in 1.023(b). % Mg = (Titer EDTA std soln for Mg) x [(mL EDTA std soln in Ca + Mg titrn) - (mL EDTA std soln in Ca titrn)] x 2/g sample. (e) For magnesium.-(For agricultural limestones contg 2-4%
- 27. AOAC METHODS (1980) ELEMENTAL ANALYSIS 5 Mg.) Pipet 10 mL aliquot (0.5-1.0 mg Mg) into 300 mL erlenmeyer and add exact vol. of 0.4% EDTA soln required for Ca detn. Titr. with 0.1% EDTA soln as in 1.023(b). % Mg = (Titer EDTA std soln for Mg) x mL EDTA std 0.1% soln x 2/g sample. Colorimetric Methods (72)-Official Final Action (Carry reagent blanks thru detn with stds and samples. Treat aliquots of blank soln (corresponding to aliquot sizes of sample solns taken for analysis) as in Determination for appropriate element and correct values for samples accordingly.) Det. AI, Fe, Mn, P, and Ti in solns prepd by HCIO. digestion, 1.016-1.017, or NaOH fusion, 1.025. Det. Si only in soln prepd by NaOH fusion. 1.025 Preparation of Sample Solution by Sodium Hydroxide Fusion Prep. samples as in 1.003, preferably in agate mortar. Grind samples to pass No. 100 sieve and dry at 105". (a) Sample Soln X.-(0.005 9 limestone orO.002 9 silicate/mL.) Place 0.5 9 limestone or 0.2 9 silicate in 75 mL Ni crucible. If sample contains org. matter, place uncovered crucible in cold furnace, raise temp. gradually to 900", and hold 15 min. Remove crucible from furnace and let cool. Mix 0.3 9 KN03 with sample and add 1.5 9 NaOH pellets. Cover crucible with Ni cover and heat 5 min at dull redness over gas flame. (Do not fuse in furnace.) Remove from flame and swirl melt around sides. Cool, add ca 50 mL H20, and warm to disintegrate fused cake. Transfer to 150 mL beaker contg 15 mL 5N HCIO. (1(60%)+1). Scrub crucible and lid with policeman, and wash any residue into beaker. Transferto 100 mL vol. flask and dil. to vol. (Sample Soln X). (This soln is acidic and is normally clear and free of insol. matter. Occasionally particles of oxidized Ni from crucible appear. When this occurs, let particles settle before taking aliquots.) (b) Sample Soln Y.-(0.00015 9 limestone or 0.00004 9 silicate/mL.) Oil. 15mL limestone Sample SolnX or 10 mL silicate Sample Soln X to 500 mL with H20. Aluminum 1.026 Reagents (a) Aluminum std solns.-(1) Stock soln.-l00 JLg AI/mL. To 0.1000 9 pure AI metal in 30 mL beaker, add 6 mL HCI (1+1). Cover with watch glass and warm gently until AI completely dissolves. Oil. to 1 L with H20. (2) Working soln.-4 JLg AI/mL. Oil. 20 mL stock soln to 500 mL. (b) Aluminon soln.-Dissolve sep. in H20: 0.5 9 NH. aurintri- carboxylate in 100 mL; 10 9 acacia (gum arabic) in 200 mL; and 100 9 NH.OAc in 400 mL. Filter acacia soln. Add 56 mL HCI to NH.OAc soIn and adjust pH to 4.5 with HCI or NH.OH. Combine 3 solns and dil. to 1 L with H20. (e) Antifoam soln.-Disperse 0.03 9 silicone defoamer (Dow Corning Corp. Antifoam A) in 100 mL H20. (d) Thioglycolic acid soln.-Dil. 1 mL HSCH2COOH to 100 mL with H20. 1.027 Preparation ofStandard Curve Transfer aliquots of std soln contg 0, 4, 20, 40, 60, and 80 JLg AI to 100 mL vol. flasks and proceed as in detn. Prep. std curve by plotting % T against JLg AI on semilog paper. 1.028 Determination Use Sample SoIn X for limestones contg <0.2% or silicates contg <0.8% AI and adjust pH of aliquot to 4.5 with NH40H. For materials contg greater concns of AI, use Sample Soln Y and omit pH adjustment. Transfer aliquot (,,;20 mL contg <80 JLg AI) of Sample Soln X or Y to 100 mL vol. flask. Oil. to 20 mL with H20. Add 2 mL thioglycolic acid soln, 0.5 mL antifoam soln, and 10 mL aluminon soln. Place flask in boiling H20 20 min (250 mL beaker contg 125 mL H20 holds 100 mL vol. flask conveniently). Remove flask from H20 and let cool ca 30 min. Oil. to 100 mL with H20. Use o JLg AI soln, 1.027, to set 100% T at 525 nm. Read % T for sample soln and det. JLg AI from std. curve. Calc. % AI in sample. Iron 1.029 Reagents (a) Iron std solns.-(1) Stock soln.-l00 JLg Fe/mL. Dissolve 0.1000 9 pure Fe metal in 5 mL 2N HCI and dil. to 1 L with H20. (2) Working soln.-5 JLg Fe/mL. Oil. 25 mL stock soln to 500 mL. (b) 2A,6-Tripyridyl-s-triazine (TPTZ) soln.- (Available from G. Frederick Smith Chemical Co.) Dissolve 0.500 9 TPTZ in few drops HCI and dil. to 1 L with H2 0. (e) Hydroxylamine hydrochloride soln.-Dissolve 50 9 NH20H.HCI in H20. Add 10 mL TPTZ soln and 0.5 9 NaCIO•.H20, and dil. to 500 mL with H20. Transfer to separator, add 25 mL nitrobenzene, and shake several min. Let phases sep. and discard lower nitrobenzene phase contg Fe. Repeat extn 2 or 3 times. (d) Acetate buffer soln.-Dissolve 164 9 anhyd. NaOAc in H20. Add 115 mL HOAc, 10 mL NH20H.HCI soln, 0.05 9 TPTZ, and 1 9 NaCIO•.H20, and dil. to 1 L with H20. Transfer to separator, add 25 mL nitrobenzene, and shake several min. Let phases sep. and discard lower nitrobenzene phase. Repeat extn 3 or 4 times. 1.030 Preparation ofStandard Curve Treat aliquots of std soln contg 0, 5, 50, and 100 JLg Fe as in detn. Prep. std curve by plotting % T against /-tg Fe on semilog paper. 1.031 Determination Use Sample Soln X (,,;5 mL) for limestones contg <0.05% or silicates contg <0.2% Fe and Sample Soln Y for materials contg greater concns of Fe. Transfer aliquot «100 /-tg Fe) of Sample Soln X or Y to 100 mL vol. flask. Add 3 mL NH20H.HCI soln and 10 mL TPTZ soln. Add NH.OH dropwise until Fe derivative remains violet on mixing. Add 10 mL buffer soln and dil. to 100 mL. Use 0 JLg Fe soln, 1.030, to set 100% Tat 593 nm. Read % T for sample soln and det. JLg Fe from std curve. Calc. % Fe in sample. Manganese 1.032 Reagents (a) Manganese std soln.-50 JLg Mn/mL. Dissolve 0.0500 9 pure Mn metal in 20 mL 0.5N H2SO. and dil. to 1 L with H20. (b) Acid mixture.-Add 800 mL HN03 and 200 mL H3P04 to H20 and dil. to 2 L. 1.033 Preparation of Standard Curve Treat aliquots of std soln contg 0, 50, 100,300, and 500 JLg Mn as in detn. Prep. std curve by plotting %T against /-tg Mn on semilog paper.
- 28. 6 1. AGRICULTURAL liMING MATERIALS AOAC METHODS (1980) 1.034 Determination Transfer aliquot «500 ILg Mn) of Sample Soln X to 150 mL beaker. Add 25 mL acid mixt. and 0.3 9 KIO•. Bring to bp and keep near boiling temp. 10 min after color develops. Let cool, transfer to 50 mL vol. flask, dil. to vol., and mix. Use 0 ILg Mn soln, 1.033, to set 100% Tat 525 nm. Read %T for sample soln and det. ILg Mn from std curve. Calc. % Mn in sample. Phosphorus (Do not clean glassware with detergents contg P.) 1.035 Reagents (a) Phosphorus std solns.-(1) Stock soln.-l00 ILg P/mL. Dissolve 0.4393 9 KH2P04 in H20 and dil. to 1 L. (2) Working soln.-5 ILg P/mL. Oil. 25 mL stock soln to 500 mL. (b) Ammonium molybdate soln.-Dissolve 20 9 (NH4)6M0702• .4H20 in 500 mL H20. Add 285 mL H2SO., cool, and dil. to 1 L with H20. (c) Hydrazine sulfate soln.-Dissolve 2 9 N2H•.H2S04 in H20 and dil. to 1 L. 1.036 Preparation of Standard Curve Treat aliquots of std soln contg 0, 5, 50, and 75 ILg P as in detn. Prep. std curve by plotting %T against ILg P on semilog paper. 1.037 Determination Transfer aliquot (,.,-;15 mL contg <75 ILg P) of Sample Soln X to 100 mL vol. flask. Add 5 mL NH, molybdate soln and mix. Add 5 mL N2H4.H2S04 soln, dil. to 70 mL with H20, and mix. Place flask in boiling H20 9 min. Remove, cool rapidly, and dil. to vol. Use 0 ILg P soln, 1.036, to set 100% Tat 827 nm. Read %T for sample soln and det. ILg P from std curve. Calc. % P in sample. Titanium 1.038 Reagents (a) Titanium std solns.-(1) Stock soln.-l00 ILg Ti/mL. Place 0.16689 Ti02and 2 9 K2S20 7in Ptcrucible. Heat covered crucible gently at first and then at dull red ca 15 min. Dissolve melt in 50 mL H2S04(1 + 1) and dil. to 1 L with H20. (2) Working soln.- 5 ILg ~i/mL. Oil. 25 mL stock soln to 500 mL. (b) 1cetate buffer soln.-pH 4.7. Dissolve 41 9 anhyd. NaOAc in H2C add 30 mL HOAc, and dil. to 1 L. (c) Disodium-l,2-dihydroxybenzene-3,5-disulfonate (Tiron) soln.-Dissolve 4 g Tiron in H20 and dil. to 100 mL. 1.039 Preparation ofStandard Curve Treat aliquots of std soln contg 0, 5, 50, and 75 ILg Ti as in detn, but do not add dithionite to stds. Prep. std curve by plotting % T against ILg Ti on semilog paper. 1.040 Determination Transfer aliquot «75ILg Ti) ofSample Soln X to 50 mL beaker. Oil. to ca 25 mL with H20. Add 5 mL Tiron soln and then NH.OH (1 +9) dropwise until soln is neut. to Congo Red paper. (Tiron soln must be added before pH is adjusted.) Transfer to 50 mL vol. flask, add 5 mL buffer soln, dil. to vol. with H20, and mix thoroly. Add 25 mg dithionite (Na2S204) and dissolve with min. agitation (to avoid reappearance of blue). Use 0 ILg Ti soln, 1.039, to set 100% Tat 410 nm. Read %T for sample soln '!Vithin 15 min after adding dithionite. Det. 1L9 Ti from std curve. Calc. % Ti in sample. Silicon (Clean all glassware with HCI (1 + 1).) 1.041 Reagents (a) Silicon std soln.-20 ILg Si/mL. Place 0.0428 9 pure Si02in 75 mL Ni crucible and treat as in 1.025(a), but dil. with H20 to 1 L instead of 100 mL. (b) Tartaric acid soln.-Dissolve 50 9 tartaric acid in H20 and dil. to 500 mL. Store in plastic bottle. (c) Ammonium molybdate soln.-Dissolve 7.5 9 (NH4)6M07024.4H20 in 75 mL H20, add 10 mL H2S04 (1+1), and dil. to 100 mL with H20. Store in plastic bottle. (d) Reducing soln.-Dissolve 0.7 9 Na2S03 in 10 mL H20. Add 0.15 9 l-amino-2-naphthol-4-sulfonic acid and stir until dis- solved. Dissolve 9 9 NaHS03 in 90 mL H20, add to first soln, and mix. Store in plastic bottle. 1.042 Preparation ofStandard Curve Treat a/iquots of std soln contg 0, 20, 100, and 200 ILg Si as in detn. Prep. std curve by plotting %T against ILg Si on semilog paper. 1.043 Determination Transfer 10 mL Sample Soln Y to 100 mL vol. flask (use Sample Soln X for limestones contg <0.2% Si) and add 1 mL NH. molybdate soln with swirling. Mix well, and let stand 10 min. Add 4 mL tartaric acid soln with swirling, and mix well. Add 1 mL reducing soln with swirling, dil. to vol., mix well, and let stand ~30 min. Use 0 ILg Si soln, 1.042, to set 100% Tat 650 nm. Read %T for sample soln and det. 1L9 Si from std curve. Calc. % Si in sample. SELECTED REFERENCES (1) JAOAC 7, 252(1924). (2) JAOAC 7,252(1924); 55, 539(1972). (3) JAOAC 38,240(1955). (4) Ind. Eng. Chem. 20, 312(1928); JAOAC 11, 153 (1928); 14, 283(1931). (5) JAOAC 38, 413(1955). (6) JAOAC 27, 74, 532(1944); 28, 310(1945); 31, 71(1948). (7) JAOAC 31, 715(1948). (8) JAOAC 46, 603(1963); 47,1019(1964). (9) U.S. Geol. Survey Bull. 700, p. 106; Ind. Eng. Chem. 9, 1114(1917). (10) Washington, "Chemical Analysis of Rocks," 3rd Ed., 1919, p. 181. (11) JAOAC 45,1(1962); 46,611(1963); 50, 190(1967). (12) JAOAC 47,1019(1964).
- 29. 2. Fertilizers Sampling-Official Final Action 2.001 Solid Fertilizers (1) (a) Bagged fertilizers.-Use slotted single or double tube trier with solid cone tip, constructed of stainless steel or brass. (Do not use unplated brass for samples on which micronutrients are to be detd.) Trier length, exclusive of handle, should be approx. length of filled bag to be sampled, but >25"; length of slot, >23"; width of slot ;;.0.5"; and id ;;.%". Take sample as follows: Lay bag horizontally and remove core diagonally from end to end. From lots of ;;.10 bags, take core from each of 10 bags. When necessary to sample lots of <10 bags, take 10 cores but at least 1 core from each bag present. For small packages (,,;;10 Ib), take 1 entire package as sample. (b) Bulk fertilizers, including railroad car-size lots.-Use trier of design represented in Table 2:01. Table 2:01 Trier specifications Compartments Trier Length, in. od, in. id, in. No. Size, in. Missouri 59 1'Is Ys 8 3 552 Grain' 63 1% 1'Is 11 3'h Missouri "0"· 52 1'!. 1 1 45 Triers available from: , Seedboro Equipment Co., 618 W Jackson Blvd, Chicago, IL 60606. b American Tool and Die, Inc., 1105 Maple St, West Des Moines, IA 50265. Draw 10 vertical cores distributed in std concentric sampling pattern (Fig. 2:01) of such design that each core represents approx. equal fractions of lot. Bulk shipments may be sampled at time of loading or un- loading by passing sampling cup, Fig. 2:02 (mouth dimensions: width %", length 16" or as long as max. diam. of stream), thru entire stream of material as it drops from belt or chute. Make 7 8 FIG. 2:01-8ampling pattern OPEN MOUTH FIG. 2:02-8ampllng cup 9 7 sampling such as to assure ;;.10 equal-timed-spaced passes thruout transfer operation. Stream samples are not applicable unless uniform continuous flow of fertilizer is maintained for >3 min while lot is being sampled. (c) Preparation of sample.-Place composite sample in air- tight container and deliver entire sample to laboratory. Reduce composite sample in laboratory, using riffle. 2.002 Liquid Fertilizers (2) (In Absence of Free Ammonia) (a) Clear solns.-(Mixed liqs and N solns.) Secure sample directly from mixing vat, storage tank, or delivery tank after thoro mixing. Take sample from surface or thru direct tap. Flush direct tap, or delivery line and faucet, and collect sample in glass or polyethylene container. Alternatively, lower sample container into well mixed material thru port in top of tank and let fill. Seal container tightly. (b) Fluid fertilizers with suspended material.-(Salt suspen- sions and slurries.) Agitate material in storage until thoroly mixed (15 min usually adequate) before taking sample. Sample directly as in (8), or use 500 mL Missouri or Indiana sampling bottle, Fig. 2:03. Lower sampling bottle from top opening to bottom of tank and raise slowly while filling. Transfer to sample bottle and seal tightly. Alternatively, secure sample from tap on recirculation line after agitating and recirculating simultaneously until thoroly mixed. Draw sample while recirculating. If recirculation line is I II/ I bY Missouri Air escape hole 1/8" _Nylon cord 1/4" for solutions 3/8" for suspensions and slurries 500 ml _ _ polyethylene bottle Air escape hole 3mm Fluid intake tube 7mm Stainless steel jacket FIG. 2:03--Missouri and Indiana weighted restricted-fill fluid fertilizer sampling bottles designed to fill while being lowered (and raised) in storage tanks
- 30. 8 2. FERTILIZERS AOAC METHODS (1980) attached to manifold delivery line, allowing cross-contamination, pump ca 30 cm (1 ') or 2000 L (500 gaL) into temporary storage tank, then sample from recirculation line as above or from delivery line. Transfer to sample bottle and seal tightly. Ammoniacal Solutions (31 2.003 Apparatus (a) Container.-Polyethylene reagent-form bottle with but- tress-type cap, 1 L (1 qt) capacity. (b) Sample flow controlapparatus.-Construct from following fittings: 1'12 x W' reducing bushing; '.4" tee; '.4" nipple 12-18" long (length not critical); two W' stainless steel, blunt-nose needle valves with hose connections (Hoke No. 328; Hoke Inc., 1 Tenakill Pk, Cresskill, NJ 07626). All fittings except valves can be either AI or stainless steel. (See Fig. 2:04.) Attach valves directly to tee which is then attached to reducing bushing thru nipple. To both valves attach '.4" id Tygon tubing (Hoke No. 314A hose connection), 12" length to sample valve and sufficient length to vent valve to reach disposal area or container. To free end of sample tubing attach 3" length of '.4" glass or stainless steel tubing inserted thru No.4 rubber stopper. To exit end of metal tube attach addnl6" length of Tygon tubing. Make certain all connections are tight. App. can be attached directly to tank cars, but requires addnl coupling, which varies with installation, to attach to storage tanks. 1'12" "quick coupler" (Ever-Tite Coupling Co., 254 W 54th St, New York, NY 10019) suffices in most cases. 2.004 Sampling Prep. sample bottle in laboratory by adding ca 500 mL H20, replacing cap, and weighing accurately (±0.1 g). Attach sampling app. to car or tank and, with sample valve closed, flush line thru vent valve. Partially collapse sample bottle, insert sample tube with stopper, and seat tightly. With sample tube dipping below surface of H20 in bottle, throttle vent valve to maintain small flow of soln and partially open sample valve, collecting ca 100 mL sample. (Bottle should not expand to full size during this VENt NEEDLE VALVE - BLUNT ~ I NEEDLE SAMPLE /' ~ )f'''.REDUCING BUSHING 1------ ~ rfi1I ~ COUPLER FIG. 2:04--Sampling apparatus for ammoniacal solutions, including "quick coupler" for attaching to storage tanks time.) Close sample valve, remove sample tube, partially col- lapse bottle, and cap tightly. Reweigh (±0.1 g) and calc. wt sample. Cool to 20°, transfer to 1 or 2 L vol. flask, dil. to vol. with' H2 0, mix thoroly, and take aliquots for analysis. Anhydrous Ammonia (31 (Caution: Use extreme care in handling anhyd. NH3. Suitable gas mask and rubber gloves are required. See 51.032.) 2.005 Sampling Use sample tube of thermal shock-resistant glass calibrated to contain 100 mL and graduated in 0.05 mL subdivisions up to 0.5 mL. (Dupont special oil centrf. tube or ASTM long-form oil tube is satisfactory.) Flush line and fill tube to 100 mL mark with sample in such manner that condensing moisture will not enter sample tube. (Skirt attached to end of sample line will drain moisture away.) 2.006 Water and Nitrogen Immediately close sample tube with tight-fitting rubber stop- per into which is inserted tight-fitting piece of 6 mm id glass tubing 5-8 cm long, bent at its exit from outer end of stopper to let gases escape but to exclude entrance of moisture or moisture-laden air. Place in H20 bath at approx. air temp. and let NH3 evap. When temp. of sample tube is ca that 'of bath, remove tube, wipe outer surface, and det. vol. of residue. % H20 in sample = mL residue x C, where C = 0.74,0.70, or 0.66 for pressures in original containers of 100, 150, or 200 psi, resp. % N = (100 - % H20) x 0.8224. 2.007 Preparation of Sample (41-0fficial Final Action Reduce gross sample to amt sufficient for analysis or grind ;3225 g (0.5 Ib) of reduced sample without previous sieving. For fertilizer materials and moist fertilizer mixts, grind to pass sieve with 1 mm circular openings, or No. 20 sieve; for dry mixts that tend to segregate, grind to pass No. 40 sieve. Grind as rapidly as possible to avoid loss or gain of moisture during operation. Mix thoroly and store in tightly stoppered bottles. 2.008 * Mechanical Analysis of Bone, Tankage, and * Basic Slag (51-Official Final Action Sieve thru circular openings 0.5 mm diam. See 2.008, 11th ed. 2.009 Mechanical Analysis of Phosphate Rock (61 Official Final Action Apparatus (a) Water pressure control.-See Fig. 2:05. Connect valve, A, std pressure gage, B, and aerator, C, with 'Va" diam. pipe. (b) Sieves.-Nos. 100 and 200, bronze or stainless steel cloth, checked against certified sieves. Sieves 8" diam. and 2" in depth to sieve cloth are recommended for both wet and dry sieving, but other sizes may be used if detd to be suitable under conditions of method. (Other sieves in U.S. series may be used, with precaution to ensure complete sepn of sample into desired fractions. ) (c) Sieve shaker.-Ro-Tap (W. S. Tyler, Inc., 8200 Tyler Blvd, Mentor, OH 44060), Syntron (FMC Corp., Material Handling *Surplus method-see inside front cover.
- 31. AOAC METHODS (1980) PHOSPHORUS 9 FIG. 2:05--Apparatus for control of water pressure Equipment Div., Homer City, PA 15748). or other suitable ma- chine. 2.010 Reagent Dispersing agent.-Dissolve 36 g Na hexametaphosphate and 8 g Na2C03 in H20 and dil. to 1 L. 2.011 Determination (a) Ground phosphate rock.-Place 100 g sample on No. 200 sieve and wash with moderate stream of tap H20 at max. gage pressure of 0.28 kg/sq em (4 Ib/sq in.) until H20 passing sieve is clear, with care to avoid loss of sample by splashing. Dry material remaining on sieve at 105° and transfer to No. 100 sieve in series with No. 200 sieve of same diam. and depth. Shake 8 min in mech. shaker. Det. % sample passing No. 100 sieve by subtracting wt of material retained on that sieve from 100. Det. % sample passing No. 200 sieve by subtracting sum of wts of material retained on that sieve and on No. 100 sieve from 100. (b) Soft phosphate with colloidal c1ay.-Add 100 g sample to rapidly stirred soln of 50 mL dispersing agent and 450 mL tap H20, with care to avoid contact of unwetted material with shaft of stirrer and side of beaker. Stir 5 min after addn of sample is completed. Transfer slurry to No. 200 sieve and proceed as in (a). 2.012 Total Water--Official Final Action (Not applicable to samples that yield volatile substances other than H20 at drying temp.) Heat 2 g sample, 2.007, 5 hr in oven at 100±1°. In case of NaN03, (NH.I.SO., and K salts, heatto constwt at 130±1°. Report % loss in wt as H20 at temp. used. FREE WATER Vacuum-Desiccation Methods (7) 2.013 Method I~fficial Final Action Place 2 g prepd sample, 2.007, in tared weighing dish. (Weigh extremely hygroscopic or damp materials by difference in covered dishes.) Dry sample at 25-30° (precise results depend on as const a temp. as possible) in vac. desiccator over anhyd. Mg(CI0')2' P20 S, or 8aO, under ;;.50 em (20") or ~55 cm (22") vac. (20-25 cm (8-10") absolute pressure) 1~18 hr. Reweigh, and report % loss in wt as free H20. 2.014 Method lI~fficial Final Action (Not applicable to samples which yield volatile substances other than H20) Weigh 2 g prepd sample, 2.007, into tared glass weighing dish. Dry sample 2 hr ±10 min at 50±1.5° in oven under vac. of 48-53 cm (19-21") (23-28 cm (9-11") absolute pressure). (Temp. control within specified limits thruout oven chamber is essentiaL) Maintain vac. by passing desiccated air thru chamber. Cool dried sample in desiccator and reweigh. Report % loss in wt as free H20. 2.015 Alternative Extraction Method (8) Official Final Action Principle Free H20 is extd with dioxane and detd by titrn with Karl Fischer reagent. 2.016 Reagents (Keep exposure of org. reagents to air at min.) (a) Karl Fischer reagent.-Stabilized single soln (Fisher Scien- tific Co., So-K-3, or equiv.) dild ca 1 + 1 with stabilized diluent (Fisher, So-K-5, or equiv.). or soln equiv. to 2.5 mg H20/mL. Stdze daily with ca 0.2 g Na tartrate.2H20. 1 mg Na tartrate.2H20 = 0.1566 mg H20. (b) Methanol.-Low in H20. 2.017 Determination Accurately weigh 2.5 g prepd sample, 2.007, into 125 mL erlenmeyer, add 50.0 mL 1A-dioxane, stopper, mix by swirling, and let stand 15 min. Mix thoroly by swirling, and centrf. in closed tube. (Caution: See 51.005.) Transfer 10 mL aliquot to titrn vessel contg pretitrd MeOH and titr. with Karl Fischer reagent. (Discard contents of titrn vessel after 3 titrns, replace with enough MeOH to cover electrodes, and pretitr. before proceeding with next sample.) Det. blank on 10 mL dioxane as above and subtract from sample detns. Calc. and report as free H20. 2.018 * Acid-Insoluble Ash (9) * Official Final Action HCI (1 +4) digestion at 100°, ignition at 800°, and redigestion. See 2.015, 11th ed. PHOSPHORUS Total Phosphorus 2.019 Reagent Magnesium nitrate soln.-Dissolve 950 g P-free Mg(N03 }2. 6H20 in H20 and dil. to 1 L. 2.020 Preparation of Solution-Official Final Action (Caution: See 51.019, 51.026, 51.028, 51.030, and 51.069.) Treat 1 g sample by (a). (b), (c). (d), or (e), as indicated. Cool soln, transfer to 200 or 250 mL vol. flask, dil. to vol., mix, and filter thru dry filter. *Surplus methocl----5ee inside front cover.
- 32. 10 2. FERTILIZERS AOAC METHODS (1980) (a) Materials containing small quantities of organic matter.- Dissolve in 30 mL HN03 and 3-5 mL HCI, and boil until org. matter is destroyed (30 min for liqs and suspensions). *(b) Fertilizers containing much Fe or AI phosphate, and basic slag. *-Dissolve in 15-30 mL HCI and 3-10 mL HN03• (e) Organic material like cottonseed meal alone or in mix- tures.-Evap. with 5 mL Mg(N03 )2 sol n, 2.019, ignite, and dissolve in HCI. *(d) Materials or mixtures containing large amounts of or- ganic matter. *-Digestion with H2SO. and NaN03 or KN03• See 2.017(d), 11th ed. (e) All fertilizers.-Boil gently 30-45 min with 20-30 mL HN03 in suitable flask (preferably Kjeldahl for samples contg large amts of org. matter) to oxidize all easily oxidizable matter. Cool. Add 10-20 mL 70-72% HCIO•. Boil very gently until soln is colorless or nearly so and dense white fumes appear in flask. Do not boil to dryness at any time (Danger!). (With samples contg large amts of org. matter, raise temp. tI.. fuming point, ca 170°, over period of ;,1 hr.) Cool slightly, add 50 mL H20, and boil few min. Spectrophotometric Molybdovanadophosphate Method (70)-Official Final Action (Not applicable to materials yielding colored solns or solns contg ions other than orthophosphate which form colored complexes with molybdovanadate. Not recommended for basic slag.) 2.021 Apparatus Photometer.-Beckman Instruments, Inc. Model DU (current models 24/25) spectrophtr with stray light filter and matched 1 cm cells. With other photometers analyst must det. suitability for use and conditions for satisfactory performance. Means for dispelling heat from light source is desirable. 2.022 Reagents (a) Molybdovanadate reagent.-Dissolve 40 9 NH. molyb- date.4H20 in 400 mL hot H20 and cool. Dissolve 2 9 NH. metavanadate in 250 mL hot H20, cool, and add 450 mL 70% HCIO•. (Caution: See 51.028(a) and (d).) Gradually add molybdate soln to vanadate soln with stirring, and dil. to 2 L. (b) Phosphate std soln.-Dry pure KH2PO. (52.15% P20 S) 2 hr at 105°. Prep. solns contg 0.4-1.0 mg P20s/mL in 0.1 mg increments by weighing 0.0767, 0.0959, 0.1151, 0.1342, 0.1534, 0.1726, and 0.1918 g KH2PO. and dilg each to 100 mL with H20. Prep. fresh solns contg 0.4 and 0.7 mg P,Os/mL weekly. 2.023 Preparation ofStandardCurve Pipet 5 mL aliquots of 7 std phosphate solns (2-5 mg P20 S/ aliquot) into 100 mL vol. flasks and add 45 mL H20. Then, within 5 min for entire series, add 20 mL molybdovanadate reagent by buret or pipet, dil. to vol. and mix. Let stand 10 min. Select 2 absorption cells (std and sample cells) and fill both with 2 mg std. Set spectrophtr to 400 nm and adjust to zero A with std cell. Sample cell must check zero A within 0.001 unit; otherwise read A for sample cell and correct subsequent read- ings. (Choose cell showing pos. A against other as sample cell so that this pos. A is always subtracted.) Using sample cell, det. A of other stds with instrument adjusted to zero A for 2 mg std. After each detn empty and refill cell contg 2 mg std, and readjust zero to avoid error that might arise from temp. changes. Plot A against concn in mg P20s/mL std soln. *Surplus metho~ee inside front cover. 2.024 Preparation ofSolution Treat 1 g sample as in 2.020, preferably (e). when these acids are suitable solv. (Soln should be free of N oxides and NOCI.) (a) For P,Os content ";5%, dil. to 250 mL. (b) For P20S content >5%, dil. to such vol. that 5 or 10 mL aliquot contains 2-5 mg P20 S. 2.025 Determination Pipet, into 100 mL vol. flasks, 5 mL aliquots of std phosphate solns contg 2 and 3.5 mg P,Os/aliquot, resp., and develop color as in 2.023. Adjust instrument to zero A for 2 mg std, and det. A of 3.5 mg std. (It is essential that A of latter std be practically identical with corresponding value on std curve.) (a) Samples containing up to 5% P,Os.-Pipet, into 100 mL vol. flask, 5 mL sample soln, 2.024(a). and 5 mL std phosphate soln contg 2 mg P,Os. Develop color and det. A concurrently with and in same manner as for std phosphate solns in preceding par., with instrument adjusted to zero A for 2 mg std. Read P,Os concn from std curve. With series of sample solns, empty and refill cell contg 2 mg std after each detn. % P,Os in sample = 100 x [(mg P,Os from std curve - 2)/20]. (b) Samples containing more than 5% P,Os.-Pipet 5 or 10 mL sample soln, 2.024(b). into 100 mL vol. flask. Without adding std phosphate soln, proceed as in (a). % P,Os in sample = 100 x (mg P20 Sfrom std curve/mg sample in aliquot). Gravimetric Quinolinium Molybdophosphate Method (77)-Official Final Action 2.026 Reagents (Store solns in polyethylene bottles.) (a) Citric-molybdic acid reagent.-Dissolve 54 g 100% mo- lybdic anhydride (Mo03) and 12 g NaOH with stirring in 400 mL hot H,O, and cool. Dissolve 60 g citric acid in mixt. of 140 mL Hel and 200 mL H20, and cool. Gradually add molybdic soln to citric acid soln with stirring. Cool, filter, and dil. to 1 L. (Soln may be green or blue; color deepens on exposure to light.) If necessary, add 0.5% KBr03 soln dropwise until green color pales. Store in dark. (b) Quinoline soln.-Dissolve 50 mL synthetic quinoline, with stirring, in mixt. of 60 mL HCI and 300 mL H20. Cool, dil. to 1 L, and filter. (e) Quimociac reagent.-Dissolve 70 g Na molybdate.2H,O in 150 mL H20. Dissolve 60 g citric acid in mixt. of 85 mL HN03 and 150 mL H20, and cool. Gradually add molybdate soln to citric acid-HN03 mixt. with stirring. Dissolve 5 mLsynthetic quinoline in mixt. of 35 mL HN03 and 100 mL H,O. Gradually add this soln to molybdate-citric acid-HN03 soln, mix, and let stand 24 hr. Filter, add 280 mL acetone, dil. to 1 L with H20, and mix. 2.027 Preparation of Solution Treat 1 g sample as in 2.020, dilg to 200 mL. 2.028 Determination Pipet, into 500 mL erlenmeyer, aliquot contg ,,;25 mg P20 S and dil. to ca 100 mL with H20. Continue by one of the following methods: (a) Add 30 mL citric-molybdic acid reagent and boil gently 3 min. (Soln must be ppt-free at this time.) Remove from heat and swirl carefully. Immediately add 10 mL quinoline soln from buret with continuous swirling. (Add first 3-4 mL dropwise and remainder in steady stream.) Or:
- 33. AOAC METHODS (1980) PHOSPHORUS 11 (b) Add 50 mL quimociac reagent, cover with watch glass, place on hot plate in well-ventilated hood, and boil 1 min. After treatment by (8) or (b). cool to room temp., swirl carefully 3-4 times during cooling, filter into gooch with glass fiber filter paper previously dried at 250° and weighed, and wash with five 25 mL portions of H20. Dry crucible and contents 30 min at 250°, cool in desiccator to room temp., and weigh as (CgH7N)3H3[PO•. 12Mo031. Subtract wt reagent blank. Multiply by 0.03207 to obtain wt P20 S (or by 0.01400 for Pl. Report as % P20 S (or % Pl. Alkalimetric Quinolinium Molybdophosphate Method (12)-Official Final Action 2.029 Reagents (8) Quimociac reagent.-5ee 2.026(e). (b) Sodium hydroxide std soln.-(l mL = 1 mg P20 S') Dil. 366.32 mL 1N NaOH, 50.032-50.036, to 1 L with H20. (e) Nitric acid std soln.-Prep. HN03 soln equiv. to concn of (b) and stdze by titrg against (b). using phthln. (For greater precision, use HN03 soln corresponding to 1/5 concn of (b).) (d) Citric acid.-l0% (w/v). (e) Indicators.-(1) Thymol bluesoln.-(J.l%. Add 2.2 mL O.lN NaOH to 0.1 g thymol blue and dil. to 100 mL with 50% alcohol. (2) Phenolphthalein.-(J.l %. Dissolve 0.1 g phthln in 100 mL 50% alcohol. (3) Mixed indicator.-Mix 3 vols (1) and 2 vols (2). 2.030 Preparation of Sample Solution Treat 1 g sample as in 2.020, first par. and (8) or (e). 2.031 Determination (8) Precipitation.-Transfer aliquot contg ~30 mg P20 S and ~5 mL coned acid to 500 mL erlenmeyer, add 20 mL citric acid soln, and adjust to ca 100 mL. Add 60 mL quimociac reagent, immediately cover with watch glass, and place on medium temp. hot plate. After soln comes to bp, move to cooler portion of hot plate and boil gently 1 min. Let cool until flask can be handled comfortably with bare hand. (b) Filtration and washing.-Prep. pulped-paper pad ca 6 mm thick on perforated porcelain disk in funnel by adding ;;.2 approx. equal increments of H2 0 suspension of pulped paper and sucking dry with vac. between addns. Swirl flask, pour contents onto filter, and wash flask with five ca 15 mL portions H20, adding washings to funnel. Immediately after funnel has emptied, wash down sides with ca 15 mL H2 0 to remove residual acetone, which causes excessively fast drying and later lump formation if allowed to evap. Wash with 3 addnl 15 mL portions H2 0, letting funnel empty between addns. Keep drying of ppt to min. Using only jet of H20, transfer ppt and pad to pptn flask and break up pad with jet of H20. Do not smear ppt against funnel or flask. (e) Titration.-Titr. with std NaOH soln and add 3-5 mL excess. Add 1 mL mixed indicator and titr. with std HN03 soln to grey-blue end point. If overtitrd (greenish-yellow). add addnl excess std NaOH soln and titr. to grey-blue. (d) Blank.-Det. blank on all reagents, adding known amt (1-2 mg) of P20 S' Use 1 + 9 dilns of std NaOH and HN03for titrn and subtract theoretical titer equiv. to P20 S added from experimental titer. Calc. difference equiv. to 0.3663N NaOH and subtract this blank from all sample detns. Calc. and report as % P20 S' Automated Method (13)-Official First Action 2.032 Principle Samples are extd for direct available P20 S or for total P20 S detns. Destruction of coloring matter, hydrolysis of nonortho- phosphates, and elimination of citrate effect are accomplished by digestion with 4N HCIO. at 95°. Digested samples are reacted with molybdovanadate reagent, and A of resulting complex is read in flowcell at 420 nm in range 0.15-0.35 mg P20 S/mL. 2.033 Apparatus and Reagents (Caution: See 51.028.) (8) Automatic analyzer.-AutoAnalyzer with following mod- ules (Technicon Instruments Corp., or equiv.): Sampler II or IV with 40/hr (4: 1) cam; proportioning pump III; P20 S anal. cartridge (with 2 heating baths, each contg 10.6 mL coil held at 95±1°; or AAI type heating bath contg one 40' x 1.6 mm id coil and holding constant temp. of 95±1°); AAII single channel color- imeter with 15 x 1.5 or 2.0 mm id flowcell and 420 nm interference filters; voltage stabilizer; and recorder. Construct manifold as in flow diagram, Fig. 2:06. (b) Molybdovanadate reagent.-Dissolve 16.5 g NH. molyb- date.4H20 in 400 mL hot H20, and cool. Dissolve 0.6 g NH. metavanadate in 250 mL hot H2 0, cool, and add 60 mL 70% HCIO•. Gradually add molybdate soln to vanadate soln with stirring. Add 2 mL wetting agent, (e), and dil. to 2 L. (e) Perch/oric acid.-4N. Add 342 mL 70% HCIO. to 500 mL H20 in 1 L vol. flask. Add 1 mL wetting agent, and dil. to vol. (d) Sampler wash soln.-Add 1 mL wetting agent to 1 L H20, and mix well. (e) Wetting agent.-Ultrawet 60 L (Technicon No. T01-0214), or equiv. (f) Phosphorus std solns.-(1) Stock soln.-10 mg P20 S/mL. Dissolve 9.5880 g dried (2 hr at 105°) KH2PO. primary std (52.15% P20 S) in H20, and dil. to 500 mL with H20. (2) Working solns.- 0.15,0.19,0.23,0.27,0.31, and 0.35 mg P20 S/mL. Using 25 mL buret, accurately measure 7.5, 9.5, 11.5, 13.5, 15.5, and 17.5 mL stock soln into six 500 mL vol. flasks. Dil. each to vol. with H20, and mix. (3) Working soln for samples ~7% P20s.-2 mg P20 S/mL. Pipet 100 mL stock soln into 500 mL vol. flask, dil. to vol. with H2 0, and mix. 2.034 Preparation of Samples Prep. samples for direct available P20 S detn as in 2.050(a). Prep. samples for total P20 S detn as in 2.020(a) or (e). and dil. to 250 mL. 2.035 Analytical System (Technicon part numbers are given to aid in construction of manifold; equiv. coils, fittings, etc., are satisfactory.) Sample, air, and 4N HCIO. are combined thru injection fitting (No. 116-0489) and mixed in 20T coil (No. 157-0248). Stream proceeds to heating bath(s) before resample thru modified AO fitting. Resample, air, and molybdovanadate reagent are com- bined thru injection fitting (No. 116-0489). Mixing and color development takes place in two 20T coils (No. 157-0248) before measurement at 420 nm. If only total P20 S samples are to be analyzed, heating bath can be removed and 4N HCIO. soln replaced by sampler wash soln, (d). Heating bath(s) and acid soln are necessary only when analyzing samples for direct available P20 S or combination of direct available and total P20 S detns. If manifold is to be constructed following flow diagram, use clear std pump tubes for all air and soln flows. All fittings, coils, and glass transmission lines are AAII type and size. Use 1.6 mm glass transmission tubing for all connections after pump to colorimeter. Construct modified AO fitting, following heating bath, by using AO fitting, N13 stainless steel nipple connector,
- 34. 12 2. FERTILIZERS 20T MIXER SAMPLER II FLOW,ml/Min 0.32 AIR 0.60 HCI04 0.23 SAMPLE 0.32 AIR 1.00 MO-V REAGENT 0.23 DIG. SAMPLE MODIFIED AO 40/H 4:1 AOAC METHODS (1980) WASTE~______0_._6_0____________~ SAMPLER 11~~~__~2~.0~0~______~H~2~0 WASTE~______O~.~BO~____FL~O~W_C~E~L~L~ D 6 FT. LENGTH 0.030 I.D.~STETRANSMISSION TUBING COLORIMETER REa>RDER 15 mm FLOWCELL 420nm FILTERS o POSITIONED WITH CAPILLARY SIDEARM ON BOTTOM FIG. 2:06-Flow diagram for automated analysis for phosphorus and 1.3 cm length of 0.035" id Tygon tubing. Insert N13 nipple approx. halfway into 0.035" Tygon tubing. Insert tubing into side arm of AO fitting far enough 50 resample line will not pump any air. Connect 01 fitting directly to waste side of AO fitting; position D1 fitting with capillary side arm on bottom. Attach 0.6 mL/min pump tube to top arm of D1 fitting, and attach 1.8 m (6') of 0.030" id transmission tubing to bottom arm. All air segments must pass thru 0.6 mL/min tube, leaving continuous column of liq. in 1.8 m length of tubing to provide for constant back pressure on heating bath coil. Length of resample pump tube should be ,,;2.5 cm from shoulder at entrance end. 2.036 Start-Up Start automatic system, place all lines in resp. solns, and let equilibrate ~30 min. Proceed as in 2.038. 2.037 Shut-Down Pump water thru reagent lines ~30 min. Do not remove HCIO. lines from reagent until 20 min after last sample is run. 2.038 Check and Calibration After equilibration, set colorimeter to damp 1 position and pump 0.15 mg P20s/mL working std soln continuously thru system. Adjust colorimeter baseline to read 10% of full scale. Pump 0.35 mg P20S/mL std and adjust std calibration to read 90% of full scale. Range of 0.15-0.35 mg P20s/mL will expand to read 10-90% of full scale. Check of bubble flow pattern will give indication of performance of system. Perfect bubble pattern is required to obtain optimum peak shapes. Check for air bubble in flowcell if noisy conditions exist. To check system carryover, place three 0.35 mg/mL stds, followed by three 0.15 mg/mL stds thru system. Iffirst 0.15 mg/mL std following 0.35 mg/mL std is ~1 chart division higher than other 2, carryover is indicated. If carryover occurs, check entire system for poor connections. 2.039 Determination Pipet aliquot of sample soln (see Table 2:02) into 100 mL vol. flask, dil. to vol. with H20, and mix by inversion 20 times. For sample contg ,,;7% P20 S' pipet 10 mL working soln, (f)(3), into flask before diln. Place 0.15-0.35 mg P20s/mL working std solns in tray in increasing order of concn, followed by group of samples. Analyze lowest concn std in duplicate, discarding first peak. Precede and follow each group of samples with std ref. curve to correct for possible drift. If drift between first and last set of stds is ~2 chart divisions, repeat sample analysis. Prep. std curve by averaging peak hts of first and second set of stds. Plot avo peak ht of stds against mg P20s/mL contained in each std. Read mg P20s/mL for each sample from graph. % P20 S= mg P20s/mL from graph (-0.20, if spiked) x F x 100, where F = factor from Table 2.02. Table 2:02 Standard Dilutions % P20, Aliquot (mL) Expected Direct Available Total 50 + "spike" no diln 25 + "spike" 1 50 0.5 Factor 1-7 8-16 17-34 ""35 50 25 2 for direct available 25 15 1.667 for totaI
- 35. AOAC METHODS (1980) PHOSPHORUS 13 Water-Soluble Phosphorus 2.040 Preparation of Solution-Official Final Action Place 1 g sample on 9 cm filter and wash with small portions H20 until filtrate measures ca 250 mL. Add H20 in fine stream directed around entire periphery of filter paper in circular path, ensuring that H20 and solids are thoroly mixed with each addn. Let each portion pass thru filter before adding more and use suction if washing would not otherwise be complete within 1 hr. If filtrate is turbid, add 1-2 mL HN03, dil. to 250 mL, and mix. 2.041 Gravimetric Ouinolinium Molybdophosphate Method-Official Final Action Pipet aliquot contg ,,;;25 mg P20 S into 500 mL erlenmeyer. Dil., if necessary, to 50 mL, add 10 mL HN03 (1+1), and boil gently 10 min. Cool, dil. to 100 mL, and proceed as in 2.028(b). 2.042 Alkalimetric Ouinolinium Molybdophosphate Method-Official Final Action Pipet aliquot contg ,,;;30 mg P20 S into 500 mL erlenmeyer. Dil., if necessary, to 50 mL, add 10 mL HN03 (1+1). boil gently 10 min, cool, and proceed as in 2.031 (a). beginning " ... add 20 mL citric acid soln ..." 2.043 Spectrophotometric Molybdovanadophosphate Method-Official Final Action Adjust concn according to 2.024(a) or (b) and proceed as in 2.025. 2.044 Citrate-Insoluble Phosphorus (14) Official Final Action Reagents (a) Ammonium citrate soln.-Should have sp gr of 1.09 at 200 and pH of 7.0 as detd potentiometrically. Dissolve 370 g cryst. citric acid in 1.5 L H20 and nearly neutze by adding 345 mL NH.OH (28-29% NH3). If concn of NH3 is <28%, add correspondingly larger vol. and dissolve citric acid in correspondingly smaller vol. H20. Cool, and check pH. Adjust with NH.OH (1 + 7) or citric acid soln to pH 7. Dil. soln, if necessary, to sp gr of 1.09 at 200 • (Vol. will be ca 2 L.) Keep in tightly stoppered bottles and check pH from time to time. If pH has changed from 7.0, readjust. (b) Other reagents and solns.-5ee 2.019, 2.022, or 2.026. 2.045 Preparation of Extract (a) Acidulated samples, mixed fertilizers, and materials con- taining water-soluble compounds.-After removing H20-sol. P2 0 S' 2.040, transfer filter and residue, within 1 hr, to 200 or 250 mL flask contg 100 mL NH. citrate soln previously heated to 650 • Close flask tightly with smooth rubber stopper, shake vigorously until paper is reduced to pulp, and relieve pressure by removing stopper momentarily. Continuously agitate stoppered flask in const temp. app. at exactly 650 • (Action of app. should be such that dispersion of sample in citrate soln is continually maintained and entire inner surface of flask and stopper is continually bathed with soln.) Exactly 1 hr after adding filter and residue, remove flask from app. and immediately filter by suction as rapidly as possible thru Whatman No.5 paper, or equiv., using buchner or ordinary funnel with Pt or other cone. Wash with H20 at 650 until vol. filtrate is ca 350 mL, allowing time for thoro draining before adding more H2 0. If material yields cloudy filtrate, wash with 5% NH.N03 soln. Prep. citrate-insol. residue for analysis by one of following methods: (1) Dry paper and contents, transfer to crucible, ignite until all org. matter is destroyed, and digest with 10-15 mL HCI until all phosphate dissolves; or (2) treat wet filter and contents as in 2.020(a). (c), (d), or (e). Dil. soln to 250 mL, or other suitable vol., mix well, and filter thru dry paper. (b) Nonacidulated samples.-Place 1 g sample (ground to pass No. 40 sieve in case of Ca metaphosphate) on dry 9 cm paper. Without previous washing with H20, proceed as in (a). If sample contains much org. matter (bone, fish, etc.). dissolve residue insol. in NH. citrate as in 2.020(c). (d). or (e). 2.046 Determination (a) Gravimetric quinolinium molybdophosphate method (tn.-Treat 1 g sample as in 2.045(a) or (b). Transfer aliquot of citrate-insol. P20 S contg ,,;;25 mg P2 0 S and proceed as in 2.028. (b) Spectrophotometric molybdovanadophosphate method (15).-Treat 1 g sample as in 2.045(a) or (b). Adjust concn of citrate-insol. P20 S soln as in 2.024(a) or (b) and proceed as in 2.025. (c) Alkalimetric quinolinium molybdophosphate method (16).-Treat 1 g sample by 2.045(a) or (b). Transfer aliquot of citrate-insol. P20 S contg ,,;;5 mL coned acid to 500 mL erlenmeyer. Add 20 mL 10% citric acid soln and dil. to 100 mL with H20. Continue as in 2.031(a). beginning "Add 60 mL quimociac reagent, ..." 2.047 Citrate-Soluble Phosphorus-Official Final Action Subtract sum of H20-sol. and citrate-insol. P20 S from total P20 S to obtain citrate-sol. P20 S• Available Phosphorus 2.048 Indirect Method-Official Final Action Subtract citrate-insol. P20 S from total P20 S to obtain available P20 S• 2.049 Direct Method (17)--Officiai Final Action (Caution: See 51.026, 51.028, 51.030, and Acids, Chap. 51.) Reagents (a) Nitric-perchloric acid mixture.-Add 300 mL 70% HCIO. to 700 mL HN03. (b) Ternary acid mixture.-Add 20 mL H2SO. to 100 mL HN03, mix, and add 40 mL 70% HCIO•. (c) Modified molybdovanadate reagent.-Prep. as in 2.022(a) except use 250 mL 70% HCIO. instead of 450 mL. 2.050 Preparation ofSolution (a) Acidulated samples, mixed fertilizers, and materials con- taining water-soluble compounds. -( 1) Without filtration of ci- trate digest.-Remove H20-sol. P20 S as in 2.040,collecting filtrate in 500 ml vol. flask, but do not add HN03 to filtrate. Treat H20- insol. residue with NH. citrate soln as in 2.045(a). Exactly 1 hr after adding filter and residue, remove flask from app. and transfer contents to flask contg H2 0-sol. fraction. Cool to room temp. immediately, dil. to vol., mix thoroly, and let stand ~2 hr before removing aliquot. (2) With filtration ofcitrate digest.-If desired, wash by gravity into 500 mL Kohlrausch flask contg 5 mL HN03 (1 + 1), catching filtrate from insol. residue, 2.045(a). in the Kohlrausch flask contg H2 0-sol. fraction, and wash residue until vol. soln in flask is ca 500 mL. Cool, dil. to 500 mL, and mix.
- 36. 14 2. FERTILIZERS AOAC METHODS (1980) (b) Nonacidulated samples.-Place 1 g sample (ground to pass No. 40 sieve in case of Ca metaphosphate) on dry 9 cm paper. Without previous washing with H20, proceed as in (a)(1) or (2). If (2) is used, wash residue until vol. soln is ca 350 mL. Cool, dil. to 500 mL, and mix. 2.051 Alkslimetric Quinolinium Molybdophosphste Method (t6)-Officisl Final Action Treat 1g sample by appropriate modification of2.050. Transfer aliquot contg ,,;;30 mg P20 S and ,,;;10 mL NH. citrate soln, 2.044(al. to 500 mL erlenmeyer. Oil., if necessary, to 50 mL, add 10 mL HN03 (1+1), and boil gently 10 min. Cool, dil. to 100 mL, and continue as in 2.031(a), beginning "Add 60 mL quimociac reagent, ..." 2.052 Spectrophotometric Molybdovanadophosphste Method (tB)-Official Final Action (Not applicable to materials yielding colored solns or solns contg ions other than orthophosphate which form colored complexes with molybdovanadate. Not recommended for basic slag.) Prep. std curve as in 2.023, using photometer, 2.021. Pipet, into 100 mL vol. flasks, 5 mL aliquots std phosphate solns contg 2 and 3.5 mg P20 S/ aliquot, 2.022(bl. resp., add 2 mL 70% HCIO., and develop color as in 2.023. Adjust instrument to zero A for 2 mg std and det. A of 3.5 mg std. (A of latter must be practically identical with corresponding value on std curve.) Prep. sample as in 2.050. (a) Samples containing up to 5% P20 s.-Pipet 10 mL sample soln into 125 mL erlenmeyer, and treat by one of following methods (Caution: See 51.019, 51.026, and 51.028): (1) Add 5 mL 20% NaCI03soln and 10 mL HN03-HCIO. mixt., 2.049(a). Boil gently until greenish-yellow color disappears (ca 20 min), cool, and add 2 mL HCI. After vigorous reaction subsides, evap. to fumes of HCIO., and fume 2 min. (2) Add 5 mL ternary acid mixt., 2.049(b), swirl, boil gently 15 min, and digest at 150-200° until clear white salt or colorless soln remains. Evap. to white fumes and continue heating 5 min. Cool, add 15 mL H20, and boil 5 min. Transfer to 100 mL vol. flask, dil. to 50 ml, swirl, and cool to room temp. Add 5 mL std phosphate soln contg 2 mg P20 S and 20 mL modified molyb- dovanadate soln, 2.049(c). Oil. to 100 mL, and continue as in 2.025(a). (b) Samples containing more than 5% P20 s.-Dil. soln to such vol. that 5-10 mL aliquot contains 2-5 mg P20 S' Digest as in (a)( 1) or (2). Without adding std phosphate soln, continue as in (a). 2.053 Gravimetric Quinolinium Molybdophosphate Method (19)-Official Final Action (a) Solns containing no organic phosphorus.-Prep. sample as in 2.050. Pipet, into 500 mL erlenmeyer, aliquot contg ,,;;25 mg P20 S and ,,;;10 mL original NH. citrate soln. Oil., if necessary, to ca 50 mL, add 10 mL HN03 (1 +11. and boil gently 10 min. Cool, dil. to 150 mL, and proceed as in 2.028(a) or (b). (b) Solns containing organic phosphorus.-(Caution: See 51.019,51.026, and 51.028.) Select aliquot as in (a). Add 10 mL 20% NaCI03 and 10 mL HN03-HCIO. mixt., 2.049(a). Boil vigor- ously until greenish-yellow color disappears (usually ca 30 minI. cool, and add 2 mL HCI. After vigorous reaction subsides, evap. to white fumes, and continue heating 5 min. Cool, and proceed as in 2.028(a) or (b). NITROGEN 2.054 Detection of Nitrates-Official Final Action Mix 5 g sample with 25 mL hot H20, and filter. To 1 vol. of this soln add 2 vols H2SO., free from HN03 and oxides of N, and let cool. Add few drops concd FeSO.soln in such manner that fluids do not mix. If nitrates are present, junction at first shows purple, afterwards brown, or if only minute amt is present, reddish color. To another portion of soln add 1 mL 1% NaN03 soln and test as before to det. whether enough H2SO. was added in first test. Total Nitrogen (Provide adequate ventilation in laboratory and do not permit accumulation of exposed Hg.) 2.055 Reagents-OHicial Final Action (a) Sulfuric acid.-93-98% H2SO., N-free. (b) Mercuric oxide or metallic mercury.-HgO or Hg, reagent grade, N-free. (c) Potassium sulfate (or anhydrous sodium sulfate).-Re- agent grade, N-free. (d) Salicylic acid.-Reagent grade, N-free. (e) Sulfide or thiosulfate soln.-Dissolve 40 g com. K2S in 1 L H20. (Soln of 40 g Na2S or 80 g Na2S20 3.5H20 in 1 L may be used.) (f) Sodium hydroxide.-Pellets or soln, nitrate-free. For soln, dissolve ca 450 g solid NaOH in H20, cool, and dil. to 1 L. (Sp gr of soln should be ~ 1.36.) (g) Zinc granules.-Reagent grade. (h) Zinc dust.-Impalpable powder. (i) Methyl red indicator.-Dissolve 1 g Me red in 200 mL alcohol. (j) Hydrochloric or sulfuric acid std soln.--Q.5N, or 0.1N when amt of N is small. Prep. as in 50.011 or 50.039. (k) Sodium hydroxide std soln.--Q.1N (or other specified concn). Prep. as in 50.032-50.034. Stdze each std soln with primary std, Chap. 50, and check one against the other. Test reagents before use by blank detn with 2 g sugar, which ensures partial reduction of any nitrates present. Caution: Use freshly opened H2SO. or add dry P20 S to avoid hydrolysis of nitriles and cyanates. Ratio of salt to acid (wt:vol.) should be ca 1: 1 at end of digestion for proper temp. control. Digestion may be incomplete at lower ratio; N may be lost at higher ratio. Each g fat consumes 10 mL H2SO., and each g carbohydrate 4 mL H2SO. during digestion. 2.056 Apparatus-OHicial Final Action (a) For digestion.-Use Kjeldahl flasks of hard, moderately thick, well-annealed glass with total capacity ca 500-800 mL. Conduct digestion over heating device adjusted to bring 250 mL H20 at 25° to rolling boil in ca 5 min or other time as specified in method. To test heaters, preheat 10 min if gas or 30 min if elec. Add ~ boiling chips to prevent superheating. (b) For distillation.-Use 500-800 mL Kjeldahl or other suit- able flask, fitted with rubber stopper thru which passes lower end of efficient scrubber bulb or trap to prevent mech. carryover of NaOH during distn. Connect upper end of bulb tube to condenser tube by rubber tubing. Trap outlet of condenser in such way as to ensure complete absorption of NH3 distd over into acid in receiver.
- 37. AOAC METHODS (1980) NITROGEN 15 2.057 Improved Kjeldahl Method for Nitrate-Free Samples (20)-Official Final Action (Caution: See 51.030 and 51.0S5.) Place weighed sample (0.7-2.2 g) in digestion flask. Add 0.7 g HgO or 0.65 g metallic Hg, 15 g powd K2S04or anhyd. Na2S04, and 25 mL H2S04, If sample >2.2 g is used, increase H2S04by 10 mL for each g sample. Place flask in inclined position and heat gently until frothing ceases (if necessary, add small amt of paraffin to reduce frothing); boil briskly until soln clears and then ~30 min longer (2 hr for samples contg org. material). Cool, add ca 200 mL H20, cool <25°, add 25 mL of the sulfide or thiosulfate soln, and mix to ppt Hg. Add few Zn granules to prevent bumping, tilt flask, and add layer of NaOH without agitation. (For each 10 mL H2S04used, or its equiv. in dild H2S04, add 15 g solid NaOH or enough soln to make contents strongly alk.) (Thiosulfate or sulfide soln may be mixed with the NaOH soln before addn to flask.) Immediately connect flask to distg bulb on condenser, and, with tip of condenser immersed in std acid and 5-7 drops indicator in receiver, rotate flask to mix contents thoroly; then heat until all NH3 has distd (~150 mL distillate). Remove receiver, wash tip of condenser, and titr. excess std acid in distillate with std NaOH soln. Correct for blank detn on reagents. % N = [(mL std acid x normality acid) - (mL std NaOH x normality NaOH)] x 1.4007/g sample 2.058 Improved Kjeldahl Method for Nitrate-Containing Samples-Official Final Action (Not applicable to liqs or to materials with high CI:N03 ratio. Caution: See 51.030 and 51.0S5.) Place weighed sample (0.7-2.2 g) in digestion flask. Add 40 mL H2S04contg 2 g salicylic acid. Shake until thoroly mixed and let stand, with occasional shaking, ~30 min; then add (1) 5 g Na2S2 0 3.5H20 or (2) 2 g Zn dust (as impalpable powder, not granulated Zn or filings). Shake and let stand 5 min; then heat over low flame until frothing ceases. Turn off heat, add 0.7 9 HgO (or 0.65 g metallic Hg) and 15 g powd K2S04 (or anhyd. Na2S04), and boil briskly until soln clears, then ~30 min longer (2 hr for samples contg org. material). Proceed as in second par. of 2.057. 2.059 Comprehensive Nitrogen Method (21) Official Final Action (Applicable to all fertilizer samples. Caution: See 51.030 and 51.079.) Reagents (a) Chromium metal.-100 mesh, low N (Fisher Scientific Co. No. C-318 or Sargent-Welch Scientific Co. No. SC11432 is satis- factory). (b) Alundum.-Boiling stones. 8-14 mesh (Arthur H. Thomas Co. No. 1590-D18, or equiv.). (e) Dilute sulfuric acid.-Slowly add 625 mL H2S04to 300 mL H20. Oil. to ca 1 L and mix. After cooling, dil. to 1 L with H20 and mix. Avoid absorption of NH3 from air during prepn, particularly if stream of air is used for mixing. (d) Sodium thiosulfate or potassium sulfide soln.-160 g Na2S20 3.5H20/L or 80 g K2S/L. For other reagents, see 2.055. 2.0S0 Determination Place 0.2-2.0 9 sample contg ~60 mg nitrate N in 500-800 mL Kjeldahl flask and add 1.2 g Cr powder. Add 35 mL H20 or, with liqs, amt to make total vol. 35 ml. Let stand 10 min with occasional gentle swirling to dissolve all nitrate salts. Add 7 mL HCI and let stand ~30 sec but ~10 min. Place flask on preheated burner with heat input set at 7.0-7.5 min boil test, 2.05S(a). After heating 3.5 min, remove from heat and let cool. Add 22 g K2S04, 1.0 g HgO, and few granules Alundum. Add 40 mL dil. H2S04, (e). (If adequate ventilation is available, 25 mL H2S04may be added instead of dil. H2S04, If org. matter which consumes large amt of acid exceeds 1.0 g, add addnl 1.0 mL H2S04for each 0.1 g org. matter in excess of 1.0 g.) Place flask on burners set at 5 min boil test. (Pre-heated burners reduce foaming with most samples. Reduce heat input if foam fills ~% of bulb of flask. Use variable heat input until this phase is past.) Heat at 5 min boil test until dense white fumes of H2S04clear bulb of flask. Digestion is now complete for samples contg ammoniacal, nitrate, and urea N. For other samples, swirl flask gently and continue digestion 60 min more. Proceed as in 2.057, second par., substituting 2.059(d) for 2.055(e). 2.0S1 Modified Comprehensive Nitrogen Method (22) Official First Action (Applicable to all fertilizer samples) Reagents See 2.055(a), (e), (f), (i), (j), (k), 2.059(a), (b), and in addn: Copper sulfate pentahydrate (or anhydrous copper sulfate).- Reagent grade, N-free. 2.0S2 Determination (Caution: See 51.019 and 51.030.) Proceed as in 2.0S0, par. 1 and 2, using 0.2-1.6 g sample. For samples contg orgs other than urea or urea-form, use ~0.5 g sample. Add 15 g K2S04 or 12 g anhyd. Na2S04, 0.4 g anhyd. CUS04 or 0.6 g CuS04.5H20, and ca 0.8 g Alundum granules. Add 37 mL H2S04 (1 +1). (If adequate ventilation is available, 20 mL H2S04 may be added instead of H2S04 (1 +1). If org. matter other than urea exceeds 1.0 g, add addnl 1.0 mL H2 S04for each 0.1 g fat or 0.2 g other org. matter in excess of 1.0 g.) Proceed as in 2.0S0, par. 4, substituting 75 min for 60 min in last sentence. Cool flask until it can be handled without gloves, and add ca 250 mL H2 0. Swirl to dissolve contents, and cool <25°. Add ca 0.8 g Alundum granules to minimize bumping, tilt flask, and add layer of NaOH without agitation. (For each 10 ml H2S04used, or its equiv. in H2S04(1 +1), add 15 g solid NaOH or enough soln to make contents strongly alk.) Proceed as in 2.057, par. 2, beginning "Immediately connect flask to distg bulb ..." Raney Powder Method (21) Official Final Action (Applicable to all fertilizer samples except "nitric phosphates" contg nonsulfate S. Caution: See 51.030 and 51.079.) 2.063 Reagents (a) Raney catalyst powder No. 2813.-50% Ni, 50% AI (w. R. Grace & Co., Davison Chemical Division, 10 E Baltimore St, Baltimore, MD 21203). Caution: Raney catalyst powders react slowly in H20 or moist air to form alumina; avoid prolonged contact with air or moisture during storage or use. (b) Sulfuric acid-potassium sulfate soln.-Slowly add 200 mL H2S04 to 625 mL H20 and mix. Without cooling, add 106.7 9
- 38. 16 2. FERllLlZERS AOAC METHOOS (1980) K2SO. and continue stirring until all salt dissolves. Oil. to ca 1 L and mix. Cool, dil. to 1 L with H20, and mix. Avoid absorption of NH3 from air during prepn particularly if stream of air is used for mixing. For other reagents, see 2.055. 2.064 Determination Place 0.2-2.0 g sample contg ~42 mg nitrate N in 500-800 mL Kjeldahl flask (800 mL flask is preferred with samples which foam considerably, especially orgs). Add 1.7 g Raney catalyst powder, 3 drops tributyl citrate, and 150 mL H2SO.-K2SO. soln. If org. matter exceeds 0.6 g, add addnl 2.5 mL of this soln for each 0.1 g org. matter in excess of 0.6 g. Swirl to mix sample with acid and place flask on cold burner. If burner has been in use, turn off completely ;.010 min before placing flask on burner. After flask is on burner, set heat input to 5 min boil test. When sample starts boiling, reduce heat to pass 10 min boil test. After 10 min, raise flask to vertical position and add 0.7 g HgO and 15 g K2SO•. (Contents of Kel-Pak No.5 (Curtin Matheson Scientific, Inc.) without plastic container may be used.) Replace flask in inclined position and increase heat to 4-5 min boil test. (Reduce heat input if foam fills ;.0% of bulb of flask. Use variable heat input until this phase is past.) Heat at 4-5 min boil test until dense white fumes of H2SO. clear bulb of flask. Digestion is now complete for samples contg only am- moniacal, nitrate, and urea N. For other samples, swirl flask gently and continue digestion addnl 30 min. Proceed as in 2.057, second par. If 800 mL Kjeldahl flasks have been used, add 300 instead of 200 mL H20. Ammoniacal Nitrogen 2.065 Magnesium Oxide Method-Official Final Action (Not applicable in presence of urea) Place 0.7-3.5 g, according to NH3 content of sample, in distn flask with ca 200 mL H20 and ;.02 g carbonate-free MgO. Connect flask to condenser by Kjeldahl connecting bulb, distil 100 mL liq. into measured amt std acid, 2.055(j), and titr. with std NaOH soln, 2.055(kL using Me red, 2.055(i). 2.066 * Formaldehyde Titration Method * Official Final Action (Applicable to NH.N03 and (NH')2S0.) See 2.058, 11th ed. Nitrate and Ammoniacal Nitrogen 2.067 * Ferrous Sulfate-Zinc-Soda Method * Official Final Action (Not applicable in presence of org. matter, Ca cyanamide, and urea) See 2.059, 11th ed. 2.068 Devarda Method ~3)-Official Final Action (Not applicable in presence of org. matter, Ca cyanamide, and urea) Place 0.35 or 0.5 g sample in 600--700 mL flask and add 300 mL H20, 3 g Devarda alloy (Cu 50, AI 45, Zn 5), and 5 mL NaOH soln (42% by wtL pouring latter down side of flask so that it does not mix at once with contents. By means of Davisson (J. *Surplus method-,see inside front cover. Ind. Eng. Chem. 11,465(1919)) or other suitable scrubbing bulb that will prevent passing over of any spray, connect with condenser, tip of which always extends beneath surface of std acid in receiving flask. Mix contents of distg flask by rotating. Heat slowly at first and then at rate to yield 250 mL distillate in 1 hr. Collect distillate in measured amt std acid, 2.055(j), and titr. with std NaOH soln, 2.055(k), using Me red, 2.055(i). Nitrate Nitrogen 2.069 Robertson Method ~4)-Official Final Action (Applicable in presence of Ca cyanamide and urea. Caution: See 51.030 and 51.065.) (a) Det. total N as in 2.058, 2.060, or 2.064. (b) Det. H20-insol. N as in 2.072, but use 2.5 g sample. Oil. filtrate to 250 mL. (c) Place 50 mL portion filtrate in 500 mL Kjeldahl flask and add 2 g FeSO•.7H20 and 20 mL H2SO•. (If total N is >5%, use 5 g FeSO•.7H20.) Digest over hot flame until all H20 is evapd and white fumes appear, and continue digestion at least 10 min to drive off nitrate N. If severe bumping occurs, add 10--15 glass beads. Add 0.65 g Hg, or 0.7 g HgO, and digest until all org. matter is oxidized. Cool, dil., add the K2S soln, and complete detn as in 2.057. Before distn, add pinch of mixt. of Zn dust and granular "20-mesh" Zn to each flask to prevent bumping. Total N (a) - H20-insol. N (b) = H20-sol. N. H20-sol. N - N obtained in (c) = nitrate N. 2.070 Jones Modification of Robertson Method ~4) Official Final Action (Applicable when H20-sol. N need not be detd. Caution: See 51.030 and 51.065.) Weigh 0.5 g sample into Kjeldahl flask, add 50 mL H20, and rotate gently. Add 2 g FeSO•.7H20 and rotate. Add 20 mL H2SO•. Digest over hot flame. When H20 evaps and white fumes appear, add 0.65 g Hg and proceed as in 2.057. Total N - N thus found = nitrate N. 2.071 * Water-Insoluble Nitrogen in Cyanamide (25) * Official Final Action See 2.063, 11th ed. Water-Insoluble Nitrogen (See 2.079(a) and (b) for urea-formaldehyde or mixts contg such compds.) 2.072 Method I-Official Final Action Place 1 or 1.4 g sample in 50 mL beaker, wet with alcohol, add 20 mL H20, and let stand 15 min, stirring occasionally. Transfer supernate to 11 cm Whatman No. 2 paper in 60° long-stem funnel 60 mm diam., and wash residue 4 or 5 times by decanting with H20 at room temp. (20--25°). Finally transfer all residue to filter and complete washing until filtrate measures 250 mL. Det. N in residue as in 2.057. Method II ~6)-Official First Action 2.073 Apparatus Extraction tube.-Glass, 250 x 10 mm id, 12 mm od, con- stricted to 3-4 mm at one end. 2.074 Determination Weigh 3.0 g unground mixed sample and place in extn tube contg small glass wool plug. Place addnl glass wool pad on top
- 39. AOAC METHODS (1980) NITROGEN 17 of sample. Connect 250 or 500 mL separator to column with 75 mm piece of rubber tubing. Close stopcock of separator and add 250 mL deionized H20. Open stopcock and let quick rush of H20 pass thru column. After initial rush of H20, close stopcock. Adjust flow thru stopcock to ca 2 mL/min. Squeeze rubber connection to bring level of H20 ca 25 mm above column bed. System then operates as constant-head feeder. After H20 wash is complete, disconnect column from rubber tubing. Invert column over Kjeldahl flask and force contents into flask with aid of pressure bulb. Wash traces of sample from tube into Kjeldahl flask and wash sample from walls of digestion flask with min. H20. Det. N in residue as in 2.059-2.060 or 2.063-2.064. * Nitrogen Activity * 2.075 Removal of Water-Soluble Nitrogen Official Final Action (a) Mixed fertilizers.-5ee 2.058, 10th ed. (b) Raw materials.-5ee 2.058, 10th ed. 2.076 Water-Insoluble Organic Nitrogen Soluble in Neutral Permanganate Official Final Action See 2.059, 10th ed. 2.077 Water-Insoluble Organic Nitrogen Distilled from Alkaline Permanganate (27) Official Final Action See 2.060-2.061, 10th ed. Nitrogen Activity Index (AI) of Urea- Formaldehyde Compounds (28) Official Final Action (Applicable to urea-formaldehyde compds and mixts contg such compds) 2.078 Reagent Phosphate buffer soln.-pH 7.5. Dissolve 14.3 g KH2P04 and 91.0 g K2HP04 in H20 and dil. to 1 L. Oil. 100 mL of this soln to 1 L. 2.079 Determination (a) Crush sample (do not grind) to pass No. 20 sieve. (b) Det. cold H20-insol. N (WIN) as in 2.072, keeping temp. at 25±2°. Stir at 5 min intervals during 15 min standing. (c) Det. hot H20-insol. N (HWIN) in phosphate buffer soln as follows: Place accurately weighed sample contg 0.1200 g WIN in 200 mL tall-form beaker. Add ca 0.5 g CaC03 to mixed fertilizers contg urea-HCHO compds. From supply of boiling buffer soln, add 100 mL from graduate to sample, stir, cover, and immerse promptly in boiling H20 bath so that liq. in beaker is below H20 level in bath. Maintain bath at 98-100°, checked with thermometer, and stir at 10 min intervals. After exactly 30 min, remove beaker from bath and filter promptly thru 15 cm Whatman No. 12 fluted paper. If filtration takes >4 min, discard detn. Repeat detn, stirring in 1 g Celite filter-aid just before removing beaker from bath, and filter. Wash insol. residue completely onto paper with boiling H20 and continue washing until total vol. used is 100 mL. Complete washing before filtrate becomes cloudy or its temp. drops to <60°. Det. total N (HWIN) in wet paper and residue as in 2.057, using 35 mL H2S04 when CaC03 has been added. Activity index (AI) = (%WIN - %HWIN) x 100/%WIN Urea (29)-Official Final Action 2.080 Reagent Neutral urease soln.-Use fresh com. 1% urease soln, or dissolve 1 g urease powder in 100 mL H,O, or shake 1 g jack bean meal with 100 mL H,O 5 min. Transfer 10 mL soln to 250 mL erlenmeyer, dil. with 50 mL H,O, and add 4 drops Me purple (available from Fisher Scientific Co.; No. 50-1-9). Titr. with 0.1N HCI to reddish purple; then back-titr. to green with 0.1N NaOH. From difference in mL, calc. vol. 0.1N HCI required to neutze remainder of soln (usually ca 2.5 mL/100 mL). add this amount of acid, and shake well. Verify enzyme activity of urease source periodically. Discard any source which does not produce soln capable of hydrolyzing 0.1 g urea/20 mL soln. 2.081 Determination Weigh 10±0.01 g sample and transfer to 15 cm Whatman No. 12 fluted filter paper. Leach with ca 300 mL H,O into 500 mL vol. flask. Add 75--100 mL satd Ba(OH), soln to ppt phosphates. Let settle and test for complete pptn with few drops satd Ba(OH), soln. Add 20 mL 10% Na,C03 soln to ppt excess Ba and any sol. Ca salts. Let settle and test for complete pptn. Oil. to vol., mix, and filter thru 15 cm Whatman No. 12 fluted paper. Transfer 50 mL aliquot (equiv. to 1 g sample) to 200 or 250 mL erlenmeyer and add 1-2 drops of Me purple. Acidify with 2N HCI and add 2-3 drops excess. Neutze soln with 0.1N NaOH to first change in color of indicator. Add 20 mL neutral urease soln, close flask with rubber stopper, and let stand 1 hr at 20--25°. Cool flask in ice-H,O slurry and titr. at once with 0.1N HCI to full purple; then add ca 5 mL excess. Record total vol. added. Back-titr. excess HCI with 0.1N NaOH to neut. end point. % Urea = (mL 0.1N HCI - mL 0.1N NaOH) x 0.3003. Biuret Spectrophotometric Method (30) Official First Action (Applicable to urea only. Do not use for mixed fertilizers) 2.082 Reagents (a) Alkaline tartrate soln.-Dissolve 40 g NaOH in 500 mL H,O, cool. add 50 g NaKC.H.06.4H,O, and dil. to 1 L. Let stand 1 day before use. (b) Copper sulfate soln.-Dissolve 15 g CuS04.5H,O in CO2- free H20 and dil. to 1 L. (c) Biuret std soln.-1 mg/mL. Dissolve 100 mg reagent grade biuret in CO,-free H,O and dil. to 100 mL. Biuret may be purified as follows: Dissolve 10 g in 1 L absolute alcohol and conc. by gentle heating to ca 250 mL. Cool at 5° and filter thru gooch (60% recovery). Repeat crystn and dry final product in vac. oven at 80°. 2.083 Preparation of Standard Curve Transfer series of aliquots, 2-50 mL, of std biuret soln to 100 mL vol. flasks. Adjust vol. to ca 50 mL with CO2-free H,O, add 1 drop Me red, and neutze with 0.1N H,SO. to pink color. Add, with swirling, 20 mL alk. tartrate soln and then 20 mL CuSO. soln. Oil. to vol., shake 10 sec, and place in H,O bath 15 min at 30±5°. Also prep. reagent blank. Det. A of each soln against blank at 555 nm (instrument with 500--570 nm filter is also satisfactory) with 2--4 cm cell. Plot std curve. *Surplus method--5ee inside front cover.
- 40. 18 2. FERTILIZERS AOAC METHODS (1980) 2.084 Determination Continuously stir oS 109 sample contg 30-125 mg biuret in 150 mL ca 50° H20 30 min. Filter and wash into 250 mL vol. flask, and dil. to vol. Transfer 50 mL aliquot to 100 mL vol. flask and proceed as in 2.083. Atomic Absorption Spectrophotometric Method (31) Official Final Action 2.085 Apparatus and Reagents (a) Atomic absorption spectrophotometer.-IL Model 353 (In- strumentation Laboratory, Inc., 113 Hartwell Ave, Lexington, MA 02173), or equiv., with Cu hollow cathode lamp. (b) Copper sulfate soln.-Dissolve 15 g CuS04.5H20 in H20 and dil. to 1 L. (c) Buffer soln.-pH 13.4. Dissolve 24.6 g KOH and 30 g KCI in H20 and dil. to 1 L. (d) Starch soln.-Treat 1 g sol. starch with 10 mL cold H20, triturate to thin paste, and pour gradually into 150 mL boiling H20 contg 1 g oxalic acid. Boil until soln clears, cool, and dil. to 200 mL. Prep. fresh weekly. (e) Bromocresol purple indicator.-Dissolve 0.1 g bromocre- sol purple in 19 mL O.lN NaOH and dil. to 250 mL with H20. (f) Biuret.-To recrystallize, weigh ca 10 g reagent grade biuret, transfer to 800 mL beaker, add 500 mL H20, and heat on hot plate with occasional stirring until dissolved. Boil until vol. decreases to ca 250 mL. Remove, and let cool gradually to room temp. Filter thru fritted-glass funnel, transfer to evapg dish, and dry 1 hr in 105-110° oven. Remove from oven, place in desiccator, and cool to room temp. (g) Biuret std soln.-O.4 mg/mL. Dissolve 0.4000 g recrystd biuret in warm H20, cool, transfer to 1 L flask, and dil. to vol. (h) Copper std solns.-Dil. aliquots of Cu stock soln, 2.110(b), with H20 to obtain ;,,4 std solns within range of detn, 1-4 ILg Cu/mL final soln. 2.086 Determination of Calibration Factor Transfer aliquots of biuret std soln contg 4, 8, and 12 mg biuret to sep. 100 mL vol. flasks, dil. to ca 30 mL with H20, and add 25 mL alcohol to each. While stirring with mag. stirrer, add 2 mL starch soln, 10 mL CuS04 soln, and 20 mL buffer soln. Remove stirring bar, rinse, dil. to vol., mix thoroly, and let stand 10 min. With vac., filter ca 50 mL thru dry 150 mL medium porosity fritted glass funnel into dry flask. Transfer 25 mL aliquots of each filtrate to 250 mL vol. flasks, acidify with 5 mL lN HCI, and dil. to vol. with H20. Proceed as in 2.109-2.113, using std solns, 2.085(h), to det. complexed Cu in soln by AA spectrophotometry after adding equiv. amts of alcohol, KOH soln, buffer soln, and IN HCI. Take ;,,3 readings of each soln. From mean value of Cu concn, calc. factor relating mg Cu found to mg biuret added. Redet. daily. 2.087 Determination (a) In urea.-Accurately weigh sample contg <10 mg biuret, dissolve in H20, transfer to 100 mL vol. flask, add 25 mL alcohol, and proceed as in 2.086, beginning "While stirring with mag. stirrer, ..." From Cu found, calc. biuret concn, using factor. (b) In mixed fertilizers.-Transfer accurately weighed sample contg <40 mg biuret to 250 mL beaker and add 1 mL H20 for each g of sample (5 g max.). Warm, add 65 mL alcohol and 7 drops bromocresol purple, and adjust pH to first blue color (pH 6-7) with 20% KOH. Place on hot plate, heat to bp, cool, and, if pH has changed, make final adjustment to first blue. Vac.-filter thru alcohol-washed paper pulp pad into 100 mL vol. flask. (If filtrate is not clear, improper pH adjustment has been made. Add HCI and readjust to pH 6-7.) Wash pad and ppt with alcohol and dil. to vol. with alcohol. Transfer 25 mL aliquot to 100 mL vol. flask, and proceed as in 2.086, beginning "While stirring with mag stirrer, ..." From Cu found, calc. biuret concn, using factor and appropriate diln factors. (Final aliquot can be varied to give Cu concn between 1 and 4 ILg/mL.) POTASSIUM 2.088 * Lindo-Gladding Method (32) * Official Final Action Gravimetric detn as K2PtCI6• See 2.076-2.078, 11th ed. 2.089 * Wet-Digestion Method (33) * Official Final Action Pptn as K2PtCI6 after digestion with HN03 and HCI. See 2.079-2.080, 11th ed. 2.090 * Recovery of Platinum (34) * Procedure See 2.081-2.083, 11th ed. Flame Photometric Method (35) Official Final Action (Caution: See 51.007.) 2.091 Reagents and Apparatus (a) Ammonium oxalate soln.-Dissolve 40 g (NH')2C20. in 1 L H20. (b) Methyl red indicator.-Dissolve 0.2 g Me red in 100 mL alcohol. (c) Dilute nitric acid.-(l +10). (d) Anion exchange resin.-REXYN 203(OH) (Fisher Scientific Co.); Duolite A-7 or Duolite A-41 (Diamond Shamrock, 1100 Superior Ave, Cleveland, OH 44114); Permutit S-100 (Permutit Co., E49 Midland Ave, Paramus, NJ 07652); or equiv. (e) Potassium nitrate or potassium chloride.-Recrystallize reagent grade salt twice from H20 and dry 5 hr at 105°. (f) Ion exchange column.-Made from 30 cm length of std wall glass tubing, 2.5 cm od; one end closed by l-hole No.4 rubber stopper thru which is inserted 2-way stopcock or glass tubing connected to rubber tubing and compressor clamp. Do not let stopcock tubing protrude above stopper. Choose stopper large enough so that there is no space between stopper vertex and column wall. Alternatively use glass chromatgc tube 300 x 19 mm id with stopcock or valve at bottom to control flow rate (such as SGA Scientific Inc. No. C-4225). Place glass wool plug in bottom of tube, close valve, and add H20 to ht of 10 cm. Transfer portion of resin to 200 mL beaker and suspend in H20. Transfer slurry to column and adjust ht of packed resin to 20 cm, draining excess H20 until 2.5 cm head remains. Regenerate resin after 10 successive aliquots have passed thru. For Na, regenerate after 5 aliquots have passed thru. 2.092 Preparation of Resin Place ca 450 g resin in 4 L beaker and add 2 L 5% NaOH. Stir 30 min with elec. stirrer. Let resin settle, and decant NaOH soln. Repeat treatment with 5% NaOH twice, decanting NaOH soln after final treatment. Add 2 L H20 to resin, stir few min, let resin settle, and decant wash H20. Repeat 3-4 times. Resin is now in *Surplus method--5ee inside front cover.
- 41. AOAC METHODS (1980) POTASSIUM 19 free base form. Regenerate to nitrate form by treating 3 times with HN03 (1 +19)' in same manner as with NaOH soln. Wash resin with H20 until pH of washings is ;;.2 by backwashing in column or by stirring and decanting in large beaker. Store resin under H20 in stoppered bottle. 2.093 Preparation ofSolution (a) Mixed fertilizers and potassium-magnesium sulfate.- Weigh 1.5058 g sample into 250 mL vol. flask (500 mL flask if sample contains >30% K20), add 125 mL H20 and 50 mL (NH.l.C20. soln, and boil 30 min. Cool, dil. to vol., mix, and pass thru dry filter. (b) Potassium chloride and sulfate.-Dissolve 1.5058 g in H20 and dil. to 500 mL. 2.094 Preparation ofStandard Curve Dissolve 1.2931 g KN03 (or 0.9535 g KCI) in H20 and dil. to 500 mL (1000 ppm K). Prep. std solns by diln to cover range 0-80 ~pm K at intervals ,;;10 ppm, adding appropriate amt LiN03 if Internal std instrument is to be used. Prep. std curve of emission against concn, adjusting instrument so that 50 ppm K gives reading near mid-scale. Atomize portions of std solns until readings for series are reproducible. 2.095 Determination (a) Mixed fertilizers, potassium sulfate, and potassium-mag- nesium sulfate.-Transfer 10 mL aliquot of sample soln to 250 mL beaker. Add 1 drop Me red and neutze with HN03 (1+10). Adjust H20 level in column to top of resin and quant. transfer aliquot to column. Open stopcock to give flow rate of 2 dropsl sec, collecting effluent in 250 mL vol. flask. Wash aliquot into resin with 2-3 small portions H20. Collect 50-75 mL effluent; then open stopcock and collect addnl 100 mL by pouring H20 onto column, making certain that H20 level does not fall below top of resin bed. Oil. to vol. and mix (if internal std instrument is used, add required amt LiN03 before dilg to voL). Atomize portions of sample several times to obtain reliable avo reading for each soln. Det. ppm K from std curve. (Temp. of std and sample solns must not differ by >2°.) Calc. % K20 as follows: 0-30%: ppm K/2 = %K20 >30%: ppm K/1 = %K20 (b) Potassium chloride.-Proceed as in (a) but omit neutzn and resin treatment. 2.096 Instrument and Procedure Performance Test Weigh 1.5058 g K acid phthalate (primary std; %K20 = 23.0) and transfer to 250 mL vol. flask. Add ca 0.5 g (NH')2HPO. and proceed as in 2.093(a), beginning " ... add 125 mL H20 ..." Table 2:03 Aliquots and factors for potassium determination % K,O Expected Aliquot 2 no diln 3-4 150 5-6 100 7-8 75 9-13 50 14-16 40 17-20 30 21-25 25 26-30 20 31-43 15 44-65 10 Factor 0.0500 0.08333 0.1250 0.1667 0.2500 0.3125 0.4167 0.5000 0.6250 0.8333 1.250 Automated Flame Photometric Method (36) Official Final Action (Caution: See 51.007.) 2.097 Apparatus Automatic analyzer.-AutoAnalyzer with following modules (available from Technicon Corp.): Sampler II, proportioning pump, flame photometer, range expander, and recorder. As- semble app. as in Fig. 2:07. 2.098 Reagents (a) Ammonium oxalate soln.-4%. See 2.091(a). (b) Ammonium citrate soln.-Prep. as in 2.044(a). (c) Lithium nitrate soln.-Dissolve 0.6894 g dried (2 hr at 105°) LiN03 in 1 L H20. (d) Potassium std solns.-(T) Stock soln.-O.5 mg K20/mL. Dissolve 1.4447 g dried (2 hr at 105°) KH2PO. in H20 and dil. to 1 L. (2) Workingsolns.-35, 38, 41, 44, 47,50, and 55fL9 K20/mL. Accurately measure 35-55 mL stock soln into 500 mL vol. flasks contg 0.2 g (NH')2C20. if samples are prepd by 2.099(a), or 12 mL NH. citrate soln, (b), if by 2.099(b). Oil. to vol. with H20 and mix. (If std solns contg citrate must be held overnight, add 3-4 drops CHCI3.) 2.099 Preparation ofSamples (a) Ammonium oxalate extraction.-Weigh 1 g sample into 500 mL vol. flask, add 50 mL 4% (NH')2C20. and 125 mL H20, boil 30 min, and cool. Oil. to vol. with H20, mix, and filter or let stand until clear. (b) Ammonium citrate extraction from direct available phos- phorus extract.-Prep. as in 2.050. (If solns must be held overnight, add 3-4 drops CHeI3.) 2.100 Calibration Pump 55 fLg K20/mL std working soln continuously thru system. Set range expander to 1x position and adjust calibration control on flame photometer to read 85% T on recorder. Pump 35 fLg/mL soln continuously and set range expander to 2x position. Turn adjustable range positioner or range expander to obtain 23% T on recorder. Range of 35-55 fLg K20/mL will read ca 20-80% T on recorder with expander set at 2 X. (Expansion from Model II flame photometers may be <20-80% T.) 2.101 Determination Pipet aliquot sample soln as indicated in Table 2:03 into 250 mL vol. flask. Oil. to vol. with H20 and mix. Place 35-55 fL9/mL working std solns in sampler tray before samples. Place 44 fL9/mL std soln at end of series to check for drift. Read samples and stds at rate of 40/hr (2:1 sample-to-wash ratio). Prep. std curve of emission against K20 concn and read fLg K20/mL from graph. % K20 = (fL9 K20/ mL from graph) x factor from Table 2:03. 2.102 Volumetric Sodium Tetraphenylboron Methods Method I (37)-Official Final Action Reagents (a) Formaldehyde soln.-37%. (b) Sodium hydroxide soln.-20%. Dissolve 20 g NaOH in 100 mL H2 0. (c) Sodium tetraphenylboron (STPB) soln.-Approx. 1.2%. Dissolve 12 g NaB(CsHs). in ca 800 mL H20. Add 20-25 g AI(OH)3'
- 42. 20 2. FERTILIZERS AOAC METHODS (1980) SAMPLER II Rate 40 )ernour (i) 1!!2----------<. 0.6 Sam Ie 0) Waste _ _-,,",,,_2""00,-- 08 rnl/m,n RA.NCE EXPANDER FIG. 2:07--f'low schematic for K20 in fertilizers stir 5 min, and filter (Whatman No. 42 paper, or equiv.) into 1 L vol. flask. Rinse beaker sparingly with H20 and add to filter. Collect entire filtrate, add 2 mL 20% NaOH, dil. to vol. with H2 0, and mix. Let stand 48 hr and stdze. Adjust so that 1 mL STPB = 1% K20. Store at room temp. (d) Benzalkonium chloride (BAC) soln.-Approx. 0.625%. Dil. 38 mL 17% Zephiran chloride (Winthrop Laboratories; also available at local pharmacies as benzalkonium chloride) to 1 L with H20, mix, and stdze. Cetyltrimethylammonium bromide may be substituted for Zephiran chloride. If other concn is used, adjust vol. (e) Clayton Yellow (Titan Yellow; Colour Index No. 19540).- 0.04%. Dissolve 40 mg in 100 mL H20. 2.103 Standardization of Solutions (a) BAC soln.-To 1.00 mL STPB soln in 125 mL erlenmeyer, add 20-25 mL H,O, 1 mL 20% NaOH, 2.5 mL HCHO, 1.5 mL 4% (NH.),C2 0., and 6-8 drops indicator, (e). Titr. to pink end point with BAC soln, using 10 mL semimicro buret. Adjust BAC soln so that 2.00 mL = 1.00 mL STPB soln. (b) Sodium tetraphenylboron soln.-Dissolve 2.500 g KH2PO. in H20 in 250 mL vol. flask, add 50 mL 4% (NH.)2C20. soln, dil. to vol. with H20, and mix. (It is not necessary to bring to boiL) Transfer 15 mL aliquot (51.92 mg K20, 43.10 mg K) to 100 mL vol. flask; add 2 mL 20% NaOH, 5 mL HCHO, and 43 mL STPB reagent. Dil. to vol. with H20, mix thoroly, let stand 5-10 min, and pass thru dry filter. Transfer 50 mL aliquot of filtrate to 125 mL erlenmeyer, add 6-8 drops indicator, (e). and titr. excess reagent with BAC soln. Calc. titer as follows: F = 34.61/(43 mL - mL BAC) = % K20/mL STPB reagent. Factor F applies to all fertilizers if 2.5 g sample is dild to 250 mL and 15 mL aliquot is taken for analysis. If results are to be expressed as K rather than as K20, substitute 28.73 for 34.61 in calcg F. 2.104 Determination Place 2.5 g sample (1.25 g if K,O >50%) in 250 mL vol. flask, add 50 mL 4% (NH.)2C20. and 125 mL H20, and boil 30 min. (If org. matter is present, add 2 g K-free C before boiling.) Cool, dil. to vol. with H20, mix, and pass thru dry filter or let stand until clear. Transfer 15 mL aliquot sample soln to 100 mL vol. flask and add 2 mL 20% NaOH and 5 mL HCHO. Add 1 mL std STPB soln for each 1% K20 expected in sample plus addnl 8 mL excess to ensure complete pptn. Oil. to vol. with H20, mix thoroly, let stand 5-10 min, and filter thru dry paper (Whatman No. 12 or equiv.). Transfer 50 mL filtrate to 125 mL erlenmeyer, add 6-8 drops indicator, (e). and titr. excess reagent with std BAC soln. % K20 in sample = (mL STPB added - mL BAC) x F, where F = % K20/mL STPB reagent. (Multiply by 2 if 1.25 9 sample was used.) 2.105 Method /I (38)-Official Final Action (For use with sample prepd for available P detn) Reagents See 2.102(a). (b). (e). (d), and (e). 2.10S Standardization of Solutions (a) Benzalkonium chloride.-In 125 mL erlenmeyer, add 2.5 mL neut. NH. citrate soln, 2.044(a), 15-20 mL H20, 4 mL HCHO, and 2.5 mL 20% NaOH soln. Swirl; then add 4.00 mL STPB soln and 6-8 drops indicator, 2.102(e). Titr. to pink end point with BAC soln, using 10 mL semi micro buret. Adjust BAC soln so that 2.00 mL = 1.00 mL STPB soln. (b) Sodium tetraphenylboron soln.-Dissolve 1.4447 g pri- mary std KH2PO. in H20 in 500 mL vol. flask, add 100 mL neut. NH. citrate soln, 2.044(a), dil. to vol. with H20, and mix. Transfer 25 mL aliquot (25.00 mg K20, 20.75 mg K) to 100 mL vol. flask; add 8 mL HCHO and 5 mL 20% NaOH, swirl, and add 25 mL STPB reagent. Dil. to vol. with H20, mix thoroly, let stand 5-10 min, and pass thru dry filter. Transfer 50 mL aliquot of filtrate to 125 mL erlenmeyer, add 6-8 drops indicator, 2.102(e). and titr. excess reagent with BAC soln. Calc. titer as follows: F = 25 mg K20/(25 mL STPB - mL BAC) = mg K20/mL STPB reagent. If results are to be expressed as K rather than K20, substitute 20.75 for 25 in calcg F. 2.107 Preparation of Sample Prep. as in 2.050. 2.108 Determination Transfer 25 mL aliquot of sample soln to 100 mL vol. flask. (If org. matter is present, treat 100 mL portion with 1 g K-free C and filter before transferring aliquot.) Add 8 mL HCHO first and then 5 mL 20% NaOH soln, and wash down sides of flask with H20. Swirl and add 1 mL STPB for each 1.5 mg K20 expected in sample aliquot plus addnl 8 mL excess to ensure complete pptn. Dil. to vol. with H20, mix thoroly, let stand 5-10 min, and pass thru dry filter (Whatman No. 12, or equiv.). Transfer 50 mL aliquot filtrate to 125 mL erlenmeyer, add 6-8 drops indicator, 2.102(e). and titr. excess reagent with std BAC soln. % K20 in sample = (mL STPB added - mL BAC) x F x 2. Calcn applies to all fertilizers if 1 g sample is dild to 500 mL and 25 mL aliquot is taken fqr analysis. OTHER ELEMENTS Minor Nutrients by Atomic Absorption Spectrophotometry (39)-Official Final Action (Caution: See 51.00S.) 2.109 Apparatus and Reagent (a) Atomic absorption spectrophotometer.-Several com. models are available. Since each design is somewhat different,
- 43. AOAC METHODS (1980) OTHER ELEMENTS 21 with varying requirements of light source, burner flow rate, and detector sensitivity, only general outline of operating parameters is given in Table 2:04. Operator must become familiar with settings and procedures adapted to his own app. and use table only as guide to concn ranges and flame conditions. (b) Disodium EDTA soln.-2.5%. Dissolve 25 g Na2H2EDTA in L H20 and adjust to pH 7.0 with 5N NaOH, using pH meter. Table 2:04 Operating Parameters Wave- Range Element length, A Flame /Lg/mL Remarks Ca 4227 Rich Air-C,H, 2-20 1% La, 1% HCI 4227 Rich N,O-C,H, 2-20 Requires special burner Cu 3247 Air-C,H, 2-20 Fe 2483 Rich Air-C,H, 2-20 Mg 2852 Rich Air-C,H, 0.2-2 May need La Mn 2795 Air-C,H, 2-20 Zn 2138 Air-C,H, 0.5-5 2.110 Standard Solutions (Do not use <2 mL pipets or <25 mL vol. flasks. Automatic diln app. may be used. Prep. std solns in 0-20/Lg range fresh daily.) (a) Calcium solns.-(1) Stock soln.-25 /1-g Ca/mL. Dissolve 1.249 9 CaCOa in min. amt 3N HCI. Oil. to 1 L. Dil. 50 mL to 1 L. (2) Working std solns.-{J, 5,10,15, and 20 /1-g Ca/mL contg 1% La. To 25 mL vol. flasks add 0, 5, 10, 15, and 20 mL Ca stock soln. Add 5 mL La stock soln and dil. to 25 mL. (b) Copperstock soln.-l000 /1-g Cu/mL. Dissolve 1.000 9 pure Cu metal in min. amt HNOa and add 5 mL HCI. Evap. almost to dryness and dil. to 1 L with O.lN HCI. (c) Iron stock soln.-l000 /1-g Fe/mL. Dissolve 1.000 9 pure Fe wire in ca 30 mL 6N HCI with boiling. Dil. to 1 L. (d) Lanthanum stock soln.-50 9 La/L. Dissolve 58.65 9 La20 a (99.99%, Ventran Corp., Alfa Products, 8 Congress St, Beverly, MA 01915, or equiv.) in 250 mL HCI, adding acid slowly. Dil. to 1 L. (e) Magnesium stock soln.-l000 /1-g Mg/mL. Place 1.000 9 pure Mg metal in 50 mL H20 and slowly add 10 mL HCI. Dil. to 1 L. (f) Manganese stock so/n.-1000 /1-g Mn/mL. Dissolve 1.582 9 Mn02 in ca 30 mL 6N HCI. Boil to remove CI and dil. to 1 L. (g) Zinc stock soln.-l000 /1-g Zn/mL. Dissolve 1.000 9 pure Zn metal in ca 10 mL 6N HCI. Dil. to 1 L. (h) Other std solns.-Dil. aliquots of solns (b). (c), (e), (f), and (g) with 0.5N HCI to make ;;.4 std solns of each element within range of detn. 2.111 Preparation of Sample Solutions (Caution: See 51.019, 51.025, and 51.028.) (a) Inorganic materials and mixed fertilizers.-Dissolve 1.00 9 well ground sample in 10 mL HCI in 150 mL beaker. Boil and evap. soln nearly to dryness on hot plate. Do not bake residue. Redissolve residue in 20 mL 2N HCI, boiling gently if necessary. Filter thru fast paper into 100 mL vol. flask, washing paper and residue thoroly with H2 0. Measure absorption of soln directly, or dil. with 0.5N HCI to obtain solns within ranges of instrument. If Ca is to be detd, add enough La stock soln to make final diln 1% La (i.e., 5 mL La to 25 mL flask, 20 mL to 100 mL flask, etc.). (b) Fertilizers containing organic matter (tankage, corncobs, cottonseed hulls, etc. ).-Place 1.00 g sample in 150 mL beaker (Pyrex, or equiv.). Char on hot plate and ignite 1 hr at 5000 with muffle door propped open to allow free access of air. Break up cake with stirring rod and dissolve in 10 mL HCI as in (a). (c) Fertilizers containing fritted trace elements.- Dissolve ";;;1.00 g well ground sample in 5 mL HCIO. and 5 mL HF. Boil and evap. to dense HCIO. fumes. Dil. carefully with H20, filter, and proceed as in (a). Alternatively, dissolve sample in 10 mL HC!. 5 mL HF, and 10 mL MeOH. Evap. to dryness. Add 5 mL HCI and evap. Repeat HCI addn and evapn. Dissolve residue as in (a). (Normally Pt ware should be used; Pyrex or other glassware may be used if Na, K, Ca, and Fe are not to be detd.) (d) For manganese.-(1) Acid-soluble, for both Mn+2 and Mn+4 .-5ee (a). (b). and (c), and 2.139(b). (2) Acid-soluble, for Mn+2 only.-5ee 2.139(a), 2.140, and 2.141*. (3) Water-soluble, for Mn+2 only.-5ee 2.142. (e) For iron and zinc.-(1) Aqueous extraction.-Place 1.00 9 sample in 250 mL beaker, add 75 mL H20, and boil 30 min. Filter into 100 mL vol. flask, washing paper with H20. Dil. to vol. and redil. if necessary. (2) Chelation extraction.-Place 1.00 g sample in 250 mL beaker, and add 5 cm (2") mag. stirrer bar and 100 mL 2.5% EDTA soln. Stir exactly 5 min, and filter thru Whatman No. 41 paper, or equiv. If filtrate is cloudy, refilter immediately thru fine paper (Whatman No.5, or equiv.). Redil., if necessary, with 0.5N HCI. 2.112 Determination (P interferes in Ca and may interfere in Mg detn with air-C2H2 burners. Eliminate interference by adding La stock soln to std and sample solns so that final dilns contain 1% La. P does not interfere with Ca detn when N20-C2H2 flame is used.) Set up instrument as in Table 2:04, or previously established optimum settings for app. to be used. Less sensitive secondary lines (Gatehouse and Willis, Spectrochim. Acta 17, 710(1961)) may be used to reduce necessary diln, if desired. Read ;;.4 std solns within anal. range before and after each group of 6-12 samples. Flush burner with H20 between samples, and re- establish 0 absorption point each time. Prep. calibration curve from avo of each std before and after sample group. Read concn of samples from plot of absorption against /1-g/mL. 2.113 Calculations % Element = (/1-g/mL) x (F/sample wt) x 10-4 , 2.114 F = mL original diln x mL final diln/mL aliquot, if original 100 mL vol. is dild. Acid-Soluble Boron (40)-Official Final Action Apparatus Use high sensitivity glass electrode pH meter for titrn. Use assembly with burets, electrodes, and mech. stirrer, arranged for convenient use with 250 mL beaker. Use ordinary 50 mL burets for the 0.025N NaOH and 0.02N HCI. 2.115 Reagents (a) Boric acid std soln.-Dissolve 1 g HaBOa in H20 and dil. to 1 L. 1 mL = 0.1748 mg B. (b) Sodium hydroxide std soln.-C02-free, ca 0.025N. Stdze as follows: Pipet 25 mL std HaBOa soln into 250 mL beaker, add 3.0 g NaCI, acidify to Me red, dil. to 150 mL, boil to expel CO2, cool, and titr. potentiometrically as in 2.116. Det. blank by repeating titrn, substituting 25 mL H20 for HaB03 soln. Calc. B equivalence as follows: mg B/mL = 4.369/[(mL NaOH soln) - (mL blank)]. Protect from atm. CO2 by soda-lime tubes or other suitable means. (c) Methyl red indicator.--Dissolve 0.1 9 Me red in 50 mL alcohol, dil. to 100 mL with H20, and filter if necessary.
- 44. 22 2. FERTILIZERS AOAC METHODS (1980) 2.116 Determination Weigh sample within 1 mg (1.0 g for up to 0.45% B, smaller samples for above that content) and place in 250 mL beaker. Add ca 50 mL H2 0 and 3 mL HCI. Heat to bp and keep hot until carbonates are decomposed. Keep soln hot but do not boil during following phosphate removal: Add 10% Pb(N03 )2 soln usually 10 mL, or 1 mL for each 1.2% P20 S if P20 S content is known to be >12%. Add NaHC03 , little at time, until soln approaches neutrality (often observed by formation of white ppt in addn to insol. matter already present). Add few drops Me red and continue adding NaHC03 gradually until just alk. to Me red (yellow or very slightly orange). Keep mixt. hot but not boiling (H20 bath or steam bath is best) 30 min, adding addnl small amts of NaHC03 if needed to keep same indicator color. (If indicator is bleached by nitrate present, add more; if color is obscured by org. matter, use external spot tests to follow neutzn.) After neutzn and heating, 40-50 mL soln should remain. Filter hot soln into 250 mL beaker and wash solids thoroly with hot H20. Acidify filtrate with few drops HCI and boil briefly to expel most of CO2 , Neutze hot soln with 0.5N NaOH, and reacidify with 0.5N HCI, using 0.3-0.5 mL excess. Oil. to ca 150 mL and boil gently few min to expel remaining CO2, Cool to room temp. in running H20. Roughly neutze mixt., using CO2- free 0.5N NaOH, and place beaker in titrn assembly with elec- trodes and stirrer immersed. Start stirrer and adjust pH to exactly 6.30 by adding 0.025N NaOH or 0.02N HCI as required. (When properly adjusted, pH should be steady; drifting usually is due to incomplete removal of CO2,) When reading of pH 6.30 is steady, read 0.025N NaOH buret, add 20 g mannitol or cryst. D- sorbitol, and titr. with 0.025N NaOH to pH 6.30. (Conveniently done with slidewire type instrument by opening pH meter circuit when mannitol is added, leaving scale setting at 6.30, closing circuit again when indicator color shows that end point is being approached, and carefully adding std NaOH soln until galva- nometer needle returns to zero. With practice, somewhat slow approach to equilibrium, characteristic of glass electrode, can be anticipated so as not to overrun end point.) When end point is reached, read buret again. Obtain reagent blank by repeating detn with all reagents but without sample. % B = (mL NaOH soln in detn - mL blank) x (mg B/mL NaOH soln)/(10 x g sample) 2.117 Water-Soluble Boron (40)-Official Final Action (Not applicable in presence of >5% urea or urea-formaldehyde resins) Weigh 2.5 g sample into 250 mL beaker. Add 125 mL H20, boil gently ca 10 min, and filter hot thru Whatman No. 40 paper, or equiv., into 400 mL beaker. Wash solids well with 6 portions hot H2 0 and dil. to ~200 mL with H20. Heat filtrate just to bp. Add 15 mL 10% BaCl, soln to ppt sulfates and phosphates, and add powd Ba(OH)2, cautiously with stirring, until just alk. to phthln, avoiding large excess. Boil in open beaker ~60 min to expel NH3 • (Samples colored by org. matter should be boiled longer.) If necessary, add H20 to keep vol. to ~150 mL. Add and stir 1-2 teaspoonfuls Filter-Cel or other inert filtering aid, and filter with suction thru packed paper pads into 500 mL Pyrex erlenmeyer. Wash ppt 6 times with hot boiled H20. (Avoid too large wash vols which increase vol. in flask to point of dangerous bumping in next step.) Make filtrate just colorless to phthln with HCI (1 +5). add Me red, and make just pink with the acid. Add 5 or 6 boiling stones and stirring rod, cover with watch glass, and boil 5 min to remove CO2, Cool in cold H20 while covered. Wash cover glass, stirrer, and sides of flask. Titr. to yellow of Me red with std O.05N NaOH, 50.032-50.036. Add 20 g D-mannitol and 1 mL or more phthln, shake, and wash down sides of flask. Titr. to pink end point. Oet. blank in exactly same manner as sample. 1 mL 0.05N NaOH = 0.0005409 B or 0.00477 g Na2 B40 7.10H20. Or, (Titer - blank) x factor = Ib Na2B40 7.10H20/ton (factor = 3.807 for 0.05N NaOH). 2.118 * Carbonate Carbon (41)-Official Final Action * Proceed as in 1.010-1.011, using 2 g sample. Report % CO2 by wt. 2.119 Water-Soluble Chlorine (42) Official Final Action Reagents (a) Silver nitrate std soln.-Oissolve ca 5 g recrystd AgN03 in H20 and dil. to 1 L. Stdze against pure, dry NaCI and adjust so that 1 mL soln = 0.001 g CI. (b) Potassium chromate indicator.-See 50.028(b). 2.120 Determination Place 2.5 g sample on 11 cm filter paper and wash with successive portions bOiling H20 until washings total nearly 250 mL, collecting filtrate in 250 mL vol. flask. Cool, dil. to vol. with H2 0, and mix well. Pipet 50 mL into 150 mL beaker, add 1 mL K2Cr04indicator, and titr. with std AgN03 soln to permanent red of Ag2Cr04' Acid-Soluble Calcium (43)-Official Final Action 2.121 Method I Weigh 2.5 g sample into 250 mL vol. flask, add 30 mL HN03 and 10 mL HCI, and boil 30 min. Cool, dil. to vol., mix, and filter if necessary. Transfer 25 mL aliquot to beaker and dil. to 100 mL. Add 2 drops bromophenol blue, 6.019(f). Add NH40H (1 +4) until indicator changes from yellow to green (not blue). If overrun, bring back with HCI (1 +4). (This gives pH of 3.5-4.0.) Dil. to 150 mL, bring to bp, and add 30 mL satd hot (NH4),C20 4 soln slowly, stirring constantly. If color changes from green to blue or yellow again, adjust to green with HCI (1 +4). If yellow, adjust with NH40H to green. Digest on steam bath 1 hr, or let stand overnight, and cool to room temp. Filter supernate thru quant. paper, gooch, or fritted glass filter, and wash ppt thoroly with NH40H (1 +50). Place paper or crucible with ppt in original beaker and add mixt. of 125 mL H20 and 5 mL H2S04, Heat to ~70° and titr. with O.lN KMn04 until first slight pink appears. Correct for blank and calc. to Ca. 2.122 Method /I (Atomic Absorption Method) See 2.109-2.113. 2.123 * Method III * Place CaC20 4 and filter paper from 2.136 in beaker in which pptn was made and dissolve and titr. as in 2.121. Cobalt (44)-Official Final Action (Caution: See 51.026, 51.028, and Acids. Chap. 51.) 2.124 Reagents (Use H20 free of interfering elements. Check by shaking 2 drops 0.01% dithizone in CCI4 with 10 mL H20. CCI4 phase should remain green.) (a) Ternary acid mixture.-See 2.049(b). *Surplus method--5ee inside front cover.
- 45. AOAC METHODS (1980) OTHER ELEMENTS 23 (b) Ammonium hydroxide.-Use fresh stock. (Reagent be- comes contaminated with heavy metals on prolonged storage in glass.) (e) Isoamyl acetate.-Distd. (d) 2-Nitroso-1-naphthol soln.-o.05%. Dissolve 0.05 g 2-ni- troso-l-naphthol in 8 drops IN NaOH and 1 mL H20. Add 50-60 mL H20 and 6.5-7 mL NH.OH, and dil. to 100 mL with H20. Divide into 2 ca equal parts and wash each part twice in 100 mL centrf. tube with 20 mL isoamyl acetate. Shake 30 sec and centrf. after each addn. (It may be necessary to remove part of aq. phase to ensure complete removal of foreign matter at interface.) (e) Cobalt stdsolns. -( 1) Stocksoln. -200 ILg Col mL. Dissolve 0.0808 g CoCI2.6H20 in H20 and dil. to 100 mL. (2) Working soln.-2 ILg Co/mL. Oil. 1 mL stock soln to 100 mL with H20. 2.125 Determination Slowly add 20 mL ternary acid mixt. to 2.00 g pulverized, mixed fertilizer in 150 mL beaker. Cover with watch glass and digest on steam bath overnight. Transfer to hot plate and heat covered until dense white fumes appear. (At this point HNOa will have been expelled. Take care not to lose significant amts of HCIO•.) Oil. sample contg undissolved residue with H2 0, transfer to 50 mL vol. flask, and dil. to vol. Transfer to 100 mL centrf. tube and centrf. 5 min at 2000 rpm. Transfer aliquot, contg 2-51Lg Co, to 50 mL g-s centrf. tube. Add 10 mL 20% diammonium citrate soln and 2 drops phthln. Adjust pH carefully to distinct pink with NH.OH (1 + 1) and add successively 1 mL 10% Na2S2 0 3 soln, 2 mL 2-nitroso-l-naphthol soln, and 5 mL isoamyl acetate. (Only isoamyl acetate addn requires high degree of precision.) Shake mixt. 5 min and let sep. Centrf., if necessary. Draw off and discard aq. phase thru glass capillary tube attached to vac. Wash isoamyl acetate phase with two 5 mL portions IN NaOH and one 5 mL portion IN HCI. Shake 5 min after each addn, let layers sep., and draw off and discard aq. phase. Centrf. 2 min at 1500 rpm and measure A or %T at 530 nm against isoamyl acetate. Det. Co from calibration curve relating A or log %T to Co content of std solns contg 0, 2, 4, and 5 ILg Co. 2.126 Iron (45)-Official Final Action (Note: Diphenylamine may be harmful. Caution: See 51.079 and 51.084.) Reagents (a) Diphenylamine soln.-Dissolve 1 g in 100 mL H2SO•. (b) Diphenylamine sulfonate soln.-Dissolve 0.5 g in H20 in 100 mL vol. flask and dil. to vol. (c) Potassium dichromate std solns.-o.1N and 0.01N. Prep. O.lN K2Cr20 7 as in 50.024. Prep. O.OlN soln by dilg 100 mL O.lN soln to 1 L. (d) Mercuric chloride saturated soln.-Shake HgCI2 with H20 and let settle. (e) Stannous chloride soln.-Dissolve 20 g SnCI2.2H20 in 20 mL HCI, warming gently. Add 20 mL H20 and dil. to 100 mL with HCI (1 +1). Keep warm until clear; then add few granules Sn. Dispense from dropping bottle. 2.127 Preparation of Sample Solution (a) Suitable for all fertilizers.-Treat 1 g as in 2.020(e). using 15 mL HCIO•. Hold ;31 hr at ca 170° to remove HN03 completely. Oil. to 200 mL. (b) Suitable for soluble salts and oxides.-Dissolve 1 gin 10 mL HCI, warming gently. Oil. to 200 mL. 2.128 Reduction Heat aliquot of sample soln (100 mL and 50 mL, resp., for samples contg <0.5 and 0.5-4.0% Fe) to bp. Add few drops diphenylamine sulfonate soln; then SnCI2 soln dropwise until violet color is discharged and 2 drops excess. (Usually 1-6 drops are required. Larger amt may be used with samples contg large amt of Fe.) If reduction does not occur, discard and proceed as follows with second aliquot: Add few granules In, boil few min, and either filter off excess In, washing with hot H20, or let In dissolve. Heat tobp and finish reduction with SnCI2 and diphenylamine sulfonate indicator as before. Add 10 mL HCI (1+1). Adjust vol. to 75-110 mL with H20. Cool rapidly in cold H20. Add 10 mL satd HgCI2 soln, swirl gently, add 5 mL H3PO., and titr. immediately. (Small amt of HgCI must ppt to ensure complete reduction.) 2.129 Titration Add 1 drop diphenylamine indicator by pipet (no more; excess will interfere with end point if amt of Fe is small). Titr. with O.OlN K2Cr20 7soln. Since end point may be difficult to see with very small amt Fe, approach end point slowly, allowing few sec for color to develop. Titr. to permanent blue (sometimes green with very small amt Fe). For samples contg >4% Fe, use O.lN K2Cr20 7for titrn. 1 mL O.lN K2Cr207 = 0.00558 g Fe; 1 mL O.OlN = 0.000558 g Fe. 2.130 Atomic Absorption Method See 2.109-2.113. Acid-Soluble Magnesium-Official Final Action 2.131 Atomic Absorption Method See 2.109-2.113. 2.132 EDTA Titration Method (46) (Applicable to samples contg ,,;;0.25% Mn or Zn) Reagents Use reagents 1.022(a). (b). (c), (d), (f) (1 mL = 1 mg Ca, equiv. to 0.6064 mg Mg). (g), (h) (stdzd as in 2.133), and in addn: (a) Triethanolamine soln.-(l +1). (b) Potassium ferrocyanide soln.-Dissolve 4 g K.Fe(CN)6 .3H20 in 100 mL H20. (e) Ferric ammonium sulfate soln.-Dissolve 136 g FeNH.(SO')2.12H20 in H20 contg 5 mL H2SO., and dil. to 1 L. Filter if not clear. 2.133 Standardization Pipet 10 mL Ca std soln into 300 mL erlenmeyer. Add 100 mL H20, 10 mL KOH-KCN soln, 2 drops triethanolamine soln, 5 drops K.Fe(CN)6 soln, and 15±1 mg calcein indicator. Immedi- ately place flask on mag. or other mech. stirrer in front of daylight fluorescent light and white background. While stirring, titr. with EDTA soln to disappearance of all fluorescent green and until soln remains pink. Titr. ;33 aliquots. From av., calc. Ca titer in mg/mL EDTA soln. Ca titer x 0.6064 = Mg titer in mg/mL. 2.134 Preparation of Solution (Caution: See 51.019 and 51.028.) (a) Organic materials.-Weigh 1 g sample into 250 mL boiling flask or erlenmeyer. Add 5 mL HCI and 10 mL HN03, and boil on hot plate or over low flame until easily oxidized org. matter is destroyed (ca 15 min). Cool, add 5 mL 70-72% HCIO., and heat to appearance of copious fumes and momentary cessation of boiling, but not to dryness. Cool, and transfer to 250 mL
- 46. 24 2. FERTILIZERS AOAC METHODS (1980) beaker with ca 100 mL H20. Continue with pH adjustment, as in 2.135. (b) Inorganic materials and mixed fertilizers.-Weigh 1 g sample into 250 mL beaker. Add 5 mL HCI and 10 mL HN03• Cover with watch glass and heat on asbestos mat on hot plate nearly to dryness (ca 30 min). If soln remains colored from org. residues, cool, add 5 mL HCIO. (70-72%), and continue heating to copious fumes and momentary cessation of boiling, but not to dryness. 2.135 Determination Cool prepd soln to room temp. Wash watch glass and inside of beaker to ca 100 mL with H20. Using pH meter with glass electrode and mech. stirring, adjust to ca pH 3 with 30% KOH soln and finally to pH 4.0 with 10% KOH soln. Add FeNH.(SO')2 soln, 5 mL for sample <7% P20 5, 10 mL for sample 7-15% P20 5, 15 mL for sample 16-30% P20 5, and proportionate amts for samples >30% P20 5• Adjust to pH 5.0 with KOH solns as above, or with HCI (1 +4) if pH is >5.0. Cool to room temp. and transfer to 250 mL vol. flask with H20. Dil. to vol. with H20 and mix. Let stand until ppt settles. Disturbing ppt as little as possible, filter enough soln for aliquots required for titrn thru dry 11 cm fluted paper, Whatman No.1, or equiv. Pipet two equal aliquots contg <15 mg Ca + Mg (usually 25 mL) into two 300 mL erlenmeyers and dil. each to 100 mL with H20. To one (titrn 1 for Ca + Mg) add 5 mL pH 10 buffer soln, 2 mL KCN soln, 2 drops triethanolamine soln, 5 drops K.Fe(CN)6 soln, and 8 drops eriochrome black T indicator. Titr. immediately with EDTA soln, stirring and lighting as in stdzn. Color changes are wine red, purple, dark blue, to clear pure blue end point, becoming green if overtitrd. To second aliquot (titrn 2 for Cal add 10 mL KOH-KCN soln, 2 drops triethanolamine soln, 5 drops K.Fe(CN)6 soln, and 15±1 mg calcein indicator. Titr. immediately with EDTA soln as in stdzn. (Titrn 1 - Titrn 2) x Mg titer EDTA x 100/mg sample in aliquot = % Mg. Titrn 2 x Ca titer EDTA x 10/mg sample in aliquot = % Ca. 2.136 * Gravimetric Method (47) * Official Final Action Removal of Ca as oxalate, pptn as MgNH.PO., and ignition to Mg2P20 7• See 2.123, 11th ed. 2.137 * Volumetric Method-Official * Final Action Titrn of pptd MgNH.P04 with acid. See 2.124, 11th ed. 2.138 Water-Soluble Magnesium (47) Official Final Action (a) In potassium-magnesium sulfate, magnesium sulfate, and kieserite.-Weigh 1 g sample into 250 mL vol. flask, add 200 mL H20, and boil 30 min. Cool, dil. to vol. with H20, and mix. If detn is to be conducted gravimetrically, 2.136*, or volumetrically, 2.137*, see 2.125, 11th ed. (b) In other materials, including mixed fertilizers.-Weigh 1 g sample into 500 mL vol. flask, add 350 mL H20, and boil 1 hr. Cool, dil to vol., mix, and filter if necessary. If detn is to be conducted gravimetrically, 2.136*, or volumetrically, 2.137*, see 2.125, 11th ed. (c) By EDTA method.-Transfer aliquot soln prepd as in (a) or (b) to beaker and det. Mg as in 2.135, using HCI or KOH to adjust pH. * Surplus metho~ee inside front cover. Acid-Soluble Manganese--Official Final Action Atomic Absorption Spectrophotometric Method (48) 2.139 Preparation ofSample (a) Applicable to Mn+2 only.-Prep. sample soln as in 2.140, omitting the 50 mL H3P04 (1+9). Proceed as in 2.112, using std solns prepd as in 2.110(f) and (h), substituting 0.5N H2SO. for 0.5N HCI in 2.110(h). (b) Applicable to total Mn+2 and Mn+4.-Prep. sample soln as in 2.111. Proceed as in 2.112, using std solns prepd as in 2.110(f) and (h). 2.140 Colorimetric Method (49) (Applicable to samples contg Mn+2 only and with ",,5% Mn) Place 1 g sample in 200 mL wide-neck vol. flask or 250 mL beaker. Add 10 mL H2SO. and 30 mL HN03. Heat gently until brown fumes diminish; then boil 30 min. If org. matter is not destroyed, cool, add 5 mL HN03, and boil. Repeat process until no org. matter remains, and boil until white fumes appear. Cool slightly, and add 50 mL H3PO. (1 +9). Boil few min. Cool, dil. to 200 mL in vol. flask, mix, and let stand to allow pptn of CaSO•. Pipet 50 mL clear soln into beaker. Heat nearly to bp. With stirring or swirling, add 0.3 9 KID4 for each 15 mg Mn present, and hold 30--60 min at 90-100°, or until color development is complete. Cool, and dil. to measured vol. that will provide satisfactory concn for colorimetric measurement by instrument chosen (usually <20 ppm Mn). Compare in colorimeter against std KMnO. soln, 7.116, or in spectrophtr at 530 nm. Calc. to Mn. 2.141 * Bismuthate Method (50) * (Applicable to Mn+2 only) See 2.127-2.128, 11th ed. Water-Soluble Manganese 2.142 Atomic Absorption Spectrophotometric Method (48)-Official Final Action (Applicable to Mn+2 only) Place 1 g sample in 50 mL beaker, wet with alcohol, add 20 mL H20, and let stand 15 min, stirring occasionally. Transfer to 9 cm Whatman No.5 paper, and wash with small portions H20 until filtrate measures ca 230 mL. Let each portion pass thru paper before adding more. Add 3-4 mL H2S04 to filtrate. Proceed as in 2.112, using std solns prepd as in 2.110(f) and (h), substituting 0.5N H2S04 for 0.5N HCI in 2.110(h). Copper-Official Final Action 2.143 * Long Volumetric Method (51) * See 2.129-2.130, 11th ed. Short Volumetric Method (52) 2.144 Reagents (a) Sodium thiosulfate std soln.......{J.03N. Prep. daily by dilg O.lN soln, 50.037-50.038. 1 mL 0.03N Na2S203 = 1.906 mg Cu. (b) Starch soln.-Mix ca 1 g sol. starch with enough cold H2 0 to make thin paste, add 100 mL boiling H20, and boil ca 1 min while stirring. (c) Bromocresol green indicator. -Dissolve 0.1 g tetrabromo- m-cresolsulfonphthalein in 1.5 mL O.lN NaOH, and dil. to 100 mL with H20.
- 47. AOAC METHODS (1980) OTHER ELEMENTS 25 2.145 Determination Place 2 g sample in 300 mL erlenmeyer and add 10 mL HN03 and 5 mL H2S04• Digest on hot plate to white fumes. If soln darkens, owing to org. matter, cool slightly, add little more HN03, and digest again to white fumes, repeating operation if necessary until org. matter appears to be destroyed. Cool, add 50 mL H20, boil ca 1 min, and cool to room temp. Add bromocresol green, then NH40H until indicator changes to light green (pH 4). Cool again to room temp., and if indicator changes back to more acid color, add NH4 0H dropwise until indicator becomes light green again, avoiding excess. Add 2 g NH4HF2 (Toxic. Caution: See 51.084), mix well, and let stand ca 5 min. Add 8--10 g KI, mix well, and titr. with std Na2S20 3 soln to light yellow. Add ca 1 mL starch soln and continue titrg slowly until color is nearly same as just before addn of the KI and becomes no darker on standing 20 sec. Report as % Cu. 2.146 Atomic Absorption Method See 2.109-2.113. Sodium-Official First Action Flame Photometric Method (53)-Official First Action 2.147 Reagents See 2.091(aHc), and in addn: Sodium chloride.-Dry 2 hr at 105°. 2.148 Preparation ofSolution Prep. soln as in 2.093(a), using 2.5 g sample «4% Na) or 1.25 g (4-20% Na). 2.149 Preparation ofStandard Curve (a) Samples containing 1% or more sodium.-Proceed as in 2.094, using 1.2716 g NaCI, range of diln 0-40 ppm Na, intervals ~5 ppm, and full scale for 40 ppm Na. (b) Samples containing less than 1% sodium.-Proceed as in 2.094, using 1.2716 g NaCI, range of diln 0-10 ppm Na, intervals 2 ppm, and full scale for 10 ppm Na. 2.150 Determination (Caution: See 51.007.) Transfer 25 mL «4% Na) or 10 mL (4-20% Na) sample soln to 250 mL vol. flask, dil. to vol. with H20, and mix (if internal std instrument is used, add required amt LiN03 before dilg to voL). Atomize portions of sample several times to obtain reliable avo readings for each soln. Det. ppm Na from std curve (a) or (b). Calc. % Na as follows: 0-4%: ppm Na!10 = % Na 4-20%: ppm Na!2 = % Na Zinc-Official Final Action 2.151 * Gravimetric Method (54) * (For samples contg ;;.0.1 % Znj Digestion with HN03 and H2S04, sepn of interfering sulfides from acid soln with H2S, pptn ofZnS at pH 3 with H2S, and ignition to ZnO. See 2.138, 12th ed. 2.152 * Colorimetric Method (55) * (For samples contg <4% Zn) Digestion with HN03 and H2S04, and detn with dithizone. See 2.139, 12th ed. Zincon Ion Exchange Method (56) (Clean all glassware with hot chromic acid or HN03 (1 + 1). Rinse thoroly with H20. Caution: See 51.023, 51.026, and 51.030.) 2.153 Reagents (a) Anion exchange resin.-100-200 mesh, strong base, poly- styrene alkyl quaternary amine, 7% cross linkage, CGA-540 (J. T. Baker Chemical Co., No. 4602, or equiv.). (b) Zincon indicator.-Dissolve 0.12 g zincon (0-[[0:-[(2- hydroxy-5-sulfophenyl)azo] benzyl idene] hydrazino] benzoic acid, Na salt) (J. T. Baker Chemical Co., No. X690) in 5 mL 0.3N NaOH and dil. to 100 mL with H2 0. Prep. fresh weekly. (c) Hydrochloric acid solns.-(T) O.5N.-Dil. 20 mL HCI to 500 mL with H20. (2) O.25N.-Dil. 2 mL HCI to 100 mL with H20. (3) O.005N.-Dil. 2.5 mL HCI to 6 L with H20. (d) Borate buffer soln.-pH 9.8. Dissolve 4 g H3B03 in 140 mL H20. Add 5 mL NH40H by pipet and then dropwise to pH 9.8. Check daily. (e) Ammonium thiocyanate.-1M. Dissolve 0.76 g NH4 CNS in 10 mL H2 0. (f) Zinc std solns.-(T) Stock soln.-1000 ppm. Dissolve 1.000 g pure Zn metal in small amt HCI-HN03 (1+1). Evap. to small vol., add 3 mL HCI, and heat. Oil. to 1 L with H20. (2) Working soln.-10 ppm. Oil. 10 mL stock soln to 1 L with H20. (g) Sodium hydroxide soln.-{).3N. Dissolve ca 1.25 g NaOH in 100 mL H20. 2.154 Preparation of Resin Column Wash 12 g new resin in 250 mL beaker with H20 until washings are neut. Introduce resin as slurry into 25 x 2.2 cm chromatgc tube with fritted glass disk and stopcock at bottom. Mark vol. levels on column at 10,40, and 50 mL above packed resin and on 250 mL separator at 90 mL. (Keep resin wet and store under liq. when not in use.) Connect separator to top of column thru stopper. Attach inverted U-shaped glass dispensing tube to 250 mL vol. flask thru vented stopper or cork and connect with Zn- free plastic tubing to stopcock of column with stopcock grease. See Fig. 2:08. Mount reservoir (aspirator bottle or carboy) contg ;;. 1 L 0.005N HCI high enough to effect backwashing. Attach Zn-free tubing and pinch clamp. 2.155 Flow Calibration Use sweep sec hand of watch or stopwatch to establish flow rates. Det. number drops!mL leaving dispensing tube. Remove separator and vol. flask; drain and then backwash resin (see 2.157). Remove reservoir tubing, open stopcock, elute 40 drops from dispensing tube, and measure vol. Use this factor to convert 0.5 mL/min (required in Zn elution, 2.157) to drops/sec. 2.156 Preparation of Sample Remove separator and elution tubing from column. Activate resin by draining column and adding 50 mL 0.5N HCI. Drain column to 40 mL mark. (a) Samples containing 0.14% or more zinc. -Dissolve 1.000 g well-ground sample in 10 mL HCI and 5 mL HN03 in 250 mL beaker. Evap. to near dryness on hot plate. (Caution: Do not bake.) Redissolve residue in ca 40 mL 0.5N HCI, boiling gently if necessary. Filterthru Whatman No. 41 paper into 100 mL vol. flask. Thoroly wash residue and dil. filtrate to vol. with 0.5N HCI. Drain column to 10 mL mark. Tap column to pack resin. Pipet aliquot contg 0.7-0.8 mg Zn onto column. Elute sample soln at ca 5 sec/drop. * Surplus method-5ee inside front cover.
- 48. 26 2. FERTILIZERS AOAC METHODS (1980) SEAL WITH WATER VENTED~ 90 ML 50 ML _ 40 ML _ 10 ML _ FIG. 2:08-Apparatus for elution of resin column. (b) Samples containing less than 0.14% zinc.-Weigh, to nearest mg, sample contg 0.7-0.8 mg Zn into 250 mL beaker. Digest and filter sample and prep. column as in (a). Tightly attach open separator to column. Close stopcock. Transfer entire sample soln to separator, rinsing with two 10 mL portions 0.5N HCI. Open stopcock. Elute sample soln at ca 5 sec/drop. Remove empty separator, rinse twice with 20 mL 0.5N HCI, and add rinses to remaining soln in column. 2.157 Elution of Zinc After sample soln passes thru resin, immediately rinse column with 0.5N HCI at ca 1.5 sec/drop until 1 mL eluate gives clear, colorless soln with 1M NH.SCN. If Fe+3 is present, soln will turn brown. Drain resin and backwash with 0.005N HCI from reservoir thru elution tubing, forcing out air bubbles from tubing and column. Simultaneously, tap resin into suspension as it is forced up. Close column stopcock when liq. reaches 50 mL mark on column. Attach dispensing tube to vol. flask. Reopen stopcock, and raise flask until flow just stops. Continue ht adjustment until a drop remains in equilibrium at tip of dispensing tube and neither rises nor falls. Secure flask. Attach open separator with H20 seal to column and close stopcock. Add 240 mL 0.005N HCI to separator and reopen stopcock. Open column stopcock until rate of ca 0.5 mL/min is sustained 10 min. If rate decreases, increase rate slightly until nearly const. Let elution continue overnight. Then, if >90 mL remains in separator, readjust rate as above and continue elution to 90 mL mark. Finally, lower flask, fill to 250 mL mark at convenient rate from dispensing tip, and mix. Detach hose and separator from column, and drain all 3. Reactivate resin, and stopper column as in 2.156. 2.158 Determination Pipet 20 mL eluate into 50 mL vol. flask contg small piece litmus paper. Make alk. with 0.3N NaOH, then just acidic with 0.25N HCI. Pipet in 2 mL more acid, 5 mL butter, and 3 mL zincon soln. Oil. to vol. with H20. Similarly prep. 0, 1,2, and 3 ppm std solns, using 0, 5, 10, and 15 mL std Zn working soln, resp. Using 0 ppm std soln as blank, det. A at 620 nm 15-45 min after zincon addn. Plot std curve of ppm against A. % Zn = (C x F)/W, where C = ppm from std curve; W = g sample; and F = 0.0625 for samples contg <0.14% Zn or 6.25/mL aliquot pipetted onto resin for samples contg ;.0.14% Zn. 2.159 Atomic Absorption Method See 2.109-2.113. Total Sulfur (57j-Official Final Action 2.160 Reagents (a) Barium chloride soln.-l0%. Dissolve 100 g BaCI2.2H20 in 900 mL H20 and filter thru Whatman No. 42 paper, or equiv. 1 mL = 14 mg S. (b) Bromine in carbon tetrachloride.-l0%. Add 10 g Br to 90 g reagent grade CCI•. Stir until homogeneous. Store in g-s bottle. (Caution: See 51.047 and 51.049.) 2.161 Determination Weigh sample contg 50-150 mg S into 250 mL beaker, and add 20 mL 10% Br in CCI., (b). Mix by swirling beaker at 5 min intervals during 30 min. Add 15 mL HN03 and mix as before. Evap. to 1-2 mL on warm hot plate. Add 15 mL HCI and 10 mL H20. Evap. just to dryness on warm hot plate or steam bath. Add 10 mL HCI and 50 mL H20, heat to boiling, boil 5 min, and filter thru Whatman No. 42 paper, or equiv. Wash paper with ten 20 mL portions hot H20. Heat filtrate to boiling. Add 5--6 drops 10% BaCI2 soln, (a). After 1 min, add dropwise amt BaCI2 soln equiv. to expected S content plus 5 mL excess. Digest at gentle boil 1 hr. Remove from hot plate and let ppt settle 15-20 min. Filter immediately thru previously ignited and weighed gooch. Wash with hot H20 until 10 mL wash H20 shows no ppt with 3 mL 1% AgN03• Dry and ignite at 8000 to const wt. Cool in desiccator over MgCIO. and weigh. % S = g BaSO. x 0.1374 x 100/g sample. 2.162 Free Sulfur (58)-Official Final Action (Caution: See 51.011,51.047,51.048, and 51.049.) Ext 1 g sample with CS2 in Soxhlet app., letting extn thimble drain ;.12 times. Transfer ext to 250 mL beaker. Evap. CS2 in draft at room temp. Heat in oven 20 min at 60-700 ; then cool to room temp. Add 10 mL satd soln of Br in CCI., cover, and let stand ca 30 min, stirring several times. Add 15 mL HN03, cover, and let stand ca 30 min, stirring several times. Evap. on hot plate to ca 5 mL. Add 20 mL HCI and evap. to ca 5 mL. Add ca 50 mL H20, filter, and wash with HCI (1 +49). Add 2 drops bromophenol blue, 6.019(f), anr,!, then NH.OH to first color change. Add HCI dropwise until distinctly acid, then 5 drops excess; dil. to 150 mL, heat to bp, and add 10% BaCI2 soln, 2.160(a), dropwise until ca 50% excess is present. Cover beaker and digest on steam bath ;.1 hr. Cool to room temp. and filter thru asbestos on gooch previously ignited at 8000 and weighed. Wash 10 times with hot H20. Ignite in furnace at 8000 ;.20 min. Cool in desiccator and weigh as BaSO•. Calc. as S as in 2.161.
- 49. AOAC METHODS (1980) 2.163 * Acid-Forming or Nonacid-Forming * Quality (59)-Official Final Action Fusion with std amt Na2C03 followed by neutzn of melt with excess acid and back-titrn with std NaOH. See 2.141-2.142, 11th ed. PEAT (Moss, humus, and reed-sedge types) 2.164 Sampling (60)-Procedure Use slotted single or double tube or slotted tube and rod, all with pointed ends and min. 1" diam. for loose materials. Use cutting type core sampler, with plunger, for compressed ma- terials. Pennsylvania State Forage Sampler (NASCO, 901 Janes- ville Ave, Fort Atkinson, WI 53538) is satisfactory core sampler. Take representative sample from lot or shipment as follows: (a) Packaged or baled peats.-Lay bag or bale horizontally and remove core diagonally from end to end. From lots of 1-10 bags, sample all bags; from lots of ;;.11, sample 10 bags. Take 1 core from each bag sampled; except for lots of 1-4 bags, take diagonal cores from each bag and addnl cores to total ;;.5 cores. (b) Bulk samples.-Draw ;;.10 cores from different regions. (c) Small containers (10 Ib or less).-Take entire package. Working rapidly to prevent moisture losses, reduce composite sample to ~500 g (by wt) or 2 L (by vol.) by mixing on clean plastic or paper and quartering. Place sample in air-tight con- tainer. Sampling by random "grab" procedure is necessary if particle size range is to be detd or if representative sample cannot be taken with core sampler as above. 2..165 Preparation of Sample (61)-Official Final Action Place representative field sample on square rubber sheet, paper, or plastic. Reduce sample to amt required by quartering and place in moisture-proof container. Work rapidly to prevent moisture losses. Moisture (61)-Official Final Action 2.166 Method I Mix sample thoroly and place 10-12 g in ignited and weighed (with fitted heavy-duty AI foil cover) Vycor or porcelain evapg dish, ;;.75 mL capacity. Crush soft lumps with spoon or spatula. Cover immediately with AI foil cover and weigh to nearest mg. Dry, uncovered, 16 hr at 105°. Remove from oven, cover tightly, cool, and weigh. % Moisture (report to nearest 0.1 %) = (g as-received sample - g oven-dried sample) x 100/g as-received sample. 2.167 Method /I (Use when pH, N, fiber, etc., are to be detd.) Mix thoroly and weigh 100-300 g representative sample, 2.165, and spread evenly on large flat pan. Crush soft lumps with spoon or spatula and let come to moisture equilibrium with room air ;;.24 hr. Stir occasionally to maintain max. air exposure of entire sample. When wt is const, calc. loss in wt as % moisture removed by air drying. Grind representative portion air-dried sample 1-2 min in high-speed blender; use for moisture, ash, and N detns. Mix air-dried, ground sample and weigh, to nearest mg, equiv. of 10 g sample on as-received basis (g air-dried sample equiv. * Surplus method--see inside front cover. PEAT 27 to 10.0 g as-received sample = 10.0 - [(10.0 x % moisture removed)/100j). Place weighed sample in ignited and weighed (with fitted heavy duty AI foil cover) Vycor or porcelain evapg dish and proceed as in 2.166. % Moisture (report to nearest 0.1%) = (10.0 - g oven-dried sample) x 10.0. 2.168 Mechanical Analysis for Determination of Particle Size Range (60)-Procedure Apparatus Mechanical sieve shaker.-With 8" diam., Nos. 8 and 20 sieves equipped with cover and bottom pan. 2.169 Preparation ofSample Air-dryas in 2.167. 2.170 Determination Mix thoroly and place 20.0 g air-dried sample on No.8 sieve nested on No. 20 sieve. Secure sieves and shake at suitable speed 10 min. Remove and weigh foreign matter, such as sticks, stones, and glass, from No.8 fraction. Weigh fractions of peat retained on Nos. 8 and 20 sieves and portion collected in bottom pan. Convert fraction and sample wts to as-received basis and calc. in terms of %. (If foreign matter is absent, conversion to as-received basis is not necessary.) % Foreign matter = fraction removed from No.8 sieve x 100; % Coarse fiber = fraction retained on No.8 sieve x 100; % Medium fiber = fraction retained on No. 20 sieve x 100; % Fine = fraction collected in pan x 100. If mech. sieve shaker is not available, use hand sieving. Conduct sieving by appropriate lateral and vertical motions accompanied by jarring action. Continue until no appreciable change is noted in sieve fraction. pH (60)-Procedure 2.171 Apparatus and Reagents (a) pH meter.-Battery-operated or on elec. line with voltage regulator. (b) Carbon dioxide-free water.-See 50.007. (c) Acid potassium phthalate buffer soln.~.05m. See 50.007(c). (d) Phosphate buffer soln.~.025m. See 50.007(d). (e) Calcium chloride solns (Method II only).-( 1) Stock soln.- 1.0M. Dissolve 147 g CaCI2.2H20 in H20 in 1 L vol. flask, cool, dil. to vol., and mix. Oil. 15 mL of this soln to 200 mL with H20 in vol. flask and stdze by titrg 25 mL aliquot dild soln. with std 0.1N AgN03, 50.029, using 1 mL 5% K2CrO. as indicator. (2) Working soln.~.01M (pH 5.0-6.5). Oil. 20 mL stock soln. to 2 L with H20. 2.172 Determination (a) Method I (in distilled water).-Weigh ca 3.0 g air-dried peat or equiv. amt moist material into 100 mL beaker. Add 50 mL H20. (Addnl H20 may be needed for very fibrous materials such as sphagnum moss peat.) Let soak 30 min, with occasional stirring. Read on pH meter. (b) Method II (in 0.01M calcium chloride soln).- Weigh ca 3.0 g air-dried peat or equiv. amt moist material into 100 mL beaker. Add 50 mL 0.01M CaCI2 • Let soak 30 min, with occasional stirring. Read on pH meter. Report results as pH in 0.01M CaCI2 soln. (pH values in CaCl, soln. are usually ca 0.5--0.8 units lower
- 50. 28 2. FERTILIZERS AOAC METHODS (1980) than those in H20. Observed pH in Cael2 soln is virtually independent of initial amt salt present in soil, whereas pH readings in H20 can be modified by salts such as fertilizer materiaL) 2.173 Ash (61)-Official Final Action Place uncovered (retain cover for weighing) Vycor or porcelain dish contg dried sample from moisture detn in furnace. Gradually bring to 5500 and hold until completely ashed. Cover with retained AI foil cover, cool, and weigh. % Ash (report to nearest 0.1%) = g ash x 100/g as-received sample taken for moisture detn. (If moisture Method II was used, g as-received sample = 10.0.) Sand (60)---Procedure 2.174 Preparation ofSample Air-dryas in 2.167. 2.175 Determination (Caution: See 51.056.) Place 25 g air-dried, ground sample into 125 mL tall-form beaker, or equiv. Nearly fill beaker with CHCI3, stir briefly, and let settle ca 1 min. With spoon, discard most floating org. material, decant remaining org. material and CHCI3, taking care not to disturb settled portion (sand), and air-dry to remove residual CHCI3• (Stirring aids drying.) When dry, weigh settled portion and calc. as % sand (includes other minerals present such as limestone, etc.). % Sand = (g air-dried settled residue x 100)/g air-dried sample 2.176 Organic Matter (67)-Official Final Action % Org. matter = 100.0 - (% moisture + % ash). 2.177 Total Nitrogen (60)---Procedure Det. N as in 2.057, using well mixed, air-dried, ground sample equiv. to 10.0 g sample on as-received basis. Det. g air-dried sample equiv. to 10.0 g as-received sample as in 2.167. Water Capacity and Volumes (62) Official Final Action 2.178 Apparatus Dispensing apparatus.-2 dispensing burets, 250 mL in 1 mL subdivisions, ±2 mL tolerance, pinchcock type; 1-hole No.6 rubber stopper; straight polyethylene drying tube with serrated rubber tubing fittings, 15 cm long, %" od, %" id (Cenco Instru- ment Corp. No. 14782-2); and stainless steel screen circle, ca 16 mesh and 28.7 mm diam. Assemble dispensing app. as follows: Discard serrated rubber tubing fittings from polyethylene drying tube and use tube only. Center stainless steel screen on one end of tube and seal. (Soldering iron is usefuL) Adjust length of tube to match con- venient graduation of buret; then scallop end without screen to allow for H20 drainage, and insert into dispensing buret with screen side up. 2.179 Preparation ofSample See 2.165. 2.180 Determination Det. moisture content on sep. sample by 2.166 or 2.167. Weigh buret fitted with plastic tube and screen. Working rapidly to prevent moisture losses, mix sample thoroly, place on top of NO.4 screen, and shake until sieving is complete. Use only portion that has passed thru sieve for detn. Firmly pack buret with 25 cm (10") of 4 mesh sample as follows: Attach rubber stopper to delivery end of buret. Add ca 20 mL portions, firmly tapping 3 times vertically from ht of 15 cm (6") on rubber stopper, for final ht of 25 cm. (This will ensure that ht of final wet vol. is 19--25 cm.) Remove stopper; weigh buret to nearest g. Position buret to use sink as drain. Place H20 source (19 L (5 gaL) bottle) equipped with siphon device above level of buret. Connect clamped rubber tubing of siphon device to buret with glass tubing (ca 13 cm (5") long, constricted at one end) inserted into one-hole rubber stopper fitting tightly into top of buret. Attach rubber tubing with pinch clamp to delivery end of buret. Open both clamps and pass H20 thru sample ~24 hr, maintaining water reservoir over sample at all times. (Moss-type samples may float but gradually settle as sample becomes wet.) After initial soaking, regulate H20 flow thru column by adjusting screw clamp at delivery end of buret. On-flow of H20 should be ca equal to out-flow; flow of ca 1 drop/sec is suitable.) When sample is supersatd, close both clamps and let sample settle in H20 ca 5 min. Top surface of sample should be as level as possible. Raise buret and replace rubber tubing on delivery end of buret with 250 mL dispensing buret filled with H20, using rubber stopper for connection. Connect two burets tightly, with no air leaks. Remove siphon device and open outlet clamps of both burets to empty. (Suction created is equiv. to ca 38 cm (15") H20. Check for air leaks to ensure that std suction is exerted on sample. It is important to remove excess H2 0 as described.) Measure ht of wet peat. Ht should be 19--25 cm. Record vol. in mL and weigh buret, plastic tube with screen, and wet peat to nearest g. Wet sample again as above ~ 1 hr, drain by suction, record vol., and weigh. Repeat until consistent results are obtained. 2.181 Calculations (a) Saturated Volume Weights, g/mL As-recd = g as-recd sample/mL wet vol. Oven-dried = g dried sample/mL wet vol., where g dried sample = g as-recd sample x [(100 - % moisture)/100j. Wet = g wet sample/mL wet vol. (b) Water-Holding Capacity, % (1) Weight basis: As-recd = [(g wet sample - g as-recd sample) x 100]/g as-recd sample Oven-dried = [(g wet sample - g dried sample) x 100]/g dried sample (2) Volume basis: Water vol. = [(g wet sample - g dried sample) x 100j/(mL wet vol. x 1.0) (e) Dry Peat Volume, % Dry peat vol. = (g dried sample x 100)/(mL wet vol. x 1.5) (d) Air Volume, % Air vol. = 100 - (% water vol. + % dry peat voL) 2.182 Alternative Methods (60) Volume---Procedure Principle Method consists of dividing particles of peat from original container by passing them thru 12.7 mm (0.5") sieve and allowing them to fall into voL-measuring container.
- 51. AOAC METHODS (1980) 2.183 Apparatus (a) Sieve.-No. ';2" (12.7 mm). (b) Measuring box.-Steel or wood, bound with metal having one of the following sets of inner dimensions: (1) ';2 cu. ft. = 12 x 12 x 12" with line scribed 6" from bottom; (2) % cu. ft. = 12 x 12 x 12" with line scribed 9" from bottom; (3) 1 cu. ft. = 12 x 12 x 12" box, 2 cu. ft. = 16 x 16 base x 13.5" ht, 5 cu. ft = 16 x 16 base x 33.75" ht. 2.184 Determination (a) Loose peat.-Remove material from bag or container, pass it thru ';2" sieve, and place directly into measuring box. Pour contents from ca 60 em (2') into measuring box. Det. contents of bag or container only once. Fill corners of measuring box by shaking with rotary motion, 1 rotation/sec for 5 sec, without lifting box from floor or surface. When filled, level off by straightedge. Use ht of box to calc. vol. in cu. ft. (b) Baled peat.-Vol. baled material = ht x area of base. Cor- rect measurements for outside wrappers. Det. amt loose peat in bale by passing thru ';2" sieve and measuring amt loose peat, using 12 x 12 x 12" box as in (a). Report vol. of peat in cu. ft. Report total vol. of sieved peat from original container. 2.185 Volume Weight, Water-Holding Capacity, and Air Capacity of Water-Saturated Peat Materials-Procedure Apparatus (a) Hollow spray nozzle.-Monarch F-97-W, nozzle No. 4.6160 (Monarch Mfg. Works Inc., 2501 E Ontario St, Philadelphia, PA 19134). or equiv. (b) Pipe connection.-For installation of nozzle on H20 faucet in sink. (c) Containers.-Approx. 2 L (2 Ib coffee cans are suitable) fitted with plastic covers. Replace metal bottom of one with No. 20 Cu screen (test container). (d) Aluminum pie pans.-20 cm (8") diam. Drill holes in side walls of pan so that H20 depth in pan remains ca 1.3 em (0.5"). 2.186 Preparation of Sample See 2.165. 2.187 Determination Det. moisture content on sep. sample by 2.166 or 2.167. Weigh test container fitted with plastic cover, screen, and circle of filter paper (12.5 em Whatman No.4, or equiv.) which is placed on screen. Thoroly mix equal wts of H20 and peat and place in container without pressure to ht of 10 em (4"); record wt in g. (If peat is dried out, mix 1 part peat with 2 parts H20. If wet, mix 2 parts peat with 1 part H20.) Place test container in AI pan filled with H20 in sink ~30 em (12") directly under spray nozzle. Water ca 24 hr as mist to prevent compression of peat. Place cover on container, seal (tape is suitable) to prevent evapn, and let stand in AI pan, maintaining 1.3 cm H20 head 2 days. Remove from pan and drain 2 hr with container at 45° angle. Remove seal on cover, and record wt and vol. Vol. can be detd by using container identical to test container not fitted with screen, filling H20 to same ht as sample in test container, and transferring to graduate with mL markings. 2.188 Calculations (a) Saturated Volume Weights, g/mL (1) As-reed = g as-recd sample! mL wet vol., where gas-reed PEAT 29 sample = 9 total sample/2; or 9 total sample!3 if 2 parts H20 used; or (g total sample x 2)/3 if 2 parts peat used. (2) Oven-dried.-See 2.181. (3) Wet.-See 2.181. See 2.181. See 2.181. See 2.181. 2.189 (b) Water-Holding Capacity, % (c) Dry Peat Volume, % (d) Air Volume, % Cation Exchange Capacity (63) Official final Action AOAC-ASTM Method Principle Cation exchange capacity is measure of total amt exchange- able cations that can be held by peat, expressed as mequiv./ 100 g air-dried peat. Peat sample is shaken with 0.5N HCI to remove bases and to sat. sorption complex with H+. Excess acid is removed; absorbed H+ is replaced with Ba+2, titrd with O.lN NaOH, using phthln indicator, and calcd to mequiv./l00 g air- dried peat. 2.190 Reagents (a) Dilute hydrochloric acid.-O.5N. Dil. 42 mL HCI to 1 L with H20. (b) Barium acetate soln.-O.5N. Dissolve 64 g Ba(OAc)2 in H20 and dil. to 1 L. (c) Silver nitrate soln.-l%. Dissolve 1 9 AgNO, in 100 mL H20. (d) Sodium hydroxide std soln. -o.lN. Prep. and stdze as in 50.032-50.035. 2.191 Preparation of Sample See 2.165. 2.192 Determination Thoroly mix air-dried ground peat sample and place 2.00 9 in 300 mL erlenmeyer. Add ca 100 mL 0.5N HCI; stopper flask and shake vigorously periodically during 2 hr (or shake mech. 30 min). Filter thru rapid paper (24 em fluted, or equiv.) in large powder funnel. Wash with 100 mL portions H20 until 10 mL wash shows no ppt with ca 3 mL 1% AgNO,. Discard filtrate. Immediately transfer moist peat to 300 mL erlenmeyer, by puncturing apex of paper and forcing moist peat thru funnel stem into erlenmeyer, using spray from wash bottle contg ca 100 mL 0.5N Ba(OAc)2' Stopper flask and shake vigorously periodically during 1 hr (or shake mech. 15 min). Filter, and wash with three 100 mL portions H20. Discard peat, and titr. washings with O.lN NaOH, using 5 drops phthln, to first pink. Calc. mequiv./l00 g air-dried peat = (mL x normality NaOH x 100)/g sample. SELECTED REFERENCES (1) JAOAC 12, 97(1929); 33,424(1950); 38,108,541(1955); 50, 190,382(1967); 51, 859(1968); 55,709(1972). (2) JAOAC 52,592(1969). (3) JAOAC 42,500 (1959). (4) JAOAC 12, 98(1929); 24,253(1941). (5) JAOAC 3,95(1917). (6) JAOAC 40,711(1957).
- 52. 30 2. FERTILIZERS AOAC METHODS (1980) (7) JAOAC 46,582(1963); 47, 32,1040(1964). (8) JAOAC 52,1127(1969); 55,699(1972). (9) JAOAC 38, 413(1955). (10) JAOAC 41,517(1958); 42, 503(1959). (11) Z. Anal. Chern. 189, 243(1962); JAOAC 45, 40, 201, 999(1962); 46, 579(1963); 47, 420(1964). (12) Z. Anal. Chern. 189,243(1962); JAOAC 45, 40, 999(1962); 49,1201(1966); 52, 587(1969). (13) JAOAC 61, 533(1978). (14) JAOAC 5, 443, 460(1922); 6,384(1923); 14,182(1931); 19, 269(1936); 22,254(1939); 42, 512(1959). (15) JAOAC 42,503(1959). (16) JAOAC 52,587(1969). (17) JAOAC 43, 478(1960); 46, 570(1963); 60, 702(1977). (18) JAOAC 44, 233(1961). (19) JAOAC 46,570(1963); 47, 420(1964). (20) JAOAC 38,56(1955). (21) JAOAC 53, 450(1970); 57, 10(1974). (22) JAOAC 61,299(1978). (23) Chern. Ztg. 16, 1952(1892); JAOAC 6, 391(1923); 15, 267(1932). (24) JAOAC 13,208(1930); 15,267(1932). (25) JAOAC 18,62,218(1935); 19,68,279(1936). (26) JAOAC 53, 808(1970); 56,853(1973). (27) JAOAC 13,215(1930). (28) JAOAC 38, 436(1955); 44, 245(1961). (29) Ind. Eng. Chern., Anal. Ed. 7, 259(1935); JAOAC 41, 637(1958); 42,494(1959). (30) JAOAC 43, 499(1960); 57, 1360(1974); 59, 22(1976); 60, 323(1977). (31) JAOAC 59, 22(1976). (32) JAOAC 18, 237, 260,281(1935); 19,302(1936). (33) Anal. Chern. 21, 984(1949); JAOAC 35, 674(1952); 36, 649(1953). (34) JAOAC 28, 782(1945). (35) J. Agric. Food Chern. 3, 48(1955); JAOAC 41, 533(1958); 51,857(1968). (36) JAOAC 53, 456(1970); 54,646(1971). (37) Anal. Chern. 29, 1044(1957); 30, 1882(1958); JAOAC 41, 533(1958); 43,472(1960). (38) JAOAC 52, 566(1969). (39) JAOAC 48, 406, 1100(1965); 50,401(1967); 51, 847(1968); 58,928(1975). (40) JAOAC 32, 422(1949); 33, 132(1950); 36, 623(1953); 38, 407(1955). (41) JAOAC 38, 413(1955). (42) JAOAC 11,34,201(1928); 16,69(1933). (43) JAOAC 24, 302(1941). (44) Anal. Chern. 30, 1153(1958); JAOAC 48, 412(1965). (45) JAOAC 50, 397(1967). (46) JAOAC 47, 450(1964). (47) JAOAC 20, 252(1937); 22, 270(1939); 23, 249(1940); 24, 268(1941); 25, 326(1942). (48) JAOAC 55, 695(1972). (49) JAOAC 23, 249(1940). (50) JAOAC 24,268(1941). (51) JAOAC 24, 305(1941). (52) JAOAC 25, 77, 352(1942). (53) JAOAC 55,986(1972); 56,859(1973); 57, 1402(1974). (54) JAOAC 25, 77, 361(1942). (55) JAOAC 25, 78(1942). (56) JAOAC 56, 846(1973). (57) JAOAC 47, 436(1964). (58) JAOAC 25, 348(1942). (59) JAOAC 19, 284(1936); 22, 289(1939). (60) Book of ASTM Stds (1971) Pts 11, 22, and 30, ASTM 02973- 02978,02980, and 02944; JAOAC 56,154(1973). (61) JAOAC 50, 394(1967). (62) JAOAC 51,1296(1968); 52, 384(1969). (63) JAOAC 56,154(1973).
- 53. 3. Plants 3.001 Sampling (l)-Official Final Action When more than one plant is sampled, include enough plants in sample to ensure that it adequately represents avo composition of entire lot of plants sampled. (This number depends upon variability in composition of the plants.) Det. details of sampling by purpose for which sample is taken. 3.002 Preparation of Sample (1)-Official Final Action (a) For mineral constituents.-Thoroly remove all foreign matter from material, especially adhering soil or sand, but to prevent leaching, avoid excessive washing. Air- or oven-dryas rapidly as possible to prevent decomposition or wt loss by respiration, grind, and store in tightly stoppered bottles. If results are to be expressed on fresh wt basis, record sample wts before and after drying. When Cu, Mn, Zn, Fe, AI, etc. are to be detd, avoid contaminating sample by dust during drying and from grinding and sieving machinery. (b) For carbohydrates.-Thoroly remove all foreign matter and rapidly grind or chop material into fine pieces. Add weighed sample to hot redistd alcohol to which enough pptd CaCOa has been added to neutze acidity, using enough alcohol so that final concn, allowing for H20 content of sample, is ca 80%. Heat nearly to bp on steam or H20 bath 30 min, stirring frequently. (Samples may be stored until needed for analysis.) 3.003 Moisture-Official Final Action See 7.003,7.006*, or 7.007. 3.004 Ash-Official Final Action See 31.012, 31.013, or 7.009. 3.005 Sand and Silica-Official Final Action Ignite 10-50 g sample in flat-bottom Pt dish in furnace, at 500-550°, until residue is white or nearly so. (Use Pt dishes with caution in ashing plant materials high in Fe; for such materials, use well-glazed porcelain crucibles and include blank detn.) Moisten with 5-10 mL HCI, boil ca 2 min, evap. to dryness, and heat on steam bath 3 hr to render Si02 insol. Moisten residue with 5 mL HCI, boil 2 min, add ca 50 mL H20, heat on H20 bath few min, filter thru hardened paper, and wash thoroly. To this filtrate add filtrate and washings from alkali-sol. Si02 detn (b) and dil. to 200 mL. Designate as Soln I. (a) Sand.-Wash residue from filter into Pt dish and boil ca 5 min with ca 20 mL satd Na2COasoln; add few drops 10% NaOH soln, let settle, and decant thru ignited and weighed gooch. Boil residue in dish with another 20 mL portion Na2COa soln and decant as before. Repeat process. Transfer residue to gooch and wash thoroly, first with hot H20, then with little HCI (1 +4), and finally with hot H20 until CI-free. Dry filter and contents, ignite at 500-550°, and weigh as sand. Confirm by microscopic ex- amination. (b) Alkali-soluble Si02.-Combine alk. filtrate and washings from (a), acidify with HCI, evap. to dryness, add 5 mL HCI, again evap., and dehydrate by heating 2 hr at 110-120°. Moisten residue with 5-10 mL HCI, boil ca 2 min, add ca 50 mL H20, and heat on H20 bath 10-15 min. Filter thru ashless filter or ignited and weighed gooch, wash with hot H20, ignite at 500-550°, and weigh as Si02. Add filtrate to Soln I. 31 METALS Calcium, Copper, Iron, Magnesium, Manganese, Potassium, and Zinc Atomic Absorption Method (2)-Official First Action 3.006 Apparatus and Reagents Deionized H20 may be used. See 2.109-2.110, and following: (a) Potassium stock soln.-l000 j.tg K/mL. Dissolve 1.9068 g dried (2 hr at 105°) KCI in H20 and dil. to 1 L. Use following parameters for Table 2:04: 7665 A, air-C2H2 flame, and 0.04-2 j.tg/mL range. 3.007 Preparation of Sample (a) Dry ashing.-Accurately weigh 1 g sample, dried and ground as in 3.002(a), into glazed, high-form porcelain crucible. Ash 2 hr at 500°, and let cool. Wet ash with 10 drops H20, and carefully add 3-4 mL HNOa (1+1). Evap. excess HNOa on hot plate set at 100-120'. Return crucible to furnace and ash addnl 1 hr at 500°. Cool crucible, dissolve ash in 10 mL HCI (1 + 1), and transfer quant. to 50 mL vol. flask. (b) Wet ashing.-Accurately weigh 1 g sample, dried and ground as in 3.002(a), into 150 mL Pyrex beaker. Add 10 mL HNOa and let soak thoroly. Add 3 mL 60% HCIO. and heat on hot plate, slowly at first, until frothing ceases. (Caution: See 51.019.) Heat until HNOa is almost evapd. If charring occurs, cool, add 10 mL HNOa, and continue heating. Heat to white fumes of HCIO•. Cool, add 10 mL HCI (1+1), and transfer quant. to 50 mL vol. flask. 3.008 Determination To soln in 50 mL vol. flask, add 10 mL 5% La soln, and dil. to vol. Let silica settle, decant supernate, and proceed as in 2.112. 3.009 Calculations ppm Element = (j.tg/mL) x F/g sample, % Element = ppm x 10.4 • where F = (mL original diln x mL final diln)/mL aliquot if original 50 mL is dild. 3.010 Iron and Aluminum (3)-Official Final Action (Caution: See 51.030.) Take aliquot of Soln I, 3.005, contg enough Fe and AI to form ca 40 mg Fe- and AIPO•. Add few drops HNOa, Br-H20, or H20 2 to oxidize Fe. If soln does not already contain excess phosphate, add 0.5 g (NH')2HPO., stir until dissolved, and dil..to 50 mL with H20. Add few drops thymol blue soln, 22.040(el. and then add NH.OH until soln just turns yellow. Add 0.5 mL HCI and 25 mL 25% NH.OAc soln, and stir. Let stand at room temp. until ppt settles (ca 1 hr). Filter, and wash 10 times with hot 5% NH.NOa soln. Ignite at 500-550° and weigh as FePO. and AIPO•. Fuse ignited residue in Pt crucible with ca 4 g Na,COa-K,C03 (1 +1) mixt. When fusion is complete, let crucible cool, add 5 mL H,S04, and heat until copious fumes of SOa are evolved. Cool, transfer to flask, add H2 0, and digest until soln is clear. Reduce Fe with In, cool, and titr. with 0.1N KMnO•. Correct for blank and calc. as % Fe or % Fe,Oa' Calc. to FePO. and subtract from total Fe- and AIPO. to obtain AIPO•. Correct for blank and report as AI,03'
- 54. 32 3. PLANTS AOAC METHODS (1980) Methods for Iron Only Colorimetric Method (4)-Official Final Action 3.011 Reagents (a) Acetic acid.-2M. Dil. 120 g HOAc to 1 L with H20. (b) Ammonium citrate soln.-1%. Dissolve 1 g NH4 citrate in H20 and dil. to 100 mL. (c) Bromophenol blue indicator.-0.04%. Grind 0.1 g bromo- phenol blue in mortar with 3 mL 0.05N NaOH, transfer to vol. flask, and dil. to 250 mL with H20. (d) Buffer solns.-(T) pH 3.5.-Mix 6.4 mL 2M NaOAc with 93.6 mL 2M HOAc and dil. to 1 L. (2) pH 4.5.-Mix 43 mL 2M NaOAc with 57 mL 2M HOAc and dil. to 1 L. (e) Hydroquinone soln.-Dissolve 1 g hydroquinone in 100 mL pH 4.5 buffer, (d)(2). Keep in refrigerator, and discard when any color develops. (f) o-Phenanthroline soln.-Dissolve 1 g o-phenanthroline H20 in H20, warming if necessary, and dil. to 400 mL. (g) Sodium acetate soln.-2M. Dissolve 272 g NaOAc.3H20 in H20 and dil. to 1 L. (h) Iron std soln.-1 mg/mL. Dissolve 1 g electrolytic Fe in 50 mL H2S04(1 +9), warming if necessary to hasten reaction. Cool, and dil. to 1 L with H20. 3.012 Preparation of Sample (Caution: See 51.011, 51.025, and 51.030.) Use Soln I, 3.005, or if Soln I is not available, weigh samples of finely ground plant material (1-5 g, depending on Fe content) into porcelain crucibles with smooth inner surfaces, and ash overnight at 500-550° in furnace. Cool, add 5 mL HCI (1 +1), and heat on steam bath 15 min to dissolve Fe and to hydrolyze pyrophosphate. Filter into 100 mL vol. flask. Transfer insol. residue to filter and wash 5 times with 3 mL portions hot HCI (1 + 100), then with hot H20 until washings are CI-free. Ignite paper and any remaining C in Fe-free Pt crucible. Cool, add 2 drops H2S04and 1 mL HF, and carefully evap. to SO, fumes. Cool, add few drops HCI (1 + 1), and warm. Filter and wash as before into same vol. flask, dil. to vol., and mix. 3.013 Determination Pipet identical aliquots of Soln I, 3.005, or sample soln, 3.012, into 25 mL vol. flask and into test tube or small erlenmeyer. Add 5 drops bromophenol blue indicator to aliquot in test tube, and titr. with 2M NaOAc soln until color matches that of equal vol. of pH 3.5 buffer contg same amt of indicator. Add 1 mL hydroquinone soln and 2 mL o-phenanthroline soln to aliquot in vol. flask, and adjust pH to 3.5 by adding same vol. NaOAc soln found necessary for aliquot in test tube. If turbidity develops upon adjusting pH of aliquot in test tube, add 1 mL NH4 citrate soln to vol. flask before adding the NaOAc soln. Dil. to vol., mix, and let stand 1 hr for complete color development, and measure A at max., ca 510 nm. Prep. curve relating A to mg Fe in 25 mL by treating series of solns contg amts of Fe that cover usable range of instrument exactly as described for unknowns, detg their respective read- ings at max. A, ca 510 nm, and plotting these against corre- sponding concns of Fe. H20 may be used as ref., and blanks detd to correct for amt Fe in reagents used, or blank soln itself may be made basis of comparison. 3.014 Titrimetric Method (5)-Official Final Action Take appropriate aliquot of Soln I or of soln prepd as in 3.012, and oxidize Fe by adding soln of KMn04(1 + 1000) dropwise until very faint permanganate color persists. Add 5 mL 10% NH.SCN and titr. with dil. TiC/asoln until red color disappears. (To prep. appropriate TiCla soln, boil 5-10 mL 20% TiCla with 50 mL HCI few min, cool, and dil. to 1 L. Stdze against std Fe soln, keep in dark in well-filled container, and restdze each time it is used, or every few hr when many detns are being made. Discard when decomposition is indicated by loss of color and increased titer against std.) Calcium-Official Final Action 3.015 Macro Method (6) Transfer aliquot of Soln I, 3.005, to 200 mL beaker, add H20 if necessary to vol. of 50 mL, heat to bp, and add 10 mL satd (NH4)2C204 soln and drop Me red, 2.055(i). Almost neutze with NH40H and boil until ppt is coarsely granular. Cool, add NH40H (1 +4) until color is faint pink (pH 5.0), and let stand ;;.4 hr. Filter, and wash with H20 at room temp. until filtrate is oxalate-free. (Reserve filtrate and washings for Mg detn, 3.017.) Break point of filter with Pt wire, and wash ppt into beaker in which Ca was pptd, using stream of hot H20. Add ca 10 mL H2S04(1 +4), heat to ca 90°, add ca 50 mL hot H20, and titr. with 0.05N KMnO•. Finally add filter paper to soln and complete titrn. 3.016 Micro Method (7) Weigh 2 g sample into small crucible and ignite in furnace at 500-550°. Dissolve ash in HCI (1+4) and transfer to 100 mL beaker. Add 5 mL HCI and evap. to dryness on steam bath to dehydrate Si02• Moisten residue with 5 mL HCI, add ca 50 mL H20, heat few min on steam bath, transfer to 100 mL vol. flask, cool quickly to room temp., dil. to vol., shake, and filter, discarding first portion of filtrate. Pipet 15 mL aliquot into conical-tip centrf. tube contg 2 mL satd (NH.)2C20 4 soln and 2 drops Me red, 2.055(i). Add 2 mL HOAc (1 +4), rotating tube to mix contents thoroly. Add NH.OH (1 +4), while intermittently rotating tube, until soln is faintly alk.; then add few drops of the HOAc until color is faint pink (pH 5.0). (It is important at this point to rotate tube so that last bit of liq. in conical tip has required color.) Let stand ;;.4 hr; then centrf. 15 min. (Ppt should be in firm lump in tip of tube.) Remove supernate, using suction device, Fig. 3:01, taking care not to disturb ppt. Wash ppt by adding 2 mL NH40H (1 +49)' rotating tube to break up ppt. (It may be necessary to jar tube sharply.) Centrf. 10 min, again remove supernate, and wash with reagent as before. Repeat washing of ppt 3 times. After removing last supernate, add 2 mL H2S04(1 +4) to tube, break up ppt as before, heat on steam bath to 80-90°, and titr. FIG. 3:01-Suction device used in micro method for determining calcium
- 55. AOAC METHODS (1980) SODIUM 33 in tube with 0.02N KMnO., rotating liq. during titrn to attain proper end point. If tube cools to <60° during titrn, as indicated by slow reduction of KMnO., reheat in steam bath few min and complete titrn. Perform blank on identical vol. H2SO. in similar tube heated to same temp. to det. vol. KMnO. soln necessary to give end point color. Subtract this value from buret reading. 1 mL 0.02N KMnO. = 0.000400 g Ca. Report as % Ca. 3.017 Magnesium (B)-Official Final Action (Caution: See 51.026.) To combined filtrate and washings from Ca detn, 3.015, add 30 mL HN03and evap. to dryness to decompose NH. salts. Take up with 5 mL HCI and dil. to ca 100 mL with H20. Add 5 mL 10% Na citrate soln and 10 mL 10% (NH')2HPD. soln, or enough to ppt all the Mg. Add NH.OH (1 +4) with const stirring (using policeman) until soln is faintly alk. and ppt forms; then add 25 mL NH.OH, stir vigorously until ppt is granular, and keep in cool place overnight. Filter, and wash CI-free with cold NH.OH (1 +10). Incinerate in furnace at 500--550° until all C is oxidized, then at 900--950° ca 4 hr to form Mg2P20 7. Cool, and weigh as Mg,P,07. (If sample is excessively high in Mn, dissolve residue in HN03, det. Mn as in 3.018, and correct Mg,P207 for Mn2P20 7.) Report as % Mg. 3.018 Manganese (9)-Official Final Action To aliquot of Soln I, 3.005, contg 0.2-0.5 g ash, add 15 mL H2SO. and evap. to ca 30 mL. Add 5--10 mL HN03and continue evapn. (Do not evap. until dense fumes appear, because Fe2(SO')3 then dissolves with difficulty. HN03 may be present, but not Hel.) Add H20, little at time, heat until Fe salts dissolve, and dil. to ca 150 mL. Add 0.3 g KID., or its equiv. in HIO., in small portions, boil few min or until color of KMnO. shows no further increase in intensity, and let cool. Prep. std as follows: To vol. H20 equal to sample add 15 mL H2SO. and enough pure Fe(N03)" free from Mn, to equal approx. amt of Fe in sample. Add measured vol. O.lN KMnO. until color is slightly darker than sample, then add 0.3 g KIO., and boil few min. When cool, transfer sample and std to 250 mL vol. flasks and dil. to vol. with H20. (If color is weak, it may be necessary to dil. to <250 mL.) Measure A with photometer or spectrophtr set at max., ca 530 nm. Report as % Mn. 3.019 * Potassium and Sodium * Official Final Action Ignition, removal of Fe, AI, and Pwith NH.OH, sulfate as 8aSO., Ca as oxalate, NH3 by ignition, and final weighing as NaCI + KCI. See 3.015, 11th ed. Potassium and/or Sodium Flame Photometric Method (10) Official Final Action 3.020 Reagents (a) Potassium stock soln.-l000 ppm K. Dissolve 1.907 g dry KCI in H20 and dil. to 1 L. (b) Sodium stock soln.-l000 ppm Na. Dissolve 2.542 9 dry NaCI in H20 and dil. to 1 L. (c) Lithium stock soln. -1 000 ppm Li. Dissolve 6.108 g LiCI in H20 and dil. to 1 L. (Needed only if internal std method of evaluation is to be used.) (d) Ammonium oxalate stock soln.-0.24N. Dissolve 17.0 g (NH.),C20 •.H20 in H20 and dil. to 1 L. *Surplus method-see inside front cover. (e) Extracting solns.-(1) For potassium.-For internal std method, dil. required vol. LiCI stock soln to 1 L; otherwise use H20. (2) For sodium.-To 250 mL NH. oxalate stock soln add required vol. LiCI stock soln Of internal std method is used) and dil. to 1 L. If internal std requirements are same for both Na and K detns, this reagent may be used as common extg soln. 3.021 Preparation of Standard Solutions Oil. appropriate aliquots of stock solns to prep. series of stds contg K and/or Na in stepped amts (including 0) to cover instrument range, and Li and NH. oxalate (if required) in same concns as in corresponding extg solns. (If common extg soln is used, 1 set of stds contg both K and Na suffices.) 3.022 Sample Extraction Transfer weighed portion of finely ground and well mixed sample to erlenmeyer of at least twice capacity of vol. of extg soln to be used. Add measured vol. extg soln, stopper flask, and shake vigorously at frequent intervals during ;315 min. Filter thru dry, fast paper. If paper clogs, pour contents onto addnl fresh paper and combine filtrates. Use filtrate for detn. Note: Do not make exts more concd than required for instru- ment because there is tendency toward incomplete extn as ratio of sample wt to vol. extg soln increases. Prep. sep. exts for K and Na when their concns in sample differ greatly. For K, use wt sample "';0.1 g/50 mL extg soln; for low Na concns use ;31.0 g/50 mL extg soln; and for higher concns, prep. weaker exts by reducing ratio of sample to extg soln rather than by dilg stronger exts. 3.023 Determination (Caution: See 51.007.) Rinse all glassware used in Na detn with dil. HN03, followed by several portions H20. Protect solns from air-borne Na con- tamination. Operate instrument according to manufacturer's instructions. Permit instrument to reach operating equilibrium before use. Aspirate portions of std solns toward end of warm-up period until reproducible readings for series are obtained. Run stds, covering concn range of samples involved, at frequent intervals within series of sample soln detns. Repeat this operation with both std and sample solns enough times to result in reliable avo reading for each soln. Plot curves from readings of stds, and calc. % K and/ or Na in samples. * Potassium-Official Final Action * 3.024 Platinic Chloride Method See 3.020, 11th ed. 3.025 Perchloric Acid Method (17) See 3.021, 11th ed. 3.026 Rapid Method for Potassium Only See 3.022, 11th ed. Sodium Only Uranyl Acetate Method (12)-Official Final Action 3.027 Reagent Magnesium uranyl acetate soln: (a) Uranyl acetate soln.-To 85 g U02(OAc)2 .2H20 in 1 L vol. flask add 60 g HOAc and H20 to ca 900 mL. Heat to dissolve, cool, and dil. to vol. with H20. (Caution: See 51.083.)
- 56. 34 3. PLANTS AOAC METHODS (1980) (b) Magnesium acetate soln.-To 500 g Mg(OAc)2 .4H20 in 1 L vol. flask add 60 g HOAc and H20 to ca 900 mL. Heat to dissolve, cool, and dil. to vol. with H20. Reheat (a) and (b) sep. to ca 70° until all salts dissolve. Mix the solns at this temp. and let cool to ca 30°. Place vessel contg mixed reagent in H20 bath at 20°, and hold 1-2 hr at 20°, or until slight excess of salts has crystd out. Filter thru dry filter into dry bottle. 3.028 Determination Moisten 1-10 g sample with H2SO. (1+10). dry in oven, and ignite in furnace at 500-550° to destroy org. matter. Heat residue on steam bath with 2-5 mL HCI, add ca 40 mL H20, and heat to bp. Add enough 5% CaC/2 soln to ensure pptn of all phosphates. Ppt phosphates by making slightly alk. with NH.OH. Filter, and evap. to ,,;5 mL if no salts sep. Cool, add 100 mL Mg uranyl acetate soln, place mixt. in H20 bath at 20°, and either stir vigorously 45 min or let stand 24 hr at this temp. Filter with suction, and wash with alcohol satd with Na-Mg-uranyl acetate. Dry 30 min at 105-110°, cool, and weigh. Wt Na-Mg-uranyl acetate x 0.0153 =wt Na. Cobalt-Official Final Action (Caution: See 51.011, 51.040, 51.049, and 51.068.) Nitrosocresol Method (13) 3.029 Reagents (Make all distns in Pyrex stills with $' joints. Store reagents in g-s Pyrex bottles.) (a) Redistilled water.-Distil twice, or pass thru column of ion exchange resin (iR-l00A, H-form, or equiv.) to remove heavy metals. (b) Hydrofluoric acid.-48%. Procurement in vinyl plastic bottles is advantageous. (c) Perchloric acid.-60%. No further purification necessary. (d) Hydrochloric acid.-( 1+1). Add equal vol. HCI to distd H20 and distil. (e) Ammonium hydroxide.-(l +1). Distil coned NH.OH into equal vol. redistd H20. (f) Ammonium hydroxide.-O.02N. Add 7 mL of the NH.OH (1+ 1) to 2.5 L redistd H20. (9) Carbon tetrachloride.-Distil over CaO, passing distillate thru dry, acid-washed filter paper. Used CCI. may be recovered as in 3.044(a). (h) Dithizone.-Dissolve 0.5 g dithizone in 600-700 mL CCI. (tech. grade is satisfactory). Filter into 5 L separator contg 2.5- 3.0 L 0.02N NH.OH, shake well, and discard CCI. layer. Shake with 50 mL portions redistd CCI. until CCI. phase as it seps is pure green. Add 1 L redistd CCI. and acidify slightly with the HCI (1 +1). Shake the dithizone into CCI. layer and discard aq. layer. Store in cool, dark place, preferably in refrigerator. (i) Ammonium citrate soln.-40%. Dissolve 800 g citric acid in 600 mL distd H20, and, while stirring, slowly add 900 mL NH.OH. Reaction is exothermic; take care to prevent spattering. Adjust pH to 8.5, if necessary. Oil. to 2 L and ext with 25 mL portions dithizone soln until aq. phase stays orange and CCI. remains predominantly green. Then ext soln with CCI. until all orange is removed. (j) Hydrochloric acid.-O.1N. Oil. 16.6 mL of the HCI (1+1) to 1 L with redistd H20. (k) Hydrochloric acid.-O.01N. Dil. 100 mL of the O.lN HCI to 1 L with redistd H20. (I) Sodium hydroxide soln.-1N. Dissolve 40 g NaOH in 1 L redistd H20. (m) Borate buffer.-pH 7.8. Dissolve 20 g H3B03 in 1 L redistd H20. Add 50 mL IN NaOH and adjust pH, if necessary. Equal vols borate buffer and O.OlN HCI should give soln of pH 7.9. (n) Borate buffer.-pH 9.1. To 1 L borate buffer, pH 7.8, add 120 mL IN NaOH and adjust pH, if necessary. (0) Skellysolve B.-Essentially n-hexane. Purify by adding 20-30 g silica gel/L, let stand several days, and distil. Available from Getty Refining and Marketing Co., PO Box 1650, Tulsa, OK 74102. (p) Cupric acetate soln.-Dissolve 10 g Cu(OAc)2.H20 in 1 L redistd H20. (q) o-Nitrosocresol soln.-Dissolve 8.4 g anhyd. CuCI2and 8.4 g NH20H.HCI in 900 mL H20. Add 8 mL m-cresol (Eastman Ko- dak Co., practical grade) and stir vigorously while slowly adding 24 mL 30% H20 2. Stir mech. 2 hr at room temp. (Standing for longer periods results in excessive decomposition.) Add 25 mL HCI and exto-nitrosocresol with four 150 mL portions Skellysolve B, (0). in large separator. Then add addnl 25 mL HCI and again ext with four 150 mL portions Skellysolve B. Wash combined Skellysolve B exts twice with 50-100 mL portions O.lN HCI and twice with 50-100 mL portions redistd H20. Shake o-nitrosocre- sol soln with successive 50-100 mL portions 1% Cu(OAc)2 soln until aq. phase is no longer deep blood-red. When light purple is evident, extn is complete. Discard Skellysolve B phase, acidify aq. soln of Cu salt with 25 mL HCI, and ext reagent with two 500 mL portions Skellysolve B; wash twice with 150-200 mL portions O.lN HCI and several times with 150-200 mL portions redistd H20. Store o-nitrosocresol soln in refrigerator at ca 4°. Reagent is stable ",6 months. (r) Sodium o-nitrosocresol soln.-Ext 100 mL o-nitrosocresol by shaking with two 50 mL portions borate buffer, pH 9.1, in separator. (If this is carried out as 2 extns, resulting reagent is more coned. It is important that total vol. o-nitrosocresol soln equal total vol. buffer.) (5) Cobalt std solns.-(1) Stock soln.-Heat CoSO•.7H20 in oven at 250-300° to const wt (6-8 hr). Weigh exactly 0.263 g of the CoSO. and dissolve in 50 mL redistd H20 and 1 mL H2SO•. Oil. to 1 L. (2) Working soln.-0.5 /Lg/mL. Transfer 5 mL stock soln to 1 L vol. flask and dil. to vol. with redistd H20. (t) Hydroxylamine acetate buffer.-pH 5.1 ±0.1. Dissolve 10 g NH20H.HCI and 9.5 g anhyd. NaOAc in 500 mL redistd H20. 3.030 Apparatus (a) Platinum dishes.-Approx. 70 mL; for ashing. (b) Automatic dispensing burets.-l00 mL; type that can be fitted to ordinary 5 Ib reagent bottle and filled by means of aspirator bulb is most convenient. (c) Wooden separator rack.-Twelve-unit 125 mL separator size is convenient for dithizone extns. Rack is fitted across top with removable bar padded with sponge rubber so all 12 separators can be shaken as unit. (d) Racks.-Consisting of 5 x 5 x 65 cm (2 x 2 x 25") wooden bars with holes drilled at close intervals to take 50 mL centrf. tubes fitted with No. 13 $' glass stoppers. To make these tubes, ream out necks of heavy-wall Pyrex centrf. tubes (Rockefeller Institute type) with $' C rod and grind to take $' stopper. Place tl'bes upright in one section, and place other section (fitted with sponge rubber disks 13 mm thick in bottom of holes) across their tops. Fasten 2 sections at ends with removable rubber connectors made from ordinary tubing of convenient size, so that any number of tubes can be shaken as unit. Use these tubes for reaction of Co with nitrosocresol, extn of complex into Skellysolve B, and washing of Skellysolve B soln. (e) Shaking machine.-Mech. shaker giving longitudinal stroke of 5 em at ca 180 strokes/min; use to make dithizone extns and to ext Co complex, or shake by hand.
- 57. AOAC METHODS (1980) COBALT 3.031 Cleaning of Glassware Clean 120 mL Pyrex separators for dithizone extns by initially soaking 30 min in hot HN03 and rinsing several times with H20. As added precaution, shake with several portions dithizone in CCI•. After use, clean by rinsing with H20, drain, and stopper to avoid contamination. It is not necessary to clean every time with acid. Repeat HN03 cleaning if blanks are unusually high. Clean 50 mL g-s Pyrex centrf. tubes by soaking 30 min in HN03 followed by several rinsings in H20. Completely submerge pipets in cylinder of chromic acid cleaning soln overnight, rinse several times with H2 0, and suspend upright in rack to dry. Wash all other glassware thoroly in detergent and rinse well with tap H20 followed by dip in chromic acid cleaning soln. Rinse off cleaning soln with tap H20 followed by several distd H20 rinses. Clean Pt by scrubbing with sea sand followed by boiling in HCI (1 +2) 30 min, and rinse several times with H20. 3.032 Preparation of Sample See 3.002(a}. Oven-dry all plant material 48 hr and prep. for ashing by either of following methods: (8) Grind material in Wiley mill equipped with stainless steel sieve, mix thoroly by rolling, and sample by quartering. (b) Using stainless steel shears, cut material by hand fine enough for convenient sampling. 3.033 Ashing of Samples (Caution: See 51.011, 51.025, and 51.028.) Weigh 6 g dry plant tissue into clean Pt dish. Cover with Pyrex watch glass and place in cool furnace; heat slowly to 5000 and hold at this temp. overnight. Remove sample and cool. Wet down ash carefully with fine stream redistd H20. From dis- pensing buret, slowly add 2-5 mL HCIO., dropwise at first to prevent spattering. Add ca 5 mL HF, evap. on steam bath, transfer to sand bath, and keep at medium heat until fuming ceases. Cover with Pyrex watch glass, return to partially cooled furnace, heat gradually to 600°, and keep at this temp. 1 hr. Remove sample and cool. Add 5 mL HCI (1+1) and ca 10 mL redistd H20. Replace cover glass and warm on steam bath to dissolve. (Usually clear soln essentially free of insol. material is obtained.) Transfer sample to 50 mL vol. flask, washing dish several times with redistd H20, dil. to vol., and mix thoroly. (Pt dishes can ordinarily be used several times between sand and acid cleanings.) 3.034 Dithizone Extraction (Caution: See 51.011(b). 51.028(a) and (d), and 51.049.) Transfer suitable aliquot (2-3 g dry material) to 120 mL separator (use petroleum jelly as stopcock lubricant). Add 5 mL NH. citrate soln and 1 drop phthln; adjust to pH 8.5 with NH.OH (1 +1). If ppt forms, add addnl NH. citrate. Add 10 mL dithizone in CCI. and shake 5 min. Drain CCI. phase into 100 mL beaker. Repeat as many times as necessary, using 5 mL dithizone soln and shaking 5 min each time. Extn is complete when aq. phase remains orange and CCI. phase remains predominantly green. Then add 10 mL CCI., shake 5 min, and combine with CCI. ext. Final 10 mL CCI. should be pure green. If not, extn was incomplete and must be repeated. Add 2 mL HCIO. to combined CCI. exts, cover beaker with Pyrex watch glass, and digest on hot plate until colorless. Remove cover glass and evap. slowly to dryness. (If sample is heated any length of time at high temp. when dry, losses of Co 35 may occur. Heat only enough to evap. completely to dryness. If free acid remains, it interferes with next step where pH control is important.) Add 5 mL 0.01N HCI to residue. Heat slightly to assure soln. If Cu is to be detd, transfer with redistd H20 to 25 mL vol. flask, and dil. to vol. Transfer 20 mL aliquot to 50 mL g-s centrf. tube or 60 mL separator and reserve remainder for Cu detn, 3.043. If Cu is not to be detd, transfer entire acid soln with redistd H20 to centrf. tube or separator. 3.035 Determination Add 5 mL borate buffer, pH 7.8, and 2 mL freshly prepd Na o-nitrosocresol soln to sample soln. Add exactly 5 mL Skellysolve B and shake 10 min. Remove aq. phase by moderate suction thru finely-drawn glass tube. To Skellysolve B layer add 5 mL Cu(OAc}, soln and shake 1 min to remove excess reagent. Again remove and discard aq. phase. Wash Skellysolve B by shaking 1 min with 5 mL redistd H20, removing aq. layer as before; finally shake Skellysolve B 1 min with 5 mL NH20H-NaOAc buffer to reduce Fe. Transfer Skellysolve B soln of the Co complex to 5 cm cell and read in spectrophtr as close as possible to point of max. A, 360 nm. 3.036 Blanks and Standards With each set of detns include ashing blank and Co stds of 0.0, 0.5, and 1.0 fLg. Beer's law holds for this range. A of 0.0 fLg point should be <0.05. If above, repurify o-nitrosocresol by transferring alternately to aq. phase as Cu salt and to Skellysolve B phase as free compd after acidifying aq. phase. It is also advisable to include std sample with each set of samples to detect contamination or unusual losses of Co in method. Com. buckwheat flour contg 0.05 ppm Co has proved satisfactory for this purpose. 3.037 Calculations Express results in terms of ppm Co, based upon dry wt of sample. ppm Co = (fL9 Co/mL dithizone aliquot) x (mL total soln/g dry sample) Value for fLg Co is obtained from curve minus ashing blank. Nitroso-R-Salt Method (14) 3.038 Reagents Those listed in 3.029 and following: (a) Nitroso-R-salt soln.--o.2%. Dissolve 2 9 powd nitroso-R- salt (Eastman Kodak Co., No. 1124) in redistd H2 0, 3.029(a), and dil. to 1 L. (b) Dilute nitric acid.-(1 +1). Dil. HN03 with equal vol. H20 and redistil in Pyrex app. Store in Pyrex bottles. (e) Bromine water.-Satd soln of Br in redistd H20, 3.029(a). (d) Citric acid soln.--o.2N. Use special reagent grade Pb-free citric acid. 3.039 Preparation and Ashing ofSamples Proceed as in nitrosocresol method, 3.032-3.033, thru "(Usu- ally clear soln essentially free of insol. material is obtained.)" except use 10 9 instead of 6 9 dry plant tissue. 3.040 Dithizone Extraction Transfer entire soln to 120 mL separator, and proceed as in 3.034, thru "If free acid remains ... pH control is important.)" Dissolve in 1 mL citric acid soln, (d). transfer to 25 mL vol. flask, and dil. to vol. with redistd H20, 3.029(a).
- 58. 36 3. PLANTS AOAC METHOOS (1980) 3.041 Determination Transfer suitable aliquot (ca 8 g dry material) of citric acid soln, 3.040, to 50 mL beaker. Evap. to 1-2 mL. Add 3 mL borate buffer, 3.029(n), and adjust pH to 8.0-8.5 with NaOH (check externally with phenol red). (Vol. .,;5 mL.) Add 1 mL nitroso-R- salt soln slowly with mixing. Boil 1-2 min and add 2 mL dil. HN03. Boil 1-2 min, add 0.5-1.0 mL Br-H2 0, cover with watch glass, and let stand warm 5 min. Boil 2-3 min to remove excess Br (use effective fume removal device). Cool, and dil. to 10 or 25 mL (depending on length of light path in absorption cell). Transfer to cell and read at 500 nm within 1 hr. Prep. stds contg 0.5, 1, 2, 3, and 4/kg Co and add 1 mL citric acid soln, 3.038(d), to each. Proceed as for unknowns, beginning "Evap. to 1-2 mL." Copper (14)-Official Final Action 3.042 Reagents Those listed in 3.029 and following: (a) Sodium diethyldithiocarbamate soln.-O.l %. Freshly prepd in redistd H20, 3.029(a). (b) Copper std soln.-l 09/mL. Dissolve 0.3929 g CuSO•.5H20 in redistd H20, 3.029(a). add 5 mL H2SO., dil. to 1 L, and mix. Take 10 mL aliquot, add 5 mL H2SO., dil to 1 L, and mix. 3.043 Determination Transfer aliquot (0.5-1 g dry material) from soln obtained from 3.034 or 3.040 to 125 mL separator. Add 2 mL NH4 citrate soln, 1 drop phthln, 5 mL Na diethyldithiocarbamate soln, and NH.OH (1+1). 3.029(e). until pink. Add 10 mL CCI. and shake 5 min. Drain CCI., centrf. 5 min, transfer to absorption cell, and read at max. A, ca 430 nm. Prep. std curve with 0, 1, 5, 10, 15, and 20 0g Cu treated as above. Zinc-Official Final Action Mixed Color Method (15) 3.044 Reagents (Redistil all H20 from Pyrex. Treat all glassware with HN03 (1 +1) or fresh chromic acid cleaning soln. Rinse repeatedly with ordinary distd H20 and finally with In-free H20.) (a) Carbon tetrachloride.-Use ACS grade without purifica- tion. If tech. grade is used, dry with anhyd. CaCI2 and redistil in presence of small amt CaO. (Used CCI. may be reclaimed by distn in presence of NaOH (1 +100) contg small amts of Na2S203' followed by drying with anhyd. CaCI2 and fractional distn in presence of small amts of CaO.) (Caution: See 51.011(b) and 51.049.) (b) Zinc std solns.-(1) Stock soln.-l mg/mL. Place 0.25 g pure In in 250 mL vol. flask. Add ca 50 mL H20 and 1 mL H2SO.; heat on steam bath until all Zn dissolves. Oil. to vol. and store in Pyrex vessel. (2) Working soln.-l0 09/mL. Oil. 10 mL stock soln to 1 L. Store in Pyrex vessel. (c) Ammonium hydroxide soln.-1N. With all-Pyrex app. distil NH.OH into H2 0, stopping distn when half has distd. Oil. distillate to proper concn. Store in g-s Pyrex vessel. (d) Hydrochloric acid.-1N. Displace HCI gas from HCI in glass flask by slowly adding equal vol. H2SO. from dropping funnel that extends below surface of the HCI. Conduct displaced HCI gas thru delivery tube to surface of H20 in receiving flask (no heat is necessary). Oil. to proper concn. Use of 150 mL each of HCI and H2SO. will yield 1 L purified HCI soln of concn >IN. (e) Diphenylthiocarbazone (dithizone) soln.- Dissolve 0.20 g dithizone in 500 mL CCI., and filter to remove insol. matter. Place soln in g-s bottle or large separator, add 2 L 0.02N NH.OH (40 mL IN NH.OH dild to 2 L). and shake to ext dithizone into aq. phase. Sep. phases, discard CCI., and ext ammoniacal soln of dithizone with 100 mL portions CCI. until CCI. ext is pure green. Discard CCI. after each extn. Add 500 mL CCI. and 45 mL IN HCI, and shake to ext dithizone into CCI•. Sep. phases and discard aq. phase. Oil. CCI. soln of dithizone to 2 L with CCI•. Store in brown bottle in dark, cool place. (f) Ammonium citrate soln.-O.5M. Dissolve 226 g (NH.).HC6H50 7 in 2 L H20. Add NH.OH (80-85 mL) to pH of 8.5-8.7. Add excess dithizone soln (aq. phase is orange-yellow after phases have been shaken and sepd). and ext with 100 mL portions CCI. until ext is full green. Add more dithizone if necessary. Sep. aq. phase from CCI. and store in Pyrex vessel. (g) Carbamate so/n.-Dissolve 0.25 g Na diethyldithiocarba- mate in H20 and dil. to 100 mL with H20. Store in refrigerator in Pyrex bottle. Prep. fresh after 2 weeks. (h) Dilute hydrochloric acid.-O.02N. Oil. 100 mL IN HCI to 5L. 3.045 Preparation of Solutions To reduce measuring out reagents and minimize errors due to variations in composition, prep. 3 solns in appropriate amts from reagents and store in Pyrex vessels, taking care to avoid loss of NH3 from Solns 1 and 2. Discard solns after 6-8 weeks because Zn increases slowly with storage. Det. std curve for each new set of reagents. Following amts of Solns 1 and 2 and 2 L dithizone soln are enough for 100 detns: (1) Soln 1.-Dil. 1 L 0.5M NH. citrate and 140 mL IN NH.OH to 4 L. (2) Soln 2.-Dil. 1 L 0.5M NH. citrate and 300 mL IN NH.OH to 4.5 L. Just before using, add 1 vol. carbamate soln to 9 vols NH3-NH. citrate soln to obtain vol. ofSoln 2 immediately required. Note: If Zn-free reagents have been prepd, they can be used to test chemicals for In. Certain lots of NH.OH and HCI are sufficiently free of Zn to be used without purification. 3.046 Ashing Ash 5 g finely ground, air-dried plant material in Pt dish in furnace at 500-550°. Include blank detn. Moisten ash with little H20; then add 10 mL IN HCI (more if necessary) and heat on steam bath until all substances sol. in HCI are dissolved. Add 5-10 mL hot H20. Filter off insol. matter on 7 cm paper (Whatman No. 42, or equiv., previously washed with two 5 mL portions hot IN HCI, then washed with hot H2 0 until HCI-free). and collect filtrate in 100 mL vol. flask. Wash filter with hot H20 until washings are not acid to Me red. Add 1 drop Me red, 2.055(i), to filtrate in 100 mL flask; neutze with IN NH.OH and add 4 mL IN HCI. Cool, and dil. to vol. with H20. 3.047 First Extraction (Sepn of dithizone complex-forming metals from ash soln) Pipet aliquot of ash soln contg .,;30 0g Zn into 125 mL Squibb separator. Add 1 mL 0.2N HCI for each 5 mL ash soln <10 mL taken, or 1 mL 0.2N NH.OH for each 5 mL >10 mL taken. (10 mL aliquot is usually satisfactory in analysis of plant materials.) Add 40 mL Soln 1 and 10 mL dithizone reagent. Shake vigorously 30 sec to ext from aq. phase the Zn and other dithizone complex- forming metals that may be present; then let layers sep. At this point excess dithizone (indicated by orange or yellow-orange aq. phase) must be present. If excess dithizone is not present, add more reagent until, after shaking, excess is indicated. Shake down the drop of eCI. ext from surface, and drain eCI. ext into second separator as completely as possible without letting any aq. layer enter stopcock bore. Rinse down eel. ext from surface
- 59. AOAC METHODS (1980) MOLYBDENUM 37 of aq. layer with 1-2 mL clear CCI.; then drain this CCI. into second separator without letting aq. phase enter stopcock bore. Repeat rinsing process as often as necessary to flush ext completely into second separator. Add 5 mL clear CCI. to first separator, shake 30 sec, and let layers sep. (CCI. layer at this point will appear clear green if metals that form dithizone complexes have been completely extd from aq. phase by previous extn.) Drain CCI.layer into second separator and flush ext down from surface and out of separator as directed previ- ously. If last ext does not possess distinct clear color, repeat extn with 5 mL clear CCI. and flushing-out process until complete extn of dithizone complex-forming metals is assured; then discard aq. phase. 3.048 Second Extraction (Sepn of Cu by extn of Zn into 0.02N HCI) Pipet 50 mL 0.02N HCI into separator contg CCI. soln of metal dithizonates. Shake vigorously 1.5 min, and let layers sep. Shake down drop from surface of aq. phase, and as completely as possible drain CCI. phase contg all Cu as dithizonate, without letting any aq. phase, which contains all the Zn, enter stopcock bore. Rinse down CCI. ext from surface of aq. phase, and rinse out stopcock bore with 1-2 mL portions clear CCI. (same as in first extn) until all traces of green dithizone have been washed out of separator. Shake down drop of CCI. from surface of aq. phase, and drain CCI. as completely as possible without letting any aq. phase enter stopcock bore. Remove stopper from separator and lay it across neck until small amt of CCI. on surface of aq. phase evaps. 3.049 Final Extraction (Extn of Zn in presence of carbamate reagent) Pipet 50 mL Soln 2 and 10 mL dithizone soln into 50 mL 0.02N HCI soln contg the Zn. Shake 1 min and let phases sep. Flush out stopcock and stem of separator with ca 1 mL CCI. ext; then collect remainder in test tube. Pipet 5 mL ext into 25 mL vol. flask, dil. to vol. with clear CCI., and measure A with spectrophtr set at absorption max., ca 525 nm. (Caution: Protect final ext from sunlight as much as possible and read within 2 hr.) Det. Zn present in aliquot from curve relating A and concn, correct for Zn in blank, and calc. % Zn in sample. 3.050 Standard Curve Place 0, 5, 10, 15, 20, 25, 30, and 35 mL Zn working std soln in 100 mL vol. flasks. To each flask add 1 drop Me red and neutze with IN NH.OH; then add 4 mL IN HCI and dil. to vol. Proceed exactly as for ash solns, beginning with first extn, and using 10 mL aliquots of each of the Zn solns (0,5, 10, 15,20,25, 30, and 35 f.Lg In, resp.). Construct std curve by plotting f.Lg In againstA. Single Color Method (16) 3.051 Reagents See 3.044-3.045 plus following: (a) Dilute dithizone soln.-Dil. 1 vol. dithizone soln, 3.044(e), with 4 vols CCI•. (b) Carbamate soln.-Dissolve 1.25 g Na diethyldithiocarba- mate in H20 and dil. to 1 L. Store in refrigerator and prep. fresh after long periods of storage. (c) Dilute ammonium hydroxide.-Dil. 20 mL IN NH.OH, 3.044(c), to 2 L. 3.052 Ashing Weigh 2 g sample finely ground plant material into well- glazed porcelain, Vycor, or Pt crucible, include crucible for blank detn, and heat in furnace at 500-550° until ashing is complete. Cool, moisten ash with little H20, add 10 mL IN HCI (more if necessary to ensure excess of acid), and heat on steam bath until all sol. material dissolves. Add few mL hot H20 and filter thru quant. paper into 200 mL vol. flask. Wash paper with hot H20 until washings are not acid to Me red. Add 2 drops Me red soln to filtrate, neutze with IN NH.OH, add exactly 3.2 mL IN HCI, dil. to vol. with H2 0, and mix. 3.053 Formation of Zinc Dithizonate (Removal of interferences and sepn of excess dithizone) Pipet aliquot of ash soln contg ~ 15 f.Lg In into 125 mL amber glass separator. (25 mL aliquot is usually satisfactory.) If nec- essary to use different vol., add 0.4 mL 0.2N HCI for each 5 mL less, or 0.4 mL 0.2N NH.OH for each 5 mL more, than 25 mL taken. If <25 mL of the soln is taken, add H20 to 25 mL. Add 10 mL dithizone reagent, 3.044(e), to aliquot in separator and shake vigorously 1 min. Let layers sep. and discard CCI. layer. Add 2 mL CCI. to aq. soln, let layers sep., and discard CCI•. Repeat this rinsing once. Then add 5 mL CCI., shake vigorously 15 sec, let layers sep., and discard CCI•. Rinse once more with 2 mL CCI. as above. Discard CCI. layer and let CCI. remaining on surface of soln in funnel evap. before proceeding. Add 40 mL NH. citrate Soln 1, 3.045(1), 5 mL carbamate soln, 3.051(b), and 25 mL dil. dithizone reagent, 3.051(a). Accurately add carbamate and dithizone reagents from pipet or buret. Shake vigorously 1 min. Let layers sep. and draw off aq. layer thru fine tip glass tube connected to aspirator with rubber tubing. To remove excess dithizone from CCI. layer, add 50 mL O.OlN NH.OH and shake vigorously 30 sec. 3.054 Determination Dry funnel stem with pipestem cleaner and flush out with ca 2 mL of the Zn dithizonate soln. Collect adequate portion of remaining soln in 25 mL erlenmeyer, or other suitable container, and stopper tightly. (Amber glass containers are convenient, but colorless glassware will suffice if solns are kept in dark until A readings are made.) Measure A of each soln against CCI. with spectrophtr set at absorption max., ca 535 nm. Correct for Zn in blank detns. Calc. amt In present in soln from curve relating concn and A. 3.055 Standard Curve Into 200 mL vol. flasks place 0, 2, 4, 6, 8, 10, 12, and 14 mL, resp., Zn working std soln. To each flask add 2 drops Me red soln, neutze with IN NH.OH, add 3.2 mL IN HCI, and dil. to vol. with H20. Pipet 25 mL aliquots of each of these solns, contg 0, 2.5,5,7.5, 10, 12.5, 15, and 17.5 f.Lg Zn, resp., into amber glass separators, and proceed as for ash solns, 3.053, beginning with second par. Det. A of each soln and plot values against corre- sponding amts In. Molybdenum (17)-Official Final Action 3.056 Apparatus Photoelectric colorimeter or spectrophotometer.- Capable of isolating band at ca 465 nm. (Photometer equipped with filter with max. Tat 440-460 nm and 1 cm cells of 10 mL capacity is suitable.)
- 60. 38 3. PLANTS AOAC METHODS (1980) 3.057 Reagents (a) Isoamyl alcohol.-Reagent grade 3-methyl-1-butanol, bp 128-132°. (b) Dilute hydrochloric acid.-(T) 20% soln.-Dil. concd HCI to ca 20% HCI (1+1.85). (2) 6N soln.-Stdze to second decimal place. (e) Iron std soln.-100 ,..,g/mL. Dissolve 0.7022 g Fe(NH')2(SO')2.6H20 in H20, add 1 mL H2SO., and dil. to 1 L. (d) Molybdenum std solns.-( T) Stock soln.-100 ,..,g/mL. Dis- solve 0.0920 g (NH.)6M0702•.4H20 in H20 and dil. to 500 mL. (2) Working soln.-5 ,..,g/mL. Oil. 25 mL stock soln to 500 mL. (e) Potassium thiocyanate soln.-20%. Dissolve 50 g KSCN in H20 and dil. to 250 mL. (f) Sodium fluoride saturated soln.-Add 200 mL H20 to ca 10 g NaF. Stir until satd and filter. (9) Stannous chloride solns.-(T) 20% soln.- Weigh 10 g SnCI2.2H20 into beaker, add 10 mL 20% HCI, (b)(T), and heat until completely dissolved. Cool, add granule of metallic Sn, dil. to 50 mL with H20, and store in g-s bottle. (2) 0.8% wash soln.- Oil. 4 mL 20% soln to 100 mL with H20. 3.058 Determination (Caution: See 51.019, 51.026, and 51.028.) Weigh 1-5 g finely ground sample, contg ,,;;35 ,..,g Mo, into 200 mL tall-form Pyrex beaker. To 1, 2, or 5 g samples add 10, 15, or 35 mL HN03, resp. Include 2 beakers for blanks. Cover beaker with cover glass, and let stand ca 15 min; then heat cautiously on steam bath or hot plate at ca 100°, avoiding frothing overtop.lffroth approaches cover glass, remove beaker from heat until frothing subsides; then continue heating. Digest, usually ca 2 hr, until most of solids disappear. Cool to room temp. If contents should go to dryness, add few mL HN03• Add 6 mL 70-72% HCIO., cover beaker, place on hot plate, and gradually raise temp. so that contents boil vigorously but do not bump. Continue heating until digestion is complete as indicated by liq. becoming colorless or pale yellow. If nec- essary, make repeated addns of HN03 and HCIO. and continue to digest until C is completely oxidized. After digestion is complete, place cover glass slightly to one side of top of beaker, or replace it with elevated watch glass, and evap. just to dryness or until residue appears only slightly moist. Remove beaker from hot plate, and cool. Wash down sides of beaker and underside of cover glass with few mL H20, return to hot plate, and boil few min. Remove from hot plate, cool, and again rinse sides of beaker and cover glass with small amt H20. Add 2 drops Me orange and neutze with NH.OH. Add 6N HCI, dropwise with stirring, until soln is just acid; then add 8.2 mL excess to give final concn of ca 3% HCI. Add 2 mL satd NaF soln, and 1 mL Fe soln, if sample contains <100,..,g Fe. Transfer soln to 125 mL separator and dil. to 50 mL with H20. Add 4 mL 20% KSCN soln, mix thoroly, and add 1.5 mL 20% SnCI2 soln. Mix again, and from buret or pipet, add exactly 15 mL isoamyl alcohol. Stopper separator and shake vigorously 1 min, let phases sep., and drain and discard aq. layer. Ext into alcohol without delay, since colored complex is somewhat unstable in aq. soln. Add 25 mL freshly prepd 0.8% SnCI2 wash soln, and shake gently 15 sec. Let phases sep., and drain and discard aq. layer. Transfer isoamyl alcohol soln to centrf. tube, and centrf. 5 min at ca 2000 rpm to remove H20 droplets. If alcohol layer is not clear, recentrf. Stopper tubes to prevent evapn, if A readings cannot be made immediately. Compare unknown solns with isoamyl alcohol at ca 465 nm in spectrophtr, and make appropriate corrections in A readings for Mo in blanks. Obtain Mo concn from calibration curve relating A readings to concns of series of solns of known Mo content. Prep. calibration curve for instrument used, as follows: Place aliquots of working std soln contg 0, 5, 10, 15, 20, 25, 30, and 35 ,..,g Mo, resp., into 200 mL tall-form beakers and carry them thru entire detn, beginning with digestion with HN03 and HCIO•. Plot A against corresponding Mo concns. NONMETALS Arsenic-Offieial Final Action 3.059 Preparation of Solution See 25.008. 3.060 Determination Proceed as in 25.009, or take aliquot and det. as in 6.013, beginning " ... add 3 mL H2SO., ..." Sulfur-Offieial Final Action Sodium Peroxide Method (18) (Caution: See 51.035.) 3.061 Preparation of Solution Place 1.5-2.5 g sample in ca 100 mL Ni crucible and add 5 g anhyd. Na2C03• Mix thoroly, using Ni or Pt rod, and moisten with ca 2 mL H20. Add Na20 2, ca 0.5 g at time, mixing thoroly after each addn, and continue until mixt. becomes nearly dry and quite granular (ca 5 g Na20 2). Place crucible over S-free flame or elec. hot plate and heat carefully, stirring occasionally, until contents are fused. (If material ignites, detn is worthless.) After fusion, remove crucible, let cool somewhat, and cover hardened mass with more Na20 2 to depth of ca 5 mm. Heat gradually and finally with full flame until fusion again takes place, rotating crucible occasionally to bring any particles ad- hering to sides into contact with oxidizing material. Continue heating 10 min after fusion is complete. Cool somewhat, place warm crucible and contents in 600 mL beaker, and carefully add ca 100 mL H20. After initial violent action ceases, wash material out of crucible, make slightly acid with HCI (adding small portions at time), transfer to 500 mL vol. flask, cool, dil. to vol., and filter. 3.062 Determination Oil. aliquot of prepd soln to ca 200 mL with H20 and add HCI until ca 0.5 mL free acid is present. Heat to bp and add 10 mL 10% BaCI2soIn dropwise with constant stirring. Continue boiling ca 5 min, and let stand ,,"5 hr in warm place. Decant thru ashless paper or ignited and weighed gooch. Add 15-20 mL boiling H20 to ppt, transfer to filter, and wash with boiling H20 until filtrate is CI-free. Dry ppt and filter, ignite, and weigh as BaSO•. Wt ppt x 0.1374 = S. Magnesium Nitrate Method (19) 3.063 Preparation of Solution Weigh 1 g sample into large porcelain crucible. Add 7.5 mL Mg(N031. soln, 2.019, so that all material comes in contact with soln. (It is important that enough Mg(N03)2 soln be added to ensure complete oxidn and fixation of the S present. For larger samples and for samples with high S content, proportionally larger vol. of this soln must be used.) Heat on elec. hot plate
- 61. AOAC METHODS (1980) CHLORINE 39 (180°) until no further action occurs. Transfer crucible while hot to furnace (";;500°) and let it remain until sample is thoroly oxidized. (No black particles should remain. If necessary, break up sample and return to furnace.) Remove crucible and let cool. Add H20; then HCI in excess. Bring soln to boil. filter, and wash thoroly. If preferred, transfer soln to 250 mL vol. flask before filtering and dil. to vol. with H20. 3.064 Determination Oil. entire filtered soln, 3.063, to 200 mL, or take 100 mL aliquot of the measured vol., dil. to 200 mL, and proceed as in 3.062. Phosphorus (20)-Offieial Final Action 3.065 * Macro Method * (a) For samples exceedingly high in P and low in Ca and Mg (certain seeds, grains, etc. I-Prep. soln as in 3.063, or evap. filtrate and washings from S detn, 3.062, to 50 mL, and proceed as in 8.033. (b) For other samples.-Take 50 mL aliquot of Soln 1,3.005, and proceed as in 8.033. Micro Method (21) 3.066 Reagents (a) Phosphorus std soln.--O.025 mg P/mL. Dissolve 0.4394 g pure dry KH2PO. in H20 and dil. to 1 L. Oil. 50 mL of this soln to 200 mL. (b) Ammonium molybdate soln.-Dissolve 25 g NH. molyb- date in 300 mL H20. Oil. 75 mL H2SO. to 200 mL and add to NH. molybdate soln. (c) Hydroquinone soln.-Dissolve 0.5 g hydroquinone in 100 mL H20, and add 1 drop H2SO. to retard oxidn. (d) Sodium sulfite soln.-Dissolve 200 g Na2S03in H20, dil. to 1 L, and filter. Either keep this soln well stoppered or prep. fresh each time. 3.067 Preparation of Solution To 1 or 2 g sample in small porcelain crucible add 1 mL Mg(NOJ )2 soln, 2.019, and place on steam bath. After few min, cautiously add few drops HCI, taking care that gas evolution does not push portions of sample over edge of crucible. Make 2 or 3 further addns of few drops HCI while sample is on bath so that as it approaches dryness it tends to char. If contents become too viscous for further drying on bath, complete drying on hot plate. Cover crucible, transfer to cold furnace, and ignite 6 hr at 500°, or until even gray ash is obtained. (If necessary, cool crucible, dissolve ash in little H20 or alc.-glycerol, evap. to dryness, and return uncovered to furnace 4-5 hr longer.) Cool, take up with HCI (1 +4). and transfer to 100 mL beaker. Add 5 mL HCI and evap. to dryness on steam bath to dehydrate Si02. Moisten residue with 2 mL HCI. add ca 50 mL H20, and heat few min on bath. Transfer to 100 mL vol. flask, cool immediately, dil. to vol., mix, and filter, discarding first portion of filtrate. 3.068 Determination To 5 mL aliquot filtrate in 10 mL vol. flask add 1 mL NH. molybdate soln, rotate flask to mix, and let stand few sec. Add 1 mL hydroquinone soln, again rotate flask, and add 1 mL Na2S03 soln. (Last 3 addns may be made with Mohr pipet.) Oil. to vol. with H20, stopper flask with thumb or forefinger, and shake to mix thoroly. Let stand 30 min, and measure A with spectrophtr set at 650 nm. Report as % P. 3.069 Gravimetric Quinolinium Molybdophosphate Method (22)~Hicial Final Action Preparation of Solution Accurately weigh ca 2 g plant sample in porcelain dish, and add 7.5 mL Mg(N03)2 soln, 2.019. Dry in oven 2 hr at 110-115° (or until dry). Ignite carefully over Fisher burner, or equiv., until bubbling and smoking cease. Complete ashing in furnace 4 hr at 5~00°. Dissolve ash in few mL HCI (2+1) and evap. to dryness on steam bath. Take up residue in 10-15 mL HCI (1 +9) and filter thru coarse paper into 200 mL vol. flask. Wash paper thoroly with H20 and let filtrate cool to room temp. Oil. to vol. with H20. 3.070 Determination Pipet 40 mL aliquot into 300 or 500 mL erlenmeyer and proceed as in 2.028. Chlorine (23)~ffieial Final Action (If bromides or iodides are present in significant amts, correct results accordingly.) 3.071 Preparation of Solution Verify complete retention of CI in each kind of material by trial, since losses can occur, especially with samples high in carbohydrates, if insufficient Na2C03 is present during ignition, or in any case if excessive temp. is used. Moisten 5 g sample in Pt dish with 20 mL 5% Na2C03 soln, evap. to dryness, and ignite as thoroly as possible at ,,;;500°. Ext with hot H20, filter, and wash. Return residue to Pt dish and ignite to ash; dissolve in HN03 (1 +4), filter, wash thoroly, and add this soln to H20 ext. 3.072 Gravimetric Method To prepd soln, add 10% AgN03, avoiding more than slight excess. Heat to bp, protect from light, and let stand until ppt coagulates. Filter on weighed gooch, previously heated to 140-150°, and wash with hot H20, testing filtrate to prove excess of AgN03. Dry AgCI at 140-150°, cool, and weigh. Report as % CI. Volumetric Method I (24) (Since precision of this titrn is considered to be ±0.2 mg CI, accuracy of 1.0% requires samples contg ;,,20 mg.) 3.073 Reagents (a) Silver nitrate std soln.-1 mL = 0.00355 g CI. Prep. soln slightly stronger than 0.1N, stdze as in 50.031, and adjust to exactly 0.1N. (b) Ammonium or potassium thiocyanate std soln.--O.1N. Prep. soln slightly stronger than 0.1N, stdze as in 50.030(b). and adjust to exactly 0.1N. (c) Ferric indicator.-Satd soln of FeNH.(SO.), .12H20. (d) Nitric acid.-Free from lower oxides of N by dilg HNO, with ca y.. vol. H20, and boiling until perfectly colorless. 3.074 Determination To prepd soln, 3.071, add known vol. std AgN03soln in slight excess. Stir well, filter, and wash AgCI ppt thoroly. To combined filtrate and washings add 5 mL ferric indicator and few mL HN03, and titr. excess Ag with thiocyanate std soln to permanent light brown. From mL AgN03 used, calc. % CI. * Surplus method-5ee inside front cover.
- 62. 40 3. PLANTS AOAC METHODS (1980) Volumetric Method /I (25) 3.075 Reagents (a) Potassium iodide std soln.-l mL = 1 mg CI. Weigh 4.6824 g pure (ACS) KI, dried to const wt at 105-150°, dissolve in H20, and dil. to 1 L. (b) Silver nitrate stock soln.-Approx. 0.3N. 1 mL = ca 10 mg CI. Dissolve 48 g AgN03 in H20, filter, and dil. to 1 L. (e) Silver nitrate std soln.-Oil. 100 ml reagent (b) to ea 900 mL and adjust by stdzg against reagent (a) so that 1 mL = 1 mg CI. (d) Chloride-free starch indicator.-For each 100 mL final soln take 2.5 g sol. starch and make to paste with cold H2 0. Stir out lumps, add 25-50 mL more cold H20, and stir or shake 5 min. Centrf., decant, and discard liq. Repeat extn 3 times and finally transfer residue to flask contg proper amt of boiling H2 0. Stir again, heat to bp, cover with small beaker, and cool under tap, shaking occasionally. (e) Dilute sulfuric acid.-Add 35 mL H2S04 to each 1 L H20, boil 5-10 min, and cool to room temp. (f) Iodine indicator.-To ca 20 g I in 500 mL g-s bottle add 400 mL dil. H2S04, (e). and shake 10 min. Decant and discard first soln, since it may contain iodides. Repeat process and store soln in small g-s bottles. (g) Potassium permanganate soln.-Dissolve 60 g KMn04 in 400 mL warm H20 (ca 50°) and dil. to 1 L. (h) Potassium sulfate-copper sulfate mixture.- Thoroly mix 16 parts K2S04 and 1 part CuS04.5H20. (i) Wash soln.-Mix 980 mL H20 and 20 mL HN03• 3.076 Determination (Caution: See 51.019, 51.026, and 51.080.) Weigh sample contg 10-40 mg CI into beaker. (If >4 g is taken, use proportionately more HN03 and KMn04 soln.) Add 10 mL 0.3N AgN03 and stir until sample is thoroly soaked, adding little H20 or warming if necessary. Add 25 mL HN03 , stir, add 5 mL KMn04 soln, and stir until frothing stops. Place mixt. in H20 bath or on hot plate and keep just below bp. Stir, and wash down sides of beaker at intervals with min. H2 0. After 20 min, or when reaction stops, add addnl KMn04 soln, little at time, until color begins to fade slowly. Dil. to ca 125 mL with boiling H20 and heat 10 min longer. (Beaker may stand in bath or on hot plate until ready to filter.) Filter while hot thru Whatman No.5, or equiv. paper, with suction as follows: Place disk of 30-mesh stainless steel wire gauze or No. 40 filter cloth in bottom of 3" (7.6 cm) Hirsch funnel. Fold 9 cm paper over bottom of No. 11 rubber stopper, shaping it to funnel by making 9-10 folds up side of stopper. Place paper in funnel and apply strong suction. Wet paper and keep wet while fitting into funnel so as to avoid double thicknesses of paper. Wash paper thoroly, first with H20 and then with wash soln. Discard washings and rinse out flask. Decant thru filter and transfer ppt and sample residue to filter. If filtrate is not turbid, or if it is only slightly opalescent, wash ppt thoroly, applying wash soln very gently, but keeping strong suction on filter. If combined filtrate and washings are clear, test for Ag. If turbid, reheat and pass thru filter, repeating until clear, and finally wash as above. If filtrate does not give definite test for Ag, repeat detn on smaller sample. Place paper and contents in Kjeldahl flask and add such amts of K2S04-CUSO. mixt. and H2S04 as would be appropriate for protein detn on same kind and amt of sample, and digest similarly. (For 2 g grass, 8 g sulfate mixt. and 20 mL acid are enough.) When digest is cool, add 175 mL H20, boil 5-10 min, and cool to room temp. Titr. the Ag2S04 in Kjeldahl flask with KI std soln, using 5 mL starch indicator and 30 mL I indicator. (Add latter just before titrn.) Rinse neck of flask after each addn of KI when near end point and titr. until soln stays blue after shaking. If <30 mg CI is present, add starch and I solns at beginning. If larger but unknown amt is present, add 2 mL starch and 10 mL I indicator at beginning and titr. until end point approaches. Shake vigorously to coagulate ppt, add rest of starch and I solns, and proceed to end point. If known large amt is present, titr. to within 2 mL of end point, shake as above, add indicator reagents, and continue titrn. If end point is overrun, add 5 mL std AgN03 soln and titr. again. Blank detns are not necessary after testing reagents. If blank made by using pure sugar as sample is >0.05 mg, examine filter paper, distd H20, and various reagents for CI. Fluoride Potentiometric Method (26)-Official First Action (Rinse all plastic and glass containers with HCI (1 +3) and H20 before use. Perform analyses in laboratory free from F; prep. samples in another laboratory.) 3.077 Principle F is extd from dry, pulverized foliage with HN03 followed by aq. KOH. Slurry is adjusted to pH 5.5, and complexing agent and background F are added. Potential is measured with ion selective electrode and compared against calibration curve. Method is applicable to 10-2000 /Lg F/g dry wt leaf tissue not exposed to unusual amts of AI or other F-binding agents; it is not applicable to insol. inorg. For F in org. combinations. Between-laboratory precision of individual analyses is ±20% at 30 ppm F; ±10%, ;;.100 ppm F. Accuracy is 90-100%. 3.078 Apparatus Electrometer.-Range ±200 mv with readability of 0.1 mv (Model 701 or 701A digital pH/mv meter, Orion Research Inc., or equiv.) or expanded scale pH meter with mv mode of operation, with F ion selective electrode (No. 94-09 single electrode, Orion Research Inc., or equiv.) and reference electrode (No. 90-01 single junction, Orion Research Inc., or equiv.). Check system at intervals to assure adherence to following perform- ance criteria: Using technic of 3.080, system should reach equilibrium (8E <0.2 mv/min) within 5 min with each F working std soln, checked in following sequence: 0.1, 0.2, 0.5, 2.0, and 10.0 ppm F. Replicate std solns should differ by ,,;1 mv. Cali- bration curve should be linear between 0.2 and 10.0 ppm and slope should be 57±2 mv per 10-fold change in F concn. If any parameter is not obtained, check electrodes, reagents, and electrometer. Maintain temp. control to ± 1°. 3.079 Reagents (Store all solns in tightly closed, plastic bottles.) (a) Nitric acid.-(1) 10N.-Add 63 mL HN03 to H20, cool, and dil. to 100 mL. (2) O.2N.-Dil. 5.0 mL 10N to 250 mL. (3) O.05N.- Dil. 5.0 mL 10N to 1 L. (b) Potassium nitrate soln.-D.4M. Dissolve 4.0 g KN03 in H20 and dil. to 100 mL. (e) Sodium citratesoln.-o.8M. Dissolve 58.8 g Na citrate.2H20 in 200 mL H20, adjust to pH 5.5 by dropwise addn of 10N HN03, using pH meter, and dil. to 250 mL with H20. (d) Sodium citrate with fluoride soln.-o.4M citrate with 1 ppm F. Dil. 125 mL 0.8M Na citrate soln and 25.0 mL 10 ppm F std soln to 250 mL with H20. (e) Fluoride std solns.-(1) Stock soln.-l00 ppm F. Dry ca 1 g NaF 2 hr at 110°. Accurately weigh 0.221 g NaF, dissolve in
- 63. AOAC METHODS (1980) FLUORIDE 41 Table 3:01 Preparation of Working Standard Solutions mL soln to be dild to 100 mL Conen, 0.4M 0.8M Na 100 ppm 10 ppm ppm KNO, citrate F soln F soln 10 10.0 5.0 10.0 0.0 10.0 5.0 2.0 0.0 0.5 10.0 5.0 0.0 5.0 0.2 10.0 5.0 0.0 2.0 0.1 10.0 10.0 mL Na citrate soln contg 1 ppm F H20, and dil. to 1 L. (2) Intermediate soln.-10 ppm F. Dil. 10.0 mL stock soln to 100 mL with H20. (3) Working solns.-Prep. as in Table 3:01 in 100 mL vol. flasks. Prep. 0.2 and 0.1 ppm solns fresh as needed. 3.080 Preparation of Calibration Curve Place 25.0 mL 0.1 ppm F working std soln into plastic container contg stirring bar. Insert electrodes ca 12 mm into soln and stir mag. Record mv readings at 1 min intervals until change is <0.2 mv/min. Remove electrodes, blot lightly with absorbent paper, and repeat reading with 0.2, 0.5, 2.0, and 10.0 ppm std solns. Place electrodes in 0.2 ppm std soln until samples are analyzed. (10 ppm std soln may be omitted if samples are known to contain <400 ppm F.) Plot potential (mv) on vertical arithmetic axis and F concn (/-Lg/ mL; ppm) on horizontal (logarithmic) axis of 2-cycle semilog graph paper. 3.081 Preparation of Sample Dry foliage 48 hr at 80°. Grind to pass No. 40 sieve and store in clean, dry, tightly closed plastic bottle. Rotate bottle to mix sample thoroly before removing aliquots. 3.082 Determination Accurately weigh ca 0.25 g powd sample, and place in 75-100 mL wide-mouth plastic container. Add 20 mL 0.05N HN03 and place on rotating shaker or stir mag. 20 min. Add 20 mL 0.1N KOH (5.6 gil) and agitate addnl 20 min. Add 5.0 mL Na citrate soln contg 1 ppm F, adjusted to pH 5.5, and 5.0 mL 0.2N HN03 • d e b (Samples may be stored covered ",4 hr at this point.) Det. mv readings as in 3.080 and prep. calibration curve before and after each series of samples. If sample series contains mixt. of high and low samples, make preliminary estn of F content after 2 min. Then det. F concn in samples contg <40 ppm first and in higher ones last. ppm F (/-Lg/g) = (C - 0.10) x 50/w, where C = ppm F from curve; 0.10 = ppm background F in final soln; 50 = mL final soln; and w = g sample. 3.083 ASTM-Intersociety Committee-AOAC Semiautomated Method (27) Official First Action Principle Dried and ground plant material is ashed, fused with alkali, and dild to vol. In case of leaf samples, F on external surfaces may be washed off sep. Digest and H2SO. are pumped into microdistn app. maintained at 170°. Stream of air carries acidified sample to fractionation column where F and H20 are distd into condenser, and condensate passes into small collector. Distillate is mixed continuously with alizarin F blue-lanthanum reagent, colored stream passes thru tubular flowcell of colorimeter, and A is measured at 624 nm. Interfering metal cations and inorg. phosphate are not distd, and org. substances are destroyed by ashing. Interference from remaining volatile inorg. anions is reduced with high concn acetate buffer with some reduction in sensitivity. Very large amts solid matter, particularly silicates, retard distn. Therefore, smallest sample consistent with obtaining suitable amt F should be used. Conditions must be carefully controlled, since accurate results depend upon obtaining same degree of efficiency of distn from samples as from std F solns used for calibration. Acid concn during distn is maintained at const value by using specific amts CaO and NaOH for ashing and fusion and HCIO. for transfer of fused samples. Any marked change in vac. (>0.2" Hg or 5 torr) over short time indicates either leak or block in system. Distil at same vac. each day and maintain proper ratio between air flow on line drawing liq. and solid wastes from distn coil and on line drawing HF and H2 0 vapor from distn unit h g p k FIG. 3:02-Schematic drawing of air flow system used in semiautomated analysis for fluoride
- 64. 42 3. PLANTS AOAC METHOOS (1980) (Fig. 3:02). Adjust flowmeters to keep this ratio const and to maintain higher vac. on HF line, Ct , so that min. is diverted to waste line. Method can detect 0.1 /-Lg F/mL. Normal range is 0.1-4.0 /-Lg F/mL. Oil. higher concns with NaOH-HCI04 soln, (k). If digested samples routinely exceed 4.0 /-Lg/ mL, modify anal. portion of pump manifold to reduce sensitivity, or use smaller sample aliquot (preferred). Most accurate results are obtained in middle or upper part of calibration curve. For example, to decrease sensitivity, pump sample thru 0.081" tube (2.5 mL/min) and dil. with H2 0 pumped thru 0.065" tube (1.6 mL/min) before sample enters distn app. Total vol. sample and diluent should approx. original vol. used (4.1 mL/min). If air-borne contaminants are present in laboratory, attach small drying bulb contg CaC03 granules to air inlet tube of microdistn unit. Teflon distn coil of microdistn unit must be cleaned periodically to avoid accumulation of solids which reduce sensitivity. Coefficient of variation of 20-100 ppm F is generally,,;; 10%. Samples with large amt of Si (orchard grass) or AI may present special difficulties. There should be no significant deviation from linearity with different amts sample and with different amts added F. 3.084 Apparatus (Cat. Nos refer to current Technicon equipment, except where indicated. Corresponding equipment under previous Cat. Nos is satisfactory.) (a) Automatic analyzer. -(Fig. 3:03) AutoAnalyzer, Technicon Instruments Corp., or equiv. (1) Sampler.-Sampler IV with rotary stirrer and 8.5 mL plastic sample cups. Use 10 or 20/hr cam with 1:3 sample-to-wash ratio (No. 171-A015-07). (2) Col- orimeter.-With 15 mm tubularflowcell and 624 nm interference filter (199-A001-05). (3) Recorder.-Ratio type with 2-100 mv full scale range (011-A115-01). (4) Multichannel proportioning pump and manifold cartridge.-With assorted pump tubes, Microdistillotion device nipple connectors, and glass connectors (pump III 113-A014-08; cartridge 116-8340-01). (b) Pulse suppressors.-For sample and color reagent streams. Coil 10' length of 0.035" id Teflon std tubing around 2.5" diam. tube. Force outlet end into short length of 0.045" id Tygon tubing which is then sleeved with piece of 0.081" Tygon tUbing. Slip sleeved end over "h" fitting which joins sample and reagent streams. (Pulse suppressor included with manifold cartridge.) (c) Voltage stabilizer.-161-A007-01 (also part of 199-A001- 05). (d) Rotary vacuum and pressure pump.-With continuous oiler (Gast No. 0211-V45F-G8CX pump, available from SGA Scientific, Inc.). (e) Microdistillation apparatus.-(Fig. 3:04) Major compo- nents are (Cat. Nos. are those of SGA Scientific, Inc., except as noted): (1) Bottom only of jacketed 1 L resin reaction flask with conical flange (JR-5130), modified by evacuating space between inner and outer walls and sealing off port (8); (2) resin reaction flask top with conical flange (JR-7935) modified to have one $' 29/42 center joint and four$' 24/40 side joints; (3) resin reaction flask clamp (JR-9210); (4) variable high-speed stirrer (S-6362) (d); (5) stainless steel, heavy duty stirrer stuffing box with $' 29/42 and shredded Teflon packing (JS-1160 and JS-3050); (6) 10 mm diam. stainless steel stirrer rod with propeller to fit stuffing box; (7) thermometer-thermoregulator, range 0-200° (T-5715) (c); (8) electronic relay control box (T-5905); (9) low drift immersion heater, 750 watts (H-1265) (b); (10) 30' length coil of flexible Teflon TFE tubing, Ye" id, 3/16" ad, 0.030" wall, on rigid support of such diam. that completed coil will fit into resin reaction flask (avoid kinking of tubing) (e); (11) 2 flowmeters with ranges 0-1 and 0-5 L/min, both with needle valve controls (Dwyer Instru- ment, Inc., PO Box 373, Michigan City, IN 46360); (12) vac. gage with range 0-10" Hg or 0-254 torr (mm Hg); (13) fractionation column of borosilicate glass (g; see also Fig. 3:05; 116-0635); (14) distillate collector (B2 fitting; 116-011-01); (15) H20-jacketed condenser (116-0156-01) (h); (16) Dow-Corning 200 fluid (100 centistokes at 25°) (f); and (17) condenser (116-0181-01) (j). I Upper . Lo.... e. I Level Tube Size Level I (inches) SAMPLER II CD 0.090 Water 12.90 ml/min) ~D-O------~Q)r------ 0) 0.056 Water (120 ml/min) r-----{~I}_-- CD 0.090Sample 12.90 ml/min) CD CD 0.081 Acid (2.50 ml/min) DI ,----===---------------------(11) 0.081 Impinger (2.50 ml/min) Vacuum pump Gloss Bead Mixer 0073 (S) A-C Reagent (169 mil min) ,--__~~~~~~::...~====~=== 0.045 (S) (070 ml/min) PROPORTIONING PUMP D RECORDER FIG. 3:03-Flow diagram for semiautomated analysis for fluoride
- 65. AOAC METHODS (1980) FLUORIDE 43 k d Q FIG. 3:04-Schematlc drawing of microdlstillation apparatus (f) Crucibles.-Inconel, Ni, or Pt, 40-50 mL. (g) Air flow system.-(Fig. 3:02) Draw air thru air inlet tube, (a), before Teflon microdistn coil, (b). Air sweeps thru (b) to fractionation column, and is diverted into 2 channels. In channel c" air passes thru H20-jacketed condenser, (d)' sample trap, (e), to waste bottle, (f). Air then passes thru '/s" id glass tube directed against surface of H2SO. in waste bottle, (g). Partially dehydrated air passes thru gas drying tower, (h), contg 450 g indicating silica gel. Emerging air passes thru T-tube, (i), connected to vac. gage, Ii) (0-10" Hg or 0-254 torr), thru flowmeter, (k) (0-5 L/min), thru T-tube, (I), and then to vac. pump, (m). In channel c2, air passes thru H20-jacketed waste trap, (n), to waste bottle, (0). Air leaving waste bottle flows thru drying bulb, (P), filled with indicating silica gel, and the dry air then passes thru flowmeter, (q) (0-1 L/min). Air stream then connects thru T-tube, (I), with air from first channel. 3.085 Reagents (Caution: See 51.028 and 51.030.) (Deionized H20 may be used. CaD for ashing and NaOH for fusion must be low in F.) (a) Sulfuric acid.-(1+1). Mix 500 mL H2SO. with 500 mL H20 and cool before use. (b) Acetate buffer.-2.14M (pH 4.0). Dissolve 60 g NaOAc.3H2 0 in 500 mL H20, add 100 mL HOAc, and dil. to 1 L with H20. Stable at 25°. (e) Alizarin fluorine blue color reagent stock soln....-{J.01M. Suspend 0.9634 g reagent (alizarin complexone, alizarin com- plexan; 3-amino-ethylalizarin-N,N-diacetic acid; Burdick & Jack- son Laboratories, Inc.) in ca 100 mL H20 in 250 mL vol. flask. Add 2 mL NH.OH and shake until completely dissolved. Add 2 mL HOAc and dil. to vol. with H2 0. Stable indefinitely at 4°. (d) Lanthanum nitrate stock soln....-{J.02M. Dissolve 8.6608 g La(NOa)a.6H20 in H20 and dil. to 1 L in vol. flask. (e) Wetting soln.-30% soln (w/v) polyoxyethylene lauryl ether in H20 (Brij-35, Technicon No. T21-0110). Soln is stable at 25°. (f) Working reagent.-Mix, in order listed, 300 mL acetate 12 5 em 16 5 em rounded, not abrupt FIG. 3:05-Microdistillation column butter, 244 mL H20, 300 mL acetone, 100 mL tett-butanol, 36 mL alizarin fluorine blue stock soln, 20 mL La (NOa)a stock soln, and 2 mL wetting soln. Unused reagent is stable 7 days at 4°. Before using reagent, place under vac. 10 min to remove air bubbles from soln. (g) Fluoride std solns.-(1) Stock soln.-l00 /Lg F/mL. Dis- solve 0.2207 g NaF in H20 and dil. to 1 L. (2) Working solns.- Prep. 7 solns contg 0.2, 0.4, 0.8, 1.6,2.4,3.2, and 4.0 /Lg F/mL. Before dilg to vol., add 6 g NaOH and 20 mL 70% HelD. for each 100 mL final working soln so that stds have same composition as sample solns. Oil. with H20 only for analysis of H20 samples or air samples absorbed in H20. Store working solns at 4° in polyethylene bottles; stable in presence of NaOH. (h) EDTA solns.-l%. Dissolve 1 g Na.EDTA in 99 mL H20. Prep. 0.05% and 0.01% solns by mixing 5 mL 1% soln with 95 mL H20 and 1 mL 1% soln with 99 mL H20, resp. (i) Phenolphthalein soln.-Dissolve 1 g phthln in 50 mL ab- solute alcohol or isopropanol and add 50 mL H2 0. (j) Detergent.-Alconox (Alconox, Inc., 215 Park Ave S, New York, NY 10003); available from laboratory supply firms. (k) Sodium hydroxide-perchloric acid soln.-Dissolve 6 g NaOH in H20, add 40 mL 70% HCIO. (1+1), and dil. to 100 mL with H20. Use to dil. samples when F in unknown sample exceeds std curve. 3.086 Preparation of Sample (a) Leaves.-If it is necessary to remove surface F, wash sample with aq. soln contg 0.05% detergent and 0.05% Na.EDTA in polyethylene container 30 sec with gentle agitation. Remove, drain 3-4 sec, and rinse 10 sec in each of 3 beakers of H2 0. Discard solns after use. (b) Fresh plant tissues.-Dry 24-48 hr in 80° forced-draft oven, and grind as in (e). (e) Dry plant tissues.-Grind in semi micro Wiley mill to pass No. 40 sieve, and store in plastic container. 3.087 Ashing and Fusion Accurately weigh 0.1-2.0 g well mixed dried plant tissue into crucible. Add 1OO± 10 mg low-F CaD, enough H2 0 to make loose slurry, and 4 drops phthln soln. Mix thoroly with polyethylene policeman. Final mixt. should be purple and remain purple during evapn to dryness.
- 66. 44 3. PLANTS AOAC METHODS (1980) Place crucible on cold hot plate and under IR lamp. Evap. under lamp to dryness, turn on hot plate, and char 1 hr. Transfer crucible to furnace at 600° and ash 2 hr. (Caution: To avoid flaming, place crucibles at front of furnace with door open ca 5 min to further char samples; then reposition in furnace.) Remove crucibles, add 3.0±0.1 g NaOH pellets, and replace in furnace with door closed to melt NaOH. (Caution: Avoid creeping of molten NaOH.) Remove crucibles individually and swirl to suspend particulate matter until melt is partially solid- ified. Let cool until addn of small amt H2 0 does not cause spattering. Wash down inner walls with 10-15 mL H20. Suspend melt with polyethylene policeman and transfer with H2 0 to plastic tube graduated at 50 mL. Rinse crucible with 20.0 mL 70% HCI04 (1 +1), add rinse to tube, and dil. to 50 mL with H20. Solns can be stored at this point if tightly capped. Analyze blank contg all reagents with each set of ca 10 samples. Clean crucibles as soon as possible after each use. Boillnconel crucibles 1 hr in 10% NaOH soln. Rinse with hot tap H20, detergent, and then distd H20. Immerse crucibles which held samples contg >100 ,."g F in 4N HCI 45 min before boiling in NaOH soln. Perform blank analyses on these crucibles before addnl use to check for contamination. Scrub Ni and Pt crucibles with detergent and hot H20 and rinse thoroly with H20. Briefly rinse crucibles which held samples contg >100 ,."g Fin 4N HCI before rinsing with H20. 3.088 AnalyticalSystem Place F std solns, ashed and fused samples, or impinged air samples in 8.5 mL plastic cups in sample module. Actuate sampler and pump from cup at net rate of 2.48 mL/min with air segmentation of 0.42 mL/min after sampler crook, and pump into microdistn device thru sample inlet (I, Fig. 3:04), using 0.051" id Teflon tUbing. Pump H2S04 at 2.5 mL/min thru acid inlet (m, Fig. 3:04). Cool and discard acid and solids. Pump distillate from sample trap at 2.0 mL/min thru 0.051" Teflon tubing, add color reagent at 1.69 mL/min, and mix in 4" length of 'Is" id glass tubing packed with pieces of 20 mesh broken Pyrex glass. Pass colored stream thru time delay coil of 15' of 0.035" Teflon spaghetti tubing, thru debubbler fitting where small portion of stream and bubbles are removed to waste bottle at rate of 0.70 mL/min, and thru 15 mm tubular flowcell of colorimeter. A is measured at 624 nm and plotted on recorder. Lag time from sampling to appearance of peak is ca 5 min. Time between samples is 6 min with sampling rate of 10/hr and 3 min at 20/hr. 3.089 Start-Up Turn on H20 to condenser and cooling jacket. Turn on color- imeter. Engage manifold on proportioning pump and start pump. Turn on stirring motor of microdistn unit, vac. pump adjusted for full vac., and heater of microdistn unit. Connect lines to H2S04, color reagent, and H20 bottles. Sampling tube of sampler unit should be in H20 reservoir. Equilibrate app. until silicone oil in microdistn unit reaches 170±2°. Check that all connections are secure. Adjust distn flowmeter (k, Fig. 3:02) to 2.5-3 L/min; adjust waste flowmeter (q) to 0.3 L/min. Distillate should now fill sample trap. Readjust flowmeter (k) to give reading on vac. gage of 5-6" Hg (127-150 torr). (Satisfactory setting for app. must be detd by trial and error. Once detd, use each day.) No air bubbles should be in anal. system beyond point where color reagent and distillate streams are joined. Turn on recorder, adjust baseline to desired level, and run several min to assure that all components are operating properly. Baseline should be reasonably smooth and straight. Transfer F std solns to 8.5 mL plastic cups and place in sampler. Sep. last std soln from sample solns with cup of H20. Program sampler for 10 samples/hr (90 sec sampling period, 270 :lec washout period) or 20 samples/hr (45 sec sampling period, 135 sec washout period). Prep. std curve, 3.092, before and after each day's set of samples. Net A of 0.7-0.9 should be obtained with std soln contg 4 ,."g F/mL. A of each std soln should be reproducible within 10% from day to day and std curve should be linear from 0.2 to ~3.2 ,."g/ mL. 3.090 Shut-Down Turn off chart recorder. Disconnect H2S04 line and place in H20. Disconnect color reagent line and place in 0.01% EDTA soln ca 1 min; then transfer line to H20 and let H20 pass thru system ca 5 min. Clean Teflon distn coil as in 3.091(8). Turn off heater and stirrer of microdistn unit. Turn offvac. pump. Release pump tube manifold. Turn off H2 0 to condenser and cooling trap. 3.091 Maintenance (a) Cleaning of Teflon distillation coil.-(After use with sam- ples contg particulate matter.) Briefly insert Tygon tube con- nected to air inlet line of microdistn unit into 0.01 % EDTA soln. After all deposited material has been removed, wash with 3-4 five mL portions distd H20. (b) Pump tubes.-Replace after 200 working hr or earlier if hard and inflexible or flattened. Always leave in relaxed position when not in use. Remove dirt and grease from pump plates and rollers after each day of use. (c) Indicating silica gel.-Regenerate when ca '$13 has lost normal blue color. (d) Cleaning tubing.-Clean tubing contg reagent after each daily run with 0.01% EDTA soln followed by distd H20. (e) Monthly checks.-Oil proportioning pumps monthly. Check gain on recorder monthly and adjust. 3.092 Calibration and Standards Before and after each day's set of samples, prep. std curve by transferring aliquots of each working std soln to 8.5 mL sample cups and proceed with analysis. Draw straight line connecting baseline before and after analysis. Record A of each peak and subtract A of baseline at peak. Plot net A against,."g F/mL. 3.093 Calculations ppm F in sample = (F x V x D)/W, where F = ,."g F/mL sample from std curve; V = mL sample, usually 50; D = diln factor used only when F of sample exceeds std curve = mL final vol. to which original aliquot was dild with NaOH-HCI04 soln, (k)/mL original aliquot taken; and W = g sample taken for analysis. 3.094 Check Procedure (8) Contamination.-Perform reagent and equipment blank with crucibles and reagents but without sample to detect con- tamination from previous samples, contaminated furnace, and reagents. Blank values >5 ,."g F are evidence of contamination. Perform 2 blank detns with each set of 20-40 samples. Usual blanks are 1-3 ,."g. (b) Recoveries.-Occasionally add known amts F std soln from microburet to aliquots of low F tissue. Recovery of added F should be 100±10%. Low values indicate loss of F, possibly during pretreatment; high values indicate contamination.
- 67. AOAC METHODS (1980) SELENIUM 45 (e) Linearity.-Occasionally analyze different amts (0.1-2.0 g) plant sample contg 50-65 ppm F. Linear relationship should exist between F found and amt tissue taken. Nonlinearity may indicate that some component of tissue is retarding distn or interfering with color development. (d) Calibration curves.-Prep. at least twice daily. 3.095 Trouble Shooting (a) Irregular baseline.-May result from: (1) excessive pulse pressures-check for faulty pump tubes, absence of surge suppressors, or improperly made or placed suppressors; (2) air bubbles in flowcell-check for absence of debubbler bypass, blockage in reagent pump tube, or periodic emptying of sample trap (latter results if air flow to distn trap becomes too great); (3) excessive H2SO. carryover-check for too high temp. in oil bath, improper H2SO. concn, or too high vac. on system; (4) air flow imbalances-check flowmeter settings, trapped air in tub- ing, or leak or block in system; (5) high F content in samples (baseline may not return to normal between samples)-dil. or check sampling speed and sample-to-wash ratio. (b) Irregular peaks.-Asymmetrical or double peaks or peaks with shoulders may result from: (1) baseline irregularities; (2) interfering substances from sample or impure reagents; (3) inadequate buffer concn; or (4) excessive amts solids in distn coil. Presence or accumulation of solids may be due to insuffi- cient flow of H2SO., too large sample, excess CaO or NaOH in sample, inadequate suspension of particles in samples, or lack of proper air segmentation in sample tubing. (e) Poor reproducibility.-Check for: improper sample pickup; faulty pump tubes; inadequate washing of distn coil between samples; large deviations in acid concn, temp., or air flow in distn coil; or changes in vac. on waste system. Selenium 3.096 * Gravimetric Method /;lBI-Official Final Action * (Applicable to materials contg >2 ppm Se) See 3.073, 11th ed. 3.097 Fluorometric Method /;l9)-Official Final Action (Caution: See 51.008, 51.019, 51.026, and 51.028.) Apparatus (a) Micro-Kjeldahl flasks.-30 mL Pyrex, ca 170 mm total length with $" 12/18 outer joint at mouth. (b) Air condensers.-l0 x 140 mm Pyrex tubes with $" 12/18 inner joint. (e) Micro-Kjeldah/ digestion unit with glass fume duct.-Fit rack to hold flasks and attached air condensers in nearly upright position during early stages of digestion. Use in fume hood. (d) Fluorometer.-Capable of illuminating sample at 369 nm and measuring fluoresced light at 525 nm. Turner Associates, 2524 Pulgas Ave, Palo Alto, CA 94303, Model 110 or 111 filter fluorometer equipped with std lamp No. 110-850, primary filter No. 7-60, and secondary filter No. 58 is satisfactory. Model 430 spectrofluorometer set to above wavelengths is also satisfactory. 3.098 Reagents (Use deionized H20 distd in glass for prepg solns and dilns.) (a) Nitric acid.-Redistd in glass. (b) Hydroxylamine-ethylenediaminetetraacetic acid soln.- Add ca 20 mL H20 to 1.9 g EDTA (acid form). Slowly add ca 5N NH.OH with stirring until EDTA just dissolves. Some excess *Surplus metho~ee inside front cover. NH.OH is not harmful. Dissolve 6 9 NH20H.HCI in 100 mL H20. Combine solns and dil. to 250 mL with H2 0. (e) Cresol red indicator. -Dissolve 0.1 g cresol red in 10 mL H20 and 1 drop 50% NaOH soln. Dil. to 50 mL with H20. (d) Selenium std soln.--().3 fL9 Se/mL. Add 10 mL HN03 to 30.0 mg Se (purity ~99%) and warm to dissolve. Dil. to 100 mL with H20, mix well, and transfer exactly 1 mL to micro-Kjeldahl flask. Add 2 mL 70% HCIO. and 1 glass bead. Boil gently to HCIO. fumes and cool. Add 1 mL H20 and 1 mL HCI (1 +4); heat 30 min in boiling H2 0 bath. Transfer to 1 L vol. flask and dil. to vol. with ca lN HCI. Store in all-glass container. Soln is stable several months at room temp. (e) Decalin.-Eastman Kodak No. 1905 decahydronaphthal- ene, or equiv. (f) 2,3-Diaminonaphthalene (DAN) soln.-Prep. soln in semi- darkened room or in room with only yellow light at time of detn. Protect from light and prep. fresh for each set of detns. Add 50 mL ca O.lN HCI to 0.05 g DAN (available from ICN-K&K Laboratories, Inc.). Place in 50° H2 0 bath in dark 15 min. Cool to approx. room temp. and ext twice with 10 mL decalin, shaking vigorously each time and discarding decalin. Filter thru paper satd with H2 0. For >8 detns, prep. larger amt. 3.099 Preparation of Samples Grind air-dried samples to pass No. 18 or finer sieve. Cut fresh or wet samples finely with scissors or knife, or grind in food chopper to assure representative sample. Some plants (e.g., Astragalus bisulcatus, A. racemosus, Stan- leya bipinnata, and Oonopsis condensata) contain Se in volatile form that is lost during drying. Analyze these plants without drying. With usual agricultural crops, this is not a problem if drying is performed at 60-70°. 3.100 Preparation of Fluorometric Blanks and Standard (a) Blank.-Place 1 mL H20 in micro-Kjeldahl flask. (For sam- ples contg <0.1 ppm, carry 10 mL HN03 as blank thru entire detn.) (b) Std.-Place 1.0 mL std Se soln in micro-Kjeldahl flask. Add 2 mL 70% HCIO. to each flask and continue as in detn, beginning "Mix contents of flasks ..." 3.101 Determination (a) Samples containing 4 or more ppm selenium.-Weigh .;;1 g sample (air-dried wt basis) contg .;;0.4 fLg Se into micro- Kjeldahl flask. Add 1 glass bead, previously cleaned with HN03• Add 10 mL HN03 and let stand at room temp. ~4 hr. (Use 5 mL HN03 for samples <0.5 g.) Affix air condenser and place flask in nearly upright position on micro-Kjeldahl digestion unit. Heat ca 15 min with low flame and then increase heat until HN03 condenses in lower part of condenser. Heat 10 min longer, turn off burner, and let cool 5 min. Wash down sides of flask with 2 mL 70% HCIO. thru air condenser. Swirl flask and continue refluxing 15 min. Remove condenser and continue heating, drawing off fumes in fume duct, until HCIO. fumes appear and then 15 min longer. Cool, add 1 mL H20, and again heat to HCIO. fumes and 1-2 min longer. Cool. and add 1 mL H20. Mix contents of flasks and add 1 mL HCI (1 +4) to each. Place in boiling H20 bath 30 min. Cool to ca room temp. To each flask add 5 mL NH20H-EDTA soln and 2 drops cresol red indicator. Neutze to yellow with ca 5N NH.OH and add HCI (1 +4) to orange-pink. From this point, perform al/ operations in semidarkened room or room with yellow light only. Prep. DAN soln, add 5 mL to each flask, and dil. to neck with ca O.lN HCI. Mix and place in 50° H2 0 bath in dark 25 min.
- 68. 46 3. PLANTS AOAC METHOOS (1980) Remove flasks from H20 bath and cool to ca room temp. in pan of H20. Pour solns into 125 mL separators with Teflon stopcocks and contg 10.0 mL decalin. Shake vigorously ;;.30 sec, let stand ca 1 min, and drain and discard lower layer. Wash decalin twice by shaking vigorously ;;.15 sec with 25 mL ca O.lN HCI. (VirTis, Rt 208, Gardiner, NY 12525, Extractomatic shaker with 100 mL separators may be substituted. When used, shake ext 5 min and wash 1 min periods.) Transfer decalin layer to 12 mL centrf. tubes and centrf. 2 min at moderate speed. Pour decalin soln into fluorometer tubes, zero fluorometer against decalin, and read all tubes at 525 nm within 5 min. Correct std and unknown readings for blank. ppm Se = 0.3 x sample reading/std reading x g sample. (b) Samples containing less than 4 ppm selenium.-Proceed as in (a) thru second par. Oil. digest to adequate vol. and take aliquot contg ca 0.3 /1-g Se for detn. Alternatively, digest sample in 10 vols HN03 2 hr on steam bath. Oil. to definite vol., and carry appropriate aliquot thru detn. Latter method is especially applicable when proper sampling requires large sample. Do not dil. decalin soln contg piazselenol, as this introduces errors. Boron (30j--Official Final Action Quinalizarin Method 3.102 Reagents (a) Dilute sulfuric acid.---O.36N. Oil. 10 mL H2S04 to 1 L. (b) Calcium hydroxide saturated soln.-Filter before use. (c) Quinalizarin soln.-Oissolve 45 mg quinalizarin in 1 L 95-96% H2S04, (d) Boron std soln.---O.5 mg B/mL. Dissolve 2.860 g H3B03 and dil. to 1 L with H20. Prep. working stds by further diln with H20. 3.103 Determination Place 1.00-2.00 g dry, ground plant material in Pt or Si02 dish. Add 5 mL satd Ca(OH)2 soln and dry at 105°. Carefully volatilize over burner, ash in furnace 1 hr at 600°, and cool. Add exactly 10 or 15 mL 0.36N H2S04 , break up ash with glass rod, stir gently, and filter. Transfer 2 mL filtrate to colorimeter tube, add an exact amt (e.g., 15 mL) quinalizarin reagent, stopper, and mix by swirling gently. Let stand at room temp. 24 hr (or until both unknowns and stds have cooled to same temp.). Shake tube again immediately before reading in photoelec. colorimeter (620 nm filter). Adjust colorimeter to 100% T with blank soln prepd as above but using 2 mL H20 in place of sample soln. Prep. std curve with series of stds contg 0.5-10 /1-g B/mL. 3.104 OTHER CONSTITUENTS Sugars (31)-Official Final Action Preparation ofSolution (a) General method.-Prep. fresh sample as in 3.002(b). Pour alc. soln thru filter paper or extn thimble, catching filtrate in vol. flask. Transfer insol. material to beaker, cover with 80% alcohol, warm on steam bath 1 hr, let cool, and again pour alc. soln thru same filter. If second filtrate is highly colored, repeat extn. Transfer residue to filter, let drain, and dry. Grind residue so that all particles will pass thru 1 mm sieve, transfer to extn thimble, and ext 12 hr in Soxhlet app. with 80% alcohol. Dry residue and save for starch detn. Combine alc. filtrates and dil. to vol. at definite temp. with 80% alcohol. For dried materials, grind samples finely, and mix well. Weigh sample into beaker, and continue as above, beginning " ... cover with 80% alcohol, ..." (b) Applicable when starch is not to be determined.- Prep. fresh sample as in 3.002(bl. but boil on steam bath 1 hr. Decant soln into vol. flask, and comminute solids in high-speed blender with 80% alcohol. Boil blended material on steam bath 0.5 hr, cool, transfer to vol. flask, dil. to mark with 80% alcohol at room temp., filter, and take aliquot for analysis. Grind dry material to pass No. 20 sieve or finer, transfer weighed sample to vol. flask, and add 80% alcohol and enough CaC03 to neutze any acidity. Boil 1 hr on steam bath, cool, adjust vol. at room temp. with 80% alcohol, filter, and take aliquot for analysis. 3.105 Clarification with Lead Place aliquot alc. ext in beaker on steam bath and evap. off alcohol. Avoid evapn to dryness by adding H20 if necessary. When odor of alcohol disappears, add ca 100 mL H20 and heat to 80° to soften gummy ppts and break up insol. masses. Cool to room temp. and proceed as in (a) or (b): (e) Transfer soln to vol. flask, rinse beaker thoroly with H2 0, and add rinsings to flask. Add enough satd neut. Pb(OAc)2 soln to produce flocculent ppt, shake thoroly, and let stand 15 min. Test supernate with few drops of the Pb(OAc)2 soln. If more ppt forms, shake and let stand again; if no further ppt forms, dil. to vol. with H20, mix thoroly, and filter thru dry paper. Add enough solid Na oxalate to filtrate to ppt all the Pb, and refilter thru dry paper. Test filtrate for presence of Pb with little solid Na oxalate. (b) Add twice min. amt of satd neut. Pb(OAcl. soln required to cause complete pptn, as found by testing portion of supernate with few drops dil. Na oxalate soln. Let mixt. stand only few min; then filter into beaker contg estd excess of Na oxalate crystals. Let Pb ppt drain on filter and wash with cold H20 until filtrate no longer gives ppt in oxalate soln. Assure excess of oxalate by testing with 1 drop Pb(OAc)2' Filter and wash pptd Pb oxalate, catching filtrate and washings in vol. flask. Oil. to vol. with H20 and mix. 3.106 Clarification with lon-Exchange Resins (32) Place aliquot alc. ext, 3.104, in beaker and heat on steam bath to evap. alcohol. Avoid evapn to dryness by adding H20. When odor of alcohol disappears, add ca 15-25 mL H20 and heat to 80° to soften gummy ppts and break up insol. masses. Cool to room temp. Prep. thin mat of Celite on filter paper in buchner or on fritted glass filter and wash until H20 comes thru clear. Filter sample thru Celite mat, wash mat with H20, dil. filtrate and washings to appropriate vol. in vol. flask, and mix well. Place 50.0 mL aliquot in 250 mL erlenmeyer; add 2 gAmberlite IR-120(H) analytical grade cation (replaced by REXYN 101(H) resin, Fisher Scientific Co.) and 3 g Duolite A-4(OH) anion ion exchange resins. Let stand 2 hr with occasional swirling. Take 5 mL aliquot deionized soln and det. reducing sugars as glucose as in 31.053. Glucose 3.107 Micro Method-Cfficial Final Action See 31.053. Fructose (33)-Official Final Action 3.108 Reagents (e) Glucose oxidase preparation.-Add slowly, stirring con- stantly, 100 mL H20 to 5 g glucose oxidase prepn ("DeeO L- 750" code 4633000, Miles Laboratories, Inc., 1127 Myrtle St,
- 69. AOAC METHODS (1980) STARCH Elkhart, IN 46514). Stir ca 1 min and centrf. or filter to obtain clear soln. Add ca 1 mL CHCI3and refrigerate. Soln is stable ;;.1 month. (b) Mcilvaine's citrate-phosphate buffer.-Oissolve 214.902 g Na2HPO•. 12H20 and 42.020 g citric acid in H20 and dil. to 1 L. 3.109 Determination To suitable aliquot add '4 its vol. of buffer to give pH ca 5.8. Add 30% as much glucose oxidase prepn as estd glucose content (for 500 mg glucose add 150 mg glucose oxidase, i.e., 3 mL soln), and few drops 30% H20 2 (omit if Somogyi method is to be used in detn). Let stand overnight at room temp. Oet. fructose by Somogyi micro method, 31.053, or by Munson- Walker method, 31.038-31.039, using Table 3:02. Check equivs in range of interest, using pure fructose as std, and correct as necessary. Table 3:02 Abbreviated Munson and Walker Table for Calculating Fructose (From Official and Tentative Methods of Analysis, AOAC, 5th Ed., 1940) Cuprous Oxide Cuprous Oxide mg Fructose mg mg 10 4.5 300 50 23.5 350 100 47.7 400 150 72.2 450 200 97.2 490 250 122.7 Reducing Sugars--Official Final Action 3.110 Munson-Walker General Method See 31.038. 3.111 * Quisumbing-Thomas Method * See 31.048-31.049,11th ed. Sucrose-Official Final Action 3.112 Hydrochloric Acid Inversion Fructose mg 148.6 174.9 201.8 229.2 253.9 Using aliquot of cleared soln, 3.105, proceed as in 7.079. 3.113 Invertase Inversion (a) For plants giving hydrolysis end point within 2 hours.- Pipet aliquot of cleared soln, 3.105, into 400 mL Pyrex beaker and make slightly acid to Me red with HOAc. Add 3 drops 1% soln of Wallerstein invertase scales. Let mixt. stand at room temp. 2 hr. Add reagents as in 31.035, and det. reducing power. Calc. results as invert sugar. Deduct reducing power of original soln, also expressed as invert sugar, and multiply difference by 0.95. (b) For plants giving slower hydrolysis end point.- Place aliquot of soln, 3.105, in small vol. flask. Make slightly acid to Me red with HOAc. Add 3 drops 1% soln of Wallerstein invertase scales and few drops toluene. Stopper flask and let stand overnight or longer at room temp. Oil. to vol. with H2 0 and use aliquot for reducing power as above. 3.114 Ether Extract-Official Final Action See 7.056. 3.115 Crude Fiber-Official Final Action See 7.061-7.065. Total Nitrogen (Crude Proteinj-Official Final Action 3.116 Kjeldahl Method for Nitrate-free Samples See 2.057. 3.117 Kjeldahl Method for Nitrate-Containing Samples See 2.058. 3.118 Automated Method See 7.021-7.024. 3.119 Semiautomated Method See 7.025-7.032. Starch (34j-Official Final Action 47 3.120 Reagents (a) Iodine-potassium iodide soln.-Grind 7.5 g I and 7.5 g KI with 150 mL H20, dil. to 250 mL, and filter. (b) Alcoholic sodium chloride soln.-Mix 350 mL alcohol, 80 mL H20, and 50 mL 20% NaCI soln, and dil. to 500 mL with H20. (c) Alcoholic sodium hydroxide soln.-o.25N. Mix 350 mL alcohol, 100 mL H20, and 25 mL 5N NaOH, and dil. to 500 mL with H20. (d) Dilute hydrochloric acid.-o.7N. Oil. 60 mL HCI to 1 L with H20. (e) Somogyi phosphate sugar reagent.-Dissolve 56 g an- hyd. Na2HPO. and 80 g Rochelle salt in ca 1 L H20, and add 200 mL LOON NaOH. Then slowly add, with stirring, 160 mL 10% CuSO•.5H20 soln. Dissolve 360 g anhyd. Na2SO. in this soln, transfer to 2 L vol. flask, and add exactly 200 mL 0.1N KI03soln (3.5667 giL). Oil. to vol., mix well, let stand several days, and filter thru dry paper into dry flask, discarding first 50 mL filtrate. Store reagent at 20-25°. It is 0.01N with respect to KI03; 5.00 mL is equiv. to 10 mL 0.005N Na2S203' Det. glucose factor of reagent as follows: Accurately weigh 150 mg NBS glucose SRM into 1 L vol. flask, dissolve in H20, dil. to vol., and mix well. Transfer 5 mL aliquot to 25 x 200 mm Pyrex test tube, add exactly 5 mL Somogyi reagent, stopper with size 00 crucible, and heat (together with several blanks contg 5 mL H20 and 5 mL reagent) exactly 15 min in boiling H20 bath. Titr. as in detn. From difference between blank and std titrns, calc. mg glucose equiv. to 1 mL exactly 0.005N Na2S20 3. Effective range for detn is 0.05--1.0 mg glucose in 5 mL aliquot. (f) Sodium thiosulfate std soln.-o.005N. Dissolve 2.73 g Na2S203.5H20 in H20 and dil. to 2 L. Stdze daily as follows: Add 1 mL KI soln, (g), and 3 mL 1.5N H2S04 to 5 mL Somogyi sugar reagent. Let stand 5 min, and titr. with Na2S20 3 soln, adding starch indicator, (h), just before end point. (g) Potassium iodide soln.-2.5%. Stabilize with little Na2C03. (h) Starch indicator.-Make 1.5 g sol. starch into paste with few mL H20, and add slowly, with stirring, to 300 mL boiling H20. 3.121 Determination Select sample as in 3.001, remove all foreign matter, dry, and grind to pass No. 80 sieve. Accurately weigh 0.1-1.0 g powd sample contg ca 20 mg starch into 25 x 150 mm Pyrex test tube. Add ca 200 mg fine sand and 5 mL H20, and mix well with stirring rod to wet sample. Heat tube in boiling H2 0 bath 15 min to gelatinize starch. Cool to room temp., and place in 22-25° bath. Add 5 mL 60% HCIO. rapidly with const agitation. Grind tissue against lower wall of tube with stirring rod for approx. *Surplus method-see inside front cover.
- 70. 48 3. PLANTS AOAC METHODS (1980) min at time. Repeat grinding frequently during 30 min; then without delay, transfer quant. to 100 mL vol. flask with H20. Add 3 mL 5% uranyl acetate soln to ppt protein, dil. to vol. with H20, mix well, and centrf. portion of mixt. Pipet 10 mL clear supernate into 25 x 150 mm test tube. Add ca 100 mg Celite, 5 mL 20% NaCI soln, and 2 mL I-KI reagent, and mix well. Let stand overnight, centrf., and decant. Wash starch-I ppt by suspending it in 5 mL alc. NaCI soln, centrf., and decant. Add 2 mL alc. NaOH soln to packed ppt. Gently shake and tap tube until ppt is no longer blue. (Do not use stirring rod; allow ample time for complex to decompose.) Wash walls of tube with 5 mL alc. NaCI soln, centrf. liberated starch, and wash with 5 mL alc. NaCI soln as before. Add 2 mL 0.7N HCI to ppt. Stopper tube loosely with size 00 crucible, and heat 2.5 hr in boiling H20 bath. (Bath should have cover with holes to accommodate tubes; holes not occupied by tubes must be covered.) Cool, and transfer quant. to 25 mL vol. flask. Add drop phenol red, 50.008, and neutze with IN NaOH. Discharge color with O. TN oxalic acid, dil. to vol., and mix well. Transfer 5 mL aliquot to 25 x 200 mL Pyrex test tube, add exactly 5 mL Somogyi reagent, and stopper tube with size 00 crucible. Heat together with several blanks contg 5 mL H20 and 5 mL Somogyi reagent in vigorously boiling H20 bath exactly 15 min. Remove tube from bath and cool to 25-30°. Add 1 mL 2.5% KI soln down wall of tube without agitation and then add 3 mL 1.5N H2S04 rapidly with agitation. After all Cu20 dissolves, titr. soln with 0.005N Na2S203' adding starch indicator, (h)' just before end point is reached. Treat blank solns similarly. % Starch = [50(mL blank - mL sample) x 0.90/mg sample] x (N/0.005) x G x 100, where 50 = diln factor, 0.90 = factor glucose to starch, N = actual normality Na2S203 soln, and G = mg glucose equiv. to 1 mL 0.005N Na2S20 3• Lignin (35)-Official Final Action Direct Method 3.122 Preparation ofSample Grind sample in mill to pass No. 80 sieve and dry at 105°. Ext wei~hed sample (5-10 g) 30 hr in Soxhlet app. with alcohol- benzene soln (32 parts alcohol and 68 parts benzene by wt). Dry material in oven to free it from solvs and place in flask of suitable size. Add 150 mL H20/g sample, and reflux 3 hr. Filter mixt. while still hot, preferably thru weighed fritted glass cru- cible, and transfer extd material to flask. Add 1% HCI (111 g coned HCI + 3890 mL H20) in proportion of 150 mL acid soln/g plant material, and reflux 3 hr. Filter mixt. while still hot thru fritted glass crucible previously used, wash with H20 until acid- free, dry at 105°, and weigh. Calc. % total loss due to successive extn with alcohol-benzene soln, hot H20, and 1% HCI. (With samples not especially rich in carbohydrates and proteins, extn with hot H20 may be omitted.) 3.123 Apparatus App., Fig. 3:06, consists of: (1) 1.5 L bottle, A, to which is attached by 2-hole rubber stopper 250 mL dropping funnel, C, having lower end of stem bent as illustrated and placed close to bottom of A; (2) Drechsel gas-washing bottle, D; (3) 3 Pyrex test tubes, 38 x 300 mm diam., G, G', G", connected in parallel by device, 0, and immersed in wooden box, L, filled with crushed ice, H; and (4) bottle contg H20 for absorption of excess HCI, K. G, G', and G" are provided with 2-hole rubber stoppers; glass tube with right angle bend extends thru 1 hole nearly to bottom of test tube, and similar tube extending ca 10 mm into test tube passes thru other hole. Rubber connections and stopcocks for regulating flow of gas are provided as indicated in diagram. A is filled with ca 500 mL H2S04 and C with HCI; HCI flowing thru stoptock B into A generates HCI gas, which is dried by H2S04 in D, and flows into G, G', and G" contg samples and fuming HCI reagent. 3.124 Reagent Fuming hydrochloric acid.-(Caution: See 51.031.) Density 1.212-1.223 at 15°. To 500 g NaCI in 1 L g-s Pyrex distg flask, add cold soln of 250 mL H20 in 450 mL H2S04• Connect side tube of distg flask to glass tube passing thru H2S04 wash bottle, and connect outlet tube of H2S04 wash bottle to another glass tube, immersed in flask contg 3 L HCI. Surround flask contg HCI with crushed ice. Heat distg flask with small flame and pass HCI gas into acid soln until it attains sp gr of 1.212-1.223 at 15°. Keep " FIG. 3:06--Apparatus for determining lignin
- 71. AOAC METHODS (1980) PIGMENTS 49 reagent refrigerated at ,,;0°. If only few detns are to be made, prep. correspondingly smaller amt. 3.125 Determination Weigh three 1 g samples of extd and dried sample in weighing bottle and place in 3 large test tubes, G, G', and G". Add 20 mL of the reagent to each tube, using this acid to wash down any particles clinging to sides. When all material is wet with reagent, add addnl 30 mL reagent. Add ca 3 drops capryl alcohol to minimize foaming. Place test tubes, G, G', and G", in wooden box, L, and surround with crushed ice. Lubricate tubes F, F', and F" with drop of glycerol so that they move easily thru holes in rubber stoppers. Lead dry HCI gas from generator into reaction mixts thru tubes F, F', and F" (F' and F" are shown in top view)' which reach nearly to bottom of tubes G, G', and G". Regulate flow of gas thru reaction mixts in G, G', and G" by stopcocks shown in top view, continuing passage of gas 2 hr. (At first rather slow stream of gas passes in, but during last 15 min, flow is fairly rapid.) After reaction period, discontinue flow of gas, and disconnect long tubes F, F', and F" and outlet tubes oftest tubes G, G', and G" from a and P. Pull tubes F, F', and F" just above surface of reaction mixt., and close with short pieces of rubber tubing having one end plugged with short piece of glass rod. Similarly close off outlet tubes, N, N', and N". Place tubes contg reaction mixt. in cold room or refrigerator (8-10°) 24 hr. Transfer contents of G, G', and G" to 1 L erlenmeyers, taking care to remove any material adhering either to inside or outside of tubes F, F', and F". Oil. reaction mixts to 500 mL with H20. Connect flasks to reflux condensers and boil 1 hr. Prep. 3 gooches in usual manner, dry at 105°, and weigh. Ignite one of weighed crucibles, X, over Bunsen burner, cool in desiccator, and reweigh. Let contents of flasks cool to room temp. and filter thru weighed gooches. Wash ppts collected in gooches with hot H20, dry at 105°, and weigh in weighing bottles. Ignite crude lignin in crucible X over Bunsen flame and det. wt ash. Place one of other 2 gooches in wide-neck Kjeldahl flask and det. % N in crude lignin as in 2.057. If methoxyl in lignin is to be detd, collect ppt from one of flasks in dried (105°) fritted glass crucible and proceed as in 47.050. Wt lignin = wt crude lignin - wt ash - wt crude protein (N x 6.25). Calc. % lignin in original dry unextd material. 3.126 Indirect Method (36) (Caution: See 51.086.) Ext 1 g sample with alcohol-benzene (1 +2) 4 hr in Soxhlet or comparable app. (extn vessel may be either coarse porosity Alundum or paper thimble, closed at top with filter paper or plug of cotton). Wash sample in thimble with suction, using 2 small portions alcohol followed by 2 small portions ether. Heat at 45° in nonsparking oven to drive off ether, and transfer sample to 250 mL wide-mouth erlenmeyer. Add 40 mL 1% soln of pepsin in O.1N HCI, wetting sample well by adding small portion reagent, stirring or shaking thoroly, and finally washing down sides of flask with remaining soln. Incubate at 40° overnight. Add 20--30 mL hot H20 and filter, using filter stick. (Filter sticks are made with Pyrex fritted glass disk, 30 mm diam., medium porosity. Thin layer of pre-ashed diat. earth (Hyflo Super-Cel, or similar filter-aid) is sucked onto disk from H20 suspension. This is usually enough for easy filtration; if not, add extra Super-Cel to material being filtered. Some sticks filter slowly with some samples. It is advisable to obtain more than needed and discard slow-filtering ones. It is convenient to arrange filter sticks in set of 12 attached to vac. manifold by rubber tubing.) Repeat washing twice and then wash residue into flask by forcing 7-8 mL 5% (w/w) H2S04 downward thru filter stick, using air pressure. Wash stick further with the H2S04, finally adding enough to bring total vol. to ca 150 mL. Reflux vigorously on hot plate 1 hr, adding H2 0 occasionally to maintain original vol. Filter off acid. Wash residue with three 20--30 mL portions hot H20, two 15-20 mL portions alcohol, and two 15 mL portions ether. Leave vac. on few min to dry residue, and transfer from stick to flask by tapping and brushing. Heat to drive off any residual ether. If disk formed upon drying is difficult to break up into finely divided state (sometimes in case of immature plant samples), disperse residue in ether in flask and then boil off ether on steam bath. Add 20 mL 72% (w/w) H2S04 at 20° to residue and hold 2 hr at 20°, stirring occasionally. Add 125 mL H20, filter, wash once with 20 mL hot H20, and filter again. Wash residue from filter stick and reflux as before 2 hr, using 150 mL 3% (w/w) H2S04 • Filter residue onto gooch with asbestos pad and wash with hot H20 until acid-free. Dry at 105-110° and det. lignin by loss in wt on ignition at 600°. 3.127 PIGMENTS Chlorophyll-Offieial Final Action Photoelectric Colorimetric Method for Total Chlorophyll Only (37) Apparatus (a) Mortar and pestle.-Deep glass mortar ca 10 cm id with well-defined lip. (b) Photoelectric colorimeter.-Calibrate for chlorophyll, us- ing plant ext as in 3.129 and light filters with max. T near 660 nm. (Combination of Corning Glass Works filters CS No. 2-60 and 1-58 (Glass No. 2408 and 3965, resp.) is suitable.) (e) Wash bottles.-Type fitted with rubber bulb, permitting operation with one hand. (d) High-speed blender.-Waring Blendor, or equiv. 3.128 Reagents (a) Acetone.-(1) Undild acetone and (2) 85% aq. soln by vol. Com. acetone, tech. grade, is satisfactory. (b) Quartz sand.-Acid-washed and dried. 3.129 Determination (Caution: See 51.004, 51.040, and 51.046.) Select field material carefully to ensure representative sample. Remove representative portion from field sample, and if fresh, cut finely with hand shears and mix as thoroly as possible. Grind dried material in mill and mix thoroly. Weigh 1-5 g into mortar and add ca 0.1 g CaC03 or Na2C03 • Macerate tissue with pestle, add quartz sand, and grind short time; then add 85% acetone, little at time, and continue grinding until tissue is finely ground. Transfer mixt. to funnel, filter with suction, and wash residue with 85% acetone. Return residue to mortar with more 85% acetone and grind again. Filter and wash as before. Repeat procedure until tissue is devoid of any green, and washings are colorless. (It is advisable to grind residue at least once with undild acetone and then to add enough H20 at end to bring acetone concn to 85%. High-speed blender may be used instead of mortar to macerate and ext tissue (see 3.132), but each investigator should satisfy himself that device used exts tissue completely.) When extn is complete, transfer filtered ext to vol. flask of appropriate size and dil. to vol. Measure T of soln with photoelec. colorimeter, and read amt of chlorophyll present from curve relating T and concn. Express
- 72. 50 3. PLANTS AOAC METHODS (1980) chlorophyll values as mg/g tissue, or in other convenient manner. Calibrate photoelec. colorimeter as follows: Ext sample of fresh, green leaf material with 85% acetone, filter, wash residue, and dil. ext to vol. as above. Make series of dilns of ext and measure T of original and of each of dild solns with instrument in same manner as when chlorophyll prepn is being used as calibration std. Transfer aliquot of original ext to ether and evaluate total chlorophyll spectrophtric as in 3.132(b) and (e). From value thus obtained, calc. chlorophyll content of original ext and that of each of dild solns, and construct curve relating concn of chlorophyll with Tor A. Spectrophotometric Method for Total Chlorophyll and the a and b Components (38, 39) 3.130 Apparatus Use app. in 3.127 (except photoelec. colorimeter), plus follow- ing: (a) Scrubbing tubes for washing ether solns. -Open tubes ca 20 mm diam. to one end of each of which is sealed tube of smaller diam. drawn to fine jet at lower end. (b) Spectrophotometer.-Capable of isolating spectral region of ca 3 nm near 660 nm with negligible stray radiation. Tubulated cells with tightly fitting glass stoppers are recommended for work with ether. 3.131 Reagents Those listed in 3.128 and following: Ether.-Com. grade is satisfactory without further purification. 3.132 Determination (Wash glassware with concd Na3PO. soln to remove traces of acid that may decompose chlorophyll.) (a) Extraction of chlorophyll from tissue.-Select and prep. sample as in 3.129. Disintegrate weighed portion (2-10 g, depending on chlorophyll content) of fresh plant tissue in blender cup that contains ca 0.1 g CaC03, or by use of mortar as in 3.129. After tissue is thoroly disintegrated, filter ext thru buchner fitted with quant. paper. Wash residue with 85% acetone, 3.128(a), and if necessary, use little ether to remove last traces of pigment. If extn is incomplete, return residue and paper to blender container with more 85% acetone and repeat extn. Filter and wash, as before, into flask contg first filtrate. Transfer filtrate to vol. flask of appropriate size and dil. to vol. with 85% acetone. Pipet aliquot of 25-50 mL into separator contg ca 50 mL ether. Add H20 carefully until it is apparent that all fat-sol. pigments have entered ether layer. Drain and discard H20 layer. Place separator contg ether soln in upper rack of support. Add ca 100 mL H20 to second separator placed in rack below first. Set scrubbing tube in place, and let ether soln run thru it to bottom of lower separator and rise in small droplets thru the H20. When all soln has left upper separator, rinse it and scrubbing tube with little ether added from medicine dropper. Place scrubbing tube in upper separator and exchange its place in support with separator now contg ether soln. Drain and discard H20 in upper separator, add similar portion of fresh H20 to lower separator, and repeat washing process. Continue washing ether soln until all acetone is removed (5-10 washings). Then transfer ether soln to 100 mL vol. flask, dil. to vol., and mix. (b) ~pectrophotometric measurements.-Add ca teaspoon- ful (ca 5 mL) anhyd. Na2S0. to 60 mL reagent bottle, and fill it with ether soln of pigment. When this soln is optically clear, pipet aliquot into another dry bottle and dil. with enough dry ether to give A value of 0.2-0.8 at wavelength to be used. (Most favorable value is near 0.6 at 660 nm, since such soln yields satisfactory value at 642.5 nm.) Fill 2 clean g-s absorption cells with dry ether from pipet and polish outside surfaces of each, first with cotton wet with alcohol and then with dry cotton. Place cells in instrument, and det. whether each gives same galvanometer deflection. If not, clean again or select cells that do, and do this daily. Empty one cell, fill it with the dried ether soln, and place in instrument. Adjust entrance and exit slits until spectral region isolated is 3--4 nm at 660.0 nm. Det. whether instrument is in proper adjustment for wave- length by taking A readings thru soln against solv. at 1 nm intervals from 658 to 665 nm. Highest value should be at 660.0 nm; if not, adjust instrument until it is, or make 660.0 nm readings at wavelength setting that gave highestA. With grating instrument, apply same correction at 642.5 nm; however, with prism instrument, correction at 642.5 nm must be obtained from wavelength calibration curve for particular instrument in use. Calibrate instrument for wavelength in this way often enough to ensure that it remains in proper adjustment. Det. A at 660.0 and 642.5 nm (or corrected settings) for each unknown soln. (e) Calculation of chlorophyll concentration.- Calc. total chlorophyll and each of a and b components (mg/L) as follows: (1) Total chlorophyll =7.12A660•0 + 16.8A6• 2.•• (2) Chlorophyll a = 9.93 A 660.0 - 0.777 A6• 2.•• (3) Chlorophyll b = 17.6 A 6• 2•• - 2.81 A 660•0 • 3.133 Supplementary Information Factors involved in spectrophtric analysis of chlorophyll sys- tem have been discussed in detail by Comar and Zscheile (39). These authors used Beer's law in form: c = (log,o/o//)/a x t [= A/a x tl, where 10 is intensity of light transmitted by solv.-filled cell; I is intensity of light transmitted by soln-filled cell; c is concn of chlorophyll (g/L); a is absorptivity; t is thickness of soln layer in cm, and A is absorbance. Since, at given wavelength, observed A value of soln having 2 components represents sum of A values of each of compo- nents, following equation holds in ease of chlorophylls a and b at given wavelength: (4) ADbserved = A. + Ab If 1 cm cell is used, this equation may be expressed as: (5) ADbserved = aaCa + BbCb' Concns of chlorophylls a and b in given ether soln can now be calcd by equation (5) as follows: (a) Det. A for soln at 2 different wavelengths (660.0 and 642.5 nm have been found advantageous for this purpose). (b) From table select proper absorptivities corresponding to wavelengths used. (e) Substitute observed A value and absorptivities in equation (5) for each of the 2 wavelengths used as illustrated for 660.0 and 642.5 nm in equations (6) and (7). Solve these 2 equations simultaneously for 2 unknowns, the conens of chlorophylls a and b. (6) A 660 .0 = 102 c. + 4.50 cb • (7) A 6•2.• = 16.3 c. + 57.5 Cb' Equations (1), (2), and (3) were derived this way. Criterion for accuracy of chlorophyll values detd by spectro- phtric method is agreement between analytical results detd from measurements at different wavelengths. Comar and Zscheile (39) demonstrated that measurements at 660.0 and
- 73. AOAC METHODS (1980) TOBACCO 51 642.5 nm are convenient for routine analysis; however, readings may be made at other wavelengths to check these values. Absorptivities for chlorophylls a and b in ether soln that may be used for this purpose are presented in Table 3:03. These values may be used for calcns as follows: (a) Values for total chlorophyll and % composition may be calcd from A at 660.0 and 642.5 nm as described. (b) Check values for total chlorophyll may be calcd from A at intersection points 600.0, 581.0, and 568.0 nm. (c) Check values for % composition may be calcd from A for each of points 613.0 and 589.0 nm in combination with value of total concn obtained from (a) or (b). Table 3:03 Absorption constants used in analysis (after Comar and Zscheile (39)) Absorptivities (for Ether 501ns) Wavelength nm Chlorophyll a Chlorophyll b 660.0 642.5 600.0 581.0 568.0 613.0 589.0 102 16.3 9.95 B.05 7.11 15.6 5.90 3.134 Carotenes-Official Final Action See 43.014-43.023. TOBACCO Moisture (40)-Official Final Action 3.135 4.50 57.5 9.95 B.05 7.11 B.05 10.3 Apparatus (a) Drying oven.-Forced-draft, regulated to 99.5±OS. Sug- gested dimensions: 19 x 19 x 19" (48 cm). Approx. oven settings: fresh air intake vent 1/5 open; air control damper y.. open; air exhaust vent 'Ia open. (b) Moisture dish.-AI, diam. 45-65 mm, depth 20-45 mm, with tight-fitting cover. 3.136 Determination Accurately weigh ca 5 g sample (ground to pass",:l mm screen) into weighed moisture dish and place uncovered dish in oven. Do not exceed 1 sample/ 10 sq in. (650 sq cm) shelf space, and use only 1 shelf. Dry 3 hr at 99.5±0.5°. Remove from oven, cover, and cool in desiccator to room temp. (ca 30 min). Reweigh to nearest 1 mg and calc. % moisture. 3.137 Chlorides (41)-Official Final Action Potentiometric Method Reagent Silver nitrate std soln. -a.IN. Stdze against KCI as in detn. 3.138 Apparatus (a) pH meter.-Leeds and Northrup, Sumneytown Pike, N Wales, PA 19454, Beckman Instruments, or equiv., equipped with Ag and glass electrodes. (b) Buret.-l0 mL, graduated in 0.05 or 0.02 mL, preferably reservoir-type. 3.139 Determination Accurately weigh ca 2 g sample, ground to pass No. 40 sieve, into 250 mL electrolytic beaker. Add 100 mL H20, small amt at first to thoroly wet sample; then remainder. Let stand ~5 min at room temp., stirring intermittently. Pipet 5 mL HN03 (1 +9) into mixt. and insert clean electrodes. Start mag. stirrer and continue stirring thruout titrn at rate that produces vigorous agitation without spattering. Titr. with std O.lN AgN03 soln to potential previously established as equivalence point. Det. equiv- alence point potential graphically by making several titrns on one or more tobacco samples. Recheck occasionally, and redet. when either electrode is replaced. Record vol. of titrant and calc.: % CI = mL AgN03 x normality x 3.5453/g sample. 3.140 Nitrogen (42)-Official Final Action Kieldahl Method for Samples Containing Nitrates (For nitrate-free samples, omit salicylic acid and thiosulfate treatment.) See 2.055 and the following: Reagents (a) Sodium hydroxide-thiosulfate soln.-Dissolve 500 g NaOH pellets and 40 g Na2S203.5H20 in H20 and dil. to 1 L. (b) Indicators.-( 1) Dissolve 1 g Me red in 200 mL alcohol; or (2) prep. mixed indicator by dissolving 0.8 g Me red and 0.2 g methylene blue in 500 mL alcohol. 3.141 Apparatus See 2.056. 3.142 Determination Place weighed sample (1-2 g) in digestion flask. Add vol. H2S04 (contg 2 g salicylic acid/40 mL) corresponding to wt sample (35 mL for 1 g,40 mL for 2 g for N03-contg samples; 20 and 25 mL, resp., for N03-free samples). Shake until thoroly mixed; let stand ~30 min with occasional shaking; then add 5 g Na2S203.5H20. Shake, let stand 5 min, and heat carefully until frothing ceases. Turn off heat, add 0.7 g HgO (or metallic Hg) and 15 g K2S04, and boil briskly 1-1.5 hr after soln clears. Cool, add ca 200 mL H20, cool to ca room temp., and add few Zn granules. Tilt flask and carefully add 50 mL NaOH-thiosulfate soln without agitation. Immediately connect flask to distn bulb on condenser whose tip is immersed in 50 mL std O.lN acid in receiving flask. Then rotate digestion flask carefully to mix contents. Heat until ~150 mL distillate collects, and titr. excess acid with std base, using Me red or mixed indicator. Correct for blank detn on reagents. Potassium (43)-Official Final Action 3.143 Reagents (a) Potassium std solns.-(1) Stock soln.-l000 ppm K. See 3.020(a). (2) Working solns.-Place 0, 5, 10, 15, 20, 25, and 30 mL stock soln in seven 1 L vol. flasks, add 40 mL 3N HCI to each, and dil. to vol. with H20. (b) Diatomaceous earth.-Celite 545, acid-washed. 3.144 Apparatus (a) Flame photometer.-Natural gas-air fuel, or equiv., ade quate for K analysis. (b) Chromatographic tube.-20 x 150 mm with coarse fritted disk.
- 74. 52 3. PLANTS AOAC METHODS (1980) 3.145 Preparation of Sample Solution Accurately weigh ca 0.5 g tobacco dust into ca 40 mL weighing dish. Add ca 1 g Celite and mix intimately with spatula. Transfer quant. thru powder funnel into chromatgctube. Add addnl Celite thru funnel into tube until 2.5 cm layer accumulates on top of sample-Celite mixt. Compact sample and Celite by tapping tip of tube on table top, and insert tip of tube into neck of 1 L vol. flask. Add 40 mL 3N HCI into tube by pipet or dispenser, washing down sides, and let elute into vol. flask. When liq. level reaches top of Celite, add 25 mL H20 and let elute. Add second 25 mL portion H20, let elute by gravity, or force thru rapidly with compressed air. Rinse tip of tube into vol. flask, dil. to vol. with H20, and mix well. 3.146 Determination Det % Tfor sample eluate and K stds as specified in instruction manual of instrument. See also 3.023. Prep. calibration curve and det. ppm K of sample from curve. % K = ppm K x 0.1/g sample. % K20 = ppm K x 0.1205/g sample. 3.147 Glycerol, Propylene Glycol, and Triethylene Glycol in Cased Cigarette Cut Filler and Ground Tobacco (44)-Official First Action (Caution: See 51.018 and 51.066.) Apparatus (a) Gas chromatograph.-With programmed temp. oven and W hot wire detector; F&M Model 720 (current models 5700 series; Hewlett-Packard, Inc.), or equiv. Conditions: Detector bridge 140 ma; temps (0): injection 265, detector 280, column 90-240 at 15°/min; He 60 mL/min adjusted, if necessary, to facilitate sepns; attenuation 4, adjusted according to sensitivity to yield peaks of sufficient size for accurate measurement (use same attenuation for all stds and samples); chart speed, 12"/hr. (b) Column.-42 (105 em) x 3116" Cu tubing packed with 5% Carbowax 20M-terephthalic acid (TPA) on 60-80 mesh Chro- mosorb G AW-DMCS (Hewlett-Packard, Inc., No. 8501-6223 or Applied Science Laboratories, Inc., No. 04388). Prep. packing by placing 30.0 g Chromosorb in 500 mL l r-b flask. Add soln of 1.50 g Carbowax 20M-TPA in 150 mL CHCI3, and slurry. Remove CHCI3 under vac. in rotary evaporator and air dry overnight at room temp. Condition new column 2 hr at 240°; then inject three 30 ILL samples tobacco ext before analyzing samples. Recon- dition columns removed from app. before use. 3.148 Reagents (a) Extracting soln. -Oil. 20.0 mL 1,3-butylene glycol stock std soln, (b), to 2 L with anhyd. MeOH. (b) 1,3-Butylene glycol stock std soln.-Accurately weigh 20.00 g USP 1,3-butanediol into 100 mL vol. flask and dil. to vol. with anhyd. MeOH. (c) Glycerol stock std soln.-Accurately weigh 10.00 g USP glycerol into 100 mL vol. flask and dil. to vol. with extg soln. (d) Propylene glycol stock std soln.-Accurately weigh 5.00 g USP propylene glycol into 100 mL vol. flask and dil. to vol. with extg soln. (e) Triethylene glycol stock std soln.-Accurately weigh 5.00 g triethylene glycol into 100 mL vol. flask and dil. to vol. with extg soln. (f) Humectant std solns.-Into each of four 100 mL vol. flasks, pipet 1.0, 2.0, 3.0, and 4.0 mL, resp., glycerol, propylene glycol, and triethylene glycol stock std solns. Oil. to vol. with extg soln. Each soln contains (in mg/100 mL): 3.149 Soln 1 2 3 4 Propylene Glycol 50 100 150 200 Glycerol 100 200 300 400 Triethylene Glycol 50 100 150 200 Extraction Place 10.00 g sample in 250 mL l erlenmeyer. Pipet 100 mL extg soln into flask and stopper. Shake mech. 1 hr and let settle few min until supernate is clear. Alternatively, shake mech. 30 min and let stand overnight. 3.150 Determination Prime column by injecting two 30 ILL aliquots supernate ext. Then alternately inject 30 ,..L supernate exts and a humectant std soln until all samples and stds have been run, repeating ext injections, if necessary. (Sequence is ext" ext" std" ext" std2, ext2' std3, ext3' std., ext., std" exts, std2, etc. If <4 exts are available, distribute ext injections among those available so that sequence thru std. is run.) Det. peak hts and calc. ratios of propylene glycol, glycerol, and triethylene glycol to butylene glycol for each std and sample soln. Plot peak ht ratios against polyol concn for std solns and construct std curve for each humectant. Det. concn in mg/100 mL for propylene glycol, glycerol, and triethylene glycol in sample soln from resp. std curves. % Humectant = (mg/100 mL) x 0.01. Total Alkaloids (As Nicotine) Distillation Method (45)-Official Final Action 3.151 Apparatus (a) Distillation apparatus.-500 mL Kjeldahl flask fitted with inlet tube for steam, trap bulb, and condenser; Griffith still (Tobacco Sci. 1, 130(1957), available from Lab Glass, Inc., PO Box 5067, Kingsport, TN 37663); or other suitable steam distn app. (b) Spectrophotometer.-Beckman Instruments Model DU (replaced by Models 24/25) or other instrument capable of accurately measuring A in 200-300 nm range, equipped with 1 cm quartz cells. 3.152 Reagents (a) Alkali-salt soln.-Dissolve 300 g NaOH in 700 mL H20 and sat. with NaCI. (b) Silicotungstic acid soln (for gravimetric determination).- Dissolve 120 g Si02.12W03.26H20 in H20 and dil. to 1 L. (Soln should be clear and free from green color.) 3.153 Standardization (Caution: Nicotine is very toxic. Avoid contact with skin and eyes. See 51.011 and 51.015.) Purify best grade of nicotine com. available by successive vac. distns until center cuts from 2 successive distns have same a at 259 nm (ca 34.3). Accurately weigh ca 0.2 g purified nicotine; dissolve in and dil. to 1 L with ca 0.05N HCI. Oil. 10 mL aliquot of this soln to 100 mL with ca 0.05N HCI. Det. A at 259 nm and calc. a = A/(c x b), where c is concn of nicotine in gil and b is cell length in cm. 3.154 Distillation Accurately weigh 2-5 g tobacco sample and transfer to distn flask or app. (If final detn of nicotine is gravimetric, use sample
- 75. AOAC METHOOS (1980) TOBACCO 53 contg ~0.1 9 alkaloids; if spectrophtric, use ~2 9 sample.) (If Griffith still is used, use 0.05--0.2 9 sample.) Place 25 mL HCI (1 +4) in receiver (1 L vol. flask is desirable) and place receiver so that condenser tube dips into acid. (With Griffith still, use 10 mL HCI (1 +4) in 250 mL vol. flask.) Add 50 mL alkali-salt soln to distn flask so that sample is rinsed into bottom of flask. (With Griffith still, use 5 mL alkali-salt soln.) If large vol. of liq. is required for proper function of still, add more alkali-salt soln; do not dil. Connect flask to app. immediately and steam distil with as rapid current of steam as can be condensed efficiently. Effluent condensate should not be above room temp. Apply heat to distn flask from burner, mantle, or other heat source to keep vol. in flask approx. const. Collect ca 900 mL condensate (or distil addnl 100 mL after condensate shows no nicotine by silicotungstic acid test). (With Griffith stili, collect 225 mL.) Dil. distillate to vol. 3.155 Determination (a) Spectrophotometric.-Dil. aliquots of distillate (if neces- sary) with O.OSN HCI so that A at 259 nm is 0.5--0.8 and read A at 236, 259, and 282 nm. Calc. correctedA'259 = 1.059 x [observed A 259 - 'h (A236 + Am)] after correcting all observed A values to original distillate vol. basis. Concn, c, of alkaloids as nicotine in gjL is given by c = A'259 /(a x b), where a is absorptivity at 259 nm, and b is cell length in cm. % alkaloid (as nicotine) = c x vol. distillate (L) x 100/g sample. (b) Gravimetric.-Det. alkaloids in distillate as in 6.176, but double amt of silicotungstic acid specified, i.e., 2 mL/each 10 mg alkaloids expected. Cundiff-Markunas Method (45)-OHicial Final Action (Total alkaloids (as nicotine), tertiary alkaloids (as nicotine)' and secondary alkaloids (as nornicotine)) 3.156 Reagents (a) Benzene-chloroform soln.-Mix equal parts by vol. of benzene and CHCI3 and sat. with H20. (b) Sodium hydroxide soln.-36%. Dissolve 500 9 NaOH in H2 0 and dil. to 1 L. (c) Dilute acetic acid.-5%. Dil. 50 mL HOAc to 1 L with H20. (d) Crystal violet indicator.-Dissolve 0.5 9 crystal violet in 100 mL HOAc. (e) Perchloric acid std soln.---O.025N. Add 4.7 mL 72% HCIO. to freshly opened 51b bottle HOAc and mix. (Caution: See 51.022 and 51.028(a) and (d).) Stdze as follows: Accurately weigh 0.1 9 KH phthalate (NBS) into 125 mL erlenmeyer, add 50 mL HOAc, and heat to dissolve. Cool, add 2 drops indicator, and titr. to blue-green end point. Perform blank titrn on 50 mL HOAc and 2 drops indicator soln, and correct vol. of titrant. N = wt KH phthalate x 4.896/mL HCIO. soln. 3.157 Determination Accurately weigh 2.5 9 finely ground tobacco into 250 mL erlenmeyer. Add 15 mL 5% HOAc and swirl until tobacco is thoroly wetted. Pipet 100 mL benzene-CHCl3 soln into flask, and then 10 mL 36% NaOH soln. Stopper flask tightly and shake 20 min, using wrist-action shaker. Add 4.5-5 9 (2 teaspoonfuls) Filter-Cel, mix, and filter most of benzene layer thru Whatman No.2 paper into second flask. If filtrate has any turbidity, add 2-2.5 9 (1 teaspoonful) addnl Filter-Cel and refilter thru Whatman No.2 paper. Filtrate must be clear. Pipet 25 mL aliquots of filtrate into each of two 125 mL erlenmeyers. Pass stream of air over surface of soln in first flask 5 min, add 2 drops indicator, and titr. to green end point with 0.025N HCIO•. Add 1.0 mL Ac20 to second flask and let stand ~15 min. Add 25 mL HOAc and 2 drops indicator, and titr. to blue-green end point with 0.02SN HCIO•. Take first appearance of blue-green thruout soln as end point. For each series of analyses perform blank titrns and correct respective vols of titrant. Calc. % alkaloids as follows: % total alkaloids (as nicotine) = V, x N x 32.45/wt sample; % tertiary alkaloids (as nicotine) = (2V2 - V,) x N x 32.45/wt sample; % secondary alkaloids (as nornicotine) = 2(V, - V2 ) x N x 29.64/wt sample; where V, = vol. titrant for nonacetylated aliquot; V2 = vol. titrant for acet- ylated aliquot; and N = normality HCIO•. 3.158 Nicotine on Cambridge Filter Pads Gas-Liquid Chromatographic Method (46) Official First Action Apparatus and Reagents (a) Gas chromatograph.-With flame ionization detector, heated injection port, and thermostated column oven. Following conditions have been found satisfactory: Column, 1.8 m (6') x 'Is" stainless steel; packing, 2% KOH and 10% Carbowax 20M (based on final packing wt) on 45-60 mesh calcined diat. earth (such as Chromosorb W, or equiv.), resieved before use to mesh range to remove fines and lumps; temps (0): column 165, detector and injection port 200-250; carrier gas flow, ca 40 mL/min. Adjust H and air flows for max. sensitivity and stability. Under these conditions, column should have ht equiv. to the- oretical plate (HETP) <1 mm and resolution of >2, calcd with nicotine and anethole. (b) Measuring system.-Measure peak areas with electronic integrator or other system with resolution of ~1 count/mv-sec. (e) Mechanical shaker.-Capable of extg ~99% nicotine. Bur- rell Wrist-Action shaker has been found satisfactory. (d) Extracting soln.-2-Propanol contg 1 mg anethole/mL as internal std for nicotine. If H20 is also to be detd, add 20 mg ::tOH/mL 2-propanol as addnl internal std. (e) Nicotinestdsolns.-( 1) Stockso/n.-Weigh 2.500 9 nicotine, 3.153, or equiv. amt of nicotine salt. Transfer quant. into 100 mL vol. flask, and dil. to vol. with extg soln. (2) Working std solns.- Pipet 1,2,3,4, and 5 mL stock soln into five 100 mL vol. flasks, and dil. to vol. with extg soln (0.25, 0.50, 0.75, 1.00, and 1.25 mg nicotine/mL). (Caution: See precaution in 3.153.) 3.159 Extraction Place Cambridge filter material in flask or serum bottle ac- comodated by shaker used, add 10.00 mL extg soln, stopper, and shake until ~99% of nicotine is extd (usually ca 15 min). 3.160 Standardization Prime column with aliquots of 1.25 mgjmL std soln. Let baseline stabilize, inject 1 ILL each std soln in succession, and repeat sequence 3 times. Det. area ratio (nicotine:anethole) for each injection, and calc. slope and intercept of response curve, preferably by method of least squares (See Definition of Terms and Explanatory Notes No. (24)). Correlation coefficient should be ~0.99 and intercept ,,;;0.05 mgjmL. 3.161 Determination Prime column with aliquots of ext, 3.159. Let baseline stabilize, and inject 1 ILL of each sample soln. Calc. nicotine concn in soln (C, mg/mL) = mx + b, where m = slope of stdzn curve, b intercept, and x = area ratio of nicotine to anethole. Nicotine yield/cigaret = (C x 10.00)/(No. cigarets/pad)
- 76. 54 3. PLANTS AOAC METHODS (1980) 3.162 Menthol (47)-Official Final Action Colorimetric Method Apparatus and Reagents (a) Distillation apparatus.-8ee Fig. 3:07. (b) Spectrophotometer.-With matched cells; capable of measuring A at 550 nm. (c) Menthol std soln.-l mg/mL. Accurately weigh 100 mg USP I-menthol into 100 mL vol. flask, add alcohol to dissolve, and dil. to vol. with alcohol. (d) DMAB color reagent.-Dissolve 0.5 g p-dimethylamino- benzaldeh)-de (Eastman Kodak, white label) in 100 mL H2S04 (1.6+1). 3.163 Preparation of Calibration Curve Prep. dil. stds by pipeting aliquots contg 0, 3, 4, 6, 8, and 10 mg menthol into 100 mL vol. flasks and dilg to vol. with alcohol (1+1). Pipet 1 mL each dil. std into 10 mL test tube, add 5 mL color reagent, mix, and place in boiling H20 bath exactly 2 min. Cool in tap H20, and within 15 min det. A at 550 nm against 0 std. Prep. calibration curve by plotting A against menthol concn (mg/l00 mL). 3.164 Determination Accurately weigh 2.00-2.15 g cigaret filler and transfer to distn flask, A. Add 80 mL H20 and few boiling stones, connect flask to condenser with tube, B, attach adapter, C, to condenser, and immerse tip in 20 mL alcohol in 100 mL vol. receiving flask. Gently heat distn flask until distn begins; then increase heat and lower receiving flask, 0, so tip of adapter is no longer immersed. Distil until 20 mL distillate collects. Disconnect con- denser from tube, and wash down condenser with alcohol. Remove receiving flask, dil. distillate to ca 70 mL with alcohol, and add H2 0 almost to vol. Mix, add alcohol to vol., and mix again. Pipet 1 mL distillate into 10 mL test tube, add 5 mL color reagent, mix, and place in boiling H20 bath exactly 2 min. Cool in tap H20, and within 15 min det. A at 550 nm, using "color" soln from nonmentholated tobacco carried thru detn as blank. (If nonmentholated sample corresponding to mentholated sam- ple is not available, use reagent blank.) Use nonmentholated 20. MM o..D. TUBING '$' 24/40. 500 ML A '$' 24/40. 8MM.o.D, TUBING D 100 ML FIG. 3:07~istillation apparatus; see 3.164 for explanation of symbols tobacco blank within 15 min after color development step. Fresh nonmentholated tobacco blank soln may be required during multiple sample runs, Det. mg menthol from calibration curve. % Menthol = mg menthol!(g original sample x 10). Gas Chromatographic Method 3.165 Apparatus and Reagents (a) Gas chromatograph.-Equipped with flame ionization de- tector and thermostated injection port and column oven. Use following conditions for analysis: Column, 1.5 m (5') x '.Is" od stainless steel packed with 10% (w/w) silicone oil DC-550 on 6O-S0 mesh Chromosorb W; temps (0): column 150, detector 150, injection port 175; N carrier gas flow ca 35 mL/min. Adjust H and air flows for max. sensitivity and reasonable stability. (b) Mechanical shaker.-Wrist action. (c) Menthol-anethole std soln.-O.250 mg menthol and 0.50 mg anethole/mL. Weigh exactly 0.5000 g tech. grade anethole and wash into 1 L vol. flask with 200 mL alcohol. Transfer 0.2500 g USP I-menthol to the vol. flask with enough alcohol to bring to vol. Store soln in dark g-s bottle. Do not use >6 weeks. (d) Extracting soln.-o.50 mg anethole/mL. Dissolve 1.000 g anethole in alcohol in 2 L vol. flask, dil. to vol. with alcohol, and store in dark. 3.166 Determination of Ratio Factor Weigh ca 3 g nonmentholated control filler, contg all usual humectants but no menthol or anethole, into 125 mL rubber- stoppered flask. Pipet 50 mL std menthol-anethole soln into flask, stopper, and shake 1 hr on mech. shaker. Let solids settle 15 min and chromatograph 2 iLL aliquot of supernate. Repeat twice more to obtain total of 3 replicates of std chromatogram. For quant. results, inject both std and unknown samples by inserting 2" (5 em) needle to hilt, injecting 2 iLL rapidly, and withdrawing needle at once. (Menthol elutes in ca 3 min, anethole in ca 5 min.) After ca 10 min, all other compds are eluted and new injection can be made. Draw baselines under menthol and anethole peaks and meas- ure peak hts in mm. Using mean peak ht of menthol and anethole from 3 std chromatograms, calc. std ratio factor of menthol to anethole as follows: Std ratio factor = peak ht for menthol (0.25 mg/mL)/peak ht for anethole (0.50 mg!mL). 3.167 Determination Accurately weigh 8-8.5 g mentholated cigarette filler and place in 250 mL rubber-stoppered erlenmeyer. Pipet 100 mL extg soln into flask, stopper, and mech. shake 2 hr. Let solids settle 15 min and chromatograph 2 iLL aliquot of supernate. Draw baselines under menthol and anethole peaks and measure peak hts in mm. Calc. ratio factor of unknown menthol as follows: Ratio factorfor unknown = peak ht for unknown menthol! peak ht for anethole (0.50 mg!mL). % Menthol = (unknown ratio factor x 0.25 x 10)/(std ratio factor x g sample). SELECTED REFERENCES (1) Botan. Gaz. 73,44(1922); Proc. Am. Soc. Hort. Sci. 1927, p. 191; JAOAC 13,224(1930); 16,71(1933); 19,70(1936). (2) JAOAC 58, 436(1975). (3) JAOAC 11,203(1928); 16, 70(1933); 19, 70(1936). (4) Ind. Eng. Chern., Anal. Ed. 9, 67(1937); 10, 13(1938); JAOAC 25,555(1942); 27, 526(1944). (5) JAOAC 19,359(1936); 27, 526(1944).
- 77. AOAC METHODS (1980) SELECTED REFERENCES 55 (6) J. BioI. Chern. 7,83(1910); JAOAC4,392(1921); 16,70(1933). (7) J. BioI. Chern. 47, 475(1921); 50, 527, 537(1922); JAOAC 14,216(1931); 16,71(1933); 19,71(1936). (8) JAOAC 3, 329(1920); 4, 393(1921); 16, 71(1933); 19, 71 (1936). (9) JAOAC 4, 393(1921). (10) JAOAC 39, 419(1956). (11) JAOAC 19, 71(1936). (12) J. Am. Chern. Soc. 51, 1664(1929); JAOAC 19, 71(1936). (13) JAOAC 34, 710(1951); 36,405(1953). (14) JAOAC 36, 405(1953). (15) Ind. Eng. Chern., Anal. Ed. 13, 145(1941); JAOAC 24, 520(1941). (16) JAOAC 36, 397(1953). (17) JAOAC 36,412(1956); 41,309(1958); 43,511(1960). (18) USDA Bur. Chern. Bull. 105, p. 151; 116, p. 92; 137, p. 30. (19) JAOAC 6, 415(1923). (20) JAOAC 16,71(1933). (21) JAOAC 14,216(1931); J. BioI. Chern. 59, 255(1924). (22) JAOAC 49,284(1966). (23) JAOAC 11,209(1928); 12, 195(1929); 21,107(1938). (24) Sutton, "Systematic Handbook of Volumetric Analysis," 11th ed., 1924, p. 146; J. Am. Chern. Soc. 37,1128(1915). (25) JAOAC 18,379(1935); 19,72(1936). (26) JAOAC 58,1129(1975). (27) JAOAC 55, 991(1972); 61, 150(1978). (28) JAOAC 19, 236(1936). (29) JAOAC 52,627(1969). (30) JAOAC 41,304(1958). (31) JAOAC 14,73,225(1931); 15,71(1932). (32) JAOAC 36, 402(1953). (33) JAOAC 41, 307, 681(1958); 42,650(1959); 43, 512(1960); 44,267(1961). (34) Anal. Chern. 20, 850(1948); JAOAC 39, 423(1956). (35) JAOAC 15, 124(1932); 18,386(1935); 19, 107 (1936). (36) JAOAC 32,288(1949). (37) Ind. Eng. Chern., Anal. Ed. 12, 148(1940); 15,524(1943). (38) Ind. Eng. Chern., Anal. Ed. 14, 877(1942); JAOAC 27, 517(1944). (39) Plant Physiol. 17, 198(1942). (40) JAOAC 49,525(1966). (41) JAOAC 46, 415(1963). (42) JAOAC 42,302(1959). (43) JAOAC 49,521(1966). (44) JAOAC 54,560(1971). (45) JAOAC 43,524(1960). (46) JAOAC 62.229(1979). (47) JAOAC 51.650(1968).
- 78. 4. Disinfectants Phenol Coefficient (1)-Official Final Action (Applicable to testing disinfectants miscible with H20 that do not exert bacteriostatic effects that cannot be neutzd by one of subculture media specified, or overcome by suitable subtransfer procedures. The 95% confidence limits are ±12%.) 1. Using Salmonella typhi 4.001 Culture Media (a) Nutrient broth.-Boil 5 9 beef ext (Difco), 5 9 NaCI, and 10 9 peptone (Anatone, peptic hydrolysate of pork tissues, manu- factured by American Laboratories, Inc., 4410 S 102nd St, Omaha, NB 68127) in 1 L H20 20 min, and dil. to vol. with H20; adjust to pH 6.8. (If colorimetric method is used, adjust broth to give dark green with bromothymol blue.) Filter thru paper, place 10 mL portions in 20 x 150 mm test tubes, and autoclave 20 min at 121°. Use this broth for daily transfers of test cultures. (b) Synthetic broth.-$oln A: Dissolve 0.05 9 L-cystine, 0.37 9 DL-methionine, 0.4 9 L-arginine.HCI. 0.3 9 DL-histidine.HCI, 0.85 9 L-lysine.HCI. 0.21 9 L-tyrosine, 0.5 9 DL-threonine, 1.0 9 DL- valine, 0.8 9 L-Ieucine, 0.44 9 DL-isoleucine, 0.06 9 glycine, 0.61 9 DL-serine, 0.43 9 DL-alanine, 1.3 9 L-glutamic acid.HCI. 0.45 9 L- aspartic acid, 0.26 g DL-phenylalanine, 0.05 9 DL-tryptophan, and 0.05 9 L-proline in 500 mL H20 contg 18 mL 1N NaOH. Soln B: Dissolve 3.0 9 NaCI, 0.2 9 KCI. 0.1 9 MgS04.7H20, 1.5 9 KH 2P04, 4.0 9 Na2HP04 , 0.01 9 thiamine.HCI, and 0.01 9 niacinamide in 500 mL H20. Mix Solns A and B, dispense in 10 mL portions in 20 x 150 mm tubes, and autoclave 20 min at 121°. Before using for daily transfers of test cultures, aseptically add 0.1 mL sterile 10% glucose soln per tube. Grow cultures with tube slanted 8° from horizontal. (c) Nutrient agar.-Dissolve 1.5% Bacto agar (Difco) in nu- trient broth and adjust to pH 7.2-7.4 (blue-green with bromo- thymol blue) or in synthetic broth, tube, autoclave, and slant. (d) Subculture media.-Use (1), (2). or (3). whichever gives lowest result. (Com. dehydrated brands made to conform with preceding specifications may be used.) With oxidizing products and products formulated with toxic compds contg certain heavy metals like Hg, (2) will usually give lowest result. With products contg cationic surface active materials, (3) will usually give lowest result. See also 4.009, par. 5. (1) Nutrient broth described in (a); (2) Fluid thioglycolate medium USP XX: Mix 0.5 9 L-cystine, 0.75 9 agar, 2.5 9 NaCI, 5.5 9 glucose.H20, 5.0 9 H20-sol. yeast ext, and 15.0 9 pancreatic digest of casein with 1 L H20. Heat on H20 bath to dissolve, add 0.5 9 Na thioglycolate or 0.3 9 thioglycolic acid, and adjust with 1N NaOH to pH 7.1 ±0.2. If filtration is necessary, reheat without boiling and filter hot thru moistened filter paper. Add 1.0 mL freshly prepd 0.1 % Na resazurin soln, transfer 10 mL portions to 20 x 150 mm tubes, and autoclave 20 min at 121°. Cool at once to 25° and store at 20-30°, protected from light. (3) "Letheen broth": Dissolve 0.7 9 lecithin (Azolectin, As- sociated Concentrates, 32-30 61 st St, Woodside, NY 11377) and 5.0 9 polysorbate 80 (Tween 80, or equiv.) in 400 mL hot H20 and boil until clear. Add 600 mL soln of 5.0 9 beef ext (DifcoL 10.0 9 peptone (Anatone, (a)L and 5 9 NaCI in H20, and boil 10 min. Adjust with 1N NaOH and/or 1N HCI to pH 7.0 ±0.2 and filter thru coarse paper; transfer 10 mL portions to 20 x 150 mm tubes, and autoclave 20 min at 121°. 56 (4) Cystine trypticase agar (BBL): Suspend 29.5 9 in 1 L H20. Heat gently with frequent agitation and boil ca 1 min or until soln is complete. Transfer 10 mL portions to 20 x 150 mm tubes, and autoclave 15 min at 121b pressure. Cool in upright position and store ~25 days at 20-30°. Use for monthly transfer of stab stock cultures of Ps. aeruginosa PRD 10 (ATCC 15442). (5) Other subculture media: Use 4.001 (d)(2) with 0.7 9 lecithin (Azolectin, Associated Concentrates, Inc., 32-30 61 st St, Wood- side, NY 11377) and 5.0 9 polysorbate 80 (Tween 80, or equiv.) added; or suspend 29.8 9 prepd fluid thioglycolate medium (DifcoL 0.7 9 lecithin, and 5.0 9 polysorbate 80 in 1 L H20, and boil until soln is clear. Cool. dispense in 10 mL portions in 20 x 150 mm tubes, and autoclave 20 min at 121°. Store at 20-30°. Protect from light. 4.002 Apparatus and Reagents (a) Glassware.-1, 5, and 10 mL vol. pipets; 1, 5, and 10 mL Mohr pipets graduated to 0.1 mL or less; 100 mL g-s cylinders graduated in 1 mL divisions; Pyrex lipped test tubes, 25 x 150 mm (medication tubes); bacteriological culture tubes, 20 x 150 mm (test culture and subculture tubes). Plug medication tubes with cotton wrapped in 1 layer of cheese cloth. Sterilize all glassware 2 hr in hot air oven at 180°. Loosely plug pipets with cotton at mouth and place in closed metal containers before sterilizing. (b) Water bath.-Insulated, relatively deep H20 bath, with cover having ;;;.10 well-spaced holes which admit medication tubes but not their lips. (c) Racks.-Any convenient style. Blocks of wood (size de- pending on space in incubator) with deep holes are satisfactory. Have holes well spaced to ensure quick manipulation of tubes. It is convenient to have them large enough to admit medication tubes while dilns are being made. (d) Transfer loop.-Make 4 mm id single loop at end of 50-75 mm (2-3") Pt or Pt alloy wire No. 23 B&S gage or 4 mm loop fused on 75 mm (3") shaft (available from Matthey-Bishop, Inc., Malvern, PA 19355). Fit other end in suitable holder (glass or AI rod). Bend loop at 30° angle with stem, Fig. 4:01. (e) Test organism.-Hopkins strain 26 of Salmonella typhi (Schroeter) Warren and Scott, ATCC No. 6539 (formerly called Bac. typhosus and Eberthella typhosa). Maintain stock culture on nutrient agar slants by monthly transfers. Incubate new stock transfer 2 days at 37°; then store at 2-5°. From stock culture inoculate tube of nutrient broth and make at least 4 consecutive daily transfers (~30) in nutrient broth, incubating at 3r, before using culture for testing. (If only 1 daily transfer has been missed, it is not necessary to repeat the 4 consecutive transfers.) Use 22-26 hr culture of organism grown in nutrient broth at 3r in test. Shake, and let settle 15 min before using. With Ps. aeruginosa PRD 10, proceed as in 4.011. (f) Phenol stock soln.-5% (w/v). Weigh 50 9 USP phenol, which congeals at ;;;.40°, in beaker. Dissolve in H20, rinse soln into 1 L vol. flask, and dil. to vol. Stdze with 0.1N KBr-KBr03 soln, (g), as follows: Transfer 25 mL stock soln to 500 mL vol. flask and dil. to vol. with H20. Transfer 15 mL aliquot of dild soln to 500 mL I flask and add 30 mL std KBr-KBr03 soln. Add 5 mL HCI and immediately insert stopper. Shake frequently during 30 min and let stand 15 min. Remove stopper just enough to quickly add 5 mL 20% KI soln, taking care that no Br vapors escape, and immediately stopper flask. Shake thoroly, remove
- 79. AOAC METHODS (1980) PHENOL COEFFICIENT 57 FIG. 4:01-Transfer loop and manner of using In phenol coefficient technic stopper, and rinse it and neck of flask with little H20 so that washings flow into flask. Titr. with 0.1N Na2S20 3, using starch indicator, 6.005(f). 1 mL 0.1N KBr-KBr03 = 0.001569 g phenol. % phenol in stock soln = (30 - mL 0.1N Na2S20 3 soln from titrn) x 0.001569 x 1333 x 100/1000; where 30 = mL 0.1N KBr-KBr03 soln added, 0.001569 = g phenol equiv. to 1 mL 0.1N KBr-KBr03 soln, 1333 = diln factor, and 1000 = original vol. phenol stock soln. If necessary, adjust stock soln to 5.00±0.05% phenol by adding H20 or phenol. Keep in well stoppered amber bottles in cool place, protected from light. (g) Potassium bromide-bromate soln.-0.1N. Prep. as in 50.020. Stdze as follows: Transfer 30 mL to I flask, and add 25 mL H20, 5 mL 20% KI soln, and 5 mL HCI. Shake thoroly and titr. with 0.1N Na2S20 3, using starch indicator, 6.005(f). 4.003 Operating Technic Make 1% stock diln of substance to be tested (or any other convenient diln, depending on anticipated concn) in g-s cylinder. Make final dilns, from 1% stock diln, directly into medication tubes and remove all excess >5 mL. (Range of dilns should cover killing limits of disinfectant in 5-15 min and should at same time be close enough for accuracy.) From 5% stock phenol soln (1-20) dil. further to make 1-90 and 1-100 dilns, and place in medication tubes. Place these tubes, contg 5 mL each of final dilns of disinfectant and of phenol, and tube contg test culture in H20 bath at 20° and leave 5 min. Add 0.5 mL test culture to each of dilns at time intervals corresponding to intervals at which transfers are to be made. (Thus, by time 10 tubes have been seeded at 30 sec intervals, 4.5 min has elapsed, and 30 sec interval intervenes before transference to subculture begins.) Add culture from graduated pipet large enough to seed all tubes in anyone set. In using Ps. aeruginosa PRD 10 (ATCC 15442), proceed as in 4.011. In inoculating medication tubes, hold them in slanting position after removal from bath, insert pipet to just above surface of disinfectant, and run in culture without letting tip touch disin- fectant. After adding culture, agitate tubes gently but thoroly to insure even distribution of bacteria, and replace in bath; 5 min after seeding first medication tube, transfer 1 loopful of mixt. of culture and dild disinfectant from medication tube to corre- sponding subculture tube. To facilitate transfer of uniform drops of medication mixt., hold tube at 60° angle, and withdraw loop so that plane of loop is parallel with surface of liq. (Fig. 4:01). After 30 sec, transfer loopful from second medication tube to second subculture tube and continue process for each successive diln; 5 min after making first transfer, begin second set of transfers for 10 min period, and finally repeatfor 15 min period. Gently agitate medication tubes before taking each interval loop subsample for transfer to subculture medium. Before each transfer, heat loop to redness in flame and flame mouth of every tube. Sterilize loop immediately after each transfer (before replugging tubes) to allow time for cooling. Use care in trans- ferring and seeding to prevent pipet or needle from touching sides or mouth of medication tube, and see that no cotton threads adhere to inner sides or mouths of tubes. Incubate subculture 48 hr at 3r and read results. Thoroly agitate individual subculture tubes before incubation. Macroscopic examination is usually sufficient. Occasionally 3-day incubation period, agar streak, microscopic examination, or agglutination with antity- phoid serum may be necessary to det. feeble growth or sus- pected contamination. 4.004 Calculation Express results in terms of phenol coefficient number, or highest diln killing test organism in 10 min but not in 5 min, whichever most accurately reflects germicidal value of disinfec- tant. Phenol coefficient is number obtained by dividing numerical value of greatest diln (denominator of fraction expressing diln) of disinfectant capable of killing S. typhi in 10 min but not in 5 min by greatest diln of phenol showing same results. Example: Disinfectant (X): Diln 5 Min 10Min 15 Min 1-300 0 0 0 1-325 + 0 0 1-350 + 0 0 1-375 + + 0 1-400 + + + Phenol: 1- 90 + 0 0 1-100 + + + Phenol coefficient would be 3 9 5 0 0 =3.89. Test is satisfactory only when phenol control gives one of following readings: Phenol 1- 90 1-100 5 Min + or 0 + 10Min + or 0 + 15 Min o + or 0 If none of dilns of disinfectant shows growth in 5 min and killing in 10 min, est. hypothetical diln only when any 3 consec- utive dilns show following results: first, no growth in 5 min; second, growth in 5 and 10 min but not in 15 min; and third, growth in 5, 10, and 15 min. Example: Diln 1-300 1-350 1-400 Disinfectant (X): 5 Min 10 Min o 0 + + Phenol: + + 15 Min o o + 1- 90 0 0 0 1-100 + + 0 . . 325 Phenol coeffiCient would be 95 =3.42.
- 80. 58 4. DISINFECTANTS AOAC METHODS (1980) To avoid giving impression of fictitious accuracy, calc. phenol coefficient to nearest 0.1. Thus, in examples cited above, phenol coefficients would be reported as 3.9 and 3.4, instead of 3.89 and 3.42. Note: Although it is commonly accepted criterion that disin- fectants be at diln equiv. in germicidal efficiency to phenol against S. typhi by calcg 20 x S. typhi coefficient to det. number of parts H20 in which 1 part disinfectant may be mixed, this should be regarded as presumptive and is subject to confir- mation by Use-Diln Method. 4.005 2. Using Staphylococcus aureus (1) Proceed as in 4.001-4.004, except change phenol dilns and test organisms. Use 22-26 hr culture of Staph. aureus FDA 209, ATCC No. 6538, having at 20° at least resistance indicated by following: Phenol 1-60 1-70 5 Min + or 0 + 10 Min + or 0 + 15 Min o + Note: Calc. results as in 4.004. If conversion 20 x Staph. aureus coefficient is used to det. number of parts H20 in which 1 part germicide may be incorporated to disinfect where py- ogenic organisms are objective, this diln is subject to confir- mation by Use-Diln Method. 4.006 3. Using Pseudomonas aeruginosa Official First Action Proceed as in 4.001-4.004. Use 22-26 hr culture of Ps. aeru- ginosa PRO 10 (ATCC 15442). having resistance to phenol at 20° at least as follows: Phenol 1-80 1-90 5 Min + or 0 + 10 Min + or 0 + 15 Min o + Use-Dilution Method (2)-Official Final Action (Applicable to testing disinfectants miscible with H20 to confirm phenol coefficient results and to det. max. dilns effective for practical disinfection) 1. Using Salmonella choleraesuis 4.007 Reagents (a) Culture media. -See 4.001. (b) Test organism, Salmonella choleraesuis.- (ATCC 10708). Maintain stock culture on nutrient agar slants by monthly transfers. Incubate new stock transfer 2 days at 37°; then store at 2-5°. From stock culture inoculate tube of nutrient broth and incubate at 3r. Make 3 consecutive 24 hr transfers; then inoculate tubes of nutrient broth (2 for each 10 carriers to be tested). using one loop of inoculum with each tube; incubate 48-54 hr at 3r. (c) Phenol.-See 4.002(f). (d) Sterile distilled water.-Prep. stock supply of H20 in 1 L flasks, plug with cotton, sterilize 20 min at 121°, and use to prep. dilns of medicants. (e) Asparagine soln. -Make stock supply of 0.1 % asparagine ("8acto") soln in H20 in erlenmeyer of convenient size, plug with cotton, and sterilize 20 min at 121°. Use to cover metal carriers for sterilization and storage. (f) Sodium hydroxide soln.-Approx. 1N (4%). (For cleaning metal carriers before use.) 4.008 Apparatus (8) Glassware.-As in 4.002(8). Also: straight side Pyrex test tubes,20 x 150 mm; 15 x 110 mm petri dishes; 100 mL, 300 mL, and 1 L erlenmeyers. Sterilize petri dishes in closed metal containers. (b) Water bath and racks.-See 4.002(b) and (c). (c) Transfer loops and needles.-(1) See 4.002(d). (2) Make 3 mm right angle bend at end of 50-75 mm nichrome wire No. 18 B&S gage. Have other end in suitable holder (glass or AI rod). (d) Carriers.-Polished stainless steel cylinders (penicillin cups), 8±1 mm od, 6±1 mm id, length 10±1 mm, of type 304 stainless steel, SS 18-8. (Obtainable from S. & L. Metal Products Corp., 58-29 57 Drive, Maspeth, NY 11378.) (e) Petri dishes.-Have available ca 6 sterile petri dishes matted with 2 layers of S&S No. 597 or Whatman No.2, 9 cm fi Iter paper. 4.009 Operating Technic Soak ring carriers overnight in 1N NaOH, rinse with tap H20 until rinse H20 is neut. to phthln, then rinse twice with distd H20; place cleaned ring carriers in multiples of 10 in cotton- plugged erlenmeyers or 25 x 150 mm cotton plugged Pyrex test tubes, cover with asparagine soln, 4.007(e), sterilize 20 min at 121°, cool. and hold at room temp. Transfer 20 sterile ring carriers, using flamed nichrome wire hook, into 20 mL 48-54 hr nutrient broth test culture in sterile 25 x 150 mm medication tube. After 15 rnin contact period remove cylinders, using flamed nichrome wire hook, and place on end in vertical position in sterile petri dish matted with filter paper, 4.008(e). Cover and place in incubator at 37° and let dry :;.20 min but :;;60 min. Hold broth culture for detn of its resistance to phenol by phenol coefficient method, 4.003. From 5% stock phenol soln (1-20) make 1-90 and 1-100 dilns directly into medication tubes. Place tube for each diln in H20 bath and let come to 20". Make stock soln of germicide to be tested in sterile g-s cylinder. From this soln make 10 mL dilns to be tested, depending upon phenol coefficient found and/or claimed against S. typhi at 20°, directly into each of ten 25 x 150 mm medication tubes; place the 10 tubes in H20 bath at 20° and let come to temp. Det. diln to be tested by multiplying phenol coefficient number found and/or claimed by 20 to det. number of parts H20 in which 1 part germicide is to be incorporated. This detn is not required when disinfectant under test yields phenol coefficient that cannot be converted validly to presump- tive use-diln, or when analyst dets that use-diln range can be found without resort to phenol coefficient test. Add 0.5 mL of test culture suspension to 1-90 diln of phenol control; after 30 sec interval, add 0.5 mL to 1-100 diln of control, using sterile cotton-plugged pipets. After adding culture, agitate tubes gently but thoroly to distribute bacteria evenly, and replace in bath; 5 min after seeding first medication tube, transfer 1 loopful of mixt. of culture and dild phenol from medication tube to corresponding subculture tube. After 30 sec, transfer loopful from second medication tube; 5 min after making first set of transfers begin second set of transfers for 10 min period; and finally repeat for 15 min period. Use technic of loop sampling, flaming loop and mouths of tubes, and agitating medication and subculture tubes as in phenol coefficient method, 4.003. Incubate subcultures 48 hr at 3r and read results. Resistance in 48-54 hr culture of S. choleraesuis should fall within range specified for 24 hr culture of S. typhi in phenol coefficient method. Add 1 contaminated dried cylinder carrier at 1 min intervals to each of the 10 tubes of use-diln of germicide to be tested. Thus, by time 10 tubes have been seeded, 9 min will have elapsed, plus 1 min interval before transfer of first carrier in series to individual tube of subculture broth. This interval is const for each tube with prescribed exposure period of 10 min.
- 81. AOAC METHODS (1980) AVAILABLE CHLORINE GERMICIDAL EQUIVALENT 59 The 1 min interval between transfers allows adequate time for flaming and cooling nichrome wire hook and making transfer in manner so as to drain all excess medication from carrier. Flame lips of medication and subculture tubes in conventional manner. Immediately after placing carrier in medication tube, swirl tube 3 times before placing it back in bath. Thoroly shake subculture tubes, incubate 48 hr at 3r, and report results as + (growth) or - (no growth) values. Where there is reason to suspect that lack of growth at conclusion of incubation period may be due to bacteriostatic action of medicant adsorbed on carrier that has not been neutzd by subculture medium used, transfer each ring to new tube of sterile medium and reincubate for addnl 48 hr at 37°. Where soln under test is such that material adsorbed on ring carriers and transferred into subculture medium makes it unsuitable for growth of test organism, as may be case with concd acids and alkalies, products carrying antibiotics, and wax emulsions, trans- fer each ring to new tube of sterile medium 30 min after initial transfer and incubate both primary and secondary subculture tubes 48 hr at 37°. Results showing no growth on all 10 carriers will confirm phenol coefficient number found. Results showing growth on any of the 10 carriers indicate phenol coefficient number to be unsafe guide to diln for use. In latter case, repeat test, using lower dilns (higher concns) of germicide under study. Max. diln of germicide which kills test organism on 10 carriers in 10 min interval represents presumed max. safe use-diln for practical disinfection. 4.010 2. Using Staphylococcus aureus Proceed as in 4.009 except change phenol dilns and test organism to those specified in 4.005. Use 48-54 hr culture of Staph. aureus FDA 209, ATCC No. 6538, having at least resistance specified for 24 hr culture at 20° in phenol coefficient method, 4.005. Results showing growth on any of 10 carriers indicate that diln is too high for use in disinfecting where pyogenic bacteria must be killed. In such cases repeat test, using lower dilns (higher concns). Max. diln of germicide which kills both this test organism and S. choleraesuis on 10 carriers in 10 min interval represents max. presumed safe use-ailn for disinfecting in hospitals, clinics, and other places where pyogenic bacteria have special significance. Note: While killing in 10 of 10 replicates specified provides reasonably reliable index in most cases, killing in 59 out of 60 replicates is necessary for confidence level of 95%. 4.011 3. Using Pseudomonas aeruginosa Official First Action Proceed as in 4.009. Use 48-54 hr nutrient broth culture Ps. aeruginosa PRO 10 (ATCC 15442). Carry stock culture on BBl CTA (cystine trypticase agar) in stab culture incubated 48 hr at 3r and stored at 5° with transfer every 30 days. Transfer nutrient broth test cultures daily for 30-day intervals with incubation at 3r. Make fresh transfer from stock culture every 30 days. Do not shake 48-54 hr test culture but decant liq. culture aseptically, leaving pellicle behind, to obtain 20 ml culture for inoculating 20 carriers in medication tube. Available Chlorine Germicidal Equivalent Concentration (3l-Official Final Action (Applicable to H20-miscible disinfectants for detg available CI germicidal equiv. concns with products offered for use as sanitizing rinses for previously cleaned nonporous surfaces, especially where speed of action and capacity are essential considerations) 4.012 Reagents Use reagents specified in 4.001 and 4.002(e} and (f). and in addn: (a) Sterile distilled H2D.-See 4.007(d). (b) Sterile phosphate buffer soln.-pH 8.0. Add 97.5 mL soln contg 11.61 g anhyd. K2HPO. in 1 l H20 to 2.5 ml soln contg 9.08 g anhyd. KH2PO. in 1 l H20 and autoclave 20 min at 121° in cotton-plugged erlenmeyer. (c) NaDel std stock soln.-Approx. 5%. Store NaOCI stock soln in tightly closed bottle in refrigerator, and det. exact available CI concn at frequent intervals by As20 3 titrn, 6.112. (d) Test organisms.-Use S. typhi ATCC No. 6539 or Staph. aureus ATCC No. 6538 or both. 4.013 Apparatus See 4.002. 4.014 Operating Technic Det. resistance of test culture to phenol as in 4.001-4.005, and use cultures with resistance specified. Prep., in sterile g-s cylinders, NaOCI solns contg 200, 100, and 50 ppm available CI in sterile buffer soln, 4.012(b). Transfer 10 ml of each soln to 25 x 150 mm medication tubes, place tubes in 20° H20 bath, and let come to temp. Starting with tube contg 200 ppm available CI, add 0.05 ml test culture prepd as in 4.002(e). shake, and return to H2 0 bath. After 1 min, make transfer to tube of appropriate subculture medium, 4.001(d). using flamed 4 mm loop. At 1.5 min, add another 0.05 mL culture to the 200 ppm CI soln, shake, and return to bath. After addnl 1 min interval (2.5 min in test). make second subculture in same manner, and in 30 sec, or at 3 min time in test, add another 0.05 ml culture, shaking and returning to H20 bath. After another 1 min interval (4 min in test). make another transfer to tube of subculture medium. Repeat operation to give total of 10 added increments. This requires total time of 14.5 min for each soln and addn of 0.5 ml total culture with subculture at std 1 min intervals after addn of culture aliquots. At conclusion of test shake all subculture tubes and incubate 48 hr at 3r. Repeat operation with solns contg 100 and 50 ppm available CI. Prep. soln of germicide to be tested at concn recommended Table 4:01 Example for Determination of Chlorine Germicidal Equivalent Concentration Subculture Series Concn, ppm Germicide Avail. CI 2 3 4 5 6 7 8 9 10 200 + + + + + NaOCI control 100 + + + + + + + 50 + + + + + + + + 25 + + + + + Unknown (X) 20 + + + + + + 10 + + + + + + + + + - = No growth + = growth
- 82. 60 4. DISINFECTANTS AOAC METHODS (1980) or selected for study in sterile H2 0 in g-s graduate. Transfer 10 mL to 25 x 150 mm medication tubes, place in H20 bath, and let come to temp. Repeat operation with this soln. To be considered equiv. in disinfecting activity to 200 ppm available CI, unknown germicide must show absence of growth in as many consecutive tubes of subculture tube series as 200 ppm available CI std. Det. activity equiv. to 100 and 50 ppm available CI in same manner. See example, Table 4:01. In this example, 25 ppm soln of germicide X could be considered equiv. to 200 ppm soln of available CI, and 20 ppm soln equiv. to 100 ppm of available CI, but 10 ppm soln of germicide X would not be considered equiv. in germicidal activity to 50 ppm of available CI. Draw conclusions relative to germicidal equiv. concns only when resistance of test culture to NaOCI control is such that ;,,1 neg. increment is obtained at 50 ppm concn and 1 pos. increment is obtained at 200 ppm level. Sporicidal Test (4)--Official Final Action (Suitable for detg sporicidal activity of liq. and gaseous chems. Applicable to germicides for detg presence or absence of sporicidal activity against specified spore-forming bacteria in various situations and potential efficacy as sterilizing agent.) 4.015 Reagents (a) Culture media.-( 1) Soil extract nutrient broth.-Ext 1 Ib garden soil in 1 L H20, filter several times thru S&S No. 588 paper, and dil. to vol. (pH should be ;"5.2). Add 5 g beef ext. (Difco), 5 g NaCl, and 10 g peptone (Anatone, 4.001(a)). Boil 20 min, dil. to vol., adjust with IN NaOH to pH 6.9, and filter thru paper. Dispense in 10 mL portions into 25 x 150 mm tubes, and autoclave 20 min at 121°. Use this broth to propagate test culture of Bacilli. (2) Nutrient agar.--See 4.001(c). Use slants of this medium to maintain stock culture of Bacilli. (3) Modified fluid thioglycolate medium USP Xx.-Prep. as in 4.001 (d)(2), except add 20 mL IN NaOH to each L before dispensing for sterilization. Use this medium to subculture spores exposed to 2.5N HCI. For spores exposed to unknown germicides, use fluid thioglycolate medium, 4.001 (d)(2). (4) Soil extract-egg-meat medium.-Add 1.5 g Bacto Egg- Meat Medium dehydrated (Difco) to 25 x 150 mm tube; then add 15 mL garden soil ext, (1). and sterilize 20 min at 121°. Use this medium to propagate test cultures ofClostridia and maintain stock cultures of species of this genus. (b) Test organisms.-Use Bacillus subtilis, ATCC No. 19659, or Clostridium sporogenes, ATCC No. 3584, for routine evalu- ation. Method is also applicable for use with strains of B. anthracis, CI. tetani, or other spore forming species. (c) Dilute hydrochloric acid.-2.5N. Use to det. resistance of dried spores. Stdze and adjust to 2.5N as in 50.012. 4.016 Apparatus (a) Glassware.-Bacteriological culture tubes, unflared, 25 x 150 mm; 100 mL g-s cylinders graduated in 1 mL divisions; 65 mm id funnels; supply of 15 x 110 mm petri dishes matted with 2 sheets 9 cm S&S No. 597 or Whatman No. 2 filter paper. Sterilize all glassware and matted petri dishes 2 hr in air oven at 180°. (b) Water bath.--See 4.002(b). (c) Racks.--See 4.002(c). (d) Transfer loop, hook, and forceps.--See 4.008(c). (e) Tissue grinder.-Arthur H. Thomas Co., No. 3431-E20, Size B, or equiv. (f) Suture loop carrier.-From spool of size 3 surgical silk suture, prep. std loops by wrapping the silk. around ordinary pencil 3 times, slipping coil so formed off end of pencil, and holding it firmly with thumb and index finger of left hand while passing another piece of suture through coil, knotting, and tying securely. Then shear off end of coil and knotted suture to within 2 mm. This should provide overall length of ca 65 mm of suture in 2-loop coil that can be conveniently handled in ordinary aseptic transfer. procedure. Ext loops in groups of 20 by immersion in 10 mL pet ether in stoppered, unflared test tube, shaking frequently during 30 min at room temp., and hold overnight (18-24 hr) at 2-5°. Shake, remove loops, drain, and dry. (g) Cylinder carriers.-"Penicylinders," porcelain, 8±1 mm od, 6±1 mm id, 10±1 mm long. (Available from Fisher Scientific Co., No. 7-907.) Sterilize 2 hr in 180° air oven. Wash used Penicylinders with Triton X-l00 and rinse with H20 4 times. 4.017 Operating Technic Grow all Bacilli in soil ext nutrient broth and all Clostridia in soil ext-meat-egg medium. Inoculate 3 tubes, using 1 loop stock culture, and incubate 72 hr at 3r. Place supply of suture loops and cylinder carriers in sep. petri dishes matted with filter paper, and sterilize 20 min at 121°. Use new loops for each test. Penicylinders must be free from chips or cracks. Filter CI. sporogenes thru funnel contg 2 x 5 x 5 cm sq piece of moist cotton or glass wool into sterile 25 x 150 mm test tubes, using same funnel. In prepg B. subtilis culture, pour tube of 72 hr culture into tissue grinder and macerate to break up pellicle. Filter thru sterile funnel contg moist cotton or glass wool into sterile 25 x 150 mm tube, repeating operation for other 2 tubes. Place 10 sterile suture loops or Penicylinders into each of 3 tubes contg 10 mL filtrate from 72 hr culture of CI. sporogenes, agitate, and let stand 10-15 min. Using this technic, contaminate 35 loops or cylinders. Place contaminated suture loops and/or cylinders into petri dish matted with 2 layers of filter paper. Drain. Proceed similarly for B. subtilis. Place the 35 suture loops or cylinders contaminated with Ct. sporogenes or B. subti/is in vac. desiccator contg CaCI2 and draw vac. of 69 cm (27") Hg for 20 min. Dry 24 hr under vac. (Spores dried and held under these conditions will retain re- sistance ;,,7 days.) Transfer 10 mL 2.5N HCI, 4.015(c). into sterile 25 x 150 mm tube. Place tube in 20° const temp. H20 bath and let come to temp. Rapidly transfer 4 dried, contaminated loop or cylinder carriers to acid tube. Transfer remaining dried, contaminated suture loop or cylinder carriers to tube of thioglycolate subcul- ture medium, 4.015(a)(3), as viability control. After 2, 5, 10, and 20 min, withdraw individual loops or cylinders from acid and transfer to individual tubes of subculture medium. Rotate each tube vigorously 20 sec and resubtransfer. Incubate 21 days at 37°. Test spores should resist HCI ;,,2 min, and many may resist HCI for full 20 min. When testing sporicidal or sterilizing activity of gas, place carriers in polyethylene bags or in petri dishes with lids ajar. Certain gases may require rehydration of spores before exposure to gas. Rehydrate spores on carriers by 1 hr immersion in H20, using ",20 mL H20/6 carriers. Drain carriers 20 min on petri dishes matted with filter paper. After exposure to gas, remove carriers, using aseptic technic to subculture media as specified in next par. For aq. sporicides and sterilizers, place 10 mL product at diln recommended for use or under investigation into each of six 25 x 150 mm tubes. Place tubes in 20° H20 bath and let come to temp. Using flamed forceps, place 5 suture loops or cylinders, contaminated with CI. sporogenes or B. subtilis and dried 24 hr
- 83. AOAC METHODS (1980) SANITIZERS 61 under vac., into each of the 6 tubes contg disinfectant, using 2- min intervals for seeding each tube. Five suture loops or cylinders can be placed into each tube within 5 sec. This seeding operation will take 10 min. After contact period specified for disinfectant has been achieved, remove suture loops or cylin- ders, using sterilized needle hook, from each tube of disinfectant to subculture medium or other subculture medium specified in 4.001(d) (select medium contg most suitable neutralizer)' placing 1 suture loop or cylinder per tube. Five cylinders can be removed within each 2 min interval. Flame transfer needle hook after each carrier has been transferred to subculture medium. After completing transfer, resubtransfer each suture loop or cylinder to fresh tube of thioglycolate medium and incubate 21 days at 37°. If no growth is observed after 21 days, heat-shock tubes 20 min at 80° and reincubate 72 hr at 3r. Report results as + (growth) or - (no growth) values. Killing in 59 of 60 replicates on 1 carrier at diln and time specified is considered evidence of sporicidal efficacy against 1 test spore and for confidence level of 95%. Tests with both B. subtilis and CI. sporogenes, using 30 replicates with each of 2 carriers specified to provide min. of 120 carriers, are required to presumptively support unqualified sporicidal claim or for presumptive evidence of sterilizing activity at concn, time, and conditions specified. For sporicidal claims, no more than 2 failures can be tolerated in this 120 carrier trial. For sterilizing claims, no failures can be tolerated. 4.018 Fungicidal Test (5)-Official Final Action (Applicable for use with H20-miscible type fungicides used to disinfect inanimate objects) Using Trichophyton mentagrophytes Test Organism Use as test fungus typical strain of Trichophyton mentagro- phytes isolated from dermatophytosis of foot. Strain must sporulate freely on artificial media, presence of abundant conidia being manifested by powdery appearance on surface of 10-day culture, particularly at top of agar slant, and confirmed by microscopic examination. Conidia-bearing mycelium should peel easily from surface of glucose agar. Conidia of required resistance survive 10 min exposure at 20° to phenol diln of 1:70, but not to one of 1:60. Strain No. 640, ATCC No. 9533, is suitable. 4.019 Culture Medium Carry fungus on agar slants of following composition: Glucose 2%, Neopeptone (Difco) 1%, agar 2%, adjusted to pH 6.1-6.3. Use same culture medium to prep. cultures for obtaining conidial suspension, and use fluid medium of same nutrient composition (without agar) to test viability of conidia after exposure to fungicide. 4.020 Care of Fungus Strain Store stock culture of fungus on glucose agar slants at 2-5°. At intervals ,,;3 months, transfer to fresh agar slants, incubate 10 days at 25-30°, and store at 2-5° until next transfer period. Do not use culture that has been kept at or above room temp. >10 days as source of inoculum for culture. (Cultures may be kept at room temp. to preserve strain and to inoculate cultures if transferred at intervals ,,;10 days.) 4.021 Preparation of Conidial Suspension Prep. petri dish cultures by planting inoculum at center of agar plate and incubating culture at 25-30° for ~10, but ,,;15 days. Remove mycelial mats from surface of 5 agar plate cultures, using sterile spatula or heavy flattened wire. Transfer to heat-sterilized glass tissue grinder, 4.016(e). and macerate with 25 mL sterile physiological NaCI soln (0.85% NaCl), or to heat-sterilized erlenmeyer contg 25 mL sterile saline with glass beads, and shake thoroly. Filter suspension thru sterile absorbent cotton to remove hyphal elements. Est. density of conidial suspension by counting in hemacytometer and store at 2-10° as stock spore suspension (125-155 x 106 conidia/mL) for ,,;4 weeks for use in prepg test suspensions of conidia. Stdze test conidial suspensions as needed by dilg stock spore suspension with physiological NaCI soln so that it contains 5 x 106 conidia/mL. 4.022 Operating Technic Prep. dilns of fungicide. (Tests are similar to those described in 4.003.) Place 5 mL of each fungicide soln and of phenol control solns in 25 x 150 mm test-culture tubes, arrange in order of ascending dilns, place tubes in 20° H20 bath, and let come to temp. With graduated pipet, place 0.5 mL spore suspension in first tube of fungicidal soln, shake, and immediately replace in H2 0 bath; 30 sec later add 0.5 mL conidial suspension to second tube. Repeat at 30 sec intervals for each fungicidal diln. If more convenient, run test at 20 sec intervals. After 5, 10, and 15 min exposure to fungicide, remove sample from each conidia-fun- gicide mixt. with 4 mm loop and place in 10 mL glucose broth, 4.019. To eliminate risk of faulty results due to possible fungi- static action, make subtransfers from the initial glucose broth subculture tubes to fresh tubes of glucose broth, using the 4 mm loop before incubation, or make initial subcultures in glucose broth contg either 0.05% Na thioglycolate, 1.5% iso- octylphenoxy-polyethoxy-ethanol, or mixt. of 0.07% lecithin (Azolectin, Associated Concentrates, Inc., 32-30 61 st St, Wood- side, NY 11377) and 0.5% polysorbate 80 (Tween 80), whichever gives lowest result. Incubate inoculated tubes at 25-30°. Read final results after 10 days, altho indicative reading can be made in 4 days. Note: Highest diln that kills spores within 10 min is commonly considered as highest diln that could be expected to disinfect inanimate surfaces contaminated with pathogenic fungi. Germicidal and Detergent Sanitizers (6) Official Final Action (Suitable for detg min. concn of chem. that can be permitted for use in sanitizing precleaned, nonporous food contact surfaces. Min. recommended starting concn is 2-4x this concn. Test also dets max. water hardness for claimed concns. As control, check accuracy of hard-water tolerance results with pure C,. alkyl dimethyl benzyl NH. chloride (Onyx Chemical Co. 190 Warren St, Jersey City, NJ 07302) at 700 and 900 ppm hardness, and pure C'6 alkyl dimethyl benzyl NH. chloride (Cetalkonium Chlo- ride, Sterling Chemical Co.), at 400 and 550 ppm hardness, expressed as CaC03.) 4.023 Reagents (a) Culture media.-(1) Nutrient agar A.-Boil 3 g beef ext, 5 g peptone (Bacto or equiv.; special grades must not be used). and 15 g salt-free agar in 1 L H20. Do not use premixed, dehydrated media. Tube, and autoclave 20 min at 121°. Use for daily transfer of test culture. (2) Nutrient agar B.-Prep. as above but use 30 g agar. Use for growing test cultures in French square bottles. (3) Nutrient agar (AOAC).-See 4.001(c). Use for prepg stock culture slants. (b) Subculture media.-( 1) Use tryptone glucose ext agar (Difco). adding 25 mL stock neutralizer, (c)jL. (2) Tryptone glucose ext agar (Difco). (c) Neutralizer stock soln.-Mix 40 g Azolectin (Associated Concentrates, 32-30 61st St, Woodside, NY 11377), 280 mL
- 84. 62 4. DISINFECTANTS AOAC METHODS (1980) polysorbate 80, and 1.25 mL phosphate buffer, (e); dil. with H20 to 1 L and adjust to pH 7.2. Dispense in 100 mL portions and autoclave 20 min at 121°. (d) Neutralizer blanks.-For use with ",;200 ppm quaternary NH, compd. Mix 100 mL neutralizer stock soln, (cl, 25 mL 0.25M phosphate buffer stock soln, (e), and 1675 mL H20. Dispense 9 mL portions into 20 x 150 mm tubes. Autoclave 20 min at 121°. (e) Phosphate buffer stock soln.-O.25M. Dissolve 34.0 g KH2PO, in 500 mL H20, adjust to pH 7.2 with 1N NaOH, and dil. to 1 L. (f) Phosphate buffer dilution water.-Add 1.25 mL 0.25M phosphate buffer stock soln, (e), to 1 L H20 and dispense in 99 mL portions. Autoclave 20 min at 121°. (g) Test organisms.-Use Escherichia coli ATCC No. 11229 or Staphylococcus aureus ATCC 6538. Incubate 24 and 48 hr, resp. Maintain stock cultures on nutrient agar (AOAC), (a)(3), at refrigerator temp. 4.024 Resistance to Phenol of Test Cultures Det. resistance to phenol at least every 3 months by 4.001-4.005. Resistance of E. coli should be equiv. to that specified for S. typhi in 4.004 and that for Staph. aureus equiv. to that specified for this organism in 4.005. 4.025 Apparatus (a) Glassware.-250 mL wide-mouth erlenmeyers; 100 mL graduate; Mohr, serological, and/ or bacteriological (APHA spec- ification) pipets; 20 x 150 mm test tubes. Sterilize at 180° in hot air oven ;,,2 hr. (b) Petri dishes.-Sterile. (c) French square bottles.-175 mL, borosilicate. Use of other containers will give variable results. (d) Water bath.-Controlled at 25°. 4.026 Preparation of Culture Suspension From stock culture inoculate tube of nutrient agar A, 4.023(a)( 1), and make ;,,3 consecutive daily transfers (",;30), incubating transfers 20-24 hr at 35-37°. Do not use transfers >30 days. If only 1 daily transfer has been missed, no special procedures are required; if 2 daily transfers are missed, repeat with 3 daily transfers. Prep. 175 mL French square culture bottles contg 20 mL nutrient agar B, 4.023(a)(2), autoclave 20 min at 121°, and let solidify with bottle in horizontal position. Inoculate culture bottles by washing growth from slant with 5 mL phosphate buffer diln H20, 4.023(f)' into 99 mL phosphate buffer diln H20, and adding 2 mL of this suspension to each culture bottle, tilting back and forth to distribute suspension; then drain excess liq. Incubate 18-24 hr at 35-3]D, agar side down. Remove culture from agar surface of 4 or more bottles, using 3 mL phosphate buffer diln H20 and glass beads in each bottle to suspend growth. Filter suspension thru Whatman No.2 paper prewet with 1 mL sterile phosphate buffer, and collect in sterile tube. (To hasten filtration, rub paper gently with sterile policeman.) Stdze suspension to give avo of 10 x 109 organisms/mL by diln with sterile phosphate buffer diln H20, 4.023(f). If Lumetron colorimeter is used, dil. suspension in sterile Lumetron tube to give % T according to Table 4:02. If McFarland nephelometer and BaSO, stds are used, select 7 tubes of same id as that contg test culture suspension. Place 10 mL of each suspension of BaSO" prepd as indicated in Table 4:03, in each tube and seal tube. Stdze suspension to correspond to No.4 std. Table 4:03 Preparation of BaSO, Suspensions Corresponding to Bacterial Concentrations mL 2% BaCl, mL 1% H2SO4 Av. Bacterial Std No. Soln (v/v) Soln Count/mL 1 4.0 96.0 5.0 x 10' 2 5.0 95.0 7.5 3 6.0 94.0 8.5 4 7.0 93.0 10.0 5 8.0 92.0 12.0 6 10.0 90.0 13.5 7 12.0 88.0 15.0 4.027 Synthetic Hard Water Prep. Soln 1 by dissolving 31.74 g MgCI2 (or equiv. of hydrates) and 73.99 g CaCI2in boiled distd H20 and dilg to 1 L. Prep. Soln 2 by dissolving 56.03 9 NaHCOa in boiled distd H20 and dilg to 1 L. Soln 1 may be heat sterilized; Soln 2 must be sterilized by filtration. Place required amt Soln 1 in sterile 1 L flask and add ;,,600 mL sterile distd H20; then add 4 mL Soln 2 and dil. to 1 L with sterile distd H20. Each mL Soln 1 will give a water equiv. to ca 100 ppm of hardness caicd as CaC03 by formula: Total hardness as ppm Cae03 = 2.495 x ppm Ca + 4.115 x ppm Mg. pH of all test waters ",;2000 ppm hardness should be 7.6-8.0. Check prepd synthetic waters chemically for hardness at time of tests, using following method or other methods described in 14th Ed. of Standard Methods for the Examination of Water, Sewage, and Industrial Wastes. 4.028 Hardness Method (a) EDTA std soln. -Dissolve 4.0 9 Na2H2EDTA.2H20 and 0.10 g MgCI2.6H20 in 800 mL H20 and adjust by subsequent diln so that 1 mL of soln is equiv. to 1 mg CaC03 when titrd as in (e). Check EDTA soln after prepn or, if com. purchased, against CaC03 std at least every 2 months. (b) Calcium std soln.-1 mL = 1 mg CaC03 • Weigh 1.00 9 CaCOa, dried overnight or longer at 105°, into 500 mL erlenmeyer and add dil. HCI thru funnel until CaeOa is dissolved. Add 200 mL H20, boil to expel CO2, and cool. Add few drops Me red indicator and adjust color to intermediate orange with dil. NH,OH or Hel as required. Transfer quant. to 1 L vol. flask and dil. to vol. Table 4:02 Per Cent Light Transmission at Various Wavelengths Corresponding to Bacterial Concentrations % Light Transmission with Filters. nm Av. Bacterial 370 420 490 530 550 580 650 Count/mL 7.0 4.0 6.0 6.0 6.0 7.0 8.0 13.0 X 10' 8.0 5.0 7.0 7.0 7.0 8.0 9.0 11.5 9.0 6.0 8.0 8.0 8.0 9.0 10.0 10.2 10.0 7.0 9.0 9.0 9.0 11.0 11.0 8.6 11.0 8.0 10.0 10.0 10.0 12.0 13.0 7.7 13.0 9.0 12.0 12.0 12.0 13.0 15.0 6.7
- 85. AOAC METHODS (1980) GERMICIDAL SPRAYS 63 (c) Determination.-Dil. 5-25 ml sample (depending on hard- ness) to 50 ml with H20 in erlenmeyer or casserole. Add 1 ml buffer soln (67.5 g NH.CI and 570 ml NH.OH dild to 1 l with H20), 1 ml inhibitor (5.0 g Na2S.9H20 or 3.7 g Na2S.5H20 dissolved in 100 ml H20)' and 1 or 2 drops indicator soln (0.5 g Chrome Black T in 100 ml 60-80% alcohol). Titr. with EDTA std soln slowly, stirring continuously, until last reddish tinge disappears from soln, adding last few drops at 3-5 sec intervals. Hardness as mg CaC03/l = (ml std soln x 1000)/ml sample. 4.029 Preparation of Samples Use composition declared or detd as guide to sample wt required for vol. sterile H20 used to prep. 20,000 ppm soln. From this stock diln, transfer 1 ml into 99 ml of the water to be used in test to give concn of 200 ppm. In making transfer, fill 1 ml pipet and drain back into stock soln; then refill, to correct for adsorption on glass. After mixing, discard 1 ml to provide 99 ml of the test water in 4.030. 4.030 Operating Technic Measure 99 ml water to be used in test, contg bactericide at concn to be tested, into chem. clean, sterile, 250 ml wide-mouth erlenmeyer and place in const temp. bath until it reaches 25°, or ~20 min. Prep. duplicate flasks for each germicide to be tested. Also prep. similar flask contg 99 ml sterile phosphate buffer diln H20, 4.023(f), as "initial numbers" control. Add 1 ml culture suspension to each test flask as follows: Whirl flask, stopping just before suspension is added, creating enough residual motion of liq. to prevent pooling of suspension at point of contact with test water. Add suspension midway between center and edge of surface with tip of pipet slightly immersed in test soln. Avoid touching pipet to neck or side of flask during addn. Transfer 1 ml portions of this exposed culture to neutralizer blanks exactly 30 and 60 sec after addn of suspension. Mix well immediately after transfer. For "numbers control" transfer, add 1 ml culture suspension to 99 ml sterile phosphate diln H20 in same manner. In case of numbers control, plants need be made only immediately after adding and mixing thoroly .;;30 sec. (It is advantageous to use milk pipets to add culture and withdraw samples.) Plate from neutralizer tube to agar, using subculture medium 4.023(b)( n for quaternary NH. compds and 4.023(b)(2) with numbers control. Where 0.1 ml portions are planted, use 1 ml pipet graduated in 0.1 ml intervals. For dilns to give countable plates, use phosphate buffer diln H20, 4.023(f). For numbers control, use following diln procedure: Transfer 1 ml exposed culture (1 ml culture suspension transferred to 99 ml phosphate buffer diln H20 in H20 bath) to 99 ml phosphate buffer diln H20, 4.023(f), (diln 1). Shake thoroly and transfer 1 ml diln 1 to 99 ml phosphate buffer diln H20, 4.023(f), (diln 2). Shake thoroly and transfer 1 ml diln 2 to 99 ml phosphate buffer diln H20 (diln 3). Shake thoroly and transfer four 1 ml and four 0.1 ml aliquots from diln 3 to individual sterile petri dishes. For test samples, use following diln procedure: Transfer 1 ml exposed culture into 9 ml neutralizer, 4.023(d). Shake and transfer four 1 ml and four 0.1 ml aliquots to individual sterile petri dishes. For numbers control, use subculture medium 4.023(b)(2); for tests with quaternary NH. compds, use medium 4.023(b)( 1). Cool agar to solidify, and then invert and incubate 48 hr at 35° before counting. 4.031 Results To be considered valid, results must meet std effectiveness: 99.999% reduction in count of number of organisms within 30 sec. Report results according to actual count and % reduction over numbers control. Counts on numbers control for germicide test mixt. should fall between 75 and 125 x 10·/ml for % reductions to be considered valid. 4.032 Sterility Controls (a) Neutralizer.-Plate 1 ml from previously unopened tube. (b) Water.-Plate 1 ml from each type of water used. (c) Sterile distilled water.-Plate 1 ml. After counting plates, confirm that surviving organisms are E. coli by transfer to brilliant green bile broth fermentation tubes or lactose broth and EMB agar; confirm Staph. aureus by microscopic examination. Germicidal Spray Products (7)-Official Final Action (Suitable for detg effectiveness of sprays and pressurized spray products as spot disinfectants for contaminated surfaces) 4.033 Reagents Use culture media and reagents specified in 4.001, 4.002(e) and (f), and 4.007 except that test organism Salmonella typhi is not used. Use as test organisms Trichophyton mentagrophytes ATCC No. 9533, prepd as in 4.021, to which has been added 0.02 ml octylphenoxy-polyethoxy-ethanol (Triton X100, Rohm & Haas)/10 ml suspension to facilitate spreading, Salmonella choleraesuis ATCC No. 10708, 4.007(b)' Staphylococcus aureus ATCC No. 6538, maintained as in 4.007(b)' and Pseudomonas aeruginosa ATCC No. 15442, maintained as in 4.011. 4.034 Apparatus Use app. specified in 4.002 and 4.008, and in addn: (a) Capillary pipets.-{).1 ml, graduated to deliver 0.01 ml. Sterilize in air oven 2 hr at 180°. (b) Microscope slides.-Non-corrosive, 25 x 25 mm (1 x 1"), or 18 x 36 mm glass slide. Sterilize by placing individual slides in petri dish matted with 2 pieces 9 cm filter paper (Whatman No.2, or equiv.) in air oven 2 hr at 180°. (c) Bacteriological culture tubes.-Pyrex, 32 x 200 mm. (d) Metal forceps.-Sharp points, straight, 115 mm long. 4.035 Operating Technic Thoroly shake 48 hr nutrient broth cultures of S. choleraesuis and Staph. aureus and let settle 10 min. With sterile capillary pipet or sterile 4.0 mm loop, transfer 0.01 ml culture onto 1 sq in. sterile test slide in petri dish and immediately spread uni- formly over entire area. Cover dish immediately and repeat operation until 12 slides have been prepd for each organism. (Use 2 slides as controL) Dry all slides 30-40 min at 3r. Spray 10 slides for specified time and distance. Hold each slide 10 min, drain off excess liq., and transfer slide to individual 32 x 200 mm tube contg 20 ml appropriate subculture medium, 4.001(d), with flamed forceps. Shake culture thoroly. If broth appears cloudy after 30 min, make subculture to fresh individual tubes of subculture broth. Transfer 2 unsprayed slides, as viability controls, to individual subculture tubes in same manner. Incubate all tubes used for primary and secondary transfers 48 hr at 37°. Read as + (growth) or - (no growth). Killing of test organisms in 10 of 10 trials is presumptive evidence of disin- fecting action. Det. resistance of S. choleraesuis as in 4.003; with S. aureus as in 4.005; with Ps. aeruginosa as in 4.006; and with T. mentagrophytes as in 4.018.
- 86. 64 4. DISINFECTANTS AOAC METHODS (1980) If there is reason to believe that lack of growth in subtransfer tubes is due to bacteriostasis, inoculate all incubated subculture tubes with loop needle inoculation of respective test culture and reincubate. Growth of these inocula eliminates bacteriostasis as cause of lack of growth. If there is question as to possibility of contamination as source of growth in subculture tubes, make gram stains and/ or subculture for identification, according to respective test culture. If fungicidal activity as well as germicidal activity is involved, use test suspension of T. mentagrophytes spores, 4.021, and prep. 12 slides, using 0.01 mL std spore suspension, spraying and subculturing exactly as above. Make subcultures in glucose broth, 4.019, incubating 7 days at 25-30°. Water Disinfectants for Swimming Pools (8) Official Final Action (Suitable for presumptive evidence of acceptability of products for disinfecting swimming pool water) 4.036 Test Culture Media (a) Nutrient Agar A.-See 4.023(a)(1). (b) NutrientAgarB (Trypticase Soy Agar, BBL). -See 4.037(b). (c) Nutrient Agar c.-Prep. as in 4.001(c). 4.037 Subculture Media (a) Tryptone glucose extract agar (Difco).- Dissolve 24 g in 1 L freshly distd H20 and heat to bp to dissolve completely. Autoclave 15 min at 121°. Use for plate counts ofE. coli survivors. (b) Trypticase soy agar (BBL).-Suspend 40 g powder in 1 L H20. Let stand 5 min and mix thoroly. Heat gently with occasional agitation and boil ca 1 min or until soln is complete. Autoclave 15 min at 121°. Let cool and reautoclave 15 min at 121°. Use for plate counts of S. faecalis survivors. (c) Fluid thioglycolate medium (Difco).-See 4.001(d)(2). (d) Lactose broth (Difco).-Dissolve 19 gin 1 L H20. Dispense 10 mL portions into tubes with fermentation vials. Autoclave 15 min at 121°. Use for detg presence of E. coli survivors. (e) Eosin methylene blue agar (Difco).-Suspend 36 g in 1 L H20 and heat to bp to dissolve completely. Autoclave 15 min at 121°. Use for confirming E. coli survivors. (f) S-F agar (Difco ).-Dissolve 36 g in 1 L H20. Add 15 g agar and heat to bp to dissolve completely. Autoclave 15 min at 121°. Use for confirming S. faecalis survivors. 4.038 Neutralizer Stock Solns (a) Sodium thiosulfate soln.-Dissolve 1 g Na2S20 3 in 1 L H20. Dispense in 100 mL portions and autoclave 20 min at 15 lb. (b) Azolectin soln.-See 4.023(c). (c) Other preparations.-Prepns found to be suitable and necessary, depending upon nature of germicidal prepns to be tested. 4.039 Neutralizer Blanks (a) With 0.6 ppm residual chlorine or less.-Dil. 10 mL neu- tralizer stock soln, 4.038(a), with 90 mL sterile H20. Dispense aseptically in 9 mL portions into sterile 25 x 150 mm tubes. (b) With quaternary ammonium compounds and phenolic derivatives.-Mix 10 mL neutralizer stock soln, 4.038(b), 2.5 mL 0.25M phosphate buffer stock soln, 4.040(a), and 167.5 mL H20. Dispense in 9 mL portions into 20 x 150 mm tubes. Autoclave 20 min at 121°. (c) Other preparations.-Use dilns of 4.038(c) as suitable. 4.040 Reagents (a) Phosphate buffer stock soln.--o.25M. See 4.023(e). (b) Phosphate buffer dilution water.-See 4.023(f). (c) Sodium thiosulfate std solns.-(1) 0.1N. Dissolve exactly 24.820 g Na2Si03.5H20 in H20 and dil. to 1 L. Stdze as in 50.038. (2) 0.001N.-Dil. 10 mL soln (1) to 1 L with H20. (d) Starch indicator soln.-See 6.005(f), except use few drops CHCI3 instead cif Hg as preservative. (e) Sterile phosphate buffer stock solns.-(1) Dissolve 11.61 g anhyd. K2HPO. in 1 L H20 and autoclave 20 min at 121°. (2) Dissolve 9.08 g anhyd. KH2PO. in 1 L H20 and autoclave 20 min at 121°. (f) NaOCI stock soln.-Approx. 5%. Store NaOCI stock soln in tightly closed bottle in refrigerator and det. exact available CI at frequent intervals by As20 3 titrn, 6.112. (g) Test organism.-Use Escherichia coli ATCC 11229 and Streptococcus faecalis PRO (Microbiology Lab., Benefits and Field Services Div., EPA, Beltsville, MD 20705). Maintain, by monthly transfer, stock cultures of E. coli on Nutrient Agar C, 4.001(c), and S. faecalis on Nutrient Agar B, 4.037(b); store at 4-5°. 4.041 Apparatus (a) Glassware.-500 mL wide-mouth erlenmeyers; 100 mL graduates; Mohr pipets; milk pipets; 20 x 150 mm tubes; Board of Health tubes; 200, 500, and 1000 mL vol. flasks. Wash in strong, fresh chromic acid cleaning soln, and fill and drain with H20 ;.3 times. Heat ;.2 hr at 180° in hot air oven. (b) Petri dishes.-Sterile. (c) Water bath.-Controlled at 20 or 25°. 4.042 Preparation of Culture Suspension From stock culture, inoculate tube Nutrient Agar A for E. coli and Nutrient Agar B for S. faecalis; make ;.3 consecutive daily transfers (";;30), incubating transfer 20-24 hr at 35-3r. Do not transfer >30 days. If only 1 daily transfer has been missed, no special procedures are required; if 2 daily transfers are missed, repeat with 3 daily transfers. Remove culture from agar surface, using 5 mL phosphate buffer diln H20, 4.040(b). Transfer culture suspension to sterile centrf. tube and centrf. 1-2 min at speed necessary to settle agar particles. Transfer supernate to another sterile centrf. tube and centrf. to obtain complete sepn of cells. Discard supernate and resuspend cells in 5 mL buffer diln H20. With S. faecalis, centrf., discard supernate, and resus- pend cells in 5 mL buffer diln H20 2 addnl times. Finally, stdze suspension to give avo of 2.0 x 10" organisms/mL by diln with sterile phosphate diln H20. If Lumetron is used, dil. suspension in sterile Lumetron tube to give % T according to Table 4:04. Make serial diln plate count of each culture suspension before use, using phosphate buffer diln H20, 4.040(b). and subculture medium, 4.037(a). with E. coli, and (b) with S. {aecalis. Incubate diln plates in inverted position 48 hr at 35-3r. Use Quebec Colony Counting Chamber and report results in terms of number of bacteria/mL suspension. Table 4:04 Per Cent Light Transmission at Various Wave- lengths Corresponding to Bacterial Concentrations % Light Transmission Av. with Filter, nm Bacterial Count 370 420 490 530 580 650 jmL E. coli 90 88 89 88 91 92 2.0 x 10' S. faeca/is 86 82 85 85 87 89 2.0 x 10'
- 87. AOAC METHODS (1980) TUBERCULOCIDAL ACTIVllY 65 Count of 2.0 x 10" is desired so that 1 mL test culture suspension + 199mL test soln will provide soln contg 1 x 1()6 organisms! mL. Permitted variation in test culture suspension is +500,000 and -100,000/mL of 200 mL test soln. Use actual count for calcg zero time count in later tests. 4.043 Determining Chlorine Demand of Freshly Distilled Test Water Place 200 mL H20 in each of five 500 mL erlenmeyers. To flasks 1-5, resp., add 0.025, 0.05, 0.075, 0.1, and 0.15 mL of 200 ppm available CI prepd from NaOCI soln, 4.040(f). Shake each flask, and let stand several min. Add crystal KI and 1 mL HOAc, and swirl. Add 1 mL starch soln, 4.040(d). Flask showing perceptible blue indicates CI demand has been satisfied. 4.044 Operating Technic Place ca 600 mL freshly sterilized distd H20 in 1 L vol. flask. Add ca 1.5-3.0 mL K2HPO. buffer, 4.040(e)( 1). and 0.5 mL KH2PO., 4.040(e)(2). and dil. to 900 mL. Add enough NaOCI from suitable diluent of std stock soln, 4.040(f). to satisfy CI demand of 1 L test H20, 4.043, and to provide ca 0.6 ppm residual available CI. Oil. to vol. (Example: If CI demand of H20 is 0.1 ppm, add 3.5 mL of 200 ppm soln of available CI made from std stock NaOCI soln, 4.040(f). and dil. to vol. This should provide soln with ca 0.6 ppm residual available CI at pH 7.5 ± 0.1.) Transfer 199 mL of this test soln to each of three 500 mL erlenmeyers and place in H20 bath at either 20 or 25°. Let come to temp. To first flask, add 1 mL boiled distd H20 and det. residual available CI as follows: Add small crystal KI and 1 mL HOAc; then add 1 mL starch soln, 4.040(d). Blue soln indicates presence of CI. Titr. with 0.001N Na2S203, 4.040(c)(2), until color disap- pears; mL 0.001N Na2S20 3 x 0.1773 = ppm residual available CI. This represents available CI at 0 time in test. Result should be ;,.0.58 but ,,;;0.62. To each of remaining flasks add 1 mL test culture suspension, 4.042, as follows: Swirl flask, stopping just before suspension is added, to create enough centrifugal motion to prevent pooling of suspension at point of contact with test H20. Add suspension midway between center and edge of liq. surface, immersing tip of pipet slightly below surface of H20. Avoid touching pipet to neck or side of test flask during operation. From one of these 2 flasks transfer 1 mL aliquots to neutralizer blanks, 4.039(8), after intervals of 0.5, 1, 2, 3, 4, 5, and 10 min. Shake neutralizer blank thoroly immediately after adding sam- ple. Prep. serial diln plate counts from neutralizer blanks, using phosphate buffer diln H20, 4.040(b). and subculture medium, 4.037(8) for E. coli, and (b) for S. faecalis. After prepg diln plate counts, inoculate 5 lactose broth tubes, 4.037(d). with 1.0 mL aliquots from each neutralizer blank tube for each time interval when E. coli is used as the test organism, and 5 thioglycolate broth tubes, 4.037(c). with 1.0 mL aliquots from each neutralizer blank tube for each time interval when S. faecalis is test organism. Incubate all diln plates in inverted position and subculture tubes 48 hr at 3r. Use Quebec Colony Counting Chamber in reading diln plates and report results in terms of number of surviving bacteria/mL test H20. Absence of colony growth on diln plates and absence of growth in all 5 lactose or thioglycolate tubes, as case may be, is necessary to show complete kill oftest organism. Immediately after transferring 10 min interval sample from second flask to neutralizer blank tube, remove third flask from H20 bath and det. residual available CI exactly as specified for first flask. Results should represent residual available CI present at 10 min exposure interval. To be acceptable, concn of available CI in this flask should be >0.4 ppm. Results in CI control test described above should show complete kill of E. coli and S. faecalis within 0.5 min. With unknown sample, prep. 2 flasks contg 199 mL each of soln at concn recommended or to be studied, using CI demand- free, unbuffered, freshly distd H2 0 previously prepd in 1 L vol. flask where CI demand, as detd above, has been satisfied by addn of NaOCI soln. Place flasks in H20 bath at 20 or 25°; let come to temp. Inoculate 1 flask with 1 mL std test culture suspension of E. coli and other with 1 mL std test culture suspension of S. faecalis. Subculture at exactly same time intervals and in same manner used with NaOCI control except vary composition of neutralizer blank depending upon nature of chem. or mixt. of chems under investigation. For example, mixt. of CI-contg chem. and quaternary NH. compd would require special neutralizer blank prepd by using both neutralizer stock solns, 4.038(a) and (b). Where no concn ofchem. under study has been recommended and objective of study is to det. concn of unknown necessary to provide result equiv. to that obtained with CI control std, use series of three or four 500 mL flasks contg 199 mL of various concns of chem. and 1 mL stdzd culture suspension with each test organism. Report results as log (number of survivors) at each time interval both for CI controls and various concns of unknown under test. Lowest concn of unknown germicide or germicidal mixt. providing results equiv. to those obtained with NaOCI as CI std is considered lowest concn which could be expected to provide acceptable disinfecting activity in swimming pool water. Tuberculocidal Activity (9)-Official Final Action (Suitable for detg max. tuberculocidal diln of disinfectants used on inanimate surfaces) I. Presumptive In Vitro Screening Test using Mycobacterium smegmatis 4.045 Reagents (8) Test organism.-Mycobacterium smegmatis (PRO No.1) (available from Microbiology Lab., Benefits and Field Services Div., EPA, Beltsville, MD 20705). Maintain on nutrient agar slants by monthly transfers. Incubate new stock transfer 2 days at 3r; then store at 2-5°. From stock culture inoculate tubes of Pros- kauer-Beck broth, (b)(1). incubate 48 hr in slanting position, carry 30 days, using 48 hr transfers, and use these 48 hr cultures to start test cultures. Inoculate 1 or 2 tubes of Proskauer-Beck broth. Incubate 6-7 days at 3r. Incubate tubes 48 hr in slanting position to provide max. surface aeration and then in upright position 4-5 days. Add 1.5 mL sterile 2.0% Bacto-Gelatin soln and homogenize culture with sterilized glass tissue grinder, 4.016(e). Adjust to 20% T at 650 nm with sterile Proskauer-Beck broth for use in testing. (b) Culture media.-(1) Modified Proskauer-Beck broth.-Dis- solve 2.5 g KH2P04 , 5.0 g asparagine, 0.6 g MgS04 .7H2 0, 2.5 g Mg citrate, 20.0 mL glycerol, 0.0046 g FeCI3, and 0.001 g ZnSO•.7H20 in 1 L H20. Adjust to pH 7.2-7.4 with 1N NaOH. Filter thru paper, place 10 mL portions in sep. 20 x 150 mm tubes, and sterilize 20 min at 121°. Use for propagating 48 hr test starter cultures and 6-7 day test cultures. (2) Subculture media.-Use (1) with addn of suitable neutzg agents such as purified lecithin (Azolectin) or Na thioglycolate, where necessary. (3) Nutrient agar.-Prep. as in 4.001(c). Use to maintain stock culture. (4) Sterile distilled water.-See 4.007(d).
- 88. 66 4. DISINFECTANTS AOAC METHODS (1980) 4.046 Apparatus (a) Glassware, water bath, transfer loops and needles, and petri dishes.-See 4.008(a). (b). (e). and (e). (b) Carriers.-See 4.016(g). 4.047 Operating Technic Transfer 20 sterile Penicylinder carriers, using flamed ni- chrome wire hook, into 20 mL 6-7 day homogenized stdzd broth culture, 4.045(a), in sterile 25 x 150 mm medicant tube. After 15 min contact, remove cylinders and place on end in vertical position in sterile petri dish matted with filter paper, 4.008(e). Cover and place in incubator at 37° and let dry ;;.20 min but ",,60 min. This will provide dried test carriers in groups of 20 in individual petri dishes. With each group of 20 carriers, add 1 dried cylinder at 30 sec intervals to each of 20 tubes contg 10 mL diln of germicide to be tested (at 20° in H20 bath). Flame lips of medicant and subculture tubes. Immediately after placing carrier in medicant tube, swirl tube 3 times before placing it back in H20 bath. (Thus, by time 20 tubes have been seeded, 9 min and 30 sec have elapsed, leaving 30 sec interval prior to subculturing series at 10 min exposure for each carrier. The 30 sec interval between transfers allows adequate time for flaming and cooling transfer hook and making transfer in manner so as to drain all excess medicant from carrier.) Transfer carrier to 10 mL subculture media, 4.045(b)(2). Shake all subculture tubes thoroly and incubate 12 days at 3r. Report results as + (growth) or - (no growth). Where there is reason to suspect that results may be affected by bacteriostatic action of medicant carried over in subculture tubes, use suitable neutralizer in subculture media. Make ;;.30 carrier exposures at each of 3 relatively widely spaced dilns of germicide under test between no response and total response diln levels. Calc. % of carriers on which organism is killed at each diln. Using log.% probit paper (3 cycle logarithmic normal No. 32.376, Codex Book Co., Inc., Norwood, MA 02062). locate % kill points on diln lines employed (log scale). Draw best fitting straight line thru these 3 points and extend to intercept 99% kill line. Read diln line (log scale) at point of intercept. This is presumed 95% confidence end point for product. (Do not use presumptive test organism for checking validity of this presumptive end point.) II. Confirmative In Vitro Test for Determining Tuberculocidal Activity 4.048 Reagents (a) Culture media.-(1) Modified Proskauer-Beck medium.- Prep. as in 4.045(b)( 1), and in addn, place 20 mL portions in 25 x 150 mm tubes. Use 10 mL portions for daily transfers of test cultures and 20 mL portions for subculturing porcelain cylinders. (2) Middlebrook 7H9 Broth Difco A.-Dissolve 4.7 g in 900 mL H20 contg 2 mL glycerol and 15.0 g agar. Heat to bp to dissolve completely. Distribute in 180 mL portions and autoclave 15 min at 121°. To each 180 mL sterile medium at 45°, add 20 mL Middlebrook ADC Enrichment (Difco) under aseptic conditions and distribute in 10 mL portions in sterile 20 x 150 mm tubes. Slant. Use to maintain test culture. (3) Middlebrook 7H9 Broth Difco B.-Dissolve 4.7 g in 900 mL H2 0 contg 2 mL glycerol and 1.0 g agar. Heat to bp to dissolve completely. Distribute in 18 mL portions in 25 x 150 mm tubes, and autoclave 15 min at 121°. To each 18 mL sterile medium at 45° add 2 mL Middlebrook ADC Enrichment under aseptic conditions. Use to subculture for survival. (4) Kirchners Medium Difco.-Dissolve 13.1 gin 1 L H20 contg 20 mL glycerol and heat to bp to dissolve completely. Distribute in 18 mL portions in 25 x 150 mm tubes and autoclave 15 min at 121°. To each 18 mL sterile medium at 50--55° add' 2 mL Middlebrook ADC Enrichment under aseptic conditions. Use to subculture for survival. (5) TB Broth Base Difco (without polysorbate 80). -Dissolve 11.6 g in 1 L H20 contg 50 mL glycerol and 1.0 g agar. Heat to bp to dissolve completely. Distribute in 18 mL portions in 25 x 150 mm tubes,· and autoclave 15 min at 121°. To each 18 mL sterile medium at 50° add 2 mL Dubos Medium Serum (Difco) under aseptic conditions. Use to subculture for survival. (b) Test organism.-Mycobacterium bovis (BCG) (available from ITR Biomedical Research, University of Illinois Medical Center, 904 W Adams St, Chicago, IL 60607). Maintain stock cultures on culture medium (a)(2) by monthly or 6 weeks transfer. Incubate new stock transfer 15-20 days at 37° until sufficient growth is indicated; then store at 2-5°. From stock culture, inoculate tube of culture medium (a)(1) and incubate 21-25 days at 3r. Shake gently once daily for 9 days; then allow to remain quiescent until 21-25th day. Make daily transfers from 21 day cultures. Transfer culture to heat-sterilized glass tissue grinder, add 1.5 mL sterile 2% Bacto-gelatin soln (Difco), grind, and dil. with culture medium (a)(1) to give 20% Tat 650 nm. Use to inoculate porcelain cylinders used in test. Tests will be satisfactory only when organism is killed on all 10 carriers by aq. phenol (1 +50) and shows survival after exposure to aq. phenol (1 + 75) control. Prep. dilns from 5% std phenol soln, 4.002(f). (e) Sterile distilled water.-See 4.007(d). (d) Sterile normal horse serum without preservative.-Difco Laboratories or Microbiological Associates, 4733 Bethesda Ave, Bethesda, MD 20014. 4.049 Apparatus (a) Glassware, water bath, transfer loops and needles, and petri dishes.-See 4.008(a). (b), (e), and (e). (b) Carriers.-See 4.016(g). 4.050 Operating Technic Soak ring carriers overnight in 1N NaOH; rinse with tap H20 and then with distd H20 until distd H20 is neut. to phthln; then rinse twice with distd H20. Place clean ring carriers in multiple of 10 or 20 in capped erlenmeyer or 20 x 150 mm tubes. Autoclave 20 min at 121°, cool, and hold at room temp. Transfer 10 sterile ring carriers, using flamed wire hook, into enough (ca 15-20 mL) 21-25 day stdzd test culture, 4.048(b)' in 25 x 150 m m medication tube. After 15 min contact period, remove cylinders, using flamed wire hook, and place on end in vertical position in sterile petri dish matted with filter paper, 4.008(e). Cover, place in incubator at 37°, and let dry ;;.20 min but ",,60 min. Let 10 tubes contg 10 mL use-diln germicide sample to be tested come to 20° in H20 bath and add 1 contaminated cylinder carrier at either 30 sec or 1 min intervals to each tube. Imme- diately after placing carrier in medication tube, swirl 3 or 4 times before plaCing tube back in bath. (Thus, by time 10 tubes have been seeded, 9 min will have elapsed, plus 1 min interval before transfer of first carrier in series to individual tube of 10 mL serum, 4.048(d). or 10 mL neutralizer blank, 4.023(d). if 1 min intervals are used. This interval is constant for each tube with prescribed exposure period of 10 min. Interval between transfers allows adequate time for flaming and cooling wire hook and making transfer in manner so as to drain all excess medication from carrier.) Transfer carrier to 10 mL serum, 4.048(d), after exactly 10 min contact. Shake tube contg carrier in serum thoroly and place carrier in tube contg 20 mL broth, 4.048(a)( 1). From
- 89. AOAC METHODS (1980) LAUNDRY ADDITIVES 67 same tube, take 2 mL portions serum and place in any 2 of the subculture media, 4.048(a)(3), (4), (5). Repeat this with each of the 10 carriers. Incubate 1 tube of each subculture medium with 2 mL sterile serum as control. Where there is reason to suspect that germicide to be tested may possess bacteriostatic action, use suitable neutralizer in lieu of serum. Shake each subculture tube thoroly, incubate 60 days at 37°, and report results as + (growth) or - (no growth). If no growth or only occasional growth is observed in subculture, incubate addnl30 days before making final reading. Max. diln of germicide which kills test organism on the 10 carriers, and no growth in each of the 2 mL aliquots for 2 extra media, represents max. safe use-diln for practical tuberculocidal disinfection. Bacteriostatic Activity of Laundry Additives (10) Official First Action (Applicable to antimicrobial products, recommended for use during laundering operations, which are intended to provide residual bacteriostatic treatment to laundered fabric. Method includes treatment of fabric with product and subsequent bac- teriostatic testing of treated fabric.) 4.051 Reagents (a) Culture media.-(1) Nutrient broth.-See 4.001(a). (2) Nutrient agar A.-See 4.001(c). Use for monthly transfer of stock cultures. (3) Nutrient agar B.-80il 3 g beef ext, 5 g peptone (Anatone), 8 g NaCI, and 10 g agar (Oifco) in 1 L H20. Transfer 100 mL portions to erlenmeyers, and autoclave 20 min at 121°. Use for agar plate tests to evaluate bacteriostatic activity of treated fabric. See also (c). (b) Test organisms.-Use Staphylococcus aureus ATCC No. 6538 and Klebsiella pneumoniae, aberrant ATCC No. 4352 (formerly Escherichia coli), and maintain as in 4.002(e). (c) 2,3,5-Triphenyl tetrazo/ium chloride.-Use as optional bi- ological indicator. With S. aureus, use 0.15% soln; with K. pneumoniae, aberrant, use 0.25% soln. Autoclave each 20 min at 121°. Apply as in 4.054. (d) Alkaline nonionic wetting agent.-Prep. aq. soln contg 0.5% alkyl phenol polyglycol ether wetting agent and 0.5% Na,C03• Use to scour test fabric. 4.052 Apparatus ! (a) Test fabric.~O x 80 threads/sq in. plain weave cotton print cloth, completely desized, bleached, and without bluing or optical brighteners (available from Test Fabrics, Inc., 55 Van Dam St, New York, NY 10013). Scour before use by boiling ca 300 g 1 hr in 3 L H20 contg 1.5 g nonionic wetting agent and 1.5 g Na2C03• Then rinse fabric, first in boiling H20 and then in cold H2 0, until all visual traces of wetting agent are removed. Air-dry and cut into long strip 5 cm (2") wide and weighing exactly 15 g. (b) Stainless steel spindle.-Fabricate from single continuous piece of stainless steel wire '/'6" diam. and bent to contain 3 horizontal extensions 5 cm (2") long connected by 2 vertical sections ca 5 cm (2") long. Shape so that vertical sections form 150° angle, and sharpen free ends of 2 outer horizontal exten- sions to point (see Fig. 4:02). Use as carrier for test fabric. Primary objective of spindle is to prevent wadding or lodging of test fabric during agitation in exposures to test chem. solns. (c) Exposure chamber.--Clean, dry 1 pt Mason jar with rubber washer or gasket and metal screw cap. (d) Agitator.-Oevice to rotate Mason jar thru 360° vertical orbit of 10-20 cm (4-8") diam. at 45-60 rpm for 5 min. Launder- ometer or Tumble Jar described in AATCC70 8-1967, 43,8154, 8155, or ASTM 0583-63 is adequate. r--- 2" Outer Ext " Hori:!:ntal Extensions TOP 1 SIDE VIEW ~ VIEW ~~ __ ...1"'_ '= 5/16 J --1- "- '" ~.++ r 1 ·Points pencil Horizontal sharp or Extensions canted ""Outer Ext. FIG. 4:02-$tainless steel spindle for winding test fabric (e) Water bath.-Thermostatically controlled at 25°. (f) Petri dishes.-Sterile, 100 x 15 mm. (9) Glassware.-See 4.002(a). (h) Transfer loops and needles.-See 4.002(d). 4.053 Preparation of Fabric (a) Fabric mounting.-Pierce one end of prescoured, 15 g test fabric strip and secure onto an outer horizontal extension of test spindle; then wind strip around 3 horizontal extensions with enough tension to obtain 12 (but not 13) entire laps. Secure final end of test fabric strip to previous laps with stainless steel safety pin. (b) Fabric treatment with product.-Oil. product as directed to 75 mL (most frequently, use directions are based on dry wt of laundry fabric equiv. to 15 g test fabric), add to Mason jar (exposure chamber), and maintain in H2 0 bath at 25°. Add addnl materials to Mason jar as required by use directions for product. These are: (1) Product recommended as final rinse additive in industrial laundering operation.-Add no addnl materials; 5:1 (v/w) treat- ment product soln to dry fabric ratio is representative of indus- trial laundering operations. (2) Product recommended as final rinse additive in home or coin-operated laundering operations.-Add 150 mL H2 0 to Ma- son jar. Resultant 10:1 (v/w) treatment product soln to dry fabric ratio is representative of home and coin-operated laundering operations. (3) Product recommended as final rinse additive in both industrial and home laundering operations.- Prep. 2 jars contg product soln according to (1) and (2) so that 2 test fabric strips may be treated at different treatment product soln to dry fabric ratios (5:1 and 10:1 (v/w)). (4) Product recommended as final rinse additive and de- scribed as compatible with adjunct chemicals which may be used in this cycle (sours, bleaches, opticalbrighteners, softeners, etc).-Prep. so that required vol. of product treatment soln contains adjunct chemicals according to description and amts specified on product label or advertising literature. 4.054 Operating Technic Place test spindle with test fabric in Mason jar contg product soln. Secure rubber gasket and Mason jar cap, remove from H2 0 bath, place jar in agitator, and rotate 5 min. Addnl manip- ulation with test spindle is required if use directions do not specify addn of product in final rinse phase of laundry cycle. In this instance, to det. durability of antimicrobial agent in fabric, execute 3 rinse operations as follows: Immediately after end of
- 90. 68 4. DISINFECTANTS AOAC METHODS (1980) initial 5 min agitation, drain treatment soln from Mason jar and replace with 100 mL H20. Secure Mason jar contg test spindle, return to agitator, and rotate 2 min. Repeat operation twice more. Following all required fabric treatment operations, remove test spindle from Mason jar and unwind test fabric strip from spindle. Let test fabric strip air dry with long axis of strip in horizontal position. When test strip is dry, remove 1 sq in. bacteriological test samples. Five test samples are required for single bacteriostatic test against 1 test organism. In each such instance, at least 2 test samples must be removed from middle 20% of length of test strip. Perform bacteriostatic agar plate tests as follows: Prep. 5 replicate plates in each test for each organism. Sep. inoculate flasks contg 100 mL sterile, liq. (~400) nutrient agar B with 1 mL 24 hr nutrient broth culture of S. aureus and K. pneumoniae, aberrant. Immediately thereafter, if desired, add 1 mL appro- priate soln of 2,3,5-triphenyl tetrazolium chloride to inoculated nutrient agar B. Vigorously swirl contents of erlenmeyers to ensure complete mixing. Add 10 mL portions of inoculated agar to 100 mm sterile petri dishes, distribute evenly, and let cool and harden. As soon as plates harden, implant single 1 sq in. treated fabric test sample on center of 1 test agar plate surface. Using blunt forceps, press each fabric test sample onto agar surface to ensure complete and uniform contact. Incubate test plates 48 hr at 3r. If desired, test plates may be refrigerated 18-20 hr before incubation. Following incubation, examine test plates to det. presence or absence of zones of inhibition along each side of test fabric sample. 4.055 Interpretation Use clear zone of inhibition adjacent to each side of test fabric sample as index of bacteriostatic activity. Size of zone is not considered important, but zone is required to extend along entire edge to be acceptable. Score zone of inhibition along single side of sq test fabric samples as 1, so that for 5-replicate plate test, a score of 20 shows that bacteriostasis occurs along all 4 sides of each sample. Total score of 18/20 sides demon- strating bacter'iostasis is required for effective demonstration of residual bacteriostatic activity of laundry fabric treated with antimicrobial laundry additive product during laundering op- eration. Unless qualified residual bacteriostatic claim is made, residual treatment must be bacteriostatic against both S. aureus and K. pneumoniae, aberrant. SELECTED REFERENCES (1) J. Roy. Sanit. Inst. 24,424(1903); Am. J. Public Health 3, 575(1913); U.S. Dept. Agr. Circ. 198 (1931); JAOAC 32, 408(1949); 38, 465(1955); Soap Chem. Spec. 34, No. 10, 79(1958). (2) J. Bacteriol. 49, 526(1945); Am. J. Vet. Res. 9, 104(1948); JAOAC 36, 466(1953). (3) Soap Sanit. Chem. 27, No.2, 133(1951); JAOAC 38, 274(1955); 40,755(1957). (4) JAOAC 36, 480(1953); 39, 480(1956); 40, 759(1957); 49, 721 (1966). (5) Arch. Dermatol. Syphilol. 28, 15(1933); J. Bacteriol. 42, 225(1941); 47, 102(1944); JAOAC 37, 616(1954); 38, 274(1955). (6) Am. J. Public Health 38, 1405(1948); J. Milk Food Technol. 19, 183(1956); Fed. Regist. 21, 7020(1956); JAOAC 41, 541(1958). (7) JAOAC 44, 422(1961); 50,763(1967); Soap Chem. Spec. 38(2),69(1962). (8) JAOAC 47,540(1964); 48,640(1965). (9) JAOAC 48,635(1965); 50, 767(1967). (10) JAOAC 52, 836(1969).
- 91. 5. Hazardous Substances PAINT lead (1)-Official Final Action 5.001 Reagents and Apparatus (a) Lead std salns.-(1) Stock saln.-1 mg Pb/mL 1% HN03 • Dissolve 159.9 mg Pb(N03)2 in HN03 (1 +99) and dil. to 100 mL with HN03 (1 +99). (2) Intermediatesaln.-300 p,g/mL dil. HN03 · Oil. 15 mL stock soln to 50 mL with 0.5 mL HN03 and H20. (3) Working salns.-To each of seven 100 mL vol. flasks contg 1 mL HN03, add resp. 0, 1, 2, 3, 4, 5, and 6 mL intermediate soln and dil. to vol. with H20 (0,3,6,9,12,15, and 18 p,g Pb/mL). (b) Atomic absorption spectrophatameter.-With Pb hollow cathode lamp and 4" single slot or 3 slot Boling burner head, capable of detecting 0.5p,g Pb/mL, such as Perkin-Elmer Model 403. Operating conditions: 283.3 nm, 0.7 nm band width slit, recorder response (if used) 0.2~1 sec time constant, air-C2H2 flame, with gas flows adjusted according to directions of man- ufacturer. (e) Heater for digestion.-Drill 7.5 cm AI block to hold ;;.16 test tubes, 16 x 150 mm. Place on hot plate capable of main- taining medium at 160-170° (Corning PC 35, or equiv.). Sand bath may be used instead of AI block. (d) Boiling chips.-Unglazed boiling chips, 1.5 mm diam., Pb- free. 5.002 Determination of Solids Thoroly mix samples manually for 10 min or mech. for 5 min. Accurately weigh 0.3-0.4 g into weighed AI dish, 63 mm diam. Add 3-5 mL hexane or pet ether to oil-based paints or H20 to latex paints and swirl to disperse. Warm on hot plate while swirling until solv. has evapd and film is formed. Heat in oven 4 hr at 105°, cool, and weigh. % Solids = g dried sample x 100/g sample. 5.003 Determination of Lead Introduce ca 0.6 g (0.3 mL) thoroly mixed sample near bottom of 16 x 150 mm test tube with syringe and weigh accurately. Add 5±0.2 mL HN03 and 2 boiling chips to each, including blanks. Place in block or bath at 90-100° so that liq. surface is slightly above heated surface. (Use hood.) After initial fuming has subsided, increase temp. until vapors are condensing in top 1-2 cm of tube (bath temp., 160-170°) and maintain at this temp. 3 hr. Cool to 50-60°, transfer to 25 mL vol. flask, including chips and any ppt, and rinse with four 4 mL portions H20, transferring as much residue as possible. Oil. to vol. with H20 and let settle 0.~1 hr. Floating residue may be removed by aspiration thru disposable pipet. Aspirate solns and stds into AA spectrophotometer, avoiding introduction of ppt. If A of sample is greater than highest std, dil. sample and re-aspirate. Det. p,g Pb/mL from std curve. % Pb in paint solids = (p,g Pb/mL) x F x 10-2/ (g sample x % solids in sample) F (diln factor) = 1/[{1/25) (b/c) (die) ...J, where 25 = vol. original sample digest, b = aliquot of original 25 mL dild to c mL; d = aliquot of c (mL) dild to e mL; etc. For dry paint films, % solids in sample = 100. 69 PREPARATIONS CONTAINING FLUORIDES Fluoride (2)-Official Final Action 5.004 Apparatus (a) pH meter.-With expanded mv scale (digital Model 110, Corning Scientific Instruments, Medfield, MA 02052, or equiv.). fluoride ion-selective electrode (Model 94-09, Orion Research Inc., 11 Blackstone St, Cambridge, MA 02139, or equiv.), and single junction ref. electrode, plastic sleeve-type (Model 90-01, Orion Research Inc., or equiv.). (b) Magnetic stirrer.-With Teflon-coated stirring bar. Use asbestos or foam mat to insulate sample from motor heat. (c) Beakers.-4.5 oz (135 mL). polypropylene, or equiv. (d) Graph paper.-Linear or semi-antilog, vol. corrected No. 90-00-90 Gran's plot paper (Orion Research Inc., or equiv.). 5.005 Reagents (a) Buffer soln.-pH 6.0. Add 77.0 g NH40Ac and 0.452 g NH4 citrate to 1 L H20. Adjust to pH 6.0 with HOAc. (b) Fluoride std soln.-1 mg F/mL. Prep. 2.2108 g NaF (reagent grade, dried 4 hr at 105°)/L buffer soln. (Caution: See 51.084.) Store in leakproof plastic bottles. Compare with 1 mg F/mL soln prepd from USP Ref. Std; equiv. reading of ±1 mv is satisfactory. 5.006 Determination (Stir all solns constantly at same rate thruout titrns. Let electrodes equilibrate ;;.2 min before addn of F std soln and 30 sec after each addn of F std soln.) (a) Blank.-Record mv values (E') of 100 mL buffer soln after addn of 4 mL std F soln from 10 mL buret and after each addnl mL up to 10 mL. (Preliminary mv values will not fall on linear range of response curve.) Vol. std soln added = V'. (b) Samples.-Est. molarity of samples from direct reading. Oil. samples, if necessary, to ca 0.001M F. Transfer 50 mL sample soln to beaker and add 50 mL buffer soln. Record initial mv reading, using expanded scale (Eo). If initial reading is <-50 mv, soln is too concd. Oil. sample to avoid asymptotic slope. Record mv values (E) after each mL F std soln is added up to 10 mL. Rinse electrodes with H20 between samples. Vol. std soln added = V. 5.007 Calculations (a) Linear graph paper.-For each addn of F std soln and corresponding E value, calc. for blank: Z' = antilog [log(Vo + V') - 0.017(E')], where Va is original vol. soln to which F std soln was added (100mL) and E' is treated algebraically (+ or - as read). PlotZ' against mL (mg) F std soln added and extrapolate to intersection of mL (mg) Faxis to obtain mL (mg) F in blank, Ve'. In graph, assign horizontal axis to mL (mg) F, with 0 at center and mL (mg) F increasing in both directions to left and right. Assign Z values to vertical axis. Plot actual readings of mL (mg) F on right portion of horizontal axis so that extrapolation will fall on left portion of axis.
- 92. 70 5. HAZARDOUS SUBSTANCES AOAC METHODS (1980) Similarly, for original readings and each addn of F std soln and corresponding E value, calc. for sample: Z = antilog [log(Vo + V) - 0.017(E}j, where Vo is original vol. soln to which F std soln was added (100 mL). Plot Z against mL (mg) F std soln on same graph as blank and extrapolate to intersection of mL (mg) Faxis to obtain mL (mg) F in sample, Ve' (b) Semi-antilog paper.-Plot E directly for both blank and sample, descending 5 mv for each major line crossing vertical axis. At top of vertical axis place most neg. E reading which still allows extrapolation of Ve on left portion of mL (mg) side of horizontal axis. Obtain Ve and V,' by extrapolation to left side of 0 mL (mg) F. % F = (Ve - V.') x (8 x 100)/[W x C x 1000 (mg/g)]. where 8 = vol. of diln, W = mL or g sample, and C = aliquot (50 mL max.) buffered to 100 mL. PREPARATIONS CONTAINING METHANOL Methanol (3)-Official Final Action (Applicable in presence of acetone, BuOAc, EtOH, isopropanol, hexane, MeEt ketone, CH2CI2, Me Cellosolve, paraffin, toluene, and H20. This includes many paint removers, fuels, liq. sanders, antifreezes, and paint products.) 5.008 Apparatus and Reagents (a) Gas chromatograph.-With flame ionization detector and oven capable oftemp. changes ~5°/min near 160° or preferably temp. programmer. Column.-1.8 m (6') x 4 mm id packed with 120-150 mesh Porapak R (Waters Associates, Inc., PO Box 246, Milford, MA 01757); condition 2 hr at 235°. Conditions: Temps (0): injection ca 200, column ca 160, detector ca 210; N flow ca 25 mL/min; set electrometer so that 8 ILL std soln provides at least half scale peak. Adjust column temp. and N flow so that MeOH retention time is ca 5-7 min. (b) Methanol std soln.--O.4% (v/v). Oil. 4.00 mL MeOH to 100 mL with dioxane; dil. 10.0 mL of this soln to 100 mL with dioxane. Rinse pipet into flask before dilg to vol. with dioxane. Prep. fresh daily. 5.009 Preparation of Sample (a) For asphalt-base tar compounds and viscous adhesives.- Refrigerate unopened sample container ~3 hr (longer for larger containers) at 1-10°, open container, and mix well; close con- tainer and refrigerate 30 min more. Transfer 1.5-3 g sample to tared, 250 mL, wide-mouth g-s erlenmeyer (tared with stopper in place). Let sample reach room temp. in stoppered erlenmeyer and weigh. Refrigerate 30 min and quickly add 100.0 mL dioxane. Stopper and shake mech. 1 hr. Refrigerate 30 min and filter thru rapid paper (S&S sharkskin, or equiv.). Filter as quickly as possible, covering funnel with watch glass and placing funnel against neck of narrow-mouth g-s receiver. Proceed as in 5.010, dilg with dioxane, if necessary. (b) For other less viscous products.-Prep. soln with pipets and vol. flasks to contain ca 0.4% (v/v) MeOH, dilg with dioxane. Avoid excessive shaking of semiviscous products and do not fill pipet above mark. (Use safety pipet filler to draw liq. to mark and hold until transfer.) Wash pipet with dioxane and add washings to soln. If MeOH concn is unknown, prep. 2% soln. Prep. addnl dilns as needed. 5.010 Determination Inject portion std soln with 10 ILL syringe. Note vol. At RMA (retention time relative to MeOH) ca 0.5, inject portion sample soln. Note vol. At RMA ca 2 (from second injection), repeat injection of std soln. At RMA ca 0.5 (from third injection), repeat injection of sample soln. After MeOH from fourth injection elutes, increase column temp. to 235° as rapidly as possible for time ca 4 x R';'A until all dioxane (RMA ca 5) is removed from column. Cool column to 160° and repeat sequence for subse- quent sample. Modify injection time if necessary to sep. MeOH from other peaks. (Note: Injection sequence is used only to save time; it need not be used if desired.) 5.011 Calculation Oet. retention areas for each MeOH peak by mUltiplying peak ht by retention distance. Average retention areas for sample (RA) and for std (RA 'I. Presence of solv. in column changes retention times, requiring use of retention areas in calcn. % MeOH (w/v) in sample soln = F x (RA/RA') x (V'/V) x C x 0.79, where F = diln factor, C = % (v/v) std soln, V and V' = vol. sample and std soln injected, resp., and 0.79 = density of MeOH. Volatile Denaturants in Alcoholic Products (4) Official First Action 5.012 Apparatus and Reagents (a) Gas chromatograph and integrator.-See 19.001(a) and (b). (b) Std solns.-fJ% (v/v). Oil. 6.00 mL of each denaturant of interest to 100 mL with anhyd. alcohol in sep. vol. flasks. Approx. slopes and retention times relative to n-PrOH are given in Table 5:01. Table 5:01 Approximate Slopes and Retention Times Relative to n-Propyl Alcohol (RT) for Denaturants Compound Acetone Benzene n-Butyl alcohol sec-Butyl alcohol Chloroform Ethyl acetate Ethylene glycol monoethyl ether Ethylene glycol monomethyl ether Isopropanol Methanol Methyl isobutyl ketone Toluene Slope 0.207 0.464 0.269 0.246 0.058 0.192 0.187 0.151 0.210 0.130 0.275 0.454 RT 0.694 2.309 2.283 1.621 1.543 1.640 3.868 2.071 0.727 0.266 5.436 5.302 5.013 Determination Pipet 25 mL of each expected denaturant std soln into sep. flasks and add 1.00 mL n-PrOH as internal std. Proceed as in 19.002, starting with "Cap immediately ...", except inject 0.3 ILL portions. Oet. peak areas and calc. slope for each compd as: Sx = (PA x/PA i )/6.00, where PAx and PA i = peak areas of compd X in std soln and of n-PrOH internal std, resp., and 6.00 = % compd X in std soln. Slopes and retention times should approximate those of Table 5:01. % Compd X in sample = (PA/PA i ) = (1/Sx), where PA = peak area of compd X in sample.
- 93. AOAC METHODS (1980) SELECTED REFERENCES 71 PREPARATIONS CONTAINING PHENOL Phenol Method I (5)-Official Final Action (Applicable to com. cresols, saponified cresol solns, coal tar dips, and disinfectants, and to kerosene solns of phenols in absence of salicylates or !3-naphthol) 5.014 Reagents (Caution: See 51.026 and 51.065.) (a) Dilute nitric acid.-Aerate HN03 until colorless and dil. 1 vol. with 4 vols H20. (b) Millon reagent.-To 2 mL Hg in 200 mL erlenmeyer under hood, add 20 mL HN03. After first violent reaction, shake as needed to disperse Hg and maintain action. After ca 10 min, when action practically ceases even in presence of undissolved Hg, add 35 mL H20, and if basic salt seps, add enough dil. HN03 to dissolve it. Add 10% NaOH soln dropwise with thoro mixing until curdy ppt that forms after adding each drop no longer redissolves but disperses as permanent turbidity. Add 5 mL dil. HN03and mix well. Prep. fresh daily. Millon reagent is danger- ously poisonous and should not be transferred with ordinary pipet and mouth suction unless protective trap is used. (c) Phenol std soln.-Dissolve weighed amt pure phenol (congealing point ;;.40°) in enough H20 to make ;;.1% soln. On day it is to be used, dil. to make 0.025% aq. soln (final std). (d) Formaldehyde soln.-Dil. 2 mL 37% HCHO soln to 100 mL with H20. (e) Methyl orange indicator.-o.5% aq. soln. 5.015 Apparatus (a) Nessler cylinders.-50 mL tall-form, matched. (b) Test tubes.-Approx. 180 x 20 mm, with rubber stoppers, marked at 25 mL. (c) Water bath for heating test tubes.-Beaker contg disk of wire gauze raised ca 2.5 cm from bottom may be used. 5.016 Preparation of Sample (a) Commercial cresol.-Weigh by difference ca 2.5 g sample into 250 mL vol. flask, dissolve in 10 mL 10% NaOH soln, and dil. to vol. with H20. (b) Saponified cresol solns, coal tar dips and disinfectants, kerosene solns of phenols, etc.-Weigh by difference ca 5 g sample (or use 5 mL and calc. wt from density) into 250 mL vol. flask and dil. to vol. with H20. With products consisting largely of kerosene, bring H20 level to mark and take aliquots from aq. portion only. 5.017 Determination Transfer 5 mL aliquot prepd soln to 200 mL vol. flask and promptly dil. to ca 50 mL. Add 1 drop Me orange, (e), and then dil. HN03 until soln is practically neut. Oil. to vol. and shake well. Place 5 mL dild soln in each of 2 marked test tubes; in each of 2 addnl test tubes place 5 mL std phenol soln. Flow 5 mL Millon reagent down side of each tube, mix, and place tubes in boiling H20 bath; continue boiling exactly 30 min, cool imme- diately and thoroly by immersion in bath of cold H20 ;;.10 min, and add 5 mL dil. HN03to each tube. Mix well and add 3 mL HCHO soln to one of each pair of tubes. Oil. all tubes to 25 mL mark with H20, stopper, shake well, and let stand overnight. (Tubes contg HCHO fade to yellow; others show orange or red color.) Pipet 20 mL from each of the 2 phenol tubes to 100 mL vol. flasks; add 5 mL dil. HN03 to each, dil. to vol., and mix. (Red flask contains "phenol std," yellow flask "phenol blank.") Trans- fer these solns to burets. Pipet 10 mL of each sample soln into Nessler tubes. (The orange or red constitutes the "unknown" and the yellow the "sample blank." Mark each Nessler tube distinctly to avoid confusion.) To "sample blank" tube add measured amt of "phenol std" and add same vol. "phenol blank" to "unknown." Agitate thoroly (aided by insertion of rubber stoppers, if necessary), and compare colors. When tubes are brought to match, each mL phenol std used = 1% phenol if sample weighing exactly 5 g was used, or 2% if exactly 2.5 g was used. Note.-Take following precautions: Pair of phenol tubes pro- vides enough final solns to assay several unknowns, but all the latter must have accompanied phenol solns thruout entire process with identical reagents and treatment. If end point is inadvertently overrun it is possible to work back to it, but since mistakes may be made in this operation it is better to repeat comparison on fresh portions from original tubes. Too much delay in matching tubes must be avoided after titrn is started, otherwise excess HCHO present in blanks may have time after mixing to affect intensity of red color. 5.018 Method II (6)-Official Final Action (Applicable to detn of phenol in presence of salicylates) Weigh by difference 10 g sample into separator (or use 10 mL and calc. wt from density of sample). Add 50 mL kerosene and ext with three 100 mL portions H20. Filter aq. exts thru wet filter into 500 mL vol. flask, dil. to vol. with H2 0, and proceed as in 5.017. When tubes are brought to match, each mL phenol std used = 1% phenol if sample weighing exactly 10 g was used. SODA LYE Carbonate and Hydroxide (7)-Official Final Action 5.019 Determination Weigh ca 10 g sample from weighing bottle, dissolve in CO2- free H20, and dil. to definite vol. Titr. aliquot with 0.5N HC!, 50.011-50.012, using Me orange, 5.014(e), and note total alky found. Transfer equal aliquot to vol. flask and add enough 10% BaCI2 soln to ppt all carbonate, avoiding any unnecessary excess. Oil. to vol. with CO2-free H20, stopper, shake, and let stand. When liq. clears, pipet off one-half and titr. with the O.5N HC!, using phthln; mL 0.5N acid required for this titrn x 2 = mL 0.5N acid equiv. to NaOH present in original aliquot. Difference between this figure and mL 0.5N HCI required for total alky = mL O.5N acid equiv. to Na2C03 present in aliquot. Calc. % Na2C03 and NaOH. EARTHENWARE 5.020 Cadmium and Lead See 25.031-25.037. SELECTED REFERENCES (1) JAOAC 57, 614(1974). (2) JAOAC 56, 798(1973). (3) JAOAC 54, 558(1971); 55,242(1972). (4) JAOAC 57, 148(1974). (5) USDA Bull. 1308, p. 17; JAOAC 13, 160(1930). (6) Ind. Eng. Chem., Anal. Ed. 1, 232(1929). (7) Sutton "Systematic Handbook of Volumetric Analysis," 10th ed., p. 61 (1911).
- 94. 6. Pesticide Formulations (Pesticide ref. stds may be available from the following: Alltech Associates, 202 Campus Dr, Arlington Hts, IL 60004; Analabs, Inc.; Applied Science Laboratories; Chem Service Inc., West- chester, PA 19380; ICN-K&K Laboratories, Inc.; RFR Corp., 1 Main St, Hope, RI 02831; and Supelco.) GENERAL METHODS 6.001 Sampling-Procedure (Caution: See 51.041.) Examine shipping cases closely for code numbers, different labels, and other pertinent information. Give special attention to products subject to deterioration. Caution: Use care in sampling and transporting toxic materials to avoid personal injury and contamination of transportation facilities in case of breakage. When dealing with rodenticides and weed-killers, avoid mutual contamination with other prod- ucts during transportation. Mark each sample container according to laboratory require- ments. (a) Small package retail units.-Take one unopened unit (1 Ib if dry, 1 pt if liq.), except take min. of 2 units of small baits in cake form. Size of sample is governed by composition of material and anal. methods. (b) Large package dry products (25 Ib or more).- Sample unopened containers, using trier long enough to reach bottom of container by inserting into container at one edge or corner and probing diagonally toward opposite edge or corner. Take cores by code or batch number. Analyze cores from same code or batch number as composite or individually. Clean trier thoroly after sampling each batch. Store samples in air-tight glass, metal, plastic, or cardboard containers. (c) Large package liquid products (5 gallons or more).-Use glass, plastic tubing, or stainless steel trier with plunger, or rubber tubing for certain materials. Store samples in glass or containers of other noncorrosive material with screw top caps lined with Teflon or other inert material. Plastic containers may be used only for carefully selected products. 6.002 Sampling of Pressurized Containers (1) Official First Action Delivery assembly.-Construct U-shaped stainless steel as- sembly from Va" od tubing, with 1 arm 70 mm long with front and back ferrules (Swagelok No. SS-204-1 and SS-203-1, Crawford Fitting Co., 29500 Solon Rd, Solon, OH 44139) per- manently swaged 3 mm from end, and other arm 135 mm longer (205 mm total length) sepd by 75 mm bend. Remove spray head from can. For male-type outlet, attach assembly from above ferrule to outlet with short piece of Tygon tUbing. For female-type outlet, insert ferrule directly into outlet. Sample is released from can by carefully applying firm hand pressure to shoulder of assembly. See Fig. 6:01. Sampling.-Remove cover and spray head from can. Place can and delivery assembly in 800 mL beaker and weigh (Wo)' Shake can vigorously 5 min. Immediately after shaking attach delivery assembly to spray outlet. Immerse long end into 250 mL separator contg 100 mL CHCI3• Press shoulder to deliver ca 9 9 sample (ca 10 sec) into CHCI3 • Return can and delivery assembly to beaker and weigh. Shake can vigorously 3 min and repeat delivery of sample until total of 10-15 g is obtained in 72 CHCI3• Reweigh can and delivery assembly in beaker (W). Calc. sample wt as (Wo - W). 6.003 Preparation of Sample-Official Final Action Thoroly mix all samples before analysis. Det. H20-sol. As on samples as received, without further pulverization or drying. In case of lye, NaCN, or KCN, weigh large amts in weighing bottles and analyze aliquots of their aq. solns. 6.004 Moisture-Official Final Action (Applicable to Paris green, powd Pb arsenate, Ca arsenate, Mg arsenate, Zn arsenite, powd Bordeaux mixt., and Bordeaux mixt. with arsenicals) Dry 2 g to const wt at 105-110° and report loss in wt as moisture. ARSENIC Total Arsenic-Official Final Action Hydrazine Sulfate Distillation Method (2) (Nitrates do not interfere. Applicable to detn of total As in Paris green, Pb arsenate, Ca arsenate, Zn arsenite, Mg arsenate, and Bordeaux mixt. with arsenicals) 6.005 (a) Arsenious oxide std soln.-O.1 50.005-50.006. PRESS Reagents or 0.05N. See fiG. 6:01-Delivery assembly for sampling pressurized containers
- 95. AOAC METHODS (1980) ARSENIC 73 (b) Iodine std soln.---O.l or 0.05N. See 50.018-50.019. (c) Bromate std soln.---O.l or 0.05N. Dissolve ca 2.8 or 1.4 g KBr03 in boiled H20 and dil. to 1 L. Stdze as follows: Pipet 25 mL aliquots As20 3 soln, (a). into 500 mL erlenmeyers. Add 15 mL HCI, dil. to 100 mL, heat to 90°, and titr. with the KBr03 soln, using 10 drops Me orange, (9). Do not add indicator until near end of titrn, and agitate soln continuously to avoid local excess of KBr03 soln. Add KBr03 soln very slowly near end point; at end point soln changes from red to colorless. (d) Hydrazine sulfate-sodium bromide soln.-Dissolve 20 g N2H4.H2S04 and 20 g NaBr in 1 L HCI (1 +4). (e) Sodium hydroxide soln.-Dissolve 400 g NaOH in H2 0 and dil. to 1 L. (f) Starch indicator.-Mix ca 2 g finely powd. potato starch with cold H20 to thin paste; add ca 200 mL boiling H20, stirring constantly, and immediately discontinue heating. Add ca 1 mL Hg, shake, and let soln stand over the Hg. (9) Methyl orange indicator.---O.05%. Dissolve 0.5 g Me or- ange in H20 and dil. to 1 L. 6.006 Apparatus See Fig. 6:02. Set 500 mL distn flask on metal gauze that fits over circular hole in heavy sheet of asbestos board, which in turn extends out far enough to protect sides of flask from direct flame of burner. First receiving flask holds 500 mL and contains 40 mL H20; second holds 500 mL and contains 100 mL H20. Vol. in first flask should be .;40 mL, otherwise compd of As may sep. that is difficult to dissolve without danger of loss of AsCI3• Keep both flasks cool by placing in pan of circulating H20, or contg H20 and ice. 6.007 Determination (Caution: See 51.041 and 51.078.) Weigh sample contg ';0.4 g As and transfer to distg flask. Add 50 mL N2H4.H2S04-NaBr soln, close flask with stopper that carries funnel tube, and connect side tube with condenser. Boil 2-3 min, add 100 mL HCI from dropping funnel, and distil until vol. in distg flask is reduced to ca 40 mL; add 50 mL more HCI and continue distn until vol. is again reduced to ca 40 mL. Wash down condenser, transfer contents of receiving flasks to 1 L vol. flask, dil. to vol., mix thoroly, and proceed as in (a) or (b): (a) Pipet 200 mL aliquot into erlenmeyer and nearly neutze with NaOH soln, using few drops phthln, and keeping soln well cooled. If neut. point is passed, add HCI until again slightly acid. Neutze with NaHC03, add 4-5 g excess, and add std I soln from buret, shaking flask continuously until yellow color disappears slowly from soln. Add 5 mL starch indicator and keep adding std I soln dropwise to permanent blue. (b) Pipet 200 mL aliquot into erlenmeyer and titr. with KBr03 soln, (c). beginning " ... heat to 90° ..." Calc. % As. Report as As20 3 or As20 s, according to whether As is present in trivalent or pentavalent form. If condition of arsenic is unknown, report as As. lodimetric Method (3) (Applicable in presence of sulfides, sulfites, thiosulfates, and large amts of S or org. matter) 6.008 Reagent Sodium thiosulfate soln.-Dissolve 13 g crystd Na2S20 3.5H20 in H20 and dil. to 1 L. See 6.005 for other reagents and solns and 6.006 for app. 6.009 Determination (Caution: See 51.041 and 51.078.) Weigh sample contg ';0.4 g As and transfer to distg flask. Add 50 mL N2H4.H2S04-NaBr soln, 6.005(d), and distil as in 6.007. Dil. distillate to vol. in 1 L vol. flask, mix thoroly, and transfer 200 mL aliquot to 400 mL Pyrex beaker or porcelain casserole. Add 10 mL HN03 and 5 mL H2S04, evap. to sirupy consistency on steam bath, and then heat on hot plate to white fumes of H2S04, Cool, and wash into 500 mL erlenmeyer. If vol. H2S04 is appreciably lessened by fuming, add enough H2S04 to make total vol. ca 5 mL. Dil. to 100-150 mL, add 1.5 g KI, and boil until vol. is reduced to ca 40 mL. Cool under running H20, dil. to 100-150 mL, and add Na2S20 3 soln, 6.008, dropwise until I color just disappears. Nearly neutze H2S04 with NaOH soln, 6.005(e). finish neutzn with NaHC03, add 4-5 g excess, and titr. with std I soln as in 6.007(a). From mL std soln used, calc. % As in sample. Report as As20 3, As20 s, or As as in 6.007. FIG. 6:02--Apparatus for distilling arsenious chloride
- 96. 74 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) Ion Exchange Method (4)-Official Final Action (Applicable to inorg. arsenates and arsenites) 6.010 Apparatus Ion exchange column.-Use Allihn filter tube 10 x 2.7 cm od with coarse filter disk. Attach piece of rubber tubing to bottom of filter tube and regulate flow with Hoffman clamp. To tube add aq. slurry of Dowex 50W-X8, 50-100 mesh, using resin bed vol. of 12 mL, and place 500 mL separator above tube. Regenerate resin bed before each run by first back-washing column few min with H20; then elute with 350 mL 2N HCI followed by 200 mL H20 at 20 mLjmin. 6.011 Preparation of Sample (Caution: See 51.041.) Weigh 200 mg sample (100 mg if As content is >30%) into 150 mL beaker, add 7 mL HN03, and bring to bp. Add 3 mL 2N KBr03 and evap. to dryness, avoiding spattering. Backwash and regenerate resin during this evapn. Dissolve cooled residue in 2 mL 6N HCI without heat and add 8 mL H20. Filter into separator, and wash filter with three 10 mL portions H20. (If residue dissolves completely in 2 mL 6N HCI, omit filtration, and dil. directly to 40 mL.) Pass soln thru resin column at 20 mLjmin and collect eluate in 250 mL erlenmeyer. Wash separator and column with 20 and 40 mL portions H20 into same erlenmeyer. 6.012 Determination Add 50 mL HCI to eluate to make 4N. Add 1 g NaHC03, 0.2 g at time, swirling constantly. Add 1 g KI, stopper, and swirl until all KI dissolves. After 5 min, titr., without starch indicator, with 0.05N Na2S203' 50.037--50.038, to disappearance of I. (Recog- nition of end point is facilitated by titrg on porcelain stand. In presence of starch, reaction between I and Na2S20 3 is retarded, so appreciable amt of Na2S20 3 reacts with acid. End point becomes indistinct if >30 mL Na2S20 3 is used in titrn.) 1 mL 0.05N Na2S20 3 = 1.873 mg As. Water-Soluble Arsenic--Official Final Action (Applicable to detn of H20-sol. arsenic in Pb arsenate, Ca arsenate, Zn arsenite, Mg arsenate, and Bordeaux mixt. with arsenicals) 6.013 Determination (Caution: See 51.041.) To 2 g original sample if powder, or 4 g if paste, in 1 L Florence flask, add 1 L recently boiled H20 that has been cooled to 32°. Stopper flask and place in constant temp. H20 bath at 32°. Digest 24 hr, shaking hourly 8 hr during this period. Filter thru dry filter. If filtrate is not clear, refilter thru buchner contg paper and enough Filter-Cel coating to give clear soln. Discard first 50 mL. Transfer 250-500 mL clear filtrate to erlenmeyer, add 3 mL H2S04 , and evap. to ca 100 mL on hot plate. Add 1 g KI, and continue boiling until vol. is ca 40 mL. Cool, dil. to ca 200 mL, and add Na2S203 soln, 6.008, dropwise, until I color is exactly removed. (Avoid use of starch indicator at this point.) Neutze with NaHC03, add 4-5 g excess, titr. with std I soln, shaking flask continuously, until yellow disappears slowly, add 5 mL starch indicator, 6.005(f), and continue titrn to permanent blue. Correct for amt std I soln necessary to produce same color, using same reagents and vol. From mL std I soln used, calc. % H20-sol. As in sample. LEAD (5)-OFFICIAL FINAL ACTION (Applicable to such prepns as Bordeaux-Pb arsenate, Bordeaux- Zn arsenite, Bordeaux-Paris green, and Bordeaux-Ca arsenate) (Caution: See 51.026, 51.031, 51.041, 51.059, and 51.078.) 6.014 Determination Weigh 1 g powd sample and transfer to beaker. Add 5 mL HBr (ca 1.38 sp gr) and 15 mL HCI, and evap. to dryness to remove As. Repeat treatment; add 20 mL HCI, and again evap. to dryness. Add 25 mL 2N HCI to residue, heat to bp, filter immediately to remove Si02, and wash with boiling H20 to vol. of 125 mL. See that all PbCI2 is in soln before filtering; if it will not dissolve completely in 25 mL 2N acid, add 25 mL more and dil. filtrate to 250 mL. Pass in H2 S until pptn is complete. Filter, and wash ppt thoroly with 0.5N HCI satd with H2S. Save filtrate and washings for Zn detn. Transfer paper with sulfides of Pb and Cu to 400 mL Pyrex beaker and completely oxidize all org. matter by heating on steam bath with 4 mL H2S04 and ca 20 mL fuming HN03 in covered beaker. Evap. on steam bath, and then completely remove HN03 by heating on hot plate to copious white fumes of H2S04 , Cool, add 2-3 mL H20, and again heat to fuming. Cool, add 50 mL H20 and 100 mL alcohol, and let stand several hr (preferably overnight). Filter thru gooch, previously washed with H20, then with acidified alcohol (100 parts H20, 200 parts alcohol, and 3 parts H2S04 ), and finally with alcohol, and dried at 200°. Wash ppt of PbS04 in crucible ca 10 times with acidified alcohol, and then with alcohol, to remove H2S04, Retain filtrate and washings for Cu detn, if desired. Dry at 200° to const wt, keeping crucible covered to prevent loss from spattering. From wt PbS04, calc. % Pb in sample, using factor 0.6832. COPPER (5)-OFFICIAL FINAL ACTION (Applicable to such prepns as Bordeaux-Pb arsenate, Bordeaux- Zn arsenite, Bordeaux-Paris green, and Bordeaux-Ca arsenate) 6.015 Electrolytic Method Evap. filtrate and washings from PbS04 pptn, 6.014, to fuming; add few mL fuming HN03 to destroy org. matter, and continue evapn to ca 3 mL. Take up with ca 150 mL H20, add 5 mL HN03, and filter if necessary. Wash into 250 mL beaker, adjust vol. to 200 mL, and electrolyze, using rotating anode and weighed gauze cathode with current of 2-3 amp. After all Cu has apparently deposited (ca 30 min), add 15-20 mL H20 to electro- lyte and continue electrolysis few min. If no further deposition occurs on newly exposed surface of electrode, wash with H20 without breaking current either by siphoning or quickly replacing beaker with electrolyte successively with 2 beakers of H20. Interrupt current, rinse cathode with alcohol, dry few moments in oven, and weigh. Calc. % Cu in sample. 6.016 Volumetric Thiosulfate Method Proceed as in 6.015 to point at which filtrate and washings from PbS04 pptn are treated with fuming HN03 and evapd to vol. of ca 3 mL. Take up in ca 50 mL H20, add NH4 0H in excess, and boil to expel excess NH3, as shown by color change in liq. and partial pptn. Add 3-4 mL HOAc (4+1), boil 1-2 min, cool, add 10 mL 30% KI soln, and titr. with std Na2S203 soln, 31.040, until brown color becomes faint. Add starch indicator, 6.005(f), and continue titrn cautiously until blue color due to free I entirely disappears. From mL std Na2S203 soln used, calc. % Cu in sample.
- 97. AOAC METHODS (1980) FLUORINE 75 ZINC (6)-OFFICIAl FINAL ACTION (Applicable to such prepns as Bordeaux-Pb arsenate, Zn arsenite, Bordeaux-Zn arsenite, Bordeaux-Paris green, and Bordeaux-Ca arsenate) 6.017 Reagent Mercury-thiocyanate soln.-(Caution: See 51.079.) Dissolve 27 g HgCI2 and 30 g NH.SCN in H20 and dil. to 1 L. 6.018 Determination Conc. filtrate and washings from sulfide pptn, 6.014, by gentle boiling to ca 50 mL; then evap. on steam bath to dryness. Dissolve residue in 100 mL H20 contg 5 mL HCI, and add 35-40 mL Hg-thiocyanate soln with vigorous stirring. Let stand ~1 hr with occasional stirring. Filter thru weighed gooch, wash with H20 contg 20 mL Hg-thiocyanate soln/L, and dry to const wt at 105°. Calc. to % Zn, using factor 0.1312. Note: Some Fe is usually present and during Zn detn should be in ferrous condition. In pptg sulfides pass H2S into soln long enough to reduce Fe as well as to ppt Cu and Pb. ZnHg(SCN). ppt normally is white, and occluded Fe(SCNb should not give more than faint pink color. FLUORINE Total Fluorine--Offieial Final Action Lead Chlorofluoride Method (7) 6.019 Reagents (a) Fusion mixture.-Mix 30 g anhyd. Na2C03 with 40 g anhyd. K2C03• (b) Lead chlorofluoride wash soln.-Dissolve 10 g Pb(N03 )2 in 200 mL H20, dissolve 1 9 NaF in 100 ml H20 and add 2 mL Hel, and mix these 2 solns. Let ppt settle and decant. Wash ppt 4 or 5 times with 200 mL H20 by decanting; then add ca 1 L cold H20 to ppt and let stand ~1 hr, with occasional stirring. Filter and use clear filtrate. (Prep. more wash soln as needed by adding more H20 to ppt of PbCIF and stirring.) (e) Silver nitrate std soln.-{).1 or 0.2N. Stdze as in 50.031. (d) Potassium or ammonium thiocyanate std soln.-{).1N. Stdze against std AgN03 soln under same conditions as in detn. (e) Ferric indicator.-To cold satd CI-free FeNH.(SO.b.12H20 soln add enough colorless HN03 to bleach brown color. (f) Bromophenol blue indicator.-Grind 0.1 g powder with 1.5 mL 0.1N NaOH and dil. to 25 mL. 6.020 Determination (a) Samples difficult to decompose such as cryolite, and others that contain aluminum or appreciable amounts of sili- ceous material.-Mix 0.5 g sample (or less if necessary to contain 0.01-0.10 9 F) with 5 g fusion mixt. and 0.2-0.3 g powd. Si02 in Pt dish, cover with 1 9 fusion mixt., and heat to fusion over Bunsen burner. (Use of blast lamp is unnecessary since it is preferable not to heat much beyond melting temp. If much AI is present, uniform, clear, liq. melt cannot be obtained; particles of white solid will sep. in melt. Cooled melt should be colorless, or at least should not have more than gray color.) Leach cooled melt with hot H20 and when disintegration is complete, filter into 400 mL beaker. Return insol. residue to Pt dish with jet of H20, add 1 9 Na2C03, dil. to 30-50 mL, and boil few min, disintegrating any lumps with flat-end rod. Filter thru same paper, wash thoroly with hot H20, and adjust vol. of filtrate and washings to ca 200 mL. Add 1 g ZnO dissolved in 20 mL HN03 (1 +9), boil 2 min, stirring constantly, filter, and wash thoroly with hot H20. During this washing return gelatinous mass to beaker 3 times and thoroly disintegrate in wash soln because proper washing of this ppt on filter is difficult. (Mass can easily be returned to beaker by rotating funnel above beaker while cutting ppt loose from paper with jet of wash soln.) Add 2 drops bromophenol blue to filtrate, and with cover glass almost entirely over beaker, add HN03 (1 +4) until color just changes to yellow. Make soln slightly alk. with 10% NaOH soln, and with cover glass on beaker, boil gently to expel CO2. Remove from burner; add the HN03 until color just changes to yellow and then 10% NaOH until color just changes to blue; then add 3 mL 10% NaCI soln. (Vol. of soln at this point should be ca 250 mL.) Add 2 mL HCI (1 + 1) and 5 g Pb(N03)2 and heat on steam bath. As soon as Pb(N03 )2 is in soln, add 5 g NaOAc, stir vigorously, and digest on steam bath 30 min with occasional stirring. Let stand overnight, filter, and wash ppt, beaker, and paper once with cold H20, then 4 or 5 times with PbCIF wash soln, and then once more with cold H20. Transfer ppt and paper to beaker in which pptn was made, stir paper to pulp, add 100 mL HN03 (5+95). and heat on steam bath until ppt dissolves. (5 min is ample to dissolve ppt. If sample contains appreciable amt of sulfates, ppt will contain PbSO., which will not dissolve. In such case heat 5-10 min with stirring and consider PbCIF to be dissolved.) Add slight excess 0.1N or 0.2N AgN03, digest on steam bath 30 min, and cool to room temp., protecting from light; filter, wash with cold H20, and det. AgN03 in filtrate by titrn with std thiocyanate soln, using 10 mL ferric indicator. Subtract amt of AgN03 found in filtrate from that originally added. Difference is amt required to combine with CI in the PbCIF; from this difference calc. % F in sample. 1 mL 0.1N AgN03 = 0.00190 9 F. (b) Water-soluble fluorides in presence of organic matter.- In presence of ,,;50% org. matter such as flour, pyrethrum, tobacco powder, and derris or cube powders, which readily decompose without addn of powd Si02 and contain little or no sulfates, AI, or siliceous compds, mix 0.5 g sample (or less if necessary to contain 0.01-0.1 g F) with 5 g fusion mixt., cover with 1 g fusion mixt., and heat to fusion over Bunsen burner. Leach cooled melt with hot H20, and when disintegration is complete, filter into 600 mL beaker. Wash thoroly with hot H20 and proceed as in (a), third par. In presence of >50% org. matter or org. matter that is impractical to free without preliminary ashing, such as apple peel and pulp, transfer enough sample to Pt crucible to be representative of mixt. and to contain 0.01-0.1 g F. Add 15 mL H20 and enough F-free CaO (0.3-0.4 g) to make mixt. distinctly alk. to phthln, mix with glass rod, and evap. to dryness on steam bath and in oven at 105°. Ignite at low heat, preferably in furnace (,,;600°). until org. matter is thoroly charred. Pulverize, with glass rod, any lumps present in charred ash, mix with 5 g of the fusion mixt., and proceed as in (a). first par., beginning " ... cover with 1 g fusion mixt., ..." (e) Water-soluble samples in absence of organic matter and appreciable quantities ofsulfates oraluminum salts. -In absence of org. matter or other interfering substances, fusion may be omitted and detn made on aliquot of aq. soln contg 0.01-0.1 g F, as in (a). third par. In presence of AI, as in samples contg Na2SiFe and KAI(SO')2 .12H20, transfer sample to 400 mL beaker, dissolve in 150 mL hot H20, add 6 g fusion mixt., and boil. Add 1 g ZnO dissolved in 20 mL HN03 (1+9). boil 2 min with const stirring, filter into 500 mL vol. flask, and wash thoroly with hot H20. Cool to room temp. and dil. to vol. Transfer 200 mL aliquot contg 0.01-0.10 g F to 600 mL beaker and proceed as in (a). third par. (d) Sodium and magnesium fluosilicates, or samples con- taining more than 5% sulfates in absence of aluminum and
- 98. 76 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) boron with or without moderate amounts of organic matter.- With large amts of Na2SiF6 and some other more volatile fluosilicates, e.g., MgSiF6' where there is possibility of some F being evolved as SiF4 before fusion is effected, or in samples contg appreciable amts of sulfates, distil F as in 6.024, and det. F in distillate as follows: Add several drops bromophenol blue, make alk. with NaOH, and adjust vol. to ca 250 mL by gently boiling down vol. from 400 to 250 mL. Proceed as in (a), third par., beginning "Remove from burner; ..." Notes: These methods give accurate results for 0.01--0.10 g F. Below 0.01 g, results tend to be slightly low, and above 0.10 g, slightly high. Convenient sample to fuse is one contg 0.07--0.08 g F; too large sample may result in incomplete fusion. Large amts of B compds and alkali salts retard or prevent complete pptn of PbCIF. B has greater effect when amt of F is large than when it is small. In methods described B has little effect, and it may be disregarded in analysis of insecticides if amt of F to be pptd is ";0.03 g. With some prepns contg Na2B40 7 or H3B03, where it is difficultto obtain representative mixt. when extremely small sample (0.1 g) is used for analysis, take larger sample and ppt PbCIF from aliquot of fusion soln. Amt of alkali carbonates specified in fusion and in washing of insol. residue is not large enough to cause low results. If sample contains S, remove it with CS2 and det. F on air-dried residue, allowing in calcns for % S removed. (Caution: See 51.039, 51.040, and 51.048.) 6.021 Modified Travers Method (8) (Applicable in absence of B, AI, and large amts of pyrethrum powder) Reagents (a) Alcoholic potassium chloride soln.-Dissolve 60 g KCI in 400 mL H20, add 400 mL alcohol, and test with phthln; if soln is not neut., adjust to exact neutrality with NaOH or HCI soln. (b) Sodium hydroxide std soln.---O.2N. Prep. and stdze as in 50.032-50.035. 6.022 Determination Treat 0.5 g sample in small beaker with 20-25 mL H20. Add 0.3 g finely divided pptd Si02 and few drops Me orange. Add HCI dropwise until soln assumes apparently permanent pink; 1 I then add 2 mL excess, cover beaker with watch glass, and boil 1 min. Cool to room temp., add 4 g KCI, and stir until KCI dissolves. Add 25 mL alcohol and let stand 1 hr, stirring frequently. Filter thru gooch contg disk of filter paper covered with medium pad of asbestos. Wash ppt with alc. KCI soln until one washing does not destroy color made by 1 drop 0.2N NaOH and phthln (usually 3-4 washings). Transfer crucible and con- tents to 400 mL.beaker, add 100 mL recently boiled H20 and 1-2 mL phthln, heat, and titr. with std NaOH soln. Finish titrn with the F soln actively boiling. Calc. % F. 1 mL 0.2N NaOH = 0.0057 g F. 6.023 Distillation Method (9) (Applicable to H20-sol. or H20-insol. insecticides in absence of gelatinous Si02, B, and AI) Reagents (a) Sodium alizarin sulfonate indicator.-Dissolve 0.1 g Na alizarin sulfonate in 200 mL H20. (b) Thorium nitrate soln.-Approx. 0.05N. Stdze in terms of g F/mL by titrg F obtained by distn from std NaF as in 6.024. In stdzg for use with 6.024(b), add 5 mL satd KMn04 soln in addn to other reagents in distn flask. 6.024 Determination (a) In absence of organic matter.-Weigh sample contg ca 0.09 g F, and with aid of little H20 transfer to 250 mL Claisen distn flask contg 12 glass beads. Adjust to ca 30 mL and close flask with 2-hole rubber stopper, thru which pass thermometer and 4 mm glass tube, both of which extend into soln. (The 4 mm glass tube extends ca 5 cm above rubber stopper and by means of rubber tube, E, connects still with 1 L Florence flask contg H20 for steam generation. Flask is equipped with steam discharge, H, and pressure tube, G. See Fig. 6:03.) Bring H20 in steam generating flask to boil with pinchcock, F, in release tube open. Connect distg flask to condenser, and add 25 mL H2S04 thru top of 4 mm tube, using pipet or special funnel. With pinchcock, F, open, connect rubber tubing to 4 mm FIG. 6:03-Apparatus for determining fluorine
- 99. AOAC METHODS (1980) HERBICIDE VOLATILITY 77 tube. Light burner under Claisen flask. Regulate flow of steam by adjusting burner flames and pinchcock, F, so that vol. of soln is held canst and temp. in flask, B, is kept at 145-150°. Continue distn until 400 mL distillate collects. Oil. to 500 mL in vol. flask, transfer 50 mL aliquot to tall-form 150 mL beaker, and add 5 drops indicator, 6.023(a). Adjust acidity with 1% NaOH soln and HCI (1 +249) until pink just disappears. Add 2 mL of the HC!, and titr. with 0.05N Th(N03). to permanent pink, using buret graduated in 0.05 mL. (b) In presence of organic matter.-(Caution: See 51.080.) In presence of moderate amts of org. matter, transfer sample contg ca 0.09 g F and contg ",0.2 g org. matter, with aid of little H20, to 250 mL Claisen distn flask contg 12 glass beads. Add 5 mL satd KMnO. soln, adjust to ca 30 mL, and proceed as in (a), beginning " ... close flask with 2-hole rubber stopper, ..." In presence of large amts of org. matter, transfer sample to medium-size Pt dish, add 15 mL H20 and enough F-free CaO to make mixt. distinctly alk. to phthln, mix with glass rod, and evap. to dryness on steam bath and in oven at 105°. Ignite at low heat, preferably in furnace (",600°), until org. matter is thoroly charred. Pulverize any lumps present in charred ash with glass rod, transfer to 250 mL Claisen distn flask by brushing, and finally wash out dish with 30 mL H2SO. (1 +9). Except to add 22 mL instead of 25 mL H2SO., proceed as in (a), par. 2. Note: If coating of pptd Si02 forms on inside of distn flask, remove by treatment with hot concd alkali soln, as it is capable of retaining F during distn of some samples and giving it up, at least in part, in later distns. 6.025 Fluorine Present as Sodium Fluosilicate Official Final Action (B, CaO, and alum absent) Reagents Alcoholic potassium chloride and sodium carbonate so/n.- Dissolve 1.0 g Na2C03 in 100 mL alc. KCI reagent, 6.021(a). For other reagent see 6.021. 6.026 Determination Weigh 1 g sample into Pt dish, and add rapidly, with continuous stirring, 50 mL of the alc. KCI-Na2C03 reagent. Do not let soln become acid, and if necessary, use more reagent to insure alky. Continue stirring until all sol. portions of sample dissolve. Proceed as in 6.022, beginning: "Filter thru gooch "Calc. % Na2SiF6 (1 mL 0.2N NaOH = 0.009403 g Na2SiFs). CONTAMINATION BY ORGANOCHLORINE PESTICIDES (10)-OFFICIAL FINAL ACTION AOAC-CIPAC Method (Applicable to detection of contamination by 0.01 % chlorinated hydrocarbons such as aldrin, DDT, dieldrin, and endrin, and 0.05-0.10% of chlordane, Strabane, and toxaphene) 6.027 Apparatus (a) Thin layer chromatographic apparatus.-See 29.006. (b) Ultraviolet apparatus.-Sterilamp G-15T8 (Westinghouse Electric Corp., Lamp Divisions, One Westinghouse Plaza, Bloom- field, NJ 07003). 6.028 Reagents (a) Adsorbent.-Aluminum oxide G, Type E (Brinkmann In- struments, Inc.). (b) Mobile solvents. -( 1) n-Hexane, (2) n-hexane-acetone. (98+2), and (3) n-hexane-alcohol (98+2). (c) Pesticide std solns.-1 IL9/ILL EtOAc, acetone, or any convenient solv. 6.029 Preparation of Sample Ext 8 g sample with 20 mL acetone in 250 mL erlenmeyer by shaking intermittently 5 min. Let solids settle. If soln is turbid, filter or centrf. to obtain clear supernate for spotting. For samples contg large amts S, use 8 g sample and 20 mL pet ether. 6.030 Preparation of Plates Dissolve 0.1-0.15 g AgN03 in 1-2 mL H20 in 100 mL beaker, add 58 mL MeOH, and mix. Weigh 40 g adsorbent, (a), in 250 mL flask, add AgN03-MeOH soln, and shake vigorously 20 sec. Apply slurry as 0.25 mm thick layer to five 20 x 20 cm (8 x 8") plates positioned on plastic mounting board. After plates appear dry, store in desiccator over desiccant. When plate is dry, scrape 1 cm strip from side edges to ensure even solv. front. Use plate immediately after removal from desiccator. 6.031 Detection Pour n-hexane into glass chromatgc tank to depth of 10-20 mm. Place 2 paper blotters (ca 7.5 x 22 cm) on each side oftank or large blotter covering back of tank and let equilibrate ~2 hr before use. Spot 10 ILL sample ext on plate with 100 ILL syringe. Do not disturb adsorbent layer. Also spot std solns of pesticides de- clared as part of formulation. Spots should be ",6 mm diam. and placed <30 mm from bottom of plate. Place plate in chromatgc tank, and let plate develop ~10 cm. Remove plate and expose to shortwave UV, 6.027(b). (Caution: See 51.016.) Chlorinated org. pesticides should be visible as dark spots against white or light gray background. Expose plates ~1 hr. Longer exposure will not harm plates. To confirm identification of pesticide, repeat TLC step with different mobile solv., 6.028(b)(2) or (3). VOLATILITY OF ESTER FORMS OF HORMONE-TYPE HERBICIDES (11)-OFFICIAL FINAL ACTION 6.032 Material (a) Paper bags.-No. 20 to open with flat bottom. Close with paper clips. (b) Filter paper.-7 cm diam. (c) Bacteriologicalloop.--{J.01 mL. Wash with acetone after each application or heat to cherry red in flame. (d) Test plants.-Actively growing tomato seedlings 65-75 mm high in 3-4" pots. (e) Formulation to be tested.-Use 0.01 mL aliquot of 4 Ib/gal. formulation or equiv. vol. of other concns. (f) High and low volatile ester stds.-Use Bu ester of 2,4-0 as high volatile ester and tetrahydrofurfural ester of 2,4-0 as low volatile ester with same wt of acid/ gal. as formulations to be tested. 6.033 Operating Technic Open bags with flat bottom and place plant toward one side on bottom of bag. Apply 0.01 mL of formulation to middle of filter paper by means of bacteriological loop, and for controls, apply 0.01 mL solv. only. Place treated paper in bottom of bag. Do not touch treated part of paper against plant, sides of bag, or pot. Close bag by folding top, secure with clips, and let stand 24 hr at 85-110°F (29-43°C).
- 100. 78 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) Use 3 plants per treatment and 3 for controls. Repeat test on another day. Remove plants from bag, let stand 24 hr, and read curvature (stem bending, epinasty) response. (Fold and discard used bags to prevent contamination.) Rate plants according to scale as follows: (1) Normal growth of untreated check-no apparent response. (2) Epinasty 1-20° compared to normal-no curling. (3) Epinasty 21-40° compared to normal-slight curling. (4) Epinasty 41-60° compared to normal-moderate curling. (5) Epinasty 61-80° compared to normal-moderate curling. (6) Epinasty 81 to >90° compared to normal-severe formative effects. Mean response of 1 to 2.4 for all tests indicates low volatility. Mean response of 2.5 to 6 indicates volatile formulation. To detect small differences between low volatile esters, or differences between 2,4-D and 2,4.5-T types, hold plants 7 days after treatment to allow time for modified leaves or stem lesions to develop. Absence of such responses indicates that formulation was a low volatile 2,4,5-T ester. INORGANIC AND ORGANOMETALLIC PESTICIDES AND ADJUVANTS * PARIS GREEN * (Caution: See 51.041.) 6.034 Moisture-Official Final Action See 6.004. 6.035 Total Arsenic-Official Final Action See 6.007. Total Arsenious Oxide-Official Final Action (Following methods det. only As present in trivalent form (As20 3) and Sb present in trivalent form (Sb20 3) in absence of ferrous and cuprous salts.) Method 1(12) 6.036 Reagents See 4.028, 10th ed. 6.037 Determination lodometric titrn. See 4.029, 10th ed. 6.038 Method II (73) Bromate titrn. See 4.030, 10th ed. 6.039 Water-Soluble Arsenious Oxide Official Final Action lodometric titrn. See 4.031, 10th ed. Total Copper-Official Final Action 6.040 Electrolytic Method See 4.032, 10th ed. 6.041 Volumetric Thiosulfate Method (74) See 4.033, 10th ed. * Methods for this product are surplus--6ee inside front cover. LEAD ARSENATE (Caution: See 51.041 and 51.078.) 6.042 Moisture-Official Final Action (a) Powder.-Dry 2 g to canst wt at 105-110°. Report loss in wt as H20. (b) Paste.-Proceed as in (a). using 50 g. Grind dry sample to fine powder, mix well, transfer small portion to sample bottle, and again dry 1-2 hr at 105-110°. Use this anhyd. material to det. total Pb and total As. Total Arsenic-Official Final Action 6.043 Method I See 6.007. 6.044 Method II (15) (Not applicable in presence of Sb) Dissolve 1 g powd sample with HN03 (1 +4) in porcelain casserole or evapg dish, add 5 mL H2S04, and heat on hot plate to copious evolution of whIte fumes. Cool, add little H20, and again evap. until white fumes appear, to assure removal of last trace of HN03. Wash into 200 mL vol. flask with H20, cool, dil. to vol., and filter thru dry filter. Transfer 100 mL filtrate to erlenmeyer and proceed as in 6.013, beginning " ... add 1 g KI, ..." From mL std I soln used, calc. % total As as As20 s. 6.045 Total Arsenious Oxide (76)-Official Final Action Weigh 2 g powd sample and transfer to 200 mL vol. flask, add 100 mL H2S04 (1 +6). and boil 30 min. Cool, dil. to vol., shake thoroly, and filter thru dry filter. Nearly neutze 100 mL filtrate with NaOH soln, 6.005(e). using few drops phthln. If neut. point is passed, make acid again with the dil. H2S04, Continue as in 6.013, beginning "Neutze with NaHC03, ..." From mL std I soln used, calc. % As20 3. Total Arsenic Oxide (77)-Official Final Action 6.046 Reagents (a) Potassium iodide soln.-Dissolve 20 g KI in H20 and dil. to 100 mL. (b) Thiosulfate std soln.-0.05N. Prep. daily by dilg O.lN soln, 50.037-50.038. 1 mL 0.05N Na2S203 = 2.873 mg As20 s. 6.047 Determination Weigh 0.5 g powd sample and transfer to erlenmeyer. Add 25-30 mL HCI and evap. to dryness on steam bath. Add 50 mL HCI and if necessary to effect soln, heat on steam bath, keeping flask covered with watch glass to prevent evapn of acid. Cool to 20-25°, add 10 mL of the KI soln and 50 mL (or more if necessary to produce clear soln) 25% NH4CI soln, and imme- diately titr. liberated I with std Na2S203 soln. When color becomes faint yellow, dil. with ca 150 mL H20 and continue titrn carefully, dropwise, until colorless, using starch indicator, 6.005(f), near end point. From mL Na2S203 soln used, calc. % As20 s• 6.048 Water-Soluble Arsenic-Official Final Action Proceed as in 6.013, and calc. results as As20 s. 6.049 Total Lead (18)-Official Final Action In 600 mL beaker on hot plate heat 0.5 g powd sample and ca 25 mL HN03 (1 +4). Filter to remove any insol. residue. Dil. to ;;.400 mL, heat nearly to bp, and add NH40H to slight pptn, then
- 101. AOAC METHODS (1980) COPPER NAPHTHENATE 79 HN03 (1 +9) to redissolve ppt, adding 1-2 mL excess. Into this soln, kept almost boiling, pipet 50 mL hot 10% K2Cr04 soln stirring constantly. Decant while hot thru weighed gooch, pre- viously heated to 140-150°, and wash ppt several times by decanting and then on filter with boiling H20 until washings are colorless. Dry PbCr04 at 140-150° to const wt. From wt PbCr04, calc. % Pb, using factor 0.6411. (PbCr04 ppt may contain small amt PbHAs04, which will cause slightly high results, but this error rarely is >0.1--0.2%.) CALCIUM ARSENATE (Caution: See 51.041.) 6.050 Moisture--Official Final Action See 6.004. 6.051 Total Arsenic-Official Final Action See 6.007. 6.052 Total Arsenious Oxide (19)-Official Final Action (a) Not applicable in presence ofnitrates.-Weigh 1 g sample, transfer to 500 mL erlenmeyer, and dissolve in 100 mL HCI (1 +3). Heat to 90° and titr. with std KBr03 soln, 6.005(c), using 10 drops Me orange, 6.005(g). From mL std KBr03 soln used, calc. % As20 3. (b) Applicable in presence of small amounts of nitrates.- Proceed as in (a) except to titr. at room temp. 6.053 Water-Soluble Arsenic-Official Final Action Proceed as in 6.013, and calc. results as As20 s' (In testing Ca arsenate by this method, low value for H20-sol. As is not assurance against plant injury when using this product.) Total Calcium (79)-Officiai Final Action 6.054 Reagents (a) Ammonium oxalate soln.-Dissolve 40 g (NH4)2C204.H20 in 1 L H20. (b) Potassium permanganate std soln.-0.1N. Prep. and stdze as in 50.025-50,026. 6.055 Method I Dissolve 2 g sample in 80 mL HOAc (1 +3), transfer to 200 mL vol. flask, dil. to vol., and filter thru dry filter. Transfer 50 mL aliquot to beaker, dil. to ca 200 mL, heat to bp, and ppt Ca with (NH4)2C204 soln. Let beaker stand 3 hr on steam bath, filter, and wash ppt with hot H20. Dissolve ppt in 200 mL H20 contg 25 mL H2S04(1 +4), heat to ca 70°, and titr. with std KMn04soln. From mL KMn04 soln used, calc. % Ca. 6.056 Method 1/ (Not applicable in presence of Pb. Caution: See 51.078.) Weigh 2 g sample, transfer to beaker, add 5 mL HBr (ca 1.38 sp gr) and 15 mL HCI, and evap. to dryness under hood to remove As. Repeat treatment, add 20 mL HCI, and again evap. to dryness. Take up with H20 and little HCI, filter into 200 mL vol. flask, wash, and dil. to vol. Transfer 50 mL aliquot to beaker, add 10 mL HCI and few drops HN03, boil, and make slightly alk. with NH40H. Let stand few min and filter. Dissolve ppt in HCI (1 +4), reppt, filter thru same paper, and wash with hot H20. To combined filtrates and washings add 20 mL HOAc (1 +3) and adjust to ca 200 mL. Heat to bp, ppt with (NH4)2C204 soln, and let stand 3 hr on steam bath. Filter, and wash with hot H20. Ignite at 950°, and weigh as CaO; or dissolve and titr. as in 6.055. From wt CaO or mL KMn04 soln used, calc. % Ca. * ZINC ARSENITE * (Caution: See 51.041.) 6.051 Moisture--Official Final Action See 6.004. 6.058 Total Arsenic-Official Final Action Proceed as in 6.007 and calc. as As20 3. Total Arsenious Oxide--Official Final Action 6.059 Method 1(19) Bromate titrn. See 4.051, 10th ed. 6.060 Method /I lodometric titrn. See 4.052, 10th ed. 6.061 Water-Soluble Arsenic-Official Final Action Proceed as in 6.013, and calc. results as AS20 3. 6.062 Total Zinc (19)-Official Final Action Gravimetric method. See 4.054, 10th ed. * COPPER CARBONATE * Copper-Official Final Action 6.063 Electrolytic Method See 4.055, 10th ed. 6.064 Volumetric Thiosulfate Method See 4.056, 10th ed. COPPER NAPHTHENATE (Caution: See 51.041.) Copper (20)-Official First Action 6.065 Titrimetric Method Accurately weigh sample contg ca 0.2 g Cu into dry g-s flask. Add 5 mL pet ether to concd products. Add 100 mL H20, 1.5 g NH4 HF2, and 5-10 g KI. Stopper and shake vigorously until reaction is complete (usually ca 2 min). Wash stopper and sides of flask with H20 and titr. with std 0.1N Na2S203 (stdzd against CuI to light brown. Add starch indicator, 6.005(f), titr. almost to end point, add 2 g KSCN, shake to dissolve, and complete titrn to starch end point. 6.066 Electrolytic Method Accurately weigh sample contg ca 0.2 g Cu into 200 mL separator. Add 50 mL pet ether and 25 mL HN03 (1 +4), and shake 2 min. Drain aq. phase into 250 mL beaker and save. Wash pet ether with 15 and 10 mL HN03 (1 +4), and combine acid exts. Neutze with NH40H, acidify with 6 mL H2S04 and 4 mL HN03, and proceed as in 6.015, beginning " ... adjust vol. to 200 mL, ..." using ca 0.5 amp during first 10 min and 1.5-2.0 amp for ca 20 min. *Methods for this product are surplus-5ee inside front cover.
- 102. 80 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) BORDEAUX MIXTURE (Caution: See 51.041.) 6.067 Moisture-Official Final Action (a) Powder.-5ee 6.042(a). (b) Paste.-Heat ca 100 g in oven at 90-100° until dry enough to powder readily and note loss in wt. Powder this partially dried sample and det. remaining H20 in 2 g as in (a). Det. CO2 as in 6.069, both in original paste and in partially dried sample. Calc. total H2 0 by following formula: M = +(100-a)(b+c) d a 100 ' where M = % total H2 0 in original paste; a = % loss in wt of original paste during first drying; b = % loss in wt of partially dried paste during second drying; c = % CO2 remaining in partially dried paste after first drying; and d = % total CO2 in original paste. Carbon Dioxide (21)-Official Final Action 6.068 Apparatus Use 200 mL erlenmeyer with 2-hole stopper; in one hole fit dropping funnel with stem extending almost to bottom of flask, and thru other hole pass outlet of condenser that is inclined upward at 30° angle from horizontal. Connect upper end of condenser with CaCI2 tube, which in turn connects with double U-tube filled in middle with pumice fragments, previously satd with 20% CuS04.5H20 soln and subsequently dehydrated, and with CaCI2 at either end. Connect 2 weighed U-tubes to absorb CO2, first filled with porous soda-lime, and second, 'I:. with soda- lime and % with CaCI2, placing the CaCI2 at exit end of train. Attach Geissler bulb, partly filled with H2S04, to last U-tube to show rate of gas flow, and connect aspirator with Geissler bulb to draw air thru app. Connect absorption tower filled with soda- lime to mouth of dropping funnel to remove CO2 from air entering app. 6.069 Determination Weigh 2 g powder or 10 g paste into the erlenmeyer and add ca 20 mL H2 0. Attach flask to app., omitting the 2 weighed U- tubes, and draw CO2-free air thru app. until it displaces original air. Attach weighed U-tubes as in 6.068, close stopcock of dropping funnel, pour into it 50 mL HCI (1 +4), reconnect with soda-lime tower, and let acid flow into erlenmeyer, slowly if there is much CO2, rapidly if there is little. When effervescence diminishes, place low Bunsen flame under flask and start flow of H2 0 thru condenser, letting slow current of air flow thru app. at same time. Maintain steady but quiet boil and slow air current thru app. Boil few min after H2 0 begins to condense, remove flame, and continue air flow at ca 2 bubbles/sec until app. is cool. Disconnect weighed absorption tubes, cool in balance case, and weigh. Increase in wt = CO2 , Copper-Official Final Action 6.070 Electrolytic Method (Also applicable to CuC03 and CuSO.) Dissolve powd sample contg 0.2--0.25 9 Cu in 45 mL HN03 (1 +4). Filter if necessary, dil. to 200 mL, and electrolyze as in 6.015. 6.071 Volumetric Thiosulfate Method Dissolve 2 g powd sample in ca 25 mL HN03 (1 +4), dil. to 50 mL, add NH.OH in excess, and heat. Without removing ppt that has formed, boil off excess NH3, add 3-4 mL HOAc, cool, add 10 mL 30% KI soln, and titr. as in 6.016, beginning " ... titr. with std Na2S20 3 soln, ..." * BORDEAUX MIXTURE WITH PARIS GREEN * (Caution: See 51.041.) 6.072 Moisture-Official Final Action See 6.067. 6.073 Carbon Dioxide-Official Final Action See 6.069. 6.074 Total Arsenic-Official Final Action Proceed as in 6.007, using 2 g sample, and calc. results as As203• 6.075 Total Arsenious Oxide-Official Final Action lodometric titrn. See 4.067, 10th ed. 6.076 Water-Soluble Arsenious Oxide-Official Final Action lodometric titrn. See 4.068, 10th ed. Copper-Official Final Action 6.077 Electrolytic Method I See 6.015. 6.078 Electrolytic Method II-(Short Method) See 4.070, 10th ed. 6.079 Volumetric Thiosulfste Method See 6.016. * BORDEAUX MIXTURE WITH LEAD ARSENATE * (Caution: See 51.041.) 6.080 Moisture-Official Final Action See 6.067. 6.081 Carbon Dioxide-Official Final Action See 6.069. 6.082 Total Arsenic-Official Final Action Proceed as in 6.007, using 2 g sample, and calc. results as As20 s. 6.083 Water-Soluble Arsenic-Official Final Action Proceed as in 6.013 and calc. results as As20 s' Copper-Official Final Action 6.084 Electrolytic Method See 6.015. * Methods for this product are surplus---,see inside front cover.
- 103. AOAC METHODS (1980) LIME SULFUR 81 6.085 Volumetric Thiosuffate Method See 6.016. 6.086 Lead-Official Final Action See 6.014. Lead and Copper-Official Final Action 6.087 Electrolytic Method (22) (Caution: See 51.026,51.047, and 51.078.) See 4.079-4.080, 10th ed. * BORDEAUX MIXTURE WITH CALCIUM ARSENATE * (Caution: See 51.041.) 6.088 Moisture--Official Final Action See 6.067. 6.089 Carbon Dioxide--Official Final Action See 6.069. 6.090 Total Arsenic--Official Final Action Proceed as in 6.007, using 2 g sample, and calc. results as As20 s' 6.091 Water-Soluble Arsenic--Official Final Action Proceed as in 6.013 and calc. results as As20 s. Copper-Official Final Action 6.092 Electrolytic Method I See 6.015. 6.093 Electrolytic Method /I See 6.078. 6.094 Volumetric Thiosuffate Method See 6.016. * CALCIUM CYANIDE (23) * 6.095 Cyanide--Official Final Action AgN03 titrn. See 4.093-4.094, 10th ed. Chloride--Official Final Action 6.096 Method I See 4.095, 10th ed. 6.097 Method /I See 4.096, 10th ed. POTASSIUM CYANATE (24)-OFFICIAL FINAL ACTION (Caution: See 51.041.) 6.098 Reagent Wash soln.-Satd aq. soln of hydrazodicarbamide, NH2CO- NHNHCONH2 • Prep. by mixing KOCN and semicarbazide.HCI, NH2CONHNH2.HCI, in H20, filter, and wash ppt with H20. Transfer ppt to flask, add small amt H20, shake vigorously, and filter. (Solubility of ppt in H20 is ca 1 part in 6600.) 6.099 Determination Weigh sample contg 0.2-0.5 g KOCN into 100 mL beaker, add 20 mL wash soln and 1 g semicarbazide.HCI, and let stand 24 hr. Filter hydrazodicarbamide on gooch or fine fritted glass crucible, wash with 10 mL wash soln, and dry at 100° to canst wt. KOCN = wt residue x 0.6868. * SODIUM AND POTASSIUM CYANIDES * (Caution: See 51.050.) 6.100 Cyanide (25)-Official Final Action AgNOa titrn. See 4.088-4.089, 10th ed. (Caution: See 51.084.) Chloride (26)--Official Final Action 6.101 Method I Pptn with AgN03 and thiocyanate back-titrn. See 4.090-4.091, 10th ed. 6.102 Method /I Oistn, pptn with AgN03, and thiocyanate back-titrn. See 4.092, 10th ed. LIME SULFUR SOLUTIONS AND DRY LIME SULFUR Soluble Sulfur (27)-Official Final Action (Use low S reagents.) 6.103 Preparation of Sample (a) Solns.-Accurately weigh ca 10 g soln, transfer to 250 mL vol. flask, and immediately dil. to vol. with recently boiled and cooled H20. Mix thoroly and either take necessary aliquots in individual pipets in min. time for detns or transfer to small bottles, filling them completely and avoiding contact of soln with air as much as possible. Stopper bottles, seal with paraffin, and store in dark, cool place. (b) Dry lime-sulfur.-Thoroly stir 5 9 sample with ca 50 mL H20 in 250 mL beaker. Let settle and decant thru paper into 250 mL vol. flask. Repeat extn with H20 until filtrate is colorless and ca 200 mL is obtained. Transfer residue to paper, wash with hot H20, cool to room temp., and dil. to vol. Dry residue 1.5 hr at 105°, and reserve for free S and sulfite S detns in residue, if desired. (Ext S from dry residue with CS2 (Caution: See 51.039, 51.040, and 51.048), evap. on steam bath or in air current, dry 15 min at 105°, weigh, and calc. % S.) Prep. soln in min. time and keep beaker and funnel covered as much as possible. 6.104 Determination With clean, dry pipet transfer 10 mL prepd soln, 6.103(a) or (b), to 250 mL beaker. Partially cover with cover glass and add 2-3 g Na20 2 in small portions, with stirring, from tip of spatula. Continue adding Na20 2 until all S appears to be oxidized to sulfate (yellow color disappears). Add slight excess Na20 2, completely cover beaker with cover glass, and heat on steam bath, stirring occasionally, 15-20 min. Wash off cover glass and sides of beaker, acidify with HCI (1 +4), and filter if necessary. Oil. to 150-200 mL, heat to bp, and add 10% BaC/2 soln (11 mL/1 g BaS04), with const stirring, at such rate that ca 4 min is required to add necessary amt. Let stand until clear and cool, filter thru quant. paper, wash until CI- free, ignite carefully, and heat to const wt over Bunsen burner. Calc. % S from wt BaS04, using factor 0.1374. * Methods for this product are surplus--,see inside front cover.
- 104. 82 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) Thiosulfate Sulfur (27)-Official Final Action 6.112 Determination 6.105 Reagent Ammoniacal zinc chloride soln.-Dissolve 50 g ZnCI2in ca 500 mL H20, add 125 mL NH.OH and 50 g NH.CI, and dil. to 1 L. 6.106 Determination To 50 mL H20 in 200 mL vol. flask add 50 mL prepd soln, 6.103(a) or (b). Add slight excess of the ammoniacal ZnCI2soln and dil. to vol. Complete detn as rapidly as possible. Shake thoroly and filter thru dry filter. To 100 mL filtrate add few drops Me orange, 6.005(9), or Me red, 2.055(i), and exactly neutze with O.lN HCI. Titr. neut. soln with 0.05N I, 6.005(b), using few drops starch indicator, 6.005(f). From mL I soln used, calc. % thiosulfate 5 present. (Factor of I soln in terms of As20 3 x 1.296 = equiv. in thiosulfate 5.) Sulfide Sulfur-Official Final Action 6.107 Zinc Chloride Method (27) To 10-15 mL H20 in small beaker add 10 mL aliquot prepd soln, 6.103(a) or (b). Calc. amt ammoniacal ZnCI2 soln, 6.105, necessary to ppt all 5 in aliquot and add slight excess. Stir thoroly, filter, wash ppt twice with cold H20, and transfer paper and ppt to beaker in which pptn was made. Cover with H20, disintegrate paper with glass rod, and add ca 3 g Na202' keeping beaker well covered with watch glass. Warm on steam bath with frequent shaking until all S is oxidized to sulfate, adding more Na202 if necessary. Acidify slightly with HCI (1 +4), filter to remove shreds of paper, wash thoroly with hot H20, and det. S in filtrate as in 6.104. 6.108 Indirect Method Difference between sol. S and sum ofthiosulfate S and sulfate 5 = sulfide S. 6.109 Sulfate Sulfur-Official Final Action Slightly acidify soln from 6.106 with HCI (1 +4) and heat to bp. Add slowly, with const stirring, slight excess 10% BaC/2 soln, boil 30 min, let stand overnight, and filter. Calc. S from wt BaSO., and report as % sulfate S. 6.110 Total Calcium (27)--Official Final Action To 25 mL prepd soln, 6.103(a) or (b), add 10 mL HCI, evap. to dryness on steam bath, and H20 and few mL HCI (1 +4), warm until all CaCI2 dissolves, and filter to remove 5 and any Si02 present. Oil. filtrate to 200-250 mL, heat to bp, add few mL NH.OH in excess, and then add excess satd (NH.),C20. soln. Continue boiling until pptd CaC20. assumes well defined gran- ular form, let stand 1 hr, filter, and wash few times with hot H20. Ignite at 9500 in Pt crucible to const wt (CaO) and calc. % Ca. CaO x 0.7147 = Ca. SODIUM HYPOCHLORITE SOLUTIONS (28) Sodium Hypochlorite Arsenious Oxide Titration Method-Official Final Action 6.111 Reagents (a) Arsenious oxide std soln.-0.1N. Prep. as in 50.005-50.006. (b) Iodine std soln.-Prep. as in 50.018. Stdze against (a). Transfer 20 mL sample to 1 L vol. flask and dil. to vol. Pipet 50 mL aliquot of mixt. into 200 mL erlenmeyer. Add excess AS20 3soln and then decided excess NaHC03. Titr. excess As20 3 with std I soln, using starch soln, 6.005(f), or the I as its own indicator. 5ubtract vol. I soln, corrected to O.lN, from vol. AS20 3 soln used, and from this value and sp gr of soln, calc. % NaOCI. 1 mL O.lN AS20 3 = 0.003722 g NaOCI. 6.113 Available Chlorine-Official Final Action Calc. % available CI from titrn, 6.112. 1 mL O.lN AS20 3 0.003545 g available CI. 6.114 Chloride Chlorine-Official Final Action Pipet 50 mL aliquot prepd soln, 6.112, into 200 mL erlenmeyer and add slight excess AS20 3 soln, 6.111 (a), calcd from NaOCI titrn; add slight excess HN03, neutze with CaC03 , and titr. with 0.1N AgN03, 50.027-50.029, using K2CrO. 501n, 50.028(b), or the Ag3AsO. formed in soln, as indicator. Det. blank on reagents and correct for any CI found. From this corrected titrn and sp gr of sample, calc. % CI. From this value subtract 'Iz the % available CI. Difference = % chloride CI. 6.115 Sodium Hydroxide (29)-Official Final Action 5tdze pH meter equipped with calomel and glass electrodes, using std pH 6.9 buffer soln, 50.007(d). Place 50 mL 10% BaCI2.2H20 soln and 30 mL 3% H20 2 soln in 250 mL beaker. Neutze to pH 7.5 with ca O.lN NaOH, using pH meter. Add 10 mL sample from pipet, stir vigorously 1 min, and titr. to pH 7.5 with stdzd O.lN HC!, using pH meter. % NaOH = (mL HCI x normality x 4.0)/(mL sample x sp gr) 6.116 * Carbon Dioxide-Official Final Action * Evolution into std Ba(OH), soln. See 4.158-4.159, 10th ed. CALCIUM HYPOCHLORITE AND BLEACHING POWDER (28) Available Chlorine 6.117 Arsenious Oxide Titration Method Official Final Action Weigh 5-10 g thoroly mixed sample into porcelain mortar, add 30-40 mL H20, and triturate to smooth cream (high-test Ca(OC!)2 will dissolve readily and not form a cream). Add more H20, stir well with pestle, and let insol. residue settle few moments. Pour mixt. off into 1 L vol. flask, add more H20, and thoroly triturate sample and pour off as before. Repeat operation until all material is transferred to flask. Rinse mortar and pestle, catch wash H20 in flask, dil. to vol., and mix. Without letting material settle, pipet 25-50 mL aliquot into 200 mL erlenmeyer. Add excess std A5203 soln, 6.111(a), and then decided excess of NaHC03. Titr. excess AS20 3with std I soln, 6.111(b)' using starch soln, 6.005(f), or I as its own indicator. Subtract vol. I soln, corrected to O.lN, from vol. AS20 3 soln used, and calc. % available CI. 1 mL O.lN AS20 3 = 0.003545 g available CI. *Surplus method--5ee inside front cover.
- 105. AOAC METHODS (1980) CHLORAMINE T (28) Active Chlorine Arsenious Oxide Titration Method-Official Final Action 6.118 Determination Transfer 0.5 g sample to 300-500 mL erlenmeyer, dissolve in 50 mL H20, and add excess std As20 3 soln, 6.111(a). and 5 mL H2S04(1 +4). Add decided excess NaHC03and titr. excess As20 3 with std I soln, 6.111(b). using starch soln, 6.005(f). or I as its own indicator. From this titrn, calc. active CI in sample. 1 mL 0.1N As20 3 = 0.001773 g active CI. (To convert active CI to available CI, multiply active CI by 2.) 6.119 Total Chlorine-Official Final Action Dissolve 0.5 g sample in 50 mL H20 in erlenmeyer and add slight excess std As20 3 soln as calcd from active CI titrn, 6.118. Add 5 mL HN03 (1 +4). neutze with CaC03, and titr. with std AgN03, 50.027-50.029, using K2Cr04, 50.028(b). as indicator. Det. blank on reagents and correct for any CI found. From corrected titrn, calc. % total CI in sample. 1 mL 0.1N AgN03 = 0.003545 g CI. If total CI exceeds active CI, NaCI is indicated. 6.120 * Sodium-Official Final Action * From wt sulfated ash. See 6.183, 11th ed. MINERAL OILS 6.121 Unsulfonated Residue (30)-Official Final Action Reagent (Caution: See 51.030 and 51.031.) Fuming 38N sulfuric acid.-In tared g-s bottle (2.5 L acid bottle is convenient) mix fuming H2S04 (free from N oxides) (x) with H2S04(y) to obtain mixed acid (z). contg slightly >82.38% total S03' Depending on strength of fuming acid available, use following proportions of 2 acids: 100 parts x (15-20% free S03) to 50 parts y; 100 parts x (20-30% free S03) to 75 parts y; or 100 parts x (50% free S03) to 140 parts y. Mix thoroly (considerable heat is generated), let cool, and again weigh to det. amt mixed acid obtained. Det. exact strength of mixed acid (z) and also of reserve supply of acid (y) as follows: Pour ca 50 mL into small beaker and fill ca 10 mL weighing bulb or pipet by slight suction, wiping off outside of bulb with moist, then with dry, cloth. Weigh on analytical balance and let acid flow slowly down sides of neck of 1 L vol. flask into ca 200 mL cold H20. (These sizes of bulb and flask give final soln ca 0.5N.) When bulb has drained, wash all traces of acid into flask, taking precautions against loss of S03 fumes. Oil. to vol. and titr. from buret with std alkali, using same indicator as used in stdzg. Calc. S03 content of both acids, and add calcd amt of reserve acid (y) to amt of mixed acid (z) on hand to bring z to 82.38% total S03 (equiv. to 100.92% H2S04), After adding required amt of y, again analyze mixed acid to make certain it is of proper concn (±0.15% H2S04), Keep acid in small bottles or in special dispenser bottle (31) to prevent absorption of H20 from air. 6.122 Determination Pipet 5 mL sample into 6" Babcock cream bottle, 16.157(a), either 9 g 50% or 18 g 30% type. To reduce viscosity of heavy oils, warm pipet after initial drainage by passing it several times thru flame; then drain thoroly. If greater accuracy is desired, weigh measured sample and calc. exact vol. from wt and sp gr. *Surplus method-see inside front cover. SOAP 83 Slowly add 20 mL 38N H2S04, gently shaking or rotating bottle and taking care that temp. does not rise above 600 • Cool in ice- H20 if necessary. When mixt. no longer develops heat on shaking, agitate thoroly, place bottle in H20 bath, and heat 10 min at 60-65', keeping contents of bottle thoroly mixed by shaking vigorously 20 sec at 2 min intervals. Remove bottle from bath and add H2S04 until oil is in graduated neck. Centrf. 5 min (or longer if necessary to obtain canst vol. of oil) at 1200-1500 rpm. Read vol. of unsulfonated residue from grad- uations on neck of bottle and, to convert to mL, multiply reading from 9 g 50% bottle by 0.1 and reading from 18 g 30% bottle by 0.2. From result obtained, calc. % by vol. of unsulfonated residue. SOAP Moisture (32) 6.123 Toluene Distillation Method-OHicial Final Action Weigh ca 20 g sample into 300-500 mL flask; add 50 mL toluene (tech. grade is satisfactory); and, to prevent foaming, add ca 10 g lump rosin (do not use powd). Distil into Dean and Stark type distg tube receiver and continue distn until no more H20 collects in receiver. Cool contents of tube to room temp., read vol. H20 under toluene in tube, and calc. % H20. 6.124 * Sodium and Potassium (33)-Official Final Action * Removal of metal ions and P, and weighing as chlorides. See 6.094, 11th ed. MINERAL OIL·SOAP EMULSIONS Water (34) 6.125 Toluene Distillation Method-Official Final Action Weigh ca 25 g sample and proceed as in 6.123, except use less rosin. 6.126 Total Oil (35)-Official Final Action Weigh ca 10 g sample into Babcock cream bottle, 16.157(a). Oil. with ca 10 mL hot H20 and add 5-10 mL H2S04 (1+1). Heat in hot H20 bath ca 5 min to hasten sepn of oil, add enough satd NaCI soln to bring oil layer within graduated neck of bottle, centrf. 5 min at 1200 rpm, and let cool. Read vol. of oil layer, det. density, and from these values calc. wt and %. From this % value deduct % fatty acids (and phenols if present), detd sep., to obtain % oil. 6.127 Soap (34)-Official Final Action (Error will result if apparent mol. wt of fatty acids varies appreciably from that of oleic acid.) Weigh 20 g samplE) into separator, add 60 mL pet ether, and ext mixt. once with 20 mL and 4 times with 10 mL 5b% alcohol. Break emulsion if necessary by letting 1 or 2 mL 20% NaOH soln run down wall of separator. Then gently swirl separator and let stand few min. Drain alc. layers and wash successively thru pet ether contained in 2 other separators. Combine ale. exts in beaker and evap. on steam bath to remove alcohol. Dissolve residue in ca 100 mL H20 made alk. with NaOH. Transfer to separator, acidify with HCI or H2S04, ext 3 times with Et ether, and wash ether exts twice with H20. Combine ether exts, evap. in weighed beaker on steam bath, and weigh as fatty acids. From wt fatty acids, calc. % soap in sample as Na or K oleate. 6.128 Unsulfonated Residues-Official Final Action Using 5 mL of the recovered oil, 6.126, proceed as in 6.122.
- 106. 84 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) 6.129 Ash (36)-Official Final Action Evap. 10 g sample, or more if necessary, in Pt dish. Ignite, and leach charred mass with H20. Ignite residue, add leachings, evap. to dryness, ignite, and weigh. From this wt, calc. % ash. Test ash for Cu, Ca, CaF2, etc. ORGANIC MERCURIAL SEED DISINFECTANTS Mercury 6.130 * Volatilization Method (37) * Official Final Action See 4.150-4.151, 10th ed. (Caution: See 51.041 and 51.065.) 6.131 * Precipitation Method (37) * Official Final Action Digestion with H2S04 and H20, and pptn as HgS. See 6.173, 11th ed. 6.132 Titrimetric Method (38)-Official Final Action Principle Sample is digested under H20-cooled condenser with fuming H2S04-fuming HN03. Hg is detd by titrn with std SCN soln with ferric alum as indicator. Small amts of chloride are oxidized to CI and expelled thru condenser. Not applicable in presence of large amts of CI-contg materials. 6.133 Reagents (a) Ferric indicator.-Dissolve 8 g FeNH4(S04)2.12H20 in 80 mL H20. Add enough HN03 to destroy brown Fe color and dil. to 100 mL with H20. (b) Ferrous sulfate soln.-Dissolve 1 g FeS04.7H20 in H20, add 1 mL H2S04, and dil. to 100 mL with H20. Prep. fresh for each detn. 6.134 Preparation of Sample (a) Solns.-Mix thoroly and weigh, by difference, amt sample (max. 10 g) contg preferably 0.07 g Hg into 500 mL '$ erlenmeyer. (b) Dusts.-Mix thoroly and, using glass weighing dish, weigh amt sample as in (a). Transfer thru powder funnel into 500 mL '$ erlenmeyer. 6.135 Determination (Caution: ~onduct detn in well ventilated hood. Method is dangerous In presence of material which reacts violently with H2S04 and/or HN03. See 51.019, 51.026, 51.030, 51.031, and 51.079.) Connect straight-tube, H20-cooled condenser to erlenmeyer contg sample. Place flask in cold H20 bath. Carefully add 10 mL H2S04thru top of condenser and mix by swirling. Add in small portions, swirling after each addn, 30-40 mLfuming H,sO. (20% free S03) thru top of condenser, followed by 10 mL red fuming HN03 (98% HN03). Remove from bath and dry outside of flask. Heat with small flame to reflux at ca 30 drops/min with red fumes persisting in flask and condenser. Heat 30 min; if small amt chloride is present, heat 2 hr with occasional addn offuming HN03 as required. Cool, and add 100 mL cold H,O slowly thru top of condenser while cooling flask in cold H20 bath. Add 2 or 3 glass beads or boiling chips and boil until N oxides have been expelled to top of condenser (ca 2 min). Wash condenser with 50 mL cold H20, disconnect flask, and add satd KMn04 soln * Surplus method-see inside front cover. until color remains purple. (If large amts insol. material are present, filter hot soln thru medium tight asbestos mat in gooch before addn of KMn04. Wash flask and filter 5 times with hot H20, and then add KMn04.) Cool flask, and destroy KMn04with fresh 1% FeSO. soln. Add 10 mL ferric indicator and titr. with O.lN NH.SCN or KSCN, 50.003-50.004, to appearance of first permanent faint orange. 1 mL O.lN NH4SCN or KSCN = 0.01003 g Hg. AOAC-CIPAC Gravimetric Method (39)-Official Final Action (Ap~licable in presence of large amts CI-contg materials; not applicable to chloro- or nitrophenols nor to materials not de- composed by digestion mixt.) 6.136 Reagents (a) Dilute sulfuric acid.-Add 30 mL H2S04to H20 in 100 mL vol. flask, cool, and dil. to vol. with H20. (b) Sodium sulfite soln.-l0%. Dissolve 10 g Na,S03 in H20 in 100 mL vol. flask and dil. to vol. with H20. (c) Ammonium citrate soln.-pH 7.0. See 2.044(a). (d) Precipitating reagent.-Add 20 mL 1,2-propanediamine (Eastman Kodak Co., P3170) to 100 mL 1M CuS04soln. Store in g-s container. (e) Wash so/n.-Add 1 g KI and 2 mL pptg reagent to 1 L H20. 6.137 Preparation of Sample (a) Solns.-Mix thoroly and weigh, by difference, sample (max. 5 g) contg 0.02-0.08 g Hg into 125 mL '$ erlenmeyer. (b) Dusts.-Mix thoroly and, using glass weighing dish, weigh sample as in (a). Transfer thru powder funnel into 125 mL '$ erlenmeyer. 6.138 Determination (Caution: Conduct detn in well ventilated hood.) Add to sample in following order: 5 mL ethylene glycol, swirling to thoroly suspend solids, 4 g KI, 10 mL dil. H2S04, 0.4 g I, and 2 glass beads. After thoro mixing, connect straight-tube, H20-cooled condenser and, with low flame, heat to slight boil so that liq. condenses in lower portion of condenser. Swirl occasionally, avoiding excessive heat and crystn of large amt I in condenser. Reflux 1 hr and, while cooling flask in H20 bath, immediately wash warm condenser with heavy stream of ca 25 mL H20. (If dye or I persists in condenser, loosen by reheating flask contents, without H20 in condenser, until liq. refluxes slightly beyond adhering material. Wash condenser again with ca 25 mL H20, and cool flask.) Disconnect condenser and wash connections directly into flask. Add ca 2 mL 10% Na,S03 dropwise, with swirling, until I color slightly lightens. (Excess I must be present.) Neutze soln with NH.OH, using pH test paper, until very slightly alk. (pH 7.0-7.3). Cool, and filter with vac. thru retentive paper (S&S Blue Ribbon, or equiv.) in buchner into 400 mL beaker. Wash flask and paper thoroly, keeping total filtrate <150 mL. Add 50 mL NH4 citrate soln, bring mixt. just to bp, and stir in 5 mL pptg reagent. Cool and let stand ~2 hr (preferably overnight); filterthru medium porosity glass crucible, previously dried at 105° and weighed. Transfer ppt with wash soln, and wash with same soln several times. Rinse I from ppt with ca 25 mL alcohol in 5 mL portions (some samples may require up to 50 mL) until filtrate is colorless. (Let alcohol stand few min with occasional swirling after each addn before applying suction. Ppt should be suspended in liq. each time.) Wash ppt with three 5 mL portions CHCI3, suspending ppt each time as above until dye and pesticides are completely removed. Finally wash with 5 mL alcohol, dry 30 min at 105°, cool, and weigh. Wt Hg = wt ppt x 0.218.
- 107. AOAC METHODS (1980) FUMIGANTS 85 ANT POISONS AND RODENTICIDES 6.139 * Alpha-Naphthylthiourea (40) * Official First Action (Caution: See 51.039, 51.041, and 51.046.) N detn. See 4.132, 10th ed. 6.140 Thallous Sulfate (41)--Official Final Action (Caution: See 51.019, 51.026, 51.031, and 51.041.) Weigh sample contg 0.1-0.15 g TI2SO. (usually 10 g). transfer to 800 mL Kjeldahl flask, and add 25 mL H2SO. followed by 5-10 mL HNOa. After first violent reaction ceases, heat until white fumes of H2SO. appear. Add few drops fuming HNOa and continue heating and adding HNOa until org. matter is destroyed, as shown by colorless or light yellow soln. Cool, add 10-15 mL H20, again cool, and wash contents of flask into 400 mL beaker, continuing washing until vol. is 60-70 mL. Boil several min to remove all HNOa, cool, and filter into 400 mL beaker. Wash with hot H20 until vol. in beaker is 175 mL, neutze with NH.OH, and then slightly acidify with H2SO. (1 +4). Add 1 g NaHSOa to ensure reduction of thallic to thallous state. Heat to bp, add 50 mL 10% K/ so/n, stir, and let stand overnight. Filter thru tight gooch contg 2 disks S&S 589 white ribbon paper covered by medium pad of asbestos. Wash 4 or 5 times with 10 mL portions 1% KI soln, and finally with absolute alcohol. Dry to canst wt at 1050 (1-1.5 hr), and weigh as TIL % TI2S04 = (g Til x 0.7619 x 100)/g sample. Warfarin (3-(a-Acetonylbenzyl)-4-hydroxycoumarin) (42) Official Final Action (Applicable to baits contg ca 0.025% and to concs contg ;,.0.5% warfarin. Not applicable to pelleted baits or baits consisting of cracked corn treated with alc. warfarin soln and aq. sugar soln, and then dried.) 6.141 Reagents (a) Sodium pyrophosphate soln.-1%. Dissolve 5 g Na,- P20 7.10H2 0 in 500 mL H20. (b) Petroleum ether, purified.-Ext 200 mL pet ether with three 20 mL portions 1% Na,P20 7 soln. (c) Warfarin std soln.-10 J-tg/mL. Dissolve 100 mg pure warfarin (Wisconsin Alumni Research Foundation, P.O. Box 7365, Madison, WI 53707) in 100 mL 1% Na,P20 7 soln. Oil. 10 mL to 100 mL with 1% Na.P20 7 soln, and dil. 10 mL of second soln to 100 mL with 1% Na,P20 7 soln. 6.142 Determination Weigh 10 g sample (0.025%), 0.600 g (0.5%). or equiv. wt of higher concn, into 125 mL g-s flask or 100 mL centrf. tube and add 50 mL Et ether from pipet. Stopper tightly and shake mech. ca 30 min. Transfer 5 or 10 mL to centrf. tube (or centrf. directly), stopper, and centrf. 5 min at high speed or until clear. Take precautions to avoid evapn of ether. Pipet 10 mL 1% Na,P20 7soln into g-s 16 x 150 mm test tube and add 2 mL centrfd ether ext from pipet. Stopper and shake vigorously 2 min. Centrf. at high speed until aq. layer is clear. Draw off ether layer, including any emulsion that remains, using fine-tip glass tube attached to aspirator. Add ca 2 mL Et ether, shake vigorously, centrf., and completely draw off ether layer. Repeat ether extn, and then ext twice with purified pet ether in same manner. Prep. blank soln similarly, using 2 mL ether instead of 2 mL ether ext. Det. A of aq. soln in 1 cm silica cell at 308 nm against blank soln in Beckman spectrophtr, model DU (replaced by models 24/25). or equiv. Det. A' (ca 0.46) of the std warfarin soln against 1% Na'P207 soln. % Warfarin = (A/A') x (10-5 g std/mL) x [100/(g sample x (2/50)(1/10))] = (A/A') x (0.250/g sample). FUMIGANTS Fumigant Mixtures (43~fficial First Action (Applicable to org. components of CS2, CCI., (CH2)2CI2, and (CH2)2Br2 mixts. Precautions: Handle with care in hood or well- ventilated area. Mixts are volatile, poisonous, and sometimes flammable and may be fatal if inhaled or swallowed. They cause skin and eye irritation. In case of contact, immediately remove contaminated clothing and flush affected area with copious amts of H20. Do not reuse clothing until free of contamination. Do not use containers or equipment of AI, Mg, or their alloys.) 6.143 Principle Components are detd by GLC. Peak area of each component is measured and compared to stds of same fumigant mixt. Precision of method is ±0.6% for each component. 6.144 Sampling Obtain representative 1 L sample from container. Sample bulk containers by means of weighted bottle, lowered toward bottom and raised at such rate that it is % full when withdrawn. Sample drums or small containers with thief or thru tap or valve located so that sample comes from well below surface. Prevent contam- ination of product or sample. Place sample in clean, dry, and solv. vapor-tight glass bottle of such size that it is nearly filled (not above shoulder) by sample. Vapor-tight g-s bottles or screw-cap bottles with Sn foil lined caps are satisfactory. Store samples at low temp.; cool to <180 before opening for analysis. 6.145 Apparatus (a) Gas chromatograph.-With flame ionization or thermal conductivity detector. Typical operating conditions: Column temp., 110°; injection port temp., 200°; flow rate, 80 mL He/ min. (b) Recorder.-O.05-1.05 mv, full scale response. Integrator may be used. (c) Syringe.-Hamilton Co. 10 J-tL No. 701 N, or equiv. (d) Column.-l.2 m (4') stainless steel, W' od, 0.194" id, packed with reagent 6.146(a). Max. temp. is 160°. Other columns can be used but chromatgc conditions and sample size must be adjusted in accordance with column requirements. One such column is: 3 m (10') stainless, a/,6" od, 0.12" id, packed with 20% by wt N,N-bis-(2-cyanoethyl) formamide on 80-100 mesh Chro- mosorb W, acid-washed. Columns are available from com. suppliers. Criterion for use is emergence of each component of mixt. of CS2, CCI" (CH2I.CI2, and (CH2J.Br2 as sep. peak. 6.146 Reagents (a) Column packing.-30% by wt tricresyl phosphate on Chro- mosorb P, 30--60 mesh. (b) Carbon disulfide std.-ACS. (c) Carbon tetrachloride std.-ACS. (d) Ethylene dichloride std.-Purified 1,2-dichloroethane, available from laboratory supply houses, or use center cut of fractionation of com. product. (e) Ethylene dibromide (1,2-dibromoethane) std.- Purified or distd as in (d).
- 108. 86 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) 6.147 Preparation of Standards Prep. fresh stds just before analysis which approximate ex- pected composition, by wt, of fumigant mixt. Place proper wt of each component in 25 mL g-s vol. flask and mix well. Do not prep. by vol. Cool CS2to prevent loss. Adjust wt stds to detector response. Carefully fill weighed 10 mL vol. flask to mark with prepd std and weigh. Use this wt to det. g/5 ILL values for each component of std. 6.148 Determination Purge column thoroly at 110° before use. Establish 0 baseline at full sensitivity. Inject 5 ILL std fumigant mixt. into chromato- graph. Attenuate successively so that each peak is at max. % of chart scale, adjusting sample size and attenuation, if necessary. Repeat injection. Detd area for each component, corrected for any baseline drift, should differ by ""1%. Order of elution from column is: CS2, CCI., (CH2)2CI2' and (CH2)2Br2. Total analysis time is ca 21 min. Inject 5 ILL sample into chromatograph. Det. corrected area of each component from chromatogram, or note integrator read- ing. g Component = S x C/B, where S = wt component in std, B = area for component in std, and C = area for component in sample. Perform calcn for each component in sample. % Component = g component in sample x 100/ sum of g components in sample. Last equation is not applicable in presence of unmeasured contaminants. 6.149 PESTICIDES RELATED TO NATURAL PRODUCTS AND THEIR SYNERGISTS Technical Allethrin (44)-Official First Action (Caution: See 51.041.) Principle Allethrin reacts quant. with ethylenediamine to form chrysan- themum monocarboxylic acid which is detd by titrn with std NaOMe in pyridine. Chrysanthemum monocarboxylic acid, an- hydride, and acid chloride interfere quant. and are detd inde- pendently. 6.150 Reagents (a) Absolute alcohol.-SDF No. 2-B is satisfactory. (b) Methanolic hydrochloric acid std soln.--O.1N. Dil. 17 mL HCI (1+ 1) to 1 L with anhyd. MeOH. Stdze against std O.lN NaOH, using phthln. If used at temp., T, different from that at which stdzd, To, calc. corrected normality = N[l-O.001(T - Toll. (c) Sodium methylate std soln.--O.1N in pyridine. Transfer 50 mL 2N NaOMe (Caution: See 51.038) to 1 L bottle contg 75 mL anhyd. MeOH and dil. to 1 L with redistd pyridine. Stdze against NBS benzoic acid, using pyridine as solv. and thymolphthalein, (i), as indicator. Dispense from 50 mL automatic buret with vents connected to Ascarite tubes. Stdze daily against std methanolic HCI, (b). (d) Methanolic potassium hydroxide std soln.- 0.02N. Dis- solve 1.12 g KOH in 1 L MeOH. Stdze as in 50.035. (e) Morpholine soln.-Transfer 8.7 mL redistd morpholine to 1 L bottle and dil. to 1 L with anhyd. MeOH. Fit bottle with 2- hole rubber stopper; thru 1 hole insert 20 mL pipet so that tip extends below surface of liq., and thru other hole insert short piece of glass tubing to which is attached aspirator bulb. (f) Ethylenediamine.-Redistd com. grade contg <3% H20. Dispense from automatic buret with vents connected to Ascarite tubes. (g) Dimethyl yellow-methylene blue mixed indicator.-Dis- solve 1 g dimethyl yellow (p-dimethylaminoazobenzene; Cau- tion: See 51.085) and 0.1 g methylene blue in 125 mL anhyd. MeOH. (h) a-Naphtholbenzein indicator.-l % alc. soln. (i) Thymolphthalein indicator.-l% pyridine soln. 6.151 Determination of Chrysanthemum Monocarboxylic Acid Chloride Add 8-10 drops mixed indicator, (g), to ca 150 mL anhyd. MeOH and add O.lN HC!, (b), dropwise until soln appears reddish brown by transmitted light. Add 0.02N KOH, (d), dropwise until appearance of first green. Transfer 25 mL to each of three 125 mL g-s erlenmeyers, reserving 1 flask as ref. color for end point. Into each of other flasks add 1.5--2.5 g sample from weighing pipet, swirling flask while adding sample. Within 5 min, titr. with 0.02N KOH, (d), to first green end point, using blank as ref. color. Calc. milliequiv. chrysanthemum monocarboxylic acid chloride/g sample, C = V x N/g sample, where V= mL N normal KOH required; % Chrysanthemum monocarboxylic acid chloride = C x 18.67. 6.152 Determination of Chrysanthemum Monocarboxylic Acid Transfer 25 mL anhyd. alcohol to each of two 125 mL g-s erlenmeyers, add 8-9 drops a-naphtholbenzein indicator, and cool to 0° in ice bath. Neutze by adding 0.02N NaOH dropwise to bright green end point. To each flask add 1.5--2.5 g sample from weighing pipet. Immediately titr. with 0.02N NaOH, 50.034, to first bright green end point. Calc. milliequiv. chrysanthemum monocarboxylic acid and acid chloride/g sample: 0 = X x N/g sample, where X = mL N normal NaOH required; (0 - C) x 16.82 = % chrysanthemum monocarboxylic acid. 6.153 Determination of Chrysanthemum Monocarboxylic Anhydride Pipet 20 mL morpholine soln, (e), into each of four 250 mL erlenmeyers, using same pipet. Fill pipet by exerting pressure in bottle with aspirator bulb. Reserve 2 flasks for blanks; into each of other flasks add 1.5--2.5 g sample from weighing pipet. Swirl flasks and let samples and blanks stand 5 min at room temp. Add 4-5 drops mixed indicator, (g), to each flask and titr. with O.lN HCI, (bl, until color changes from green to faint red when viewed by transmitted light. Calc. milliequiv. chrysanthe- mum monocarboxylic anhydride/g sample: E = (B - Y) x N/g sample, where Y = mL N normal HCI required for sample, and B = mL N normal HCI required for blank; (E - 2C) x 31.84 = % chrysanthemum monocarboxylic anhydride. 6.154 Determination of Allethrin Add sample contg 0.8-1.1 g allethrin to each of two 250 mL erlenmeyers from weighing pipet. To each of 2 flasks as blanks and to samples add 25 mL ethylenediamine, (f)' with swirling. Let samples and blanks stand 2 hr at 25±2°. Wash down sides of flasks with 50 mL redistd pyridine. To each flask add 6-10 drops thymolphthalein indicator, (i), and titr. with O.lN NaOMe, (c), to first permanent blue-green end point. (With colorless samples, first blue end point may be used.) Calc. milliequiv. allethrin/g sample: F = (Z - B) xN/g sample, where Z = mL N normal NaOMe required for sample, and B = avo mL N nor- mal NaOMe required for blank; (F + C - 0 - E) x 30.24 = % allethrin.
- 109. AOAC METHODS (1980) NATURAL PRODUCTS 87 d-trans-Allethrin (dl-Z-Allyl-4-hydroxy-3-methyl-Z- cyclopentene-1 Ester of d-trans-Z,Z-Dimethyl-3- (Z-methylpropenyl)cyclopropanecarboxylic Acid) (45) Official Final Action 6.155 Gas Chromatographic Method (Caution: See 51.041.) Principle d-trans-Allethrin is dild in acetone contg dibutyl phthalate as internal std. Ratios of GLC peak hts of d-trans-allethrin and dibutyl phthalate in sample and std are compared for quant. detn. Method is applicable to both tech. d-trans-allethrin and various formulations of it. Not applicable to formulations contg large amt MGK Repellent 874 (2-hydroxyethyl-n-octyl sulfide). 6.156 Apparatus and Reagents (a) Gas chromatograph.-Equipped with flame ionization de- tector and 1.2 m (4') x 4 mm id glass column packed with 5% OV-1 (Analabs, Inc.) on 80-100 mesh Chromosorb W (HP). Operating conditions: temps (O)--column 165, injection port 230, detector 230; gas flows (ml/min)-N carrier gas 125, air 350-400, H 40-50; sensitivity-10-9 amp full scale, attenuation 4x for tech. material, 10-9 amp full scale, attenuation 1for formulations. Before use, condition column 2-3 hr at 275° with N flow 50 mL/min. If necessary, vary column temp. or gas flow to attain retention times of ca 4 and 7 min for internal std and d-trans- allethrin, resp. Also vary detector sensitivity or injection vol. to attain ;;.100 mm peak ht for each compd (ca 16 JLg d-trans- allethrin). Theoretical plates/ft must be >200. Calc. theoretical plates/ft (N) as follows: N = 16U/(M2 x F), where L = retention of GLC peak in mm; M = peak baseline produced by drawing tangents to points of inflection of peak; and F = length of column (ft). (b) Internal std soln.-4.0 mg dibutyl phthalate/mL acetone. (c) d-trans-Allethrin std solns.-(1) Soln 1.-Approx. 4 mg/mL. Accurately weigh ca 1.0 g d-trans-allethrin (available from McLaughlin Gormley King Co., 8810 Tenth Ave N, Min- neapolis, MN 55427) into 50 mL vol. flask and dil. to vol. with acetone. Pipet 20 mL this soln into 100 mL vol. flask, add 50 mL internal std soln by pipet, and dil. to vol. with acetone. Use this soln for detn of tech. material. (2) Soln 2.-Approx. 1 mg/mL. Pipet 25 mL Soln 1 into 100 mL vol. flask and dil. to vol. with acetone. Use this soln for detn of d-trans-allethrin in formula- tions. 6.157 Preparation of Sample (a) Technical material.-Accurately weigh sample contg ca 1.0 g d-trans-allethrin into 50 mL vol. flask and dil. to vol. with acetone. Pipet 20 mL aliquot into 100 mL vol. flask, add 50 mL internal std soln by pipet, and dil. to vol. with acetone. (b) Formulations.-Accurately weigh sample contg ca 200 mg d-trans-allethrin into 50 mL vol. flask, add 25 mL internal std soln by pipet, and dil. to vol. with acetone. Pipet 25 mL aliquot into 100 mL vol. flask and dil. to vol. with acetone. 6.158 Gas Chromatography (a) Technical material.-Inject aliquots (ca 3 JLL) std Soln 1 until ratio of d-trans-allethrin:dibutyl phthalate peak hts varies <1% for successive injections. Repeat with sample soln, fol- lowed by duplicate injections of std soln. If peak ht ratios differ > ± 1% from previous std injections, repeat series of injections. (b) Formulations.-Proceed as in (a), using std Soln 2. Repeat std injections after each series of 3 sample injections. If peak ht ratios differ >±1.5% from previous std injections, repeat injec- tions. 6.159 Calculations (a) Technical material.-Calc. peak ht ratios for duplicate std injections before and after sample injections and average the 4 values. Calc. and average peak ht ratios for sample injections. % d-trans-Allethrin = (W' x P x R)/(W x R'), where W' and W = g std and sample, resp.; P = % purity of std; and R' and R = peak ht ratios of std and sample, resp. (b) Formulations.-Calc. avo for all std peak ht ratios and for sample peak ht ratios. % d-trans-Allethrin = (W' x P x R x 2)/(W x R'), where W' = g std in final diln. DERRIS AND CUBE POWDER Rotenone Crystallization Method (46)~fficial Final Action (Caution: See 51.049 and 51.084.) 6.160 Reagents (a) Purifiedrotenone.-Dissolve rotenone in boiling eCI.; cool in refrigerator or ice bath at 0-10° until pptn of rotenone-Cel. solvate stops. Filter thru buchner and wash once or twice with ice-cold CCI•. Conc. filtrate, crystallize, and filter as before. Transfer cryst. residue to beaker, add ca twice their vol. alcohol, and heat nearly to boiling. (Crystals need not dissolve com- pletely.) Cool to room temp., filter thru buchner, and draw air thru cryst. residue until most alcohol is removed. Remove rotenone from funnel, dry in air, and finally heat 1 hr at 105°. Mp, detd in Pyrex, of purified material should be 163-164°. (Mother liquors may be concd and rotenone-CCI. solvate allowed to crystallize. Cryst. material may be used for further purification, or kept for prepn of wash solns or for seeding to induce crystn in detn.) (b) Rotenone-CCI. solvate.-Ppt rotenone from CCI. soln, filter by suction, and dry in air. (c) Rotenone-CCI. wash soln.-Sat. eel. at 0°, and keep at 0° during use. (d) Alcohol saturated with rotenone at room temp. (e) Charcoal, activated.-Norit-A neutral, or equiv. 6.161 Preparation of Solution (a) Weigh 30 g (if sample contains >7% rotenone, use amt to give 1.0-1.5 g rotenone in 200 mL aliquot) finely powd root and 10 g of the C, (e), into 500 mL g-s erlenmeyer. Add 300 mL CHCI3, measured at known room temp.; fasten stopper securely and place flask on shaking machine. Agitate vigorously ;;.4 hr, preferably interrupting shaking with overnight rest (or flask may be shaken continuously overnight). Rapidly filter mixt. into suitable flask, using fluted paper without suction and keeping funnel covered with watch glass to avoid evapn loss. Stopper flask and adjust temp. of filtrate to that of original CHCI3 • (b) Alternative extraction method.-If sample has ratio of rotenone to total ext of >0.4, use amt contg 1.0-1.5 g rotenone and successively ext 4 times with CHCI3, using 300 mL CHCI3 and 4 hr agitation for first extn as in (a) and 200 mL and 2 hr each for other extns. Filter after each extn and return marc to flask for extn with fresh solv. Finally combine exts, evap. almost to dryness, and use entire ext to det. rotenone. (c) Extraction method for formulations containing 0.75-1.0% rotenone with or without sulfur and/or pyrethrins.-Weigh two 50 g portions sample into sep. 500 mL g-s erlenmeyers. Add 5 g of the C and 300 mL CHCI3, measured at known room temp., to each. Stopper and continue as in (a).
- 110. 88 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) 6.162 Determination (Caution: See 51.011, 51.018, 51.046, 51.049, and 51.056.) Pipet 200 mL soln, 6.161 (or entire soln if alternative extn, (b), is used), into 500 mL Pyrex erlenmeyer and distil until ca 25 mL remains. (For formulations, 6.161(c): In absence of S, combine the 2 exts in one of the erlenmeyers. In presence of S, remove all CHCI3 on steam bath in air current, avoiding prolonged heating. Add 35 mL acetone to each residue and boil gently on steam bath to dissolve all resins. Remove from steam bath, stopper tightly, and hold 2 hr at 0-5°. Filter both acetone solns thru same 15 mL, medium porosity, fritted glass buchner into single 500 mL erlenmeyer. Rinse and wash with acetone at 5°. Remove acetone as CHCI3 was removed above.) Evap. almost to dryness on steam bath in current of air. Remove remainder of solv. under reduced pressure, heating cautiously on steam bath when necessary to hasten evapn. (Suction may be applied directly to flask if stopper with vent is used to release pressure, so that excessive vac. may be avoided. Use flasks with slightly convex bottoms; do not use flasks below avo wt.) Dissolve ext in 15 mL hot CCI. and again, in similar manner, remove all solv. Repeat with another 10-15 mL portion hot CCI•. (This treatment removes all CHCI3 from resins. CHCI3 ext is usually completely sol. in CCI.; if small amts of insol. material are present, purification described later will eliminate them.) Dissolve residue in ca 10 mL CCI. and transfer quant. with hot CCI. to 50 mL erlenmeyer marked at 25 mL. Adjust vol. to 25 mL by evapg on steam bath or by adding CCI•. Cool flask in ice bath several min, stopper flask, and swirl until crystn is apparent. Seed with few crystals of rotenone-CCI. solvate if necessary to induce crystn. If at this stage only small amt of cryst. material seps, add accurately weighed amt of purified rotenone, 6.160(a), estd to be enough to assure that final result, expressed as pure rotenone, is ~1 g. Then warm to dissolve completely, and again induce crystn. At same time prep. satd soln of rotenone in CCI., 6.160(c), for washing. Place flasks contg ext and washing soln in ice bath capable of holding temp. at 0°, and let stand overnight. (Store ice bath in refrigerator to keep ice from melting too rapidly.) After 17-18 hr in ice bath, rapidly filter ext thru weighed gooch fitted with filter paper disk, removing flask from ice bath only long enough to pour each fraction of ext into crucible. Rinse cryst. residue from flask and wash under suction once with the ice-cold satd rotenone-CCI. wash soln. (';;12-15 mL soln should be used for rinsing and washing.) Continue suction ca 5 min; then dry to const wt at 40° (ca 1 hr). Wt obtained is crude rotenone-CCI. solvate. Break up contents of crucible with spatula, mix thoroly, and weigh 1.000 g into 50 mL erlenmeyer. Add 10 mL alcohol previously satd with rotenone at room temp., swirl flask few min, stopper tightly, and set aside ~4 hr, preferably overnight, at same temp. Filter on weighed gooch fitted with filter paper disk. Rinse crystals from flask and wash under suction with alcohol satd with rotenone at temp. of recrystn (ca 10 mL usually required). Continue suction 3-5 min and then dry crucible at 105° to const wt (ca 1 hr). Multiply g residue by g total crude rotenone-CCI. solvate, and add 0.07 g to product as correction for rotenone held in soln in the 25 mL CCI. used in crystn. If any pure rotenone was added, subtract its wt from value obtained. This gives wt pure rotenone contained in aliquot of ext. Note: Most important precaution in using this method is to keep temp. of CCI.-rotenone wash soln and crucibles .as near 0° as possible. Keep wash soln surrounded by crushed Ice. except when actually being used. In warm weather keep crUCibles In refrigerator until ready to use. 6.163 Infrared Method (47)-()fficial First Action (Not applicable to derris products) Standardization Prep. std solns of purified rotenone, 6.160(a), in CHCI3 at concns of 5,10,15, and 20 mg/mL. Scan each std soln from 7.0 to 8.0 p'm at sP!led of 6 min/p.m and scale of 10 cm/p.m, using 0.1 mm cell and accurately matching cell filled with CHCI3 as ref. Scan each in duplicate. Obtain avo A of each concn, using 7.57 p.m as base point and 7.65 p'm as peak. Plot A against concn. 6.164 Determination Weigh sample contg 250-300 mg rotenone into 25 x 200 mm culture tube. Add 1-2 g anhyd. Na2SO., 2 g activated charcoal, and 50 mL CHCI3 by pipet. Close securely with Teflon-lined screw cap and tumble end over end 1 hr at ca 35 rpm. Filter thru medium paper, avoiding evapn losses. Transfer 20 mL aliquot to 50 mL erlenmeyer and evap. on steam bath with current of air. Transfer residue to 10 mL g-s vol. flask and dil. to vol. with CHCI3• Stopper, and mix thoroly. Scan from 7.0 to 8.0 p'm, using 0.1 mm cell and matched cell filled with CHCI3 as ref. Det. A by baseline method from 7.57 to 7.75 p'm and peak at 7.65 p'm, using same scanning speed and scale expansion as in stdzn. Calc. % rotenone from std curve and wt sample in final diln. 6.165 Total Ether Extract-Official Final Action (Caution: See 51.009, 51.039,51.054, and 51.070(b).) Ext 5 g finely powd root with ether 48 hr in Soxhlet or other efficient extn app. Conc. ext and filter off any insol. material present. Receive filtrate in tared beaker, evap. ether on steam bath, and dry in oven at 105° to const wt. 6.166 Piperonyl Butoxide (48)-Official Final Action Apparatus and Reagents (a) Photoelectric colorimeter.-Equipped with narrow band- pass interference type filter with central wavelength 630 nm. (Filter is available from: Baird-Atomic Inc.; Bausch and Lomb Optical Co., 10 Champeney Terrace, Rochester, NY 14602; and Photovolt Corp., 1115 Broadway, New York, NY 10010.) Spec- trophtr set at wavelength in range 625-635 nm may also be used. (b) Purified tannic acid.-Purify as follows: To 20 g tannic acid (USP reagent grade) add 100 mL EtOAc (99%) and stir mech. ca 1 hr. Filter by suction thru fritted glass funnel, and wash residue with three 5 mL portions EtOAc. To combined filtrate and washings add 2 g finely powd Darco G-60 (or equiv. decolorizing C), and stir mech. ca 0.5 hr. Filter by gravity thru double thickness Whatman No.1, or equiv., paper into graduated dropping funnel. Wash residue several times with EtOAc until vol. filtrate and washings is ca 125 mL. Place dropping funnel over 1 L, 3-neck, r-b flask, equipped with mech. stirrer, and with vigorous agitation in flask, add filtrate dropwise to 5 times its vol. of toluene. Purified tannic acid is pptd immediately. Filter by suction thru fritted glass funnel, and wash product thoroly with toluene, stirring solids with toluene to assure complete removal of EtOAc. Continue suction until practically all toluene is removed. Dry purified tannic acid in vac. oven at ca 40°, and place in tightly stoppered bottle. (c) Tannic acid reagent.-Completely dissolve exactly 0.025 g purified tannic acid in 20 mL HOAc by shaking at room temp. Add 80 mL H3PO. and mix thoroly. Prep. fresh daily. Store tightly stoppered, as it is hygroscopic.
- 111. AOAC METHODS (1980) NATURAL PRODUCTS 89 (d) Purified piperonyl butoxide.-Purify by low pressure frac- tional distn oftech. product. (Caution: See 51.015.) Also available from Fairfield American Corp., 3932 Salt Rd, Medina, NY 14103. (e) Piperonyl butoxide stdsoln.-50 I1-g/0.1 mL. Weigh exactly 1.000 g purified piperonyl butoxide into 100 mL vol. flask. (Hypodermic syringe and needle are convenient for adding compd to flask.) Oil. to vol. with deodorized kerosene and mix well. Pipet 10 mL of this soln into 200 mL vol. flask. Oil. to vol. with deodorized kerosene and mix well. This soln is stable for several months. Ifstd is to be used with sample contg pyrethrum, add enough pyrethrum ext to std before initial diln to give ratio piperonyl butoxide to pyrethrins similar to sample. 6.167 Preparation of Sample Accurately weigh sample contg 0.5-1.5 g piperonyl butoxide into tared 100 mL vol. flask, dil. to vol. with deodorized kerosene, and mix well. Pipet 10 mL into 200 mL vol. flask, dil. to vol. with deodorized kerosene, and mix well. 6.168 Determination Pipet 0.1 mL (from 1 mL pipet graduated in 0.1 mL) sample soln into 18 x 150 mm test tube. Add exactly 5 mL tannic acid reagent and shake vigorously 1 min. Treat std and blank, consisting of 0.1 mL deodorized kerosene, simultaneously in same manner. Place test tubes in test-tube basket and place in vigorously boiling H20 bath 5 min. Remove basket and let tubes cool to room temp. Transfer solns to colorimeter tubes and read, against H20, using 625-635 nm filter or setting. (After cooling to room temp. there is no appreciable change in A for several hr.) Subtract Ao of deodorized kerosene from readings of both sample, A, and std, A'. mg Piperonyl butoxide = A x 0.05/A'. PYRETHRINS Mercury Reduction Method (49)-Cfflcial Final Action (Caution: See 51.039, 51.054, 51.070, and 51.073.) 6.169 Reagents (a) Oeniges reagent.-Mix 5 g yellow HgO with 40 mL H20, and, while stirring, slowly add 20 mL H2S04 ; then add addnl 40 mL H20 and stir until all dissolves. Test for absence of mercurous Hg by adding few drops of (b) to 10 mL and titrg with (e) as in 6.171, par. 2, beginning "Add 50 mL previously prepd and cooled dil. HCI ..." (b) Iodine monochloride soln.-Dissolve 10 g KI and 6.44 g KI03 in 75 mL H20 in g-s bottle; add 75 mL HCI and 5 mL CHCI3, and adjust to faint I color (in CHCI3 ) by adding dil. KI or KI03 soln. If much I is liberated, use stronger soln of KI03 than O.OlM at first, making final adjustment with O.OlM soln. Keep in dark and readjust when necessary. Do not store in refrigerator. (e) Potassium iodate std soln.--O.01M. Dissolve 2.14 g pure KI03, previously dried at 105°, in H20 and dil. to 1 L. 1 mL = 0.0057 g pyrethrin I and needs no further stdzn. (d) Alcoholic sodium hydroxide soln.-( 1) 1.0N.-Dissolve 40 g NaOH in alcohol and dil. to 1 L with alcohol. (2) 0.5N-Dil. 1.0N with alcohol (1+1). (e) Petroleum ether.-Aromatic-free, bp range 30-60°. (f) Ethyl ether.-Peroxide-free, reagent grade. 6.170 Preparation of Sample (a) Pyrethrum powder.-Ext sample contg 40-150 mg total pyrethrins in Soxhlet or other efficient extn app. 7 hr with pet ether. After extn is complete, evap. pet ether to ca 40 mL, stopper flask, and place in refrigerator at O±O.so overnight. Filter cold ext thru cotton plug satd with cold pet ether, in stem of funnel, collecting filtrate in 250 mL erlenmeyer. Wash with three 15 mL portions cold pet ether. Evap. filtrate and washings on H20 bath, using air current, until <1 mL solv. remains. Add 15-20 mL 0.5N alc. NaOH to evapd ext, connect to reflux condenser, and boil gently 1-1.5 hr. Transfer to 600 mL beaker and add enough H20 to bring vol. to 200 mL. Add few glass beads, or preferably use boiling tube, and boil down to 150 mL. Transfer to 250 mL vol. flask and add 1 g Filter-Cel and 10 mL 10% BaC/2 soln. Do not shake before dilg to vol. Oil. to vol., mix thoroly, filter off 200 mL, neutze with H2S04 (1 +4), using 1 drop phthln, and add 1 mL excess. (If necessary to hold soln overnight at this point, leave in alk. condition.) (b) Pyrethrum extracts in mineral oil.-Weigh or measure sample contg 40-150 mg total pyrethrins, add 50 mL pet ether and 1 g Filter-Cel, and place in refrigerator at O±OS overnight. Filter thru gooch into 300 mL erlenmeyer and wash with three 15 mL portions cold pet ether. Evap. filtrate and washings on H20 bath, using air current, until <1 mL solv. remains. Add 20 mL IN alc. NaOH, or more if necessary, to ext, connect to reflux condenser, and boil gently 1-1.5 hr. Transfer to 600 mL beaker and add enough H20 to make aq. layer 200 mL. If >20 mL alc. NaOH soln was used, add enough H20 so that all alcohol is removed when vol. is reduced to 150 mL. Add few glass beads, or preferably use boiling tube, and boil aq. layer down to 150 mL. Transfer to 500 mL separator and drain aq. layer into 250 mL vol. flask. Wash oil layer once with H20 and add wash H20 to aq. portion. If slight emulsion still persists after draining aq. layer and washings, add 2-3 mL 10% BaCI2 soln, but do not shake vigorously after adding BaCI2 because reversed emulsion difficult to sep. may form. To aq. soln in 250 mL flask add 1 g Filter-Cel and ;;.10 mL of the BaCI2 soln. Swirl gently and let stand 30 min. Oil. to vol., mix thoroly, and filter off 200 mL. Test filtrate with BaCI2 soln to see if enough has been added to obtain clear soln. Neutze with H2S04 (1 +4), using 1 drop phthln, and add 1 mL excess. (If necessary to hold soln overnight at this point, leave in alk. condition.) 6.171 Determination of Pyrethrin I Filter acid soln from 6.170(a) or (b) thru 7 cm paper, coated lightly with suspension of Filter-Cel in H20, on buchner, and wash with three 15 mL portions H20. Transfer to 500 mL g-s separator and ext with two 50 mL portions pet ether. Shake each ext ;;.1 min, releasing pressure if necessary by inverting separator and carefully venting thru stopcock. Let layers sep. ;;.5 min or until aq. layer is clear before draining and re-extn. Reserve aq. layer for pyrethrin II detn. Do not combine pet ether exts but wash each in sequence with same three 10 mL portions H20, and filter pet ether exts thru small cotton plug into clean 250 mL separator. Wash separators and cotton in sequence with 5 mL pet ether. Ext combined pet ether solns with 5 mL O.lN NaOH, shaking vigorously;;'l min. Let layers sep. ;;.5 min before draining aq. layer into 100 mL beaker. Wash pet ether with addnl 5 mL portion O.lN NaOH and with 5 mL H20, adding washings to beaker. Add 10 mL Deniges reagent and let stand in complete darkness 1 hr at 25±2°. Add 20 mL alcohol and ppt HgCI with 3 mL satd NaCI soln. Warm to ca 60° and let stand several min until ppt coagulates and settles. Filter thru small paper, transferring all ppt to paper, and wash with ;;.10 mL hot alcohol. Wash with 2 or more 10 mL portions hot CHCI3 and place paper and contents in 250 mL g-s erlenmeyer. Add 50 mL previously prepd and cooled dil. HCI (3+2). Add 5 mL CHCI3 or CCI. and 1 mL freshly adjusted ICI
- 112. 90 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) soln, and titr. with 0.1M KI03 soln, shaking vigorously ~30 sec after each addn, until no I color remains in CHCI3 or CCI. layer. Take as end point when red color disappears from solv. layer and does not return within 1-3 min. From mL std KI03 soln used in titrn and blank on Deniges reagent, calc. % pyrethrin I. (Reactions: 2Hg2CI2 + 41CI = 4HgCI2 + 212 212 + KI03 + 6HCI = KCI + 51CI + 3H20 Addn of ICI does not change vol. relationship between mer- curous Hg and KI03 soln, and aids in detg end point in titrn of small amts of Hg.) Note: Chrysanthemum monocarboxylic acid reacts with De- niges reagent to form series of colors beginning with phthln red, which gradually changes to purple, then to blue, and finally to bluish green. Color reaction is very distinct with 5 mg monocarboxylic acid, and amts as low as 1 mg can usually he detected. Therefore no pyrethrin I should be reported if color reaction is neg. With samples contg much perfume or other saponifiable ingredients, it may be necessary to use as much as 50 mL 1N ale. NaOH. When lethanes are present, after washing HgCI ppt with alcohol and CHCI3, wash once more with alcohol and then several times with hot H20. 6.172 Determination of Pyrethrin 1/ (50) If necessary, filter aq. residue from pet ether extn thru gooch. Conc. filtrate to ca 50 mL and transfer to 500 mL g-s separator. Wash beaker with three 15 mL portions H20. Acidify with 10 mL HCI and sat. with NaCI. (Acidified aq. layer must contain visible NaCI crystals thruout following extns.) Ext with 50 mL ether, drain aq. layer into second separator, and ext again with 50 mL ether. Continue extg and draining aq. layer, using 35 mL for third and fourth extns. Shake each ext ~1 min, releasing pressure, if necessary, by inverting separator and carefully venting thru stopcock. Let layers sep. ~5 min or until aq. layer is clear before subsequent draining and extn. Combine ether exts, drain, and wash with three 10 mL portions satd NaCI soln. Filter ether exts thru cotton plug into 500 mL erlenmeyer and wash separator and cotton with addnl 10 mL ether. Evap. ether on H20 bath, and remove any fumes of HCI with air current and continued heating .;;5 min. Dry 10 min at 100°. (a) For crude pyrethrum exts.-Treat residue with 75 mL boiling H20 and filter thru 9-11 cm Whatman No.1, or equiv., paper. Wash flask and paper with five 20 mL portions boiling H20 or until filtrate from final wash is neut. to litmus. Add 1-2 drops phthln and rapidly titr. with 0.02N NaOH (1 mL = 0.00374 g pyrethrin II). Check normality of 0.02N NaOH same day sample is titrd. (b) For refined pyrethrum exts.-Add 2 mL neut. alcohol and 20 mL H20, and heat to dissolve acid. Cool, filter thru gooch if necessary, add 1-2 drops phthln, and titr. with O.02N NaOH (1 mL = 0.00374 g pyrethrin II). Check normality of 0.02N NaOH same day as sample is titrd. 6.173 Sabadilla Alkaloids (51)-Official Final Action (In dust formulations) Determination (Caution: See 51.011, 51.040, and 51.056.) Weigh 10 g mixed 50% sabadilla dust (or corresponding amt of lesser concn) into 500 mL g-s erlenmeyer. Add exactly 300 mL ether-CHCl3 (3+1). and shake 5 min. Make alk. with 10 mL NH.OH and shake mech. 2 hr. Let stand overnight; then shake 1 hr. Filter, avoiding evapn. Place 200 mL aliquot in 500 mL sepa- rator, acidify with H2SO. (3+97). and shake; withdraw small amt aq. layer and test with litmus paper, returning soln to separator. Add 50 mL of the dil. H2S04 and shake. Let sep. and transfer acid ext to second 500 mL separator. Add 50 mL pet ether to acid ext and shake. Let layers sep. and transfer acid ext to third separator. Repeat extn of soln in first separator with two 50 mL portions of the dil. H2SO., using same 50 mL pet ether in second separator for washing. Collect acid exts in third separator. Make acid exts alk. to phthln with NH.OH. Ext with three 50 mL portions CHCI3 • Wash each CHCI3 ext by shaking gently with same 100 mL portion H20 in fourth separator. (If emulsion forms, add small amt anhyd. Na2SO•. ) Filter each CHCI3 ext thru cotton into weighed 250 mL flask. Evap. CHCI3 on steam bath. Add few mL alcohol, and evap. again. Dry 1 hr at 100° and weigh sabadilla alkaloids. Calc. % total alkaloids. 6.174 Qualitative Test Add 1-2 mL H2SO. to few mg of residue, 6.173. Presence of sabadilla alkaloids is indicated by yellow that gradually becomes intensely red with greenish fluorescence. TOBACCO AND TOBACCO PRODUCTS Nicotine (Note: Nicotine is very toxic. Avoid contact with skin.) Silicotungstic Acid Method (52)-Official Final Action (Includes nornicotine) 6.175 Reagent Silicotungstic acid so/n.-Dissolve 120 g silicotungstic acid (4H20.Si02.12W03.22H20 or Si02.12W03.26H20) in H20 and dil. to 1 L. (Acid should be white or pale yellow crystals, free from green color; soln should be free from cloudiness and green color. Of the several silicotungstic acids, 4H20.Si02.1 OW03.3H20 and 4H20.Si02.12W03.20H20 do not give cryst. ppts with nicotine and should not be used.) 6.176 Determination Weigh sample contg preferably 0.1-1.0 g nicotine. If sample contains very little nicotine (ca 0.1 %). do not increase amt to point where it interferes with distn. Wash with H20 into 500 mL Kjeldahl flask, and if necessary add little paraffin to prevent frothing and few small pieces pumice to prevent bumping. Add 10 g NaCI and 10 mL NaOH so/n (30% by wt). and close flask with rubber stopper thru which passes stem of trap bulb and inlet tube for steam. Connect trap bulb to well-cooled condenser, lower end of which dips below surface of 10 mL HCI (1 +4) in suitable receiving flask. Steam distil rapidly. When distn is well under way, heat flask to reduce vol. of liq. as far as practicable without bumping or excessive sepn of insol. matter. Distil until few mL distillate shows no cloud or opalescence when treated with drop silicotungstic acid soln and drop HCI (1 +4). Confirm alky of residue in distn flask with phthln. Adjust distillate, which may total 1.0-1.5 L, to convenient exact vol. (soln may be concd on steam bath without loss of nicotine). mix well, and pass thru dry filter if not clear. Test distillate with Me orange to confirm acidity. Pipet aliquot contg ca 0.1 g nicotine into beaker. (If samples contain very small amts of nicotine, aliquot contg as little as 0.01 g nicotine may be used.) To each 100 mL liq., add 3 mL HCI (1 +4) and 1 mL silicotungstic acid for each 0.01 g nicotine supposed to be
- 113. AOAC METHODS (1980) ALDRIN, DIELDRIN, ENDRIN 91 present. Stir thoroly and let stand overnight at room temp. Before filtering, stir ppt to see that it settles quickly and is in cryst. form. Filter on either ashless paper or gooch and wash with HCI (1 +1000) at room temp. Continue washing for 2 or 3 fillings of filter after no more opalescence appears when few mL fresh filtrate is tested with few drops nicotine distillate. With paper, transfer paper and ppt to weighed Pt crucible, dry carefully, and ignite until all C is destroyed. Finally heat over Meker burner ,,;;10 min. Wt residue x 0.1141 = wt nicotine in aliquot. With gooch, dry in oven 3 hr at 105° and weigh. Wt residue x 0.1012 = wt nicotine in aliquot. 6.177 HALOGENATED PESTICIDES Aldrin, Dieldrin, and Endrin-Offieial Final Action * Total Chlorine by Sodium Biphenyl * Reduction Method (53) Principle Org. halogen compds are decomposed by Na biphenyl and liberated halide ion is titrd by Volhard method or potentiomet- rically after extn with H20 from reaction medium. Applicable to detn of aldrin, dieldrin, or endrin in dusts, granules, wettable powders, emulsifiable concs, and solns in absence of other org. CI-contg compds. More than trace amts of H20 and appreciable amts of org. compds contg labile H cause excessive consumption of Na biphenyl. Interference of S is avoided, when present, by special treatment. 6.178 Reagents (a) Dilute nitric acid.........fJ% by wt. Add 60 mL HN03 to 945 mL H20. (b) Sodium biphenyl reagent.-30% w/w. (Caution: See 51.034 and 51.038.) Place 300 mL dry toluene and 58 g Na in dry 2 L 3-neck flask equipped with adjustable speed sealed stirrer, inlet for N, and reflux condenser. With stirrer off, and with slow stream of N passing thru flask, warm until refluxing begins and Na is entirely melted. Agitate vigorously until Na is finely dispersed; then cool to <10°. Remove reflux condenser and add 1.25 L anhyd. ethylene glycol dimethyl ether. Add 390 g biphenyl with moderate stirring and with slow stream of N passing thru flask. Reaction should begin within few min, indicated by' blue or green color which gradually darkens to black. Maintain temp. at <30° with oil bath or other cooling medium not involving hazard should flask contg Na break. Reaction should be complete in 1 hr. Reagent protected from moisture and air has useful life of 1-2 months at 25°. (Premixed reagent, packed in'15 mL vials, each enough for 1 detn, is available from Southwestern Analytical Chemicals, Inc., PO Box 485, Austin, TX 78767.) (e) Toluene.-Nitration grade, CI-free. 6.179 Preparation of Sample (Caution: See 51.041 and 51.084.) (a) Technical products.-Accurately weigh ca 0.1 g sample into 125 mL separator contg 25-30 mL toluene. Cautiously add 10-14 g Na biphenyl reagent, mix by swirling, and let stand 5 min. If soln is not dark green, add addnl 10-14 g reagent. (Dieldrin and endrin require 15 min reaction time afterfinal addn of reagent.) Destroy excess reagent by dropwise addn of H2 0, shaking frequently between addns, until green color is completely re- moved. Then slowly add 25 mL dil. HN03, with intermittent swirling. Stopper separator, and mix with gentle rocking motion, venting occasionally. Avoid vigorous shaking during this first extn. Let sep., rinse stopper and walls of separator with H20, and drain aq. phase into 250 mL g-s erlenmeyer. Re-ext reaction mixt. with two 25 mL portions dil. HN03, shaking vigorously. Add aq. exts to erlenmeyer and det. CI. (b) Emulsifiable concentrates and oil spray solns.- Mix tho- roly and weigh sample contg 0.05-0.08 g CI into 250 mL separator contg 25-30 mL toluene. Proceed as in (a). beginning "Cautiously add ..." (e) Dusts, granules, and wettable powders.-Weigh sample contg 0.1-0.15 g active ingredient in paper Soxhlet extn thimble, place in extn app., and ext with ca 150 mL acetone in 300 mL flask 3 hr. Evap. ext to dryness on steam bath, dissolve residue in few mL toluene, and quant. transfer to 250 mL separator, using 25-30 mL toluene. Continue as in (a), beginning "Cau- tiously add ..." If S is brought into soln by decomposition of emulsifiers or other compds such as org. thiophosphates, remove as follows: Add 30% NaOH soln to acid soln in erlenmeyer until alk. to phthln, and add 1 mL excess. Add 5 mL 30% H20 2, heat to bp on hot plate, and boil ca 10 min. Let cool slightly, cautiously add 5 mL more 30% H20" and boil again ca 10 min. Cool, and add small flake (ca 0.05 g) hydrazine sulfate to remove last traces of H20 2• Neutze with dil. HN03 to phthln and add 2-3 mL excess. 6.180 Determination (a) Colorless solns.-To acid aq. soln add 30 mL H,O, 10 mL nitrobenzene, 3 mL ferric indicator, 6.019(e), and, from buret, 0.4-0.6 mL 0.05N KSCN. Swirling constantly, titr. with O.lN AgN03 until red is discharged, and add 2-5 mL excess. Stopper flask tightly and shake vigorously 15 sec. Without refilling buret, titr. slowly with the 0.05N KSCN until end point approaches. Stopper flask, shake vigorously 20-30 sec, and continue titrn until 1 drop produces distinct reddish color which does not fade on swirling or vigorous shaking. (b) Colored solns or chloride in presence of bromide and/or iodide.-To acid aq. soln add 30 mL H,O, transfer to 400 mL beaker, adjust vol. to 200-250 mL, and add 0.5 g Ba(N03)2' Titr. with O.lN AgN03 potentiometrically, with stirring, using cell system of either glass ref. electrode and Ag indicating electrode or Ag-AgCI electrode system, electronic voltmeter, and 10 mL buret. (e) Blank determination.-Det. blank on all reagents by adding 10-14 g Na biphenyl reagent to 25 mL toluene and continuing as in 6.179(a). 6.181 Calculations % Aldrin, dieldrin, or endrin = (net mL AgN03 - mL blank) x normality x 35.45 x F/(10 x g sample). where F is 1.61 for aldrin, 1.81 for dieldrin, and 1.74 for endrin. Net mL AgN03 = [mL 0.1N AgN03 - (mL 0.05N KSCN/2)]. (For most accurate results, det. factor F for specific batch of tech. pesticide used in formulation. Toxicant content is stenciled on drum. Calc. F = PIC, where P = % purity (toxicant content as stenciled on drum). and C = % CI by wt.) Infrared Method (53) 6.182 Principle Dieldrin and endrin in dusts, granules, wettable powders, emulsifiable concs, and solns are purified on adsorbent columns. Hexachloro-epoxy-octahydro-endo,exo-dimethanonaphthalene *Surplus method-see inside front cover.
- 114. 92 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) (HEOD) content of the purified dieldrin or of tech. dieldrin is detd by IR, using baseline technic, and dieldrin is calcd assuming 85% HEOD content. Endrin content of purified or tech. endrin is detd as hexachloro-epoxy-octa-hydro-endo,endo-dimethan- onaphthalene similarly. Aldrin is extd from dusts, wettable powders, and inorg. fertilizers on adsorbent column. Hexachloro-hexahydro- endo,exo-dimethanonaphthalene (HHDN) content of the ext or of tech. aldrin is detd by IR, using baseline technic, and aldrin is calcd assuming 95% HHDN content. Method is not applicable to emulsifiable concs or granules contg petroleum hydrocarbon solvs or to mixts contg other common pesticides or adjuvants that absorb in same wavelength region as HHDN. 6.183 Reagents and Apparatus (a) Chromatographic solvent A.-Mix 1 vol. CHCI3 with 19 vols hexane. (b) Chromatographic solvent B.-Mix 1.5 vols acetone with 98.5 vols chromtgc solvent A. (c) Extraction solvent.-Mix 1 vol. acetone with 19 vols CS2• (d) Infrared spectrophotometer.-With sealed liq. cells with NaCI windows, having optical path length of ca 0.1 mm (dieldrin and endrin) and 0.2 mm (aldrin). 6.184 Preparation of Standard Solutions (a) HEOD std soln for dieldrin.-Accurately weigh ca 100,200, 300, 400, 500, and 600 mg std hexachloro-epoxy-octahydro- endo,exo-dimethanonaphthalene (HEOD) into 10 mL vol. flasks, dissolve in CS2, and dil. to vol. Concns will be 1, 2, 3, 4, 5, and 6 gl100 mL, resp. (b) Std soln for endrin.-Accurately weigh ca 50, 100, 150, 200, 300, and 400 mg std hexachloro-epoxy-octahydro- endo,endo-dimethanonaphthalene (endrin) into 10 mL vol. flasks, dissolve in CS2, and dil. to vol. Concns will be 0.5, 1.0, 1.5,2.0,3.0, and 4.0 gl100 mL, resp. (c) HHDN std soln for aldrin.-Accurately weigh ca 100, 150, 200, 250, 300, and 350 mg std hexachloro-hexahydro-endo,exo- dimethanonaphthalene (HHDN) into 10 mL vol. flasks, dissolve in CS2, and dil. to vol. Concns will be 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 gl100 mL, resp. 6.185 Preparation of Standard Curve Fill 0.1 mm cell (0.2 mm for aldrin) with most dil. of stds solns, using hypodermic syringe. Adjust spectrophtr to optimum set- tings for gain, slit width, response, speed, and drum drive. Make duplicate scans of CS2 soln over scanning range indicated in Table 6:01 and repeat with each of other std solns at same instrument settings. For each of scans of the 6 std solns of each compd, draw line between baseline points indicated in table. Draw perpendicular from zero radiation line thru absorption peak to baseline and measure distance from 0 line to peak, P, and to baseline Po. Calc. A (= log PoIP) and plot as ordinate against concn in g/100 mL as abscissa. Since std curve intersects abscissa at pos. concn value, method is not applicable to concns below this value. Peak wavelengths given in table are characteristic for low Table 6:01 Characteristic Wavelength Points for Infrared Determination of Dieldrin, Endrin, and Aldrin, J.Lm Scanning Baseline Peak at Compound Range Points Low Concn HEOD 11.59-12.18 11.64,12.18 11.80 Endrin 11.43-12.04 11.50,11.97 11.76 HHDN 11.79-12.24 11.85, 12.24 12.01 concns and they shift at higher concns. P is always detd as distance from 0 line to point of max. absorption. 6.186 Preparation of Sample (Caution: See 51.011, 51.041, 51.046, 51.056, and 51.061.) (a) Dusts and wettable powders.-Transfer 3-20 g sample, depending on concn (75--0.5%), weighed to nearest 0.01 g, to chromatgc tube contg 25-50 mm (ca 5.5 g) Hyflo Super-Cel. (For finely divided dieldrin or endrin powder, use 3 9 activated C instead of Super-Cel.) Tamp or vibrate column slightly to settle contents. Place 250 mL wide-mouth erlenmeyer or 500 mL evapg dish under tip of column. Working in well ventilated hood, add 50 mL portions extn solv. to column (if S is present, ext with acetone instead of extn solv.), letting soIv. percolate thru column between addns, until 150 mL ext collects. Rinse tip of column with addnl 10 mL extn solv. Evap. solv. almost to dryness on steam bath under N. Dry HEOD or HHDN residues 15 min at 75°; dry endrin in vac. oven 15 min at 30° and 10 mm pressure. (Extd endrin may no longer be associated with its inhibitors. Residue must not be exposed to elevated temps and must be dissolved promptly to avoid decomposition.) Cool residue and dissolve in few mL CS2• Quant. transfer to vol. flask of such size (5-100 mL) as to give optimum concn of 3 g HEOD, 2 g endrin, or 2 g HHDN/l00 mL, dil. to vol. with CS2, and mix thoroly. If soln is cloudy from H20, add little NaCI, shake, and let settle. (b) Granules containing dieldrin or endrin.-Slurry 40 g Flo- risil in 200 mL beaker with 100 mL hexane. Transfer to chromatgc column with stream of hexane from wash bottle. Eliminate any bubbles or voids by vibration or agitation. Let hexane drain until only 2-3 mm layer remains above surface of column. Add small layer of Na2SO. to top of column. Transfer 2-10 g finely ground sample, depending on concn (10-1%), to prepd column. Rinse down column walls with three 10 mL portions chromatgc solv. A, letting each portion enter column before adding next. Add 170 mL chromatgc soIv. A, let percolate thru column, and discard. Gently flow 10 mL chromatgc soIv. B down walls of tube, avoiding disturbing surface of adsorbent. After solv. sinks into column, repeat washing with 2 addnl 10 mL portions. Add 220 mL chromatgc solv. B and let flow at rate of 2-5 mL/min, collecting eluate in 500 mL wide-mouth erlenmeyer or evapg dish. Evap. solv. to dryness on steam bath, avoiding spattering, and proceed as in (a), using 5-10 mL vol. flask. (c) Emulsifiable concentrates and solns. -Weigh 1.5 g dieldrin conc. (1.5 Ib/gal.), 1.0 g endrinconc. (1.6 Ib/gal.), or 30.0 g 0.5% dieldrin soln, and add 5 mL hexane. Transfer to prepd column and proceed as in (b). (d) Technical materials.-Transfer sample contg 1.75-4.00 g dieldrin, 1.50-3.00 g endrin, or 1.00-2.00 g aldrin, weighed to 0.01 g, to 100 mL vol. flask. Dissolve in CS2 and dil. to vol. with CS2• 6.187 Determination Fill same 0.1 mm cell (0.2 mm for aldrin) used for prepn of std curve with sample soln. Make duplicate scans, and calc. A and mean A as in prepn of std curve. From appropriate std curve, obtain g HEOD, endrin, or HHDN/l00 mL sample soln, W. % dieldrin = W x V x 1.175IS; % endrin = W x VIS; % aldrin = W x V x 1.053/S; where V = mL sample soln; S = 9 sample; 1.175 and 1.053 = conversion factors HEOD to dieldrin and HHDN to aldrin, resp.
- 115. AOAC METHODS (1980) 6.188 Amiben (3-Amino-2,5-dichlorobenzoic Acid) (54)-Official Final Action AOAC-CIPAC Method Principle Amiben contains conjugated 7T electron system of benzene which absorbs strongly in UV. Absorption is measured quant. at 297 nm. (Caution: See 51.018 and 51.041.) 6.189 Apparatus and Reagents (a) Spectrophotometer.-For use in UV, with 1 cm cells. (b) Shake-out flask.-250 mL erlenmeyer, with screw cap. (c) Amiben std solns.-(1) Stock soln.-0.38 mg/mL. Accu- rately weigh 19±2 mg amiben (Amchem Products, Inc., Ambler, PA 19002) into 50 mL vol. flask, add 25 mL 1% NaOH, agitate until dissolved, dil. to vol., and mix. (2) Working soln.-0.038 mg/mL. Pipet 5 mL stock soln into 50 mL vol. flask, dil. to vol. with 1% NaOH soln, and mix. 6.190 Preparation of Sample Mix 10 g granular sample on 12 x 12" paper by lifting alternate corners. 6.191 Determination (a) Dry granular formulations.-Add amt solid material and 1% NaOH soln specified in Table 6:02 to 250 mL shake-out flask and shake 30 min. Filter, and transfer stated aliquot to vol. flask. Oil. to vol. with 1% NaOH soln and mix. Det. A at 360 and 297 nm against 1% NaOH. Calc. ~A = A297 - A3S0' Det. ~A' of working std soln similarly. % Amiben = (~A x (mg std/mL) x F)/(~A' x g sample x 10). where F = factor in Table 6:02. (b) Liquid formulations.-Weigh amt liq. indicated in Table 6:02 into 100 mL vol. flask, dil. to vol. with 1% NaOH soln, and mix. Transfer 1 mL aliquot to 100 mL vol. flask, dil. to vol. with 1% NaOH, and mix. Proceed as in (a). Benzene Hexachloride (BHe) (Hexachlorocyclohexane) Gamma Isomer (lindane) Partition Chromatographic Method (55) Official Final Action (Caution: See 51.009, 51.011, 51.039, 51.040, 51.041, and 51.054.) 6.192 Apparatus (a) Partition column.-Column and 0 type reduction valve are shown in Fig. 6:04. Construct column of heavy-wall Pyrex tubing ca 3.5 mm thick, 90 cm long x 2.5 cm diam. Seal coarse porosity fritted glass disk in place and attach No. 18/9!f joint 5 cm below disk. Supply pressure from laboratory supply line. (Column available from SGA Scientific, Inc.; specify Cat. No. JC 1800 constructed from heavy rather than std wall tUbing.) (b) Solvent evaporator.-Fig. 6:04. Evap. fractions to dryness BHC 93 under reduced pressure at 60°, with aid of H2 0 pump. Recover solv. in trap consisting of Kjeldahl flask immersed in mixt. of NaCI and ice. (c) Melting point apparatus.-Use Thiele mp app. equipped with mech. stirrer. App. shown in Fig. 6:05, or Hershberg modification (56) (available from Ace Glass, Inc., Cat. No. 7686) is suitable. (d) Thermometer.-Precision grade, meeting NBS specifica- tions: partial immersion; range 90-120° in 0.2° subdivisions. Calibrated by NBS or against thermometer checked by NBS. (e) Melting point tubes.-1-2 mm capillary tubes of uniform wall thickness and diam. 6.193 Reagents (a) n-Hexane.-Com. grade, distd before use. (b) Nitromethane.-Reflux com. grade material 4 hr and distil. No visible residue is left after evapn of 10 mL purified material. (c) Silicic acid.-Use Mallinckrodt reagent grade (for chro- matgy) which meets following requirements: When column prepd as in 6.195 is used for detn on sample contg known amt of y-isomer, flow rate and packing characteristics should be similar to those of an H2Si03 known to be satisfactory, and recovery of y-BHC should be within ±3% of the y-BHC content. (d) Dye soln.-Dissolve 25 mg D&C Violet No.2 (1-hydroxy- 4-p-toluidino-anthraquinone) in 50 mL mobile solv. and store in g-s bottle. (Available from Aldrich Chemical Co.) (e) Mobile solvent.-Satd soln nitromethane in n-hexane. Vigorously shake 2 L n-hexane with excess nitromethane in g-s bottle. Decant mobile solvent from nitromethane as needed. 6.194 Preparation of Sample (a) Powders containing more than 10% y-BHC.-Crush and thoroly mix sample with mortar and pestle. Weigh enough sample into tared 125 mL erlenmeyer to provide ca 0.2 g y- isomer after extg and aliquoting. Add 25 mL mobile solv., heat just to bp on steam bath, and cool to room temp., shaking occasionally. Decant ext thru buchner with ca 34 mm medium porosity fritted disk into 100 mL Kohlrausch flask, with gentle suction. Re-ext residue in flask, using 10 mL mobile soIv. Wash residue and flask with five 10 mL portions cold mobile solv., decanting each wash thru buchner. Add 2 mL dye soln and dil. to vol. with mobile solv. (b) Dusts containing less than 10% y-BHC.-Weigh enough sample to provide 1.75-2.00 g y-isomer. Transfer to Soxhlet extractor and ext overnight with ether. Evap. most of ether on steam bath and evap. remainder at room temp. under vac. Ext y-isomer from residue with mobile solv. as in (a). 6.195 Preparation of Column (Caution: See 51.004 and 51.061.) Transfer 100±0.5 g H2Si03 to high-speed blender, add 300 mL mobile solv., and with mixing, add 55 mL nitromethane. Mix 15-30 sec; then pour into column thru glass funnel. Stir slurry with long glass stirring rod to displace air bubbles. Wash down Table 6:02 Parameters for sample analysis Sample Amiben, Wt, 1% NaOH, Aliquot, Final Factor Sample % g±O.l mL mL Diln (F) Dry granular 1.2 3.0 50 2 50 1,250 Dry granular 4 7.5 100 100 10,000 Dry granular 10 3.0 100 100 10,000 Liquid 21.6 1.8 100 100 10,000
- 116. 94 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) FIG. 6:04-Partition column and solvent evaporator sides of column with few mL mobile solv. and apply 5 Ib pressure to pack column and force out excess solv.; tap column gently to aid packing. When boundary between soIv. and H,Si03 remains stationary, release pressure cautiously, pipet out most of excess solv., and reapply pressure until ca 3 mm solv. remains above adsorbent. FIG. 6:0S-Melting point apparatus 6.196 Determination (Caution: See 51.015 and 51.018.) Pipet 10 mL aliquot of sample soln onto column by letting it flow slowly down inside of column without disturbing H,Si03 surface. Wash down side of column with 2 mL mobile soIv. and force soln into column by applying 2-3 Ib pressure, releasing pressure when all solv. has entered column. Add 10 mL mobile solv. and force into column. Release pressure and slowly add mobile solv. to within 7-12 cm from top of column. Apply enough pressure to force solv. thru column at 3-4 mL/min. Just before last trace of dye leaves column, begin to collect 10 mL fractions, alternately using two 10 mL graduates. Transfer each fraction to 125 mL erlenmeyer and evap. to dryness, using solv. evaporator. (Evap. fractions without boiling; if boiling begins, raise flask momentarily from H20 bath.) Appearance of y-isomer upon evapn is recognized by its tendency to cover bottom of flask as white residual film with typical crystal formation. When first residue of y-isomer is recognized, begin to collect 10 mL fractions until all ')I-isomer is obtained (usually ,,;8 fractions). Dissolve residue in each flask with 5 mL n-hexane and transfer to weighed flask, rinsing flasks successively with 5 mL portions n-hexane. Evap. solv., using soIv. evaporator. Evacuate flask ca 20 min at room temp. with vac. pump. (There is little danger in evacuating 125 mL erlen- meyer; larger size erlenmeyer, however, is likely to collapse under vac.) Release vac., wipe with clean, moist towel, and let stand 5 min. Weigh, and calc. % ')I-benzene hexachloride in original sample. 6.197 Melting Point Determination of the Gamma Fraction Dissolve residue in min. amt acetone and transfer quant. to 10 mL beaker. Evap. acetone at 40°, using filtered air stream. Scrape residue from beaker for mp detn. (Beaker may be set on piece of solid CO2 to ensure prepn of finely powd product.) Place material in agate mortar and mix thoroly with pestle. Select 2 clean, dry capillary tubes and fill with sample. Be sure material is well packed into bottom of tube to ensure max. contact between sample and wall of tube. Insert tubes and
- 117. AOAC METHODS (1980) BALAN OR TRIFLURALIN 95 thermometer bulb in Thiele tube so that samples and thermom- eter bulb touch. Start stirrer and heater, and adjust heating rate to 1°/min at 90°. Continue heating until sample melts or reaches 106°. Reduce heating rate to 0.5°/min and continue heating until sample melts. Sample mp is corrected temp. of bath when last solid disap- pears into the clear melt. If mp is <108°, check result by IR method,6.198-6.201. Infrared Spectrophotometric Method (57) Official Final Action (Applicable to tech. BHC. Caution: See 51.018, 51.041, and 51.048.) 6.198 Apparatus Infrared spectrometer.-With matched pair of liq. absorption cells, 0.5-1.1 mm thick. 6.199 Calibration of Cells Oet., in spectrometer, difference between deflections of the 2 cells filled with CS2 • Plainly mark one cell to be used as sample cell for reading I. Correct values of '0 obtained with other cell by adding or subtracting difference between cells and refer to this as cell factor F. Check factor every 10-14 days. 6.200 Preparation of Standards and Working Curves Obtain a, f3, y, and 0 isomers of BHC, either by fractional crystn from tech. material or as sepd materials, and recrystallize several times from solvs that have been redistd from all-glass app. Recrystallize from following solvs until mps by capillary tube method become const: a isomer from benzene followed by MeOH (mp ca 158°); f3 isomerfrom toluene (mp ca 210.5", sealed capillary); y isomer from MeOH (mp ca 113°); and 0 isomer from CCI. followed by CHCI3 (mp ca 138.5"). Confirm purity of each isomer as follows: Evap. to dryness enough mother liquor from last crystn to yield ~1 g dissolved solids, grind residue, and dry overnight in evacuated desiccator. Weigh and dissolve in enough CS2 to make 4 g/100 mL soln. Prep. corresponding soln of recrystd isomer as std. Compare solns of residue and std in spectrometer at wavelength points used for analysis of other isomers. Consider purity of isomer satisfactory if A of residue soln is not significantly greater than that of std at these points. Prep. working curves of the isomers by detg T of their solns in CS2 at various concns as in 6.201. Calc. A and plot against concn in g/L. 6.201 Determination Reduce sample of tech. BHC to ca 2 g by grinding and quartering, and dry 24 hr in vacuo at room temp. Weigh 1.5000 g dried material into 50 mL vol. flask and dil. to vol. with CS2 (equiv. to 30 g/L). Shake vigorously to dissolve (f3 isomer is not completely sol. and will settle out). Pipet 25 mL of this sample soln into another 50 mL vol. flask and again dil. to vol. with CS2 (equiv. to 15 g/L). Fill sample cell with the concd soln for reading I, and fill blank cell with CS2, place in spectrometer, and read T in duplicate at following wavelengths: Alpha Beta Gamma Delta Epsilon Wavelength, iJ-m 12.58 13.46 14.53 13.22 13.96 Average duplicates for calcns. Repeat readings with dil. soln (15 g/L) at a and y wavelengths. Calc. A of each of isomers at the various wavelengths from T measurements by equation: Lo (F x ' b) - (F x Ib x % Set) = A g Is - (F X Ib x % Sct) , where F = cell factor, Ib = reading of blank cell, % Set = % scatter, Is = reading of sample cell, and A = absorbance. Obtain approx. concns from working curves, 6.200. Correct A at each wavelength for absorption of interfering components. (Altho f3 isomer has low solubility in CS2, this isomer interferes with 0 analytical point; therefore det. A of f3 isomer in CS2 at this point and apply as correction.) Since these new values are overcorrected, make repeated evaluations until successive val- ues are const, within desired precision. 6.202 * Radioactive Tracer Method (58) * Official First Action See 6.257-6.260, 11th ed. N-Butyl-N-ethyl-a.a.a-trifluoro-2.6-dinitro-p-toluidine (8alan®) or Trifluralin (a,a.a-Trifluoro-2.6-dinitro-N.N- dipropyl-p-toluidine (59)--Official Final Action Ultraviolet Method 6.203 Principle Trifluralin or Balan is extd from solid carrier or dissolved in n-hexane if liq., purified by chromatgy on Florisil, and detd by UV spectrometry at 376 nm. 6.204 Reagents (a) Florisil.-100-200 mesh. Test elution characteristics of Florisil by adding 5 mL std soln to prepd column. Proceed as in 6.207. Elution vol. should be ~80 mL but <100 mL. If elution vol. does not fall within this range, adjust H20 content of Florisil by trial and error to obtain proper elution (add H20 to decrease elution time; dry at 130° to increase it). (b) Std soln.-1.25 mg/mL. Weigh 0.125 g trifluralin or Balan Ref. Std (Elanco Products Co.l. into 100 mL vol. flask, dil. to vol. with n-hexane, and mix. 6.205 Preparation of Column Insert glass wool plug in bottom of 25 x 400 mm glass tube with Teflon stopcock. Add, with const tapping of column, 5 g anhyd. Na2SO., stdzd Florisil, (al. to ht of 50 mm, and 5 g anhyd. Na2SO•. With stopcock open, add 50 mL n-hexane and let drain to top of column. Close stopcock. 6.206 Preparation of Sample (a) Dry formulations (containing more than 1% trif/uralin or 8alan).-Weigh sample contg 0.25 g trifluralin or Balan into Soxhlet extn thimble (33 x 80 mm), cover with glass wool, and ext with CHCI3 1 hr beyond time when no further color is extd. Quant. transfer ext to 200 mL vol. flask with CHCI3, dil. to vol. with CHCI3, and mix. Transfer 5 mL to rob flask and evap. just to dryness on rotary evaporator. (b) Dry formulations (containing 1% or less trif/uralin or 8alan).-Weigh sample contg 0.05 g trifluralin or Balan, ext, transfer to 200 mL vol. flask, and dil. as in (a). Transfer 25 mL to rob flask and evap. just to dryness on rotary evaporator. (c) Liquid formulations.-Weigh sample contg 0.12 g triflur- alin or Balan into 100 mL vol. flask. Oil. to vol. with n-hexane and mix vigorously. Proceed as in 6.207. *Surplus method-see inside front cover.
- 118. 96 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) 6.207 Determination Transfer 5 mL soln from (e) or residue from (a) or (b). with aid of n-hexane, to Florisil column. Transfer 5 mL std soln to second Florisil column. Wash sample into column with small portions n-hexane. Let each portion drain to top of column before adding next. Fill column with n-hexane, discarding eluate until band has moved ca % length of column. Collect eluate contg trifluralin or Balan band (first yellow-orange band to elute) in 100 mL vol. flask. (If band requires >100 mL vol. to elute, replace vol. flask with rob flask, evap., and transfer quant. to 100 mL vol. flask.) (Caution: See 51.011(a) and 51.061.) Dil. to vol. with n-hexane and mix. Det. A of sample and std solns in 1 cm cells at 376 nm against n-hexane as ref. 6.208 Calculations % Trifluralin or Balan = (A x g std x F x P)/(A' x g sample). where A and A' refer to sample and std solns, resp.; P = % purity of std; and F = 2, 0.4, or 1 for sample prepns (a). (b), or (e), resp. Gas-Liquid Chromatographic Method 6.209 Principle Trifluralin or Balan is extd from solid carrier, or dissolved in acetone if liq., and detd by GLC. 6.210 Reagents (a) Diisobutyl phthalate internal std soln.-Weigh 0.625 g diisobutyl phthalate (lCN-K&K Laboratories, Inc.) into 250 mL vol. flask, dil. to vol. with acetone, and mix. (b) Std soln.-l.6 mg/mL. Weigh 0.16 g trifluralin or Balan Ref. Std into 100 mL vol. flask, dil. to vol. with acetone, and mix. 6.211 Apparatus (a) Gas chromatograph.-Equipped with flame ionization de- tector; capable of programmed column temp. from 135 to 190° at 8°/min. Approx. instrumental conditions: inlet 205°, detector 2750 , N carrier gas 60 mL/min. (b) Column.-l.5 m (5') x 'Is or '14" od, stainless steel or Pyrex glass tube packed with 5% DC 200, 12,500 cstokes (Analabs, Inc.) on 80-100 mesh Chromosorb W (HP). Condition newly prepd column at 230° overnight with N carrier gas. 6.212 Preparation of Sample (a) Dry formulations (containing more than 1% trifluralin or Balan).-Weigh sample contg 0.16 g trifluralin or Balan into Soxhlet extn thimble (33 x 80 mm). cover with glass wool, and ext with acetone 1 hr beyond time when no further color is extd. (Caution: See 51.011(a) and 51.046.) Evap. to ca 60 mL on steam bath with stream of air directed into flask. Transfer quant. to 100 mL vol. flask with acetone. Dil. to vol. with acetone and mix. (b) Dry formulations (containing 1% or less trifluralin or Balan).-Weigh sample contg 0.04 g trifluralin or Balan, ext, and evap. as in (a). Transfer quant. to 100 mL vol. flask with acetone and proceed as in 6.213 without dilg, beginning, " ... add 10 mL internal std soln, ..." (e) Liquid formulations.-Weigh sample contg 0.16 g triflur- alin or Balan into 100 mL vol. flask, dil. to vol. with acetone, and mix. 6.213 Determination Pipet 25 mL acetone soln, 6.212(a) or (e), and 25 mL std soln, (b). into sep. 100 mL vol. flasks, add 10 mL internal std soln, dil. to vol. with acetone, and mix. Inject 2.5 ,ttL trifluralin or Balan std soln and start temp. program to give symmetrical peak ca 70% scale deflection and retention time 5.5 min. Diisobutyl phthalate internal std peak appears ca 2 min after std peak. Repeat injection of std soln until ratio of trifluralin or Balan peak area to internal std peak area is reproducible. Without changing conditions inject 2.5 ,ttL sample soln. 6.214 Calculations Calc. areas ot'trifluralin or Balan and diisobutyl phthalate peaks. Divide area oftrifluralin or Balan peak by area of diisobutyl phthalate internal std peak to det. ratio, R. % Trifluralin or Balan = (R x W' x P)/(R' x W x Fl. where Rand R' = ratio for sample and std solns, resp.; Wand W' = g sample and std, resp.; P = % purity of std; and F = 1, 1, or 4 for sample prepns (al. (e), or (b), resp. 6.215 Captan (N-(Triehloromethylthio)-4-eyelohexene- 1,2-diearboximide) (60)-Offieial First Action AOAC-CIPAC Method (Caution: See 51.039 and 51.041.) Principle Captan is extd from inerts with soln contg dieldrin in dioxane. Ratio of captan peak ht to dieldrin peak ht in gas chromatgy is measured and compared to ratio from std captan prepd similarly. Method applies to tech. and dry formulated products contg captan as only active ingredient. 6.216 Reagents (a) Extracting soln.-Weigh ca 5.0 g dieldrin (ca 85% HEOD) into 500 mL vol. flask, add ca 300 mL lA-dioxane, shake to dissolve dieldrin, and dil. to vol. with dioxane. (b) Captan std so/n.-Accurately weigh, by difference, 0.25 g captan ref. std into glass vial. Pipet 25 mL extg soln into vial, stopper, and shake until dissolved. Prep. fresh after 24 hr. 6.217 Gas Chromatography Use any app. that will completely sep. captan from HEOD and with following conditions: Sample inlet port 10-30° higher than column; thermal conductivity or H flame detector maintained ~20° higher than column (typical column temp. 220°); 1.5 m (5') x 'I." od glass column packed with 3% XE-60 silicone nitrile gum rubber on Chromosorb G, acid-washed and dimethyl dichlorosilane-treated (allowable variations are 0.6-1.8 m (2-6'). 'Is-'l." od, 2-10% liq. loading, Chromosorb W). Condition newly prepd column at 230° overnight while purging with carrier gas (He for thermal conductivity or N for H flame detectors). 6.218 Preparation of Sample Accurately weigh, by difference, well mixed portion of sample contg ca 0.25 g captan into 30 mL glass vial with plastic-lined screw cap. Into vial pipet 25 mL same extg soln used for prepn of std soln. Stopper and shake mech. 15 min. Centrf. if necessary to ppt inerts. Sample supernate with syringe for captan detn. Prep. fresh after 24 hr. 6.219 Determination Adjust column temp., carrier gas flow, injection size, and recorder attenuation so that captan and HEOD peaks are com- pletely sepd in <10 min and so that ht of HEOD peak, which elutes first, is ca % full scale. Repeat injections of std soln until peak ht ratio captan:HEOD of 2 consecutive injections varies
- 119. AOAC METHODS (1980) CHLORDANE 97 <2%. Without changing conditions, inject supernate from sam- ple. If sample ratio differs by > ± 10% from std ratio, reweigh sample. For detn, inject std, sample in duplicate, and std. Measure HEOD and captan peak hts. When solv. peak tailing extends into region of HEOD and captan peaks, draw curved extension as baseline. Det. peak ht ratio captan:HEOD and average the 2 values (Rs) for std injections just before and after sample injections. Calc. and average peak ht ratio of the 2 samples (Rx). % Captan = (Rx/Wx) x (Ws x P/Rs), where Ws = g std, Wx = g sample, and P = % purity of captan std. Technical Chlordane Total Chlorine Method (61}-Offlcial Final Action (Caution: See 51.011, 51.018, 51.038, 51.039, 51.040, 51.041, and 51.045.) 6.220 Standardization of Standard Solutions (a) Sodium chloride std soln.-0.1N. Dissolve 5.845 g NaCI, previously dried 2 hr at 105°, in H2 0, and dil. to 1 L in vol. flask. (b) Silver nitrate std soln.-0.1N. Prep. as in 50.027. To 250 mL g-s erlenmeyer add 15.00 mL O.lN NaCI, (a), 50 mL H20, 10 mL HN03 (1 +1), boiled to expel oxides of N, and 25.00 mL of the AgNOj soln. Add 3 mL nitrobenzene, stopper, and shake vigorously 15 sec. Add 5 mL ferric indicator, 6.019(e), and back- titr. with O.lN KSCN, (c), to reddish-brown end point. (Potenti- ometric titrn using Ag indicator electrode and Ag-AgCI or glass ref. electrode may be substituted for indicator method, but must be used in both stdzn and detn.) (c) Potassium thiocyanate std soln.-0.1N. Prep. and titr. against AgN03 soln, (b), as in 50.030(b). Calc. F = mL AgN03 soln/mL KSCN soln. Normality AgN03 soln = mL NaCI soln x 0.1000/(mL AgN03 soln --; mL KSCN soln x F). 6.221 Preparation of Sample (a) Emulsifiable concentrate formulations.-Accurately weigh sample contg 0.5±0.05 g tech. chlordane into 50 mL vol. flask, dissolve, and dil. to vol. with toluene. Transfer 5 mL aliquot to 125 mL separator, add 15 mL or g Na biphenyl reagent, 6.178(b), and swirl. If soln is not dark green, add more reagent. Let stand 3 min and add 3-5 mL H20 dropwise. With stopper removed, swirl soln gently to decompose excess reagent. Add 25 mL H20, stopper, and mix with gentle rocking motion. (Do not shake vigorously.) Let layers sep. and drain lower aq. layer into 250 mL erlenmeyer. Re-ext solv. layer with two 25 mL portions 3N HN03 and combine aq. solns in erlenmeyer. (b) Dusts, granular impregnates, and wettable powders.- Accurately weigh sample contg 0.5±0.05 g tech. chlordane into Soxhlet extn thimble. Ext with 80 mL benzene in Soxhlet app. 1 hr. Transfer to 100 mL vol. flask, washing with several 3 mL portions benzene. Dil. to vol. with benzene and transfer 10 mL aliquot to 125 mL separator. Proceed as in (a). 6.222 Determination Add 15.00 mL O.lN AgN03 and 3 mL nitrobenzene to erlen- meyer, stopper, and shake vigorously 15 sec. Rinse stopper, add 5 mL ferric indicator, 6.019(e), and back-titr. with O.lN KSCN to reddish-brown end point. (Designate mL KSCN as D.) Det. blank on reagents by pipetting 5 mL toluene into 125 mL separator, add 15 mL or g Na biphenyl reagent, and proceed as in 6.221(a), thru combining aq. solns in erlenmeyer. Add 15.00 mL O.lN NaCI, 25.00 mL O.lN AgN03, and 3 mL nitrobenzene, and proceed as above. Calc. blank correction factor, C = mL KSCN used in stdzn of AgN03 - mL KSCN used in blank detn. % Chlorine = [15 - (C + D) x F] x normality AgN03 x 3.545/g sample. % Tech. chlordane = % CI x 1.56. Colorimetric Method (62}-Offlcial Final Action (Method is empirical; all conditions must be reproduced exactly to attain good precision. Temp., reaction time, and vol. of reagents affect color intensity.) 6.223 Apparatus (a) Constant temperature bath.-Capable of maintaining 100±1° and holding twelve 20 x 150 mni test tubes. (b) Cuvets.-l0 or 2 mm light path (available from Pyrocell Mfg. Co., 91 Carver Ave, PO Box 176, Westwood, NJ 07675). (c) Spectrophotometer.-Capable of accepting cuvets, (b). 6.224 Reagents (a) Methanol.-90% (by voL). (b) Methanol-benzene.-Mix 7 vols MeOH with 3 vols ben- zene. (c) Diethanolamine.-Purify by vac. distn at ca 20 mm Hg and take middle fraction. (Caution: See 51.011 and 51.015.) (d) Diethanolamine-KOH soln (Davidow reagent). -Mix 1 vol.- reagent (c) with 2 vols 1.0N KOH in MeOH. (e) Chlordane std solns.-l.5, 2.5, and 3.5 mg/mL. Ref. grade (available from Velsicol Chemical Corp.). Dissolve tech. chlor- dane in reagent (b). Discard stds after 2 weeks. 6.225 Preparation of Sample (a) Liquid formulations.-Transfer weighed sample contg 200-300 mg tech. chlordane to 100 mL graduate and dil. to 100 mL with MeOH-benzene. (b) High concentration solid formulations (10% chlordane or more).-Treat as in (a) and shake vigorously several min. Let settle 1 hr. (c) Low concentration solid formulations (less than 10% chlor- dane).-Transfer weighed sample contg 200-300 mg tech. chlor- dane to Soxhlet and ext 1 hr with pentane. (Caution: See 51.039 and 51.074.) Evap. pentane on steam bath and transfer ext to 100 mL g-s graduate. Dil. to 100 mL with MeOH-benzene. 6.226 Determination Pipet 2 mL aliquot prepd sample to 20 x 150 mm test tube. Add No.8 grit SiC boiling chip and 2 mL Davidow reagent, and place in 100° const temp. bath. Remove after exactly 45 min and cool immediately in beaker of cold H20. Transfer to 10 mL vol. flask and dil. to vol. with 90% MeOH. Transfer aliquot of soln to 2 mm cell and read A at 550 nm within 15 min with 90% MeOH as ref. (Comparable results are obtained by dilg soln to 50 mL and using 1 cm cello) Treat 2 mL each std soln with each set of samples. (Read 1 std soln before samples, 1 after half the samples are read, and 1 after last sample is read.) 6.227 Calculations Calc. absorptivity (a) for each of 3 stds, and use avo in subsequent calcns (expected a is ca 0.25): a =A '/W, where A' = A std soln and W = mg tech. chlordane (2 x concn std soln in mg/mL). % Tech. chlordane in sample = A x 5000/(a x mg sample).
- 120. 98 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) Hexachlorocyclopentadiene (HEX) (63)-Official Final Action (Applicable to tech. chlordane, but not to formulations) 6.228 Reagent Hexachlorocyc!opentadiene (HEX) std solns.- Stock soln.- 0.1 g/100 mL. Weigh 0.1000 9 hexachlorocyclopentadiene Ref. Std (available from Velsicol Chemical Corp.) in 100 mL vol. flask, dil. to vol. with MeOH, and shake to dissolve. Std soln 1.-0.005 g/100 mL. Oil. 5 mL stock soln to 100 mL with MeOH. Std soln 2.-0.002 g/100 mL. Oil. 2 mL stock soln to 100 mL with MeOH. Method I 6.229 Calibration With MeOH in both ref. and sample cells (matched 1 cm silica). adjust 0 and 100% settings on UV spectrophtr at 324 nm. Empty sample cell, rinse several times with, and then fill with, std soln 1, and read A. Empty sample cell, rinse with MeOH, then rinse and fill with std soln 2, and read A. Calc. A factor, K, for each std soln = (g std HEX/l00 mL)/A. Average the two K values. 6.230 Determination Weigh 0.5 9 sample in 100 mL vol. flask, dil. to vol. with MeOH, and shake to dissolve. Proceed as in 6.229, treating sample soln in same manner as stds. % HEX in sample = (A of sample soln x 100 x K)/(g sample/l00 mL). 6.231 Method /I (Includes corrections for other components of chlordane which absorb at 324 nm) Proceed as in 6.229-6.230, except det. A of all solns at 300, 324, and 350 nm. Settings of 0 and 100% must be repeated at 300, 324, and 350 nm for A readings at those points. Calc. K = (g std HEX/laO mL)/ [A 32• - 0.5(A300 + A3S0))' % HEX in sample = [A 32• - 0.5(A300 + A 3S0)) x 100 x K/(g sample/lOa mL). AG Chlordane (Octachloro-4,7-methanotetra- hydroindane) (64)-Official Final Action a- and y-Isomers in Technical Products-Infrared Method (Not applicable to tech. chlordane or its formulations) 6.232 Apparatus and Reagent (a) Infrared spectrophotometer.-Double beam, with matched NaCI cells, 0.1 and 0.2 mm. (b) Std soln.-Into tared 10 mL vol. flask, weigh 1.00:!:0.05 g ref. std a-chlordane and 0.38±0.02 9 ref. std y-chlordane (Velsicol Chemical Corp.). dissolve in CS2, and dil. to vol. with CS2• 6.233 Preparation of Sample Melt entire sample in 1000 oven and mix. Weigh 1.5:!:0.02 g into tared 10 mL vol. flask, dissolve in CS2, and dil. to vol. with CS2 • 6.234 Determination (a) a-Chlordane.-Fill ref. cell with CS2• Scan std and sample solns from 750 to 710 cm-1 (13.3 to 14.1 JLm). using 0.1 mm NaCI cells. Construct baseline from 738 to 715 cm-1 (13.6 to 14.0 JLm) and draw line from point of max. A ca 725 cm-1 (13.8 /Lm), to intersect baseline. (b) y-Chlordane.-Fill ref. cell with CS2• Scan std and sample solns from 1390 to 1290 cm-1 (7.19 to 7.75 JLm). using 0.2 mm NaCI celis. Proceed as in (a). using min. at 1370 and 1310 cm-1 (7.30 and 7.63 JLm), and det. A at max. 1320 cm-1 (7.58 JLm). 6.235 Calculations Wt % a(a-chlordane) or y(y-chlordane) = (A x F x 100)/Ws F = [W(a or y in std) x % purity in std)/A '(a or y of std), where W = wt (9). and A and A' refer to sample and std, resp. (Wts given are for cell thicknesses specified. For other cells, adjust wts to yield peak A between 0.2 and 0.5 (30-65% T).) AG Chlordane in Granular Formulations-Infrared Method (Not applicable to tech. chlordane or its formulations. Caution: See 51.011, 51.041, 51.046, and 51.048.) 6.236 Apparatus (a) Infrared spectrophotometer.-See 6.232. (b) Soxhlet extraction apparatus.-With 25 x 80 mm What- man cellulose thimble. (c) Vigreux distilling tube.-15 mm long. (d) Vials.-5 dram, with plastic-lined screw caps. 6.237 Reagents (a) Acetone.-Spectral grade. (b) Std soln.-Into tared 5 dram vial, weigh 1.00:!:0.05 g Ref. Std a-Chlordane and 0.38±0.02 g Ref. Std y-Chlordane (Velsicol Chemical Corp.) and pipet in 10 mL CS2• 6.238 Preparation of Sample Into tared thimble, weigh sample equiv. to wt of std and cover with glass wool. Insert into extn app. and attach to 250 mL flat- bottom l flask contg boiling chips. Add 125 mL acetone, attach extractor to condenser, and ext 1 hr. Rinse extn app. with acetone. Sep. flask from extractor and condenser, attach distg tube to flask, and evap. acetone on steam bath. Remove tube, add 5 mL CS2, and evap. carefully. Repeat addn and evapn of CS2 4 more times. (All residual acetone must be removed because acetone interferes with IR measurement.) Ory residue further, using forced air, until cryst. solid appears. Pipet 10 mL CS2 into flask, and swirl carefully to dissolve solid. Release stopper pressure. 6.239 Determination Proceed as in 6.234. 6.240 Calculation Proceed as in 6.235 for calcn of wt %. % Total AG chlordane = % a-chlordane + % y-chlordane. Generally, factor representing specification grade of 95% AG chlordane may be used. Then, % AG chlordane = % total (a + y) x 1.053. Heptachlor in AG Chlordane-Gas Chromatographic Method (Not applicable to tech. chlordane or its formulations) 6.241 Apparatus and Reagents (a) Gas chromatograph.-Equipped with flame ionization de- tector and 1.5 m (5') x '!a" id glass column packed with 5% silicone OC 200 (Viscosity 12500, Analabs, Inc.) on 130-140 mesh
- 121. AOAC METHODS (1980) DIMETHYLUREAS 99 Anakrom AS5. Operating conditions: temps (O)-column 165, injector 215, detector 220; N carrier gas 30 mL/min (ca 80 psig at inlet); and chart speed 0.5"/min. (b) Stds.-Ref. 5td a-Chlordane, y-Chlordane, and Heptachlor (Velsicol Chemical Corp.) and hexachlorobenzene (C6CI6 ) internal std (Eastman Kodak Co.), recrystd from benzene. (e) Std so/n.-Accurately weigh following components into 10 mL vol. flask, dissolve in C52, and dil. to vol.: 0.48 g a- chlordane, 0.18 g y-chlordane, 0.010 g C6C16, and 0.010 g heptachlor. 6.242 Preparation of Sample Melt entire sample in 100° oven and mix. Accurately weigh 0.73 g sample and 0.010 g C6CI6 into 10 mL vol. flask, and dil. to vol. with C52 • 6.243 Determination Inject 1.5 /-tL sample soln into gas chromatograph; retention times for C6CI6 and heptachlor are ca 2 and 4 min, resp. Also inject 1.5 /-tL std soln to det. response factor (RF). Use attenuation (ca 2 x) to keep internal std peak on scale and include in calcns. 6.244 Calculations Calc. each peak area by any convenient means. Built-in integrators or planimeters provide most accurate method of detg areas where peaks are not perfectly symmetrical. % Heptachlor = (PH x RF x WI x 100)/(PI x W) RF = (PI x W')/(PH' x WI), where PH, PH', and PI = peak areas of sample, std heptachlor, and internal std, resp.; W, W', and W/ = g sample, std heptachlor, and internal std, resp. Chlorotoluron [3-(3-Chloro-4-methylphenyl)-1,1-dimethyl- urea), Chloroxuron [3-[4-(4-chlorophenoxy)phenyl)-1,1- dimethylurea), or Metoxuron (Dosanex@1[3-(3-Chloro-4- methoxyphenyl)-1,1-dimethylurea) (65)-Official Final Action CIPAC-AOAC Method 6.245 Principle Pesticide is extd from formulations with CH2CI" free amines are removed with acid, and ext is hydrolyzed by alkali to Me,NH which is distd and titrd. Related byproducts, 3-(3-chloro-4-meth- ylphenyl)-l-methylurea (I), 3-(4-methylphenyl)-1, l-dimethylurea (II) (from chlorotoluron), 3-[4-(4-chlorophenoxy)phenyl)-1-meth- ylurea (III) and 3-(4-chlorophenyl)-1,1-dimethylurea (IV) (from chloroxuron), and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (V), 3-(3-chloro-4-hydroxyphenyl)-1,1-dimethylurea (VI), and 3-(4- methoxyphenyl)-l,l-dimethylurea (VII) (from metoxuron), which may interfere, are detd by semiquant. TLC. Limit of detection for TLC is 0.1 % for each byproduct. On same TLC plate for chloroxuron, free amine 4-(4-chlorophenoxy)aniline (VIII) is detd by sep. detection technic. Other byproducts, 1,3-bis(3- chloro-4-methylphenyl) urea (IX), 1,3-bis[4-(chlorophenoxy)- phenyl) urea (X), and 1,3-bis(3-chloro-4-methoxyphenyl) urea (XI), do not interfere with chlorotoluron, chloroxuron, and metoxuron detns, resp. 6.246 Preparation of Sample (a) Technical formulation.-Accurately weigh ca 3 g sample (4 g for chloroxuron) and transfer, using 100 mL CH2CI2, into 250 mL separator, dissolve, and add 50 mL IN HCI. (b) Wettable powder.-Accurately weigh ca 3.5-4.0 g sample (for 80%) or 6.0-6.5 g (for 50%) into 200 mL beaker. Add 100 mL CH,CI, and stir mag. 5 min. Filter thru fritted glass crucible contg paper and 0.5 g layer of Celite, and rinse beaker and crucible with portions of CH,CI2 to total vol. of ca 200 mL. Use only slight vac. to prevent crystn of pesticide on walls of crucible. Transfer quant. to 500 mL separator, and add 50 mL IN HCI. 6.247 Determination Vigorously shake mixt. 1 min and drain lower org. layer into second separator. Add 25 mL (50 mL for chloroxuron) IN HCI, shake 30 sec, and drain lower layer into 500 mL rob flask. Wash the 2 acid layers successively with same 100 mL portion CH2CI2 (with two 50 mL portions for chloroxuron) and drain lower layer into the 500 rob flask. Discard acid. Vac.-evap. CH2CI2 in rotary evaporator to dryness at max. of 40°. Remove all solv. to prevent interference in subsequent titrn. Add 100 mL propylene glycol, 40 g KOH, and some boiling stones to residue. Immediately connect flask securely to distn app. (Fig. 6:06) whose joints are lubricated with thin film of silicone grease. Place end of condenser delivery tube (~10 mm id) in 400 mL beaker below level of absorbing soln of 0.2 g H3B03 and 1 mL mixed indicator soln (40 mg methylene blue and 60 mg Me red dissolved in 100 mL alcohol) in 150 mL H20. (To enhance end point, use 150 mL MeOH (2+1).) Gently warm flask until all particles dissolve; then boil 10 min or until propylene glycol distils into condenser. Titr. distd Me,NH continuously with stdzd IN HCI, 50.011-50.017. Complete distn by carefully adding H,O dropwise from dropping funnel at rate of 1 drop/sec. Continue titrn until end point persists 2 min (V mL). Perform blank detn (B mL) with each series. % Pesticide = [(V - B) x N x F/g sample) - % byproducts (from 6.248), where F = 21.27 for chlorotoluron, 29.07 for chloroxuron, or 22.87 for metoxuron, and N = normality of stdzd HCI. a heating bath or heating mantle b round bottom flask (500 mil distilling column, plain dropping funnel (250 mil e distilling head f distilling bridge allihn condenser beaker (400 mil I magnetic stirrer FIG. 6:06-Distillation apparatus (all dimensions in mm)
- 122. 100 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) 6.248 Determination of Byproducts (a) For chlorotoluron.-Dissolve 100 mg each of byproducts I and II (6.245) (available from Ciba-Geigy Ltd, Analytical De- partment, CH-4002 Basel, Switzerland) together in tetrahydro- furan and dil. to 50 mL in vol. flask. Dil. aliquots of 1,2,3,4, and 5 mL to 20 mL with tetrahydrofuran, equiv. to 0.2, 0.4, 0.6, 0.8, and 1.0%, resp., of each byproduct. Dissolve 1.0 g sample in tetrahydrofuran, and dil. to 20 mL with same solv. Spot 5 f.LL each of sample and std solns on 20 x 20 cm glass plates precoated with 0.25 mm layer of silica gel 60 F-254 (No. 5715, E. Merck, Darmstadt, Germany, or equiv.). and develop by ascending technic in tank, presatd 30 min with developing soIv. CHCI3-EtOAc (4+1). without filter paper linings, for ca 70 min (13 cm migration). Expose plate to 254 nm UV light and compare spots of samples with those of stds to est. concn of byproducts. Approx Rf values: chlorotoluron, 0.50; byproduct I, 0.25; by- product II, 0.35; and byproduct VI (does not interfere). 0.82. (b) For chloroxuron.-Dissolve 100 mg each of byproducts III, IV, and VIII (available from Ciba-Geigy Ltd) together in acetone and dil. to 100 mL in vol. flask. Dil. aliquots of 1, 3, 5, 8, and 10 mL to 50 mL with acetone, equiv. to 0.1, 0.3, 0.5, 0.8, and 1% resp., of each byproduct. Dissolve 1.0 g sample in acetone, and dil. to 50 mL with same solv. Proceed as in (a). but use CHCl3-dioxane (9+2) as devel- oping solv. for ca 80 min (14 cm). Approx R, values: chloroxuron 0.75; byproduct III, 0.40; byproduct IV, 0.65; and byproduct X (does not interfere). 0.90. Det. byproduct VIII on same TLC plate. Place beaker contg ca 2 g NaNO, in empty developing tank and pour ca 3 ml HCI over salt. After 2 min, insert plate into tank 3 min, remove, and dry 5 min at room temp. with hair dryer. Spray with 1% soln of N- (1-naphthyl)ethylenediamine.2HCI in 0.1N HCI and compare violet sample spots with those of stds (R" 0.85). (c) For metoxuron.-Proceed as in (a). except use 100 mg each of byproducts V, VI, and VII (available from Sandoz Ltd, Agrochemical Division, Research, CH-4002 Basel, Switzerland). Approx. Rf values: metoxuron, 0.25; byproduct III, 0.34; by- product IV, 0.08; byproduct V, 0.13; and byproduct VII (does not interfere). 0.46. 6.249 Identification (a) Technical chloroxuron.-Record IR spectrum of 1% CH2CI2 soln of sample and compare with spectrum of 1% CH2CI, soln of authentic ref. std. (b) 50% Wettable powder.-Stir ca 2 g sample and 2 g silica gel (70-230 mesh) with 100 mL CH2CI2 5 min and percolate thru fluted filter. Record IR spectrum of filtrate in NaCI cell (0.5 mm path length) from 3000 to 650 cm-1 , using blank solv. as ref. Identity is established if sample spectrum corresponds qual. to that of std. 6.250 Dichlobenil (2,6-Dichlorobenzonitrile) (66) CIPAC-AOAC Method Reagents and Apparatus (a) Dichlobenil.-~99.5% purity (Philips-Duphar B.V., Weesp, Netherlands). or equiv. (b) Methyl myristate.-Fluka AG Cat. No. 70129, ~99.5% purity (Fluka AG, Buchs, Switzerland). or equiv. (c) Mixed solvent soln.-l,2-Dichloroethane-ether (1 + 1). (d) Internal std soln.-Dissolve 0.80 g Me myristate, (b). in 100 mL mixed solv. soln, (c). (e) Calibration soln.-Accurately weigh ca 0.10 g dichlobenil, (a). into conical flask, pipet in 5 mL internal std soln, (d). and add 45 mL solv. soln, (c). (f) Gas chromatograph.-With on-column injection, flame ionization detector, injection port heating, and, preferably, de- tector heating. Pyrex column 1.83 m x 3 mm id, packed with 10% Carbowax 20M on 100-120 mesh Chromosorb P, acid washed, dimethyldichlorosilane treated (available from Analabs, Cat. No. GCP-Q09D). Operating temps (0): oven 200, injection port 210, detector 210. Carrier gas (N) flow rate 25 mLimin. Approx. retention times 7 and 12 min for Me myristate and dichlobenil, resp. 6.251 Preparation of Sample (a) Technical dichlobenil.-Accurately weigh ca 2.0 g dichlob- enil into 100 mL vol. flask. Dissolve in mixed solv. soln, (c), and dil. to vol. Pipet 5 mL aliquot into 100 mL conical flask, add 5.00 mL internal std soln, (d), and dil. to 50 mL with mixed solv., (c). (b) Wettable powders.-Accurately weigh sample contg ca 1.0 g dichlobenil into 100 mL vol. flask, add few mL mixed solv., (c). swirl, and dil. to vol. with mixed solv. Let settle, pipet 10 mL clear supernate into 100 mL conical flask, and continue as in (a). (c) Granules.-Accurately weigh ca 6.0 g sample into 100 mL conical flask. Add 20 mL dichloroethane and stir 10 min on mag. stirrer. Filter with vac. thru glass filter paper (Whatman GF 82, or equiv.), supported on fritted glass filter. Wash granules 5 times with 5 mL dichloroethane, collect filtrate in 100 mL vol. flask, and dil. to vol. with ether. Pipet aliquot of this soln, contg ca 0.1 g dichlobenil (10 mL for 20% granules, 25 mL for 7% granules). into 100 mL conical flask, add 5.00 mL internal std soln, (d), and dil. to 50 mL with mixed solv., (c). 6.252 Determination Inject 2 f.LL portions of calibration soln, (e). until response factor varies < 1% for successive injections. Inject duplicate 2 f.LL portions of sample soln, followed by 2 f.LL portions of calibration soln, (e). Measure peak areas of dichlobenil and Me myristate, either by multiplying peak ht by retention time, or by digital integration. Use avo of duplicate values. 6.253 Calculation p = (lq x r x 20)/(/, x q), where p = response factor, Iq and I, = peak areas of internal std and dichlobenil, resp., q = g internal std, r = g dichlobenil in calibration soln. (Response factor is ca 1.5.) % Dichlobenil = (ld x q X P x F x 100)/(/m x W x 20), where Id and 1m = peak areas of dichlobenil and internal std, resp., p = response factor, F = diln factor for sample (100/x, where x = mL taken to obtain final soln). and W = g sample. Dichlorodiphenyltrichloroethane (1,1,1-Trichloro-2,2- bis(p-chlorophenyl) Ethane) (DDT)-Official Final Action * Total Benzene-Soluble Chlorine Method (67) * (Applicable in absence of other org. CI compds. Use H20 2 and isoamyl alcohol-ether extn method on dispersible powders or sprays that contain surface active agents or other ingredients that react with AgN03 • Caution: See 51.034, 51.039, 51.040, 51.041, and 51.045.) 6.254 Reagents (a) Benzene.-Thiophene- and CI-free. (b) Metallic sodium.-Ribbons or small pieces. *Surplus method-see inside front cover.
- 123. AOAC METHODS (1980) (c) Decolorizing carbon.-Test for presence of CI by heating with HN03 (1 +4), filtering, and adding AgN03 soln to filtrate. If CI is present, wash with the HN03 until washings are CI-free. Note: Det. blank on all reagents, limiting 0.1N AgN03 to 5 mL. 6.255 Preparation of Solution (a) Technical grade DDT.-Weigh sample contg ca 1 g DDT and transfer to 250 mL vol. flask. Dissolve sample in 10 mL benzene; then dil. to vol. with 99% isopropanol. Transfer 25 mL aliquot to 250-500 mL l erlenmeyer. (Direct weighing of sample may be substituted, provided it does not introduce error >0.1%.) Add 2.5 g Na and shake to mix sample with isopropanol. Do not add Na thru top of condenser or get Na on ground glass joints. Connect flask to reflux condenser and boil gently ;;;.30 min, shaking occasionally. Eliminate excess Na by cautiously adding 10 mL 50% isopropanol thru condenser at rate of 1-2 drops/sec. Disconnect condenser, add 60 mL H20, boil soln ca 30 min to expel isopropanol, and proceed as in 6.256(a), (b), (c), or (d). (b) Dusting mixtures containing DDT in absence of organic matter.-Weigh sample contg ca 0.75 g DDT, transfer to 100-200 mL vol. flask, and add exactly 100 mL benzene. Shake until DDT dissolves and soln is well mixed. Let settle and transfer 10 mL aliquot to 250--500 mL l erlenmeyer. Evap. on steam bath to remove most of benzene. (Do not evap. to dryness, as DDT may decompose with loss of HCI.) Add 25 mL 99% isopropanol and proceed as in (a), second par. If free S is present, proceed as in (f)' beginning "Then add 5 mL 30% H2 0 2 , ••• " (c) Dusting mixtures in presence of organic matter (coloring matter, plant resins, etc. ).-Weigh sample contg ca 0.75 g DDT, transfer to 100--200 mL vol. flask, and add 0.5-1.0 g decolorizing C and exactly 100 mL benzene. Shake until DDT dissolves and soln is well mixed. Filter into narrow-neck flask thru fast qual. paper without suction, keeping funnel covered with watch glass to avoid evapn loss. Transfer 10 mL aliquot to 250--500 mL l erlenmeyer. Proceed as in (b)' second par. Before detg CI remove org. matter as follows: Cool, add 2-3 drops phthln, and neutze by adding HN03 (1 + 1) dropwise; then 10 mL excess. Cool, if necessary, to room temp., transfer contents of flask and aq. washings to small separator, and shake with 15 mL isoamyl alcohol-ether (1 + 1). Drain aq. layer into second separator and ext again with 15 mL isoamyl alcohol-ether (1+1). Drain aq. layer into 250 mL beaker. Wash the 2 exts successively with two 10 mL portions H2 0. Combine aq. wash solns with aq. soln in beaker. Det. CI by one of following methods: (1) Proceed as in 6.256(a), beginning "Add slight excess 0.1N AgN03, .•• " (2) Proceed as in 6.256(b), beginning "Add 0.1N AgN03 ••• " (3) Proceed as in 6.256(c), beginning "Cool flask to room temp...." (4) Add 2-3 drops phthln to sample, make alk. by adding 1N NaOH, and proceed as in 6.256(d), beginning " ... transfer contents to Pt dish." (d) Mineral oil sprays in absence of organic matter (plant extractive material, organic thiocyanates).- Transfer weighed sample contg 0.065-0.075 g DDT to 250--500 mL l flask. Add 25 mL 99% isopropanol and proceed as in (a), second par. Note: If DDT content is <2%, use isoamyl alcohol-ether extn (c), second par., to remove excess oil. ' Proceed as in 6.256(a), (b), (c), or (d). (e) Mineral oil sprays in presence of organic matter (plant extractive material from pyrethrum or derris and/or cube.)- DDT 101 Proceed as in (d), using isoamyl alcohol-ether extn, (c), to remove excess oil. (f) Mineral oil sprays in presence of organic thiocyanates with or without plant extractive material. -Transfer sample contg 0.065-0.075 g DDT to 250-500 mL "$ erlenmeyer. Add 25 mL 99% isopropanol and proceed as in (a), second par., thru " ... add 60 mL H20, ..." Then add 5 mL 30% H20 2, few drops at time, thru top of condenser, heat mixt. in flask to bp, and boil 15 min. Add addnl 5 mL H20 2 and again boil 15 min. Add 15 mL more H20 2 , disconnect reflux condenser, and boil 15-30 min to expel isopropanol. Proceed as in (c), second par. (9) Emulsions (solvent, emulsifier, and water).- Weigh well mixed sample contg ca 0.75 g DDT in weighing bottle. Wash into 100 mL vol. flask and dil. to vol. with isopropanol. Transfer 10 mL aliquot to 250-500 mL "$ erlenmeyer. Expel isopropanol and H20 on steam bath in air current. If drops of H20 still remain, add 10 mL isopropanol and repeat evapn. Add 25 mL 99% isopropanol and proceed as in (a), second par. Not<;:. If S is brought. into the soln as by decomposition of emulSifier, proceed as In (f), beginning "Then add 5 mL 30% H2 0 2 , • •• " 6.256 Determination (a) Cool flask and transfer contents to 250 mL beaker. Add 2-3 drops phthln and neutze with HN03 (1 +1); then add 10 mL excess. Add slight excess 0.1N AgN03, 50.031, and coagulate pptd AgCI by digesting on steam bath 30 min, stirring frequently. Cool, filter thru fast qual. paper, and wash thoroly with H20. Add 5 mL satd ferric indicator, 6.019(e), and det. excess AgN03 in filtrate by titrn with 0.1N KSCN, 50.030(b). Subtract amt AgN03 found in filtrate from that originally added. Difference is that required to combine with CI in the DDT. 1 mL 0.1N AgN03 = 0.003545 g CI. CI x 2 = DDT. (b) Cool flask, add 2-3 drops phthln soln, and neutze with HN03 (1+1); then add 10 mL excess. Add 0.1N AgN03 from buret in excess of amt necessary to ppt all CI; then add 5 mL nitrobenzene and 0.5 g Fe2 (S04), and swirl flask to coagulate ppt. Back-titr. excess AgN03 with 0.1N KSCN to faint pink. Cross- titr. with both std solns, crossing end point in each direction to assure results. From vol. AgN03 , calc. % DDT as in (a). (c) Cool flask, add 2-3 drops phthln, neutze with HN03 (1 +1), and add 6 mL excess. Cool flask to room temp. and transfer contents to 400 mL beaker. (Vol. should be 200-250 mL.) Titr. CI with 0.1N AgN03 potentiometrically, using Ag-AgCI electrodes (Fisher Titrimeter, or equiv.). Calc. % DDT as in (a). Note: Whe.n this method is used, decolorizing C' step in 6.255(c), and Isoamyl alcohol-ether extn in 6.255(c), (d), and (e), may be omitted. (d) Cool flask and transfer contents to Pt dish. Evap. to dryness and ignite as thoroly as possible at ";525°. Ext with hot H20, filter, and wash. Return residue to Pt dish and ignite to ash; dissolve in HN03 (1 +4), filter from any insol. residue, wash thoroly, and add this soln to aq. ext. Add 0.1N AgN03, avoiding more than slight excess. Heat to bp, protect from light, and let stand until ppt coagulates. Filter on weighed gooch, previously heated to 140-150°, and wash with hot H2 0, testing filtrate to prove excess of AgN03 • Dry AgCI at 140-150°, cool, and weigh. Calc. % CI and DDT as in (a). 6.257 Infrared Method (68) (Caution: See 51.041.) Reagent DDT std soln.-Weigh 0.250 g tech. DDT into 50 mL vol. flask or g-s container and add exactly 25 mL CS2• If sample to be analyzed contains S, add wt of S expected in portion of
- 124. 102 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) sample to be taken for analysis. Shake to dissolve and add small amt anhyd. Na2S04• Centrf. portion of soln if it is not clear. 6.258 Determination Weigh sample contg ca 0.25 g DDT into 50 mL vol. flask and add exactly 25 mL CS2and small amt anhyd. Na2SO•. Let stand ~30 min with occasional shaking. Transfer portion to g-s test tube and centrf. short time. Transfer to NaCI cell and scan with infrared spectrophtr, using 0.5 mm cell in region, 8.5-10.5 fLm. Scan std soln in same manner. Measure A of DDT peak at 9.83 JLm with baseline from 9.4 to 10.2 JLm, and calc. % DDT. Dimethyl 2,3,5,6-Tetrachloroterephthalate (Dacthal) (69)-Official Final Action (Caution: See 51.011, 51.018, 51.039, 51.040, 51.041,51.045,51.046, and 51.048.) Gas Chromatographic Method (Under conditions specified, other pesticides or ingredients may interfere with GLC analysis, e.g., aldrin has same retention time as Dacthal. Aldrin and Dacthal may be sepd at 170° column temp.) 6.259 Apparatus Gaschromatograph.-l.8 m (6') x Va" id stainless steel column contg 10% silicone UC-98 (Applied Science Laboratories, Inc.) on 80-100 mesh silanized Diatoport S (Hewlett-Packard Co., Rt 41, Avondale, PA 19311). Conditions (applicable to Hewlett- Packard F&M Model 5750)-temps(0): column 200, injection port 240, flame ionization detector 260; H, air, and He carrier flows, 115, 600, and 25 mL/min, resp.; chart speed 0.25"/min; atten- uation 4x; range setting 1()2 (10-10 amp full scale). 6.260 Preparation of Standard Curve (a) Dacthal std solns.-Weigh 0.5 g Dacthal (available from Diamond Shamrock Corp., PO Box 348, Painesville, OH 44077) into 100 mL vol. flask, add ca 90 mL acetone (soln is rapid), and dil. to vol. Pipet 5,10, and 15 mL into sep. 25 mL vol. flasks and dil. to vol. with acetone. (b) Hexachlorobenzene (HCB) std solns.-Weigh 0.5 g ref. grade HCB into 100 mL vol. flask, add 90 mL benzene, and dil. to vol. with benzene. Pipet 1, 2, and 3 mL into sep. 25 mL vol. flasks and evap. to dryness with current of dry air. Add 20 mL acetone to each flask and dil. to vol. with acetone. Inject 5 JLL each dild HCB and Dacthal std at least twice. Prep. curve of peak area or ht against concn for Dacthal and peak ht against concn for HCB. 6.261 Determination (a) Benzene extraction.-Grind granular product. Weigh por- tion contg ca 300-400 mg Dacthal into Whatman extn thimble (33 x 88 mm). Cover with glass wool. Place thimble in medium Soxhlet extractor; add 150-175 mL benzene and 3 glass beads. Ext 6 hr. Quant. transfer ext to 400 mL beaker and evap. to ca 5 mL on steam bath with dry air current; remove and evap. to dryness with air current. Add ca 150 mL acetone and let stand until soln is complete (white, flaky crystals may indicate incom- plete soln; soln may be hastened by placing flask in ultrasonic cleaner). Filter soln thru glass wool into 200 mL vol. flask. Wash beaker with acetone, transfer washings to vol. flask, and dil. to vol. (b) Alternative acetone extraction.-Substitute acetone for benzene in extn. Proceed as in (a) thru "Ext 6 hr." Continue with "Filter soln thru glass wool ..." Inject duplicate 5 JLL sample soln into gas chromatograph. Compare peak ht or peak area to std curve to det. % hexa- chlorobenzene (HCB) and Dacthal. Infrared Method 6.262 Preparation of Sample Grind granula.r product. Weigh sample contg 200-500 mg Dacthal into Whatman extn thimble. Proceed as in 6.261(a) thru " ... evap. to dryness with air current." Add 25 mL CS2, allow ca 30 min for complete soln, and transfer Quant. to 50 mL vol. flask with CS2, filtering sample thru glass wool. Dil. to vol. 6.263 Preparation of Standard Solution Weigh 1.25 9 Dacthal into 100 mL vol. flask. Add ca 90 mL CS2 (soln may be hastened by placing flask in ultrasonic cleaner) and dil. to vol. Pipet 10,15, and 20 mL into sep. 25 mL vol. flasks and dil. to vol. 6.264 Determination Set spectrophtr at optimum operating condition. Use 0.5 mm KBr (or NaCI) matched cells. Fill ref. cell with CS2. Transfer dild stds to other cell and scan slowly from 1100 to 900 cm-1 • Repeat with samples. Construct baseline from 1030 to 925 cm-1 and draw line from midpoint of max. A at ca 964 cm-1 to intersect baseline. Compute LiA at 964 cm-1 at point of intersection of stds and sample. Prep. LiA-concn curve for std; Beer's law is obeyed over concn range 2-15 mg Dacthal/ mL. Calc. % Dacthal from std curve. Dicamba (3,6-Dichloro-o-anisic Acid; 2-Methoxy-3,6- dichlorobenzoic Acid) (70)-Official Final Action 6.265 Reagents and Apparatus (a) Acetone.-Spectral grade. (b) Dimethylamine (DMA) soln.-60% (w/w). (c) Dicamba std.-Ref. grade (Velsicol Chemical Corp.). (d) Infrared spectrophotometer.-With BaF2 cells, 0.025 mm, and matched NaCI cells, 0.2 mm. 6.266 Preparation of Sample (Sample wts are for cell thicknesses specified. For other cells, adjust wts to yield peak between 30 and 60% T.) (a) Aqueous solns of DMA salt (4Ib/gal. ).-Pipet, using same pipet as for std, 5.00 mL sample into tared 25 mL vol. flask and weigh. Dil. to vol. with acetone. (Use this soln directly in 0.025 mm BaF2 cell.) (b) Solns of DMA salt (other concentrations).- Prep. as in (a), adjusting sample size to yield 2.4 g dicamba/25 mL. (c) Technical dicamba.-Weigh 0.2±0.005 g sample into tared 25 mL vol. flask and dil. to vol. with CS2. 6.267 Preparation of Standard (a) Liquid formulations.-(1) Aqueous solns of DMA salt (4 Ib/gal.): Weigh 11.98±0.02 g dicamba std into tared 50 mL beaker. Add 5 mL H20 and 4 mL 60% DMA. Adjust pH to 7.0 by titrg with 60% DMA soln, using mag. stirrer and pH meter. (All solids should be dissolved at this time.) Rinse each pH electrode with two 1 mL H20 rinses (4 mL total), collecting rinses in the 50 mL beaker. Cool soln to room temp. and transfer to tared 25 mL vol. flask. Rinse beaker twice with H20, collecting rinses in
- 125. AOAC METHODS (1980) 2,4-D flask. Dil. to vol. with H20 and mix tharoly. Weigh flask and contents to det. total wt of soln. Pipet 5.0 mL std formulation into tared 25 mL vol. flask, weigh, and dil. to vol. with acetone. (2) Aqueous solns of DMA salt (other concentrations): Prep. as in (a)(1), adjusting dicamba content to required concn. (b) Technical dicamba.-Weigh 0.2±0.005 g dicamba std into tared 25 mL vol. flask and dil. to vol. with CS2. 6.268 Determination (a) Liquid formulations.-Record spectra of std and sample between 1070 and 930 cm-1 (9.3-10.7 JLm), using BaF2 cell. Use air in ref. beam. Obtain <lA and <lA' for sample and std, resp., at 1012 cm-1 (9.89 JLm) from horizontal baseline tangent to min. between 1020 and 1070 cm-1 (9.4-9.7 JLm). (b) Technical dicamba.-Record spectra of std and sample from 1100 to 930 cm-1 (9.1-10.7 JLm), using NaCI cells. Use CS2 in ref. cell. Obtain <lA and ~A' for sample and std, resp., at 1012 cm-1 (9.89 JLm) from horizontal baseline tangent to min. between 1075 and 1035 cm-1 (9.3-9.66 JLm). 6.269 Calculations (a) Liquid formulations.-Dicamba, Ib/gal. = ~A x C/~A', where C = Ib std/gal. = (g std x % purity of std x 8.35)/25. % Dicamba by wt = (~A x F)/(g sample/25 mL), where F = [(g std/25 mL) x % purity of stdll~A'. (b) Technical dicamba.-% Oicamba by wt = ~A x F/g sample, where F = (g std x % purity of std)/~A'. Dicamba-2-Methyl-4-chlorophenoxyacetic Acid (MCPA) and Dicamba-2,4-D (71)-Official Final Action 6.270 AOAC-CIPAC Method (Caution: See 51.041.) Principle Method is applicable to aq. dimethylamine (OMA) salt for- mulations of dicamba and 2-methyl- 4-chlorophenoxyacetic acid (MCPA) or 2,4-0 (2,4-dichlorophenoxyacetic acid). Active ingre- dients are pptd by HCI and extd with CHCI3. Solv. is evapd, residue dissolved in acetone, and A measured at characteristic IR wavelengths. 6.271 Preparation of Standard Solutions (a) Dicamba-MCPA.-Accurately weigh 0.20±0.02 g dicamba and 0.60±0.02 g MCPA into tared weighing bottle. Pipet in 25 mL acetone and swirl until completely dissolved. If cells other than 0.2 mm are used, adjust wts to give A of 0.2-0.5 (30-65% T) for both std and sample solns. (b) Dicamba-2,4-D.-Prep. as in (a), using 0.20±0.02 g di- camba and 0.40±0.02 g 2,4-0. 6.272 Preparation of Sample (a) Dicamba-MCPA.-Accurately weigh sample contg 0.20±0.02 g dicamba and 0.60±0.02 g MCPA into tared weighing bottle. Add 5 mL H20 and transfer quant. to 125 mL separator with 5-10 mL H20. (b) Dicamba-2,4-D.-Prep. as in (a), using 0.20±0.02 g di- camba and 0.40±0.02 g 2,4-0. 6.273 Determination To soln add HCI dropwise with const swirling to pH 1; then add 5 drops excess. Pipet in 25 mL CHCI3 and shake to dissolve 103 ppt. Drain CHCI3ext into 125 mL erlenmeyer and re-ext with two 15 mL portions CHCI3. Add boiling chips to combined ext and evap. on steam bath to dryness. Let dry in hood overnight at room temp. (Do not dry in air or vac. oven.) Pipet in 25 mL acetone and swirl to completely dissolve residue. Add few g granular anhyd. Na2S0. ifany H20 is present. Record IR spectrum and measure ~A in matched 0.2 mm NaCI cells with acetone in ref. cell at following wavelengths: (a) Dicamba-MCPA .-Range, 1135-930 cm-1 (8.8-10.75 JLm); dicamba peak, 1012 cm-1 (9.89 JLm); MCPA peak, 1070 cm-1 (9.35 JLm); baseline, horizontal tangent to min. at 970-965 cm-1 (10.3-10.4 JLm) for both constituents. (b) Dicamba-2,4-D.-Range, 1130-945 cm-1 (8.85-10.6 JLm); dicamba peak, 1012 cm-1 (9.89 JLm); 2,4-0 peak, 1080 cm-1 (9.26 JLm); baseline, horizontal tangent to min. at 970-960 cm-1 (10.3-10.4 JLm) for both constituents. 6.274 Calculations % by wt of constituent = (~A/W)(W' x P/~A'), where <lA and ~A' = absorbance of constituent in sample and std solns, resp.; Wand W' = 9 constituent in sample and std solns, resp.; and P = % purity of constituent in ref. std. Ib/gal. = % by wt x sp gr x 8.345. 6.275 2,4-0 (2A-Dichlorophenoxyacetic Acid) Automated High Pressure Liquid Chromatographic Method (72)-Official First Action Principle Esters of 2,4-D are saponified in situ; amine salts are converted to H20-sol. K salt of 2,4-0. Ionic 2,4-D is protonated by pH 2.95 CH3CN-H20 (1 +4) eluant, and sepd from all known impurities and p-bromophenol internal std on reversed phase bonded microparticulate column. 2,4-D elutes between impurities 2,4- and 2,6-dichlorophenol. 6.276 Apparatus (a) Liquid chromatograph.-Fitted with 5000 psi pressure gage, 280 nm UV detector, line filter in eluant reservoir, and 10 mv full scale deflection strip chart recorder. Automated sampling system and computing integrator are optional. Typical operating conditions: chart speed, 0.2 cm/min; eluant flow rate, 0.9-3.0 mL/min depending upon psi range of pump; detector sensitivity, 0.64A unit full scale; temp., ambient; injection valve vol., 10 JLL. (b) Liquid chromatographic column.-No. 316 stainless steel, 250 x 4.6 (id) mm, Partisil® 10 JLm ODS column with 50 x 4.6 (id) mm Co:Pell ODS pellicular guard column (Nos. 6526-124 and 6561-404, resp., Whatman Inc., 9 Bridewell PI, Clifton, NJ 07014). Regenerate, if necessary, by pumping CH3CN thru col- umn until baseline is stable. Repack first 5 mm of guard and main columns with Co:Pell ODS if peaks begin to "tail". 6.277 Reagents (a) Eluant.-pH 2.95. CH3CN (distd-in-glass)-H20 (deionized, 0.4 J-tm filtered) (1+4) contg NaOH added from (1+1) aq. soln of known normality, 50.033(b), at final vol. concn of 0.3M. Add H3PO. to adjust pH to 2.95. (b) Saponification-internal std soln.-4 g p-Bromophenol/L 0.2N KOH in isopropanol-H20 (2+1). Add KOH from (1+1) aq. soln of known normality. (c) 2,4-D std soln.-300 mg/25 mL. Accurately weigh ca 300 mg 2,4-D anal. ref. std (99+% isomer pure; available from Dow Chemical Co., Sample Coordinator, 9001 Bldg, Midland, MI 48640), previously dried 15 min at 100°, into 1 or 2 oz glass vial
- 126. 104 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) with polyethylene-lined screw cap. Pipet in 25 mL saponification- internal std soln, and shake to dissolve. Prep. 2,4-0 std soln and sample soln, 6.278, at same time, using same pipet. 6.278 Preparation of Sample Accurately weigh sample contg ca 300 mg 2,4-0 acid equiv. into 1 or 2 oz glass vial with polyethylene-lined screw cap. Pipet in 25 mL saponification-internal std soln, and shake 15 min, warming ester formulations to 50' several min before shaking. Filter prepd sample thru 9 cm Whatman glass microfiber filter GF/A, or equiv., collecting major portion of aq. phase for chromatography. Adjust isopropanol-H20 ratio, if necessary, to obtain complete dissoln of sample; e.g., amine formulations are best prepd with isopropanol-H20 (1 + 1). 6.279 Determination Transfer ca 1 mL portions of samples and stds to automated sampler vials, and cap. Place samples and stds in position, and start automatic sampler. With programmed integrator use fol- lowing calcn program automatically: % 2,4-0 = (R/R') x (W'/W) x P, where Rand R' = peak ht or area ratios of 2,4-0 to internal std for sample and std, resp.; W' = mg 2,4-0 in std; W = mg sample; and P = % purity of std. If automated sampler and computing integrator are unavailable, inject 10 p,L samples and stds and perform calcns manually. As check on calibration, place stds in sample sequence at beginning, middle, and end. Period- ically confirm linearity by analyzing stds contg 200, 300, and 400 mg 99+% 2,4-0/25 mL saponification-internal std soln. Continuously recycle and mag. stir eluant. Replace eluant after ca 200 injections/L. Sodium Salt of Dalapon (2,2-Dichloropropionic Acid) (73)-Official Final Action (Caution: See 51.041.) 6.280 Apparatus (a) Reflux apparatus.-250 mL erlenmeyer connected thru !j 35/25 ball joint to reflux condenser. (b) Filtering apparatus.-60 mL, medium porosity fritted glass funnel attached to glass filter bell, 11 cm od, 18 cm high, with bottom gasket and slide valve. 6.281 Reagents (a) Mercuric-cupric nitrate soln.-(Caution: See 51.065.) Dis- solve 100.0 g yellow HgO and 60 g Cu(N03)2.3H20 in 500 mL 3.100±0.003N HN03, measured from vol. flask, in 1 L vol. flask, dil. to vol. with H20, and filter. (b) Potassium iodide soln.-Dissolve 150 g KI in H20, dil. to 1 L, and neutze to phthln. 6.282 Determination Accurately weigh sample contg 0.11-0.22 g Na salt of 2,2- dichloropropionic acid, transfer to erlenmeyer of reflux app., and add 100 mL Hg-Cu nitrate soln. Add some boiling chips, attach condenser, and reflux 15 min. Cool in H20 bath. Filter thru filtering app., washing flask and ppt acid-free with H20 from wash bottle. Discard filtrate and washings, and place 250 mL narrow-mouth erlenmeyer in filtering bell. Add 50 mL KI soln to erlenmeyer to dissolve any remaining ppt, transfer to funnel, and stir until ppt dissolves. Draw soln into narrow-mouth erlenmeyer with vac. Wash flask and funnel with ",,50 mL KI soln from wash bottle, adding washings to filtrate. Add few boiling chips to filtrate and boil 1 min. Cool in H20 bath. Titr. immediately with O.lN HCI, using phthln. % Na salt 2,2-dichloropropionic acid = mL O.lN HCI x 0.004499 x 100/g sample. Dicofol (Kelthane®, 4,4' -Dichloro-a-(trichloromethyl)- benzhydrol) Potentiometric Method (74)-Official First Action 6.283 Principle Dicofol is hydrolyzed in alc. KOH under reflux, and hydrolyz- able org. CI is converted to ionizable CI which is titrd potentio- metrically with std AgN03. 6.284 Apparatus (a) Condenser.-"$ 24/40 Pyrex condenser, water cooled, 400 mm long with drip tip. (b) Potentiometer.-Fisher Accumet Model 320 (new model 325) expanded scale pH meter, or equiv., with 50 mL buret graduated in 0.1 mL, Ag billet indicating electrode (Fisher No. 13-639-122). and Ag-AgCI ref. electrode (Fisher No. 13-639-53). Keep Ag electrode free from tarnish by polishing with aq. NaHC03-CaC03 (1 + 1) paste. Before each analysis, rinse Ag electrode with NH4 0H (1+1) followed by H20. 6.285 Reagents (Use deionized H20 thruout.) (a) Alcoholic potassium hydroxide soln.-O.5N. Dissolve 28.1 g KOH pellets in ca 600 mL alcohol and dil. to 1 L with alcohol. (b) Potassium chloride std soln.-0.1N. Dissolve 7.456 g KCI in H20 and dil. to 1 L with H20. (c) Silver nitrate std soln.-0.1N. Dissolve 17.00 g AgN03 in 100 mL H20, add 1.7 mL HN03, and dil. to 1 L with H20. To stdze, dil. 25 mL O.lN KCI to 200 mL with H20 in 400 mL beaker. Adjust pH to 2.0±O.2, using NH40H (1+4) and/or HN03 (1+4). and dil. to 300 mL. Titr., using potentiometer as in 6.288. Plot mv against vol. O.lN KCI and det. mL O.lN AgN03 at end point. Calc. normality of AgN03std soln. Stdze AgN03std soln daily. (Equiv. wt dicofol = 370.5/3 = 123.5.) (d) Thymol blue indicator soln.-O.l %. Dissolve 100 mg thy- mol blue in 100 mL alcohol (1+1). 6.286 Preparation of Sample (a) Kelthane technical.-Fuse sample in loosely capped jar in 100e oven and mix thoroly with glass rod. Accurately weigh ca 4-6 g molten sample into 150 mL beaker. Add 50-75 mL isopropanol and heat with occasional swirling until sample dissolves. Transfer Quant. to 500 mL vol. flask, let cool to 25°, and dil. to vol. with isopropanol. Pipet 25 mL sample soln into 300 mL "$ 24/40 Pyrex erlenmeyer. (Caution: See 51.018.) (b) Kelthane formulations.-(1) Kelthane MF and Kelthane 35.-Accurately weigh ca 1 g sample into 300 mL"$ 24/40 Pyrex erlenmeyer. (2) Kelthane EC.-Proceed as in (1). using ca 2 g sample. 6.287 Hydrolysis Transfer 50 mL alc. KOH soln to erlenmeyer contg sample. Attach condenser, seal with 2-3 drops alcohol, and reflux gently on hot plate 1.5 hr. Let cool, and rinse condenser and tip with 25 mL alcohol. Quant. transfer soln to 400 mL beaker, using 50 mL alcohol and 100 mL H20. Rinse erlenmeyer with addnl portions H20 to total vol. of 250 mL. Add 10 drops thymol blue indicator soln to beaker and, with
- 127. AOAC METHODS (1980) HEPTACHLOR 105 stirring, add HN03 (1 +1) dropwise to first pink color of indicator. Adjust pH to 2±0.2, using NH40H (1+4) and/or HN03 (1+4). Adjust total vol. to 300 mL with H,O. 6.288 Determination Place sample beaker on mag. stirrer, and adjust to rapid stirring. Titr. with AgN03 std soln to same mv end point used for stdzg AgNOJ std soln. Titr. blank (unhydrolyzed sample). % Active ingredient in tech. Kelthane = (((Vs/Sa) - (Vb/Sb)) x N x V, x 0.1235 x 100}/V; % Active ingredient in Kelthane formulations = [(Vs/Sa) - (Vb/Sb)) x N x 0.1235 x 100, where Vs and Vb = mL AgN03 std soln required to titr. sample and blank, resp.; N = normality of AgN03 std soln; Sa and Sb = g sample taken for hydrolysis and blank, resp.; V, = total vol. sample soln = 500 mL; and V = aliquot vol. sample soln = 25 mL. Fluometuron (1,1-Dimethyl-3-(a,a,a-trifluoro-m-tolyl)urea) Gas Chromatographic Method (75)-Official Final Action 6.289 Standard Solutions (a) Diethyl phthalate internal std soln.-Weigh 1.5±0.1 g tech. diethyl phthalate, dissolve in ca 100 mL alcohol-free CHCI3, dil. to 250.0 mL with CHCI3, and mix well. Std should be >98% pure and contain no impurities eluting at retention time of fluome- turon. (b) Fluometuron std soln.-Accurately weigh ca 125 mg tech. fluometuron of known purity (available from Ciba-Geigy Corp., PO Box 11422, Greensboro, NC 27409) into 2 oz round bottle with Teflon-lined or Poly-Seal screw cap. Pipet in 25 mL diethyl phthalate internal std soln and shake to dissolve. Pipet in 3 mL trifluoroacetic anhydride and shake mech. 15 min; then place bottle in 55° H,O bath 30 min. Let cool to room temp. 6.290 Preparation of Sample Accurately weigh sample contg ca 125 mg fluometuron into 2 oz round bottle with Teflon-lined or Poly-Seal screw cap. Pipet in 25 mL diethyl phthalate internal std soln and shake well. Pipet in 3 mL trifluoroacetic anhydride and shake mech. 15 min; then place bottle in 55° H,O bath 30 min. Let cool to room temp. Let insol. materials settle or centrf. portion of ext to obtain clear soln. 6.291 Gas Chromatography Use instrument equipped with flame ionization detector and 1.83 m x 2 (id) mm glass column packed with 2% OV-3 (Applied Science Laboratories, Inc.) on 80-100 mesh Gas-Chrom Q. Condition 24 hr at 240° with N or He at ca 40 mL/min. Column should have ~1500 theoretical plates. Use on-column injection to prevent decomposition of derivative. Typical operating conditions: temps (O)-inlet 150, column 115±10, detector 250; N or He carrier gas, 20-22 mL/min; air and H as specified by manufacturer; attenuation varied so that peak hts of pesticide and internal std are 60--80% full scale. Retention times forfluometuron derivative and diethyl phthalate are 3-5 and 8-10 min, resp. 6.292 Determination Proceed as in 6.433-6.434, except inject 1 ILL aliquots. 6.293 Calculations See 6.435. Folpet (N-(Trichloromethylthio)phthalimide) High Pressure Liquid Chromatographic Method (76) AOAC-CIPAC Method-Official Final Action (Applicable to dry formulations contg folpet as only active ingredient and to folpet combination formulations except those contg propargite or Me parathion. Compds insol. in CH2CI2 , e.g., maneb or inorg. salts, do not interfere.) 6.294 Apparatus (a) Liquid chromatograph.-Equipped with 254 nm UV detec- tor. Typical operating conditions: chart speed, 0.2"/min; eluant flow rate, 2 mL/min (ca 800 psi); detector sensitivity, 0.16 A unit full scale; temp., ambient; valve injection vol., 20 ILL. Adjust operating conditions to elute folpet peak in 4±1 min. Factors such as different H20 content in CH,CI, eluant can change retention times. Folpet peak must be completely resolved from dibutyl phthalate peak which normally elutes in ca 7 min. (b) Liquid chromatographic column.-Stainless steel, 300 x 4 (id) mm, packed with 10 ILm diam. silica gel particles (Waters Associates, Inc., No. 27477, or equiv.). 6.295 Reagents (a) Eluant.-Degassed CH2CI2 • (b) Internal std soln.-Accurately weigh ca 0.5 g dibutyl phthalate (MC/ B Manufacturing Chemists) into 200 mL vol. flask. Oil. to vol. with CH,CI, and mix. (c) Folpet std soln.-(100 ILg folpet + 250 ILg dibutyl phthal- ate)/mL. Accurately weigh ca 20 mg folpet ref. std, 99+% pure (Chevron Chemical Co., 940 Hensley St, Richmond, CA 94804) into glass vial, pipet 20 mL internal std soln into vial, and shake to dissolve. Pipet 1 mL into 10 mL vol. flask. Oil. to vol. with CH,CI,. 6.296 Preparation of Sample Accurately weigh sample contg 20 mg folpet into vial. Pipet 20 mL internal std soln into vial and shake 30 min. Centrf. to ppt solids. Pipet 1 mL supernate into 10 mL vol. flask, dil. to vol. with CH,CI" and mix. Sample contains ca (100 I1-g folpet + 250 ILg dibutyl phthalate)/mL. 6.297 Determination Inject 20 ILL folpet std soln onto column thru sampling valve and adjust operating conditions to give largest possible on-scale peaks with retention time of 4±1 min forfolpet. Repeat injections until ratio of folpet to dibutyl phthalate peak hts is within ±1% of previous injection. Without changing conditions, inject sample soln until its ratio is within ±1% of previous ratio for sample. Average last 2 peak ht ratios for sample and for std, resp., and calc. % folpet. % Folpet = (R/R') x (W'/W) x P, where Rand R' = avo peak ht ratios for sample and std, resp.; W' = mg folpet in std soln (ca 20 mg); W = mg sample extd for analysis; and P = % purity of std. 6.298 Heptachlor-Official Final Action Active Chlorine Method (61) Reagents (a) Dilute acetic acid.-80%. Oil. 800 mL HOAc to 1 L with H,O. (b) Silver nitrate-acetic acid std soln.-Dissolve 17 g AgN03 in 200 mL H,O, add 56 mL HN03 (1+1). and dil. to 1 L with HOAc. Stdze potentiometrically by adding 25 mL of this soln to
- 128. 106 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) 600 mL beaker contg 250 mL 80% HOAc. Immerse glass and Ag electrodes in soln and stir with mag. stirrer. Titr. with O.lN NaCI soln, 6.220(a)' to end point (max. change in mv/mL NaCI soln). Normality AgN03 = mL NaCI x normality NaCl/mL AgN03• 6.299 Preparation of Sample (a) Emulsifiable concentrate formulations .-Accurately weigh sample contg 0.3±0.05 g heptachlor in 250 mL erlenmeyer. Dissolve in 50 mL HOAc, and pipet in 25 mL 0.1N AgN03, (b). Attach reflux condenser and reflux 1 hr. (b) Granular and dust formulations.-(Caution: See 51.039, 51.041, and 51.074.) Accurately weigh sample contg 0.3±0.05 g heptachlor into 80 x 25 mm Soxhlet extn thimble. Ext 2 hr with pentane and transfer ext to 250 mL erlenmeyer. Attach short reflux column such as 3-ball Snyder or 12" (30 cm) Vigreux to flask and evap. to dryness on steam bath. (Results will be low if reflux column is not used.) Rinse down column with 50 mL HOAc, pipet in 25 mL O.lN AgN03, (b), attach reflux condenser, and reflux 1 hr. (c) Technical.-Accurately weigh 0.40±0.05 g heptachlor and proceed as in (a). 6.300 Determination Rinse tip of condenser or column with H2 0 and cool soln to room temp. Transfer quant. to 600 mL beaker, rinsing with four 10 mL portions 80% HOAc. Immerse glass and Ag electrodes in soln and stir with mag. stirrer. Titr. with 0.1N NaCI soln, 6.220(a), to end point. % Heptachlor = 37.33 x (25 x normality AgN03 soln - mL NaCI soln x normality NaCI soln)/g sample. Gas Chromatographic Method (77) 6.301 Apparatus (a) Gas chromatograph.-Equipped with H flame ionization detector; capable of accepting glass column and glass-lined sample introduction system or on-column injection. Use follow- ing conditions: Temps (0): column 175, detector 175-190, sample inlet 190; N carrier gas pressure 30 psig; recorder chart speed 2.5 cm/min. (b) Glass-stoppered tubes.-Approx. 25 and 75 mL capacity. (c) Microliter syringe.-l0 J.LL, Hamilton Co., 701-N. 6.302 Reagents (a) Heptachlor.-Ref. grade (Velsicol Chemical Corp.). (b) Aldrin.-Ref. grade (Velsicol Chemical Corp.). 6.303 Preparation of Column To 9.5 g 10D-120 mesh Gas Chrom Q in vac. flask add 0.50 g silicone GE Versilube F-50 (available from Applied Science Labs) dissolved in 50 mL CH2CI2• Shake slurry well to wet solid thoroly. Connect flask to H20 aspirator and evap. solv. with frequent shaking. When solids appear dry, complete drying by placing flask in steam bath and connecting to vac. pump until ca 4 mm pressure is attained. Remove flask from steam bath and let cool under vac. Fill 1.5 m (5') x 'Ie" od (0.067" id) Pyrex glass tube with this packing, using vac. pump and gentle tapping. Plug ends of column with glass wool. Condition column 24 hr in 190° oven while purging with N. Let column cool while still purging with N; then install in chromatograph. 6.304 Preparation of Sample (a) Liquids.-Weigh sample contg ca 750 mg heptachlor into 75 mL g-s vial and add 500 mg ref. grade aldrin. Add 75 mL fresh CS2 , stopper, and shake vigorously 2 min. (b) Solids.-Transfer weighed sample contg ca 750 mg hep- tachlor to Soxhlet and ext 2 hr with 75 mL pentane. Let cool, add 500 mg ref. grade aldrin to soln, and swirl. 6.305 Calibration Weigh 0.2500 g ref. grade heptachlor and 0.1670 g ref. grade aldrin into 25 mL g-s flask. Dissolve in 25 mL CS2 • Chromatograph this soln under conditions given in 6.301(a) 5 times to obtain accurate response correction factor. (On new column, it is sometimes desirable to inject several 5 J.LL aliquots of std soln to condition column before use.) 6.306 Determination Let instrument equilibrate as in 6.301 (a). Inject ca 1 J.LL sample soln at sensitivity setting such that ht of heptachlor peak is ca % full scale. For each analysis, allow lD-12 min for heptachlor related components to elute. Components and approx. retention times in min are: heptachlor 4.5, aldrin 5.9, chlordene 3.1, and y-chlordane 9.9. 6.307 Calculations Calc. area of heptachlor and aldrin peaks by multiplying peak ht in mm by width of peak at half ht in mm. Alternatively, use integrator. Calc. response correction factor (f, ca 0.82) for each of the 5 std injections as follows: f = (area of heptachlor peak x mg aldrin x purity of aldrin)/(area of aldrin peak x mg heptachlor x purity of hep- tachlor). Average 5 replicates and use avo to calc. % heptachlor in samples. % Heptachlor = (area of heptachlor peak x mg aldrin x purity of aldrin x 100)/(area of aldrin peak x mg sample x f). Picloram (4-Amino-3,5,6-Trichloropicolinic Acid) and 2,4-D (2,4-Dichlorophenoxyacetic Acid) High Pressure Liquid Chromatographic Method (78) Official Final Action 6.308 Apparatus (a) Liquid chromatograph.-Equipped with 280 nm UV detec- tor and injection valve. Alternatively, septum injection head may be used; however, stop-flow injection is recommended. Oper- ating conditions: eluant flow rate, 0.7 mL/min (ca 1000 psi); detector sensitivity, 0.08 A unit full scale; temp., ambient, but within ±2S. (b) Liquid chromatographic column.-No. 316 stainless steel, 1000 x 2.1 mm id, with Varian No. 96-000075-00 reducing union ('Ie" x 1/16") contg 2 J.Lm frit (regular reducing union packed with glass wool may be used instead) packed with DuPont No. 820960005 Zipax® SAX (strong anion exchange) resin. Preclean column with few mL each of CHCI3, acetone, and MeOH, and vac.-dry. Pack in small increments over 40 min period while tapping column on hard surface. 6.309 Reagents (a) Eluants.-Prep. sep. solns of 0.01M Na2840 7.10H20 (3.8 gil) and 0.002M NaCI04.H20 (0.28 gil) in previously boiled and cooled deionized H20. (b) Salicylic acid internal std so/n.-Accurately weigh ca 3.6 g USP Ref. Std Salicylic Acid into 1 L vol. flask, dil. to vol. with 0.05N NaOH in isopropanol-H,O (1 +11. and mix.
- 129. AOAC METHODS (1980) AMITROLE 107 (c) Picloram-2,4-D std soln.-(4 mg picloram + 12 mg 2,4-D + 3.6 mg salicylic acid)/mL. Accurately weigh ca 100 mg picloram ref. std, 99+% pure (Dow Chemical Co.). and ca 300 mg 2,4-0 ref. std, 99+% pure (Dow Chemical Co.l. into glass vial, pipet in 25 mL salicylic acid internal std soln, and shake to dissolve. 6.310 Preparation of Sample Accurately weigh ca 1.6 g sample into ca 10 dram glass vial, pipet in 25 mL salicyclic acid internal std soln, and shake to dissolve. 6.311 Determination Inject 2 J-LL picloram-2,4-D std soln onto column and adjust attenuation to give largest possible on-scale peaks. Repeat injections until peak ht ratios of herbicide:internal std vary ",,1% for successive injections. Without changing conditions, inject 2 J-LL aliquots sample soln until peak ht ratios vary ",,1%. Average last 2 peak ht ratios for picloram and 2,4-D and calc. % herbicide. % Herbicide = (RJRs) x (Ws/Wx) x P, where Rx and Rs = avo peak ht ratios of each herbicide to the internal std for sample and std, resp.; Ws = mg herbicide in std; Wx = mg sample; and P = % 'purity of std. 6.312 Sodium Trichloroacetate (79)--Official Final Action (Caution: See 51.011, 51.039, 51.041, and 51.070.) Apparatus and Reagent (a) Reflux apparatus.-250 mL erlenmeyer attached thru "$ 24/40 joint to 50 cm water-cooled condenser. (b) Dioxane.-Freshly distd. 6.313 Determination Dissolve 25 g sample in H20 and dil. to 100.0 mL. Pipet aliquot (usually 10 mLl. titrg ca half that of blank, into 250 mL refluxing flask, add 1 drop Me red, and neutze with ca 1N H2S04 to distinct orange-pink. pH is 5.3-5.5; usually <0.15 mL is required. If soln is acid, titr. with ca 1N NaOH. Add 25.00 mL 1N H2S04, 35 mL dioxane, and few glass beads. Boil vigorously under reflux ~60 min. Cool, add 2 drops Me red, and titr. with std 1N NaOH to sharp change from orange to yellow end point. Perform blank detn, omitting sample. % Na trichloroacetate = Net mL 1N acid x 0.1854 x 100/g sample in aliquot. 6.314 Trifluralin-Official First Action See 6.203~.214. NONHAlOGENATED PESTICIDES Aldicarb (2-Methyl-2-(methylthio)propionaldehyde-O- (methylcarbamoyll Oxime) (BO)--Official Final Action (Caution: See 51.041.) 6.315 Apparatus and Reagents (a) Infrared spectrophotometer.-Perkin-Elmer Model 337, or equiv. Adjust conditions as required by specific instrument. (b) Soxhlet extractor.-With 125 mL flask and 25 x BO mm cellulose thimble. (c) Aldicarb std soln.-D.18 g/100 mL. Accurately weigh (to 0.1 mg) 0.1B±0.01 g anal. grade aldicarb (available from Union Carbide Corp., Agricultural Products and Services) into 100 mL g-s vol. flask, add ca 80 mL CH2CI2, mix to dissolve, and dil. to vol. with CH2CI2 • 6.316 Determination Transfer accurately weighed sample contg 0.1B±0.01 g aldi- carb to extn thimble, cover with wad of surgical grade cotton, and place thimble in extractor. Add 2-3 Alundum boiling stones and ca BO mL CH2CI2 to flask, and ext at rate to provide 5 extns within 60 min. Let cool to room temp., transfer quant. to 100 mL g-s vol. flask with CH2CI2, and dil. to vol. Using matched 0.5 mm NaCI cells, scan sample and std solns from 5.2 to 6.0 J-Lm (1900 to 1600 cm-') against CH2CI2• Calc. A of sample and A' of std at 5.75 J-Lm (1740 cm-'I. using corre- sponding A at 5.4 J-Lm (1850 cm-') as 10, (A and A' should both be ca 0.45.) 6.317 Determination of Binder Correction Pipet 50 mL sample soln into 100 mL beaker and place in room temp. H20 bath in hood. Evap. to dryness, using gentle stream of clean, dry air. Add 25 mL MeOH, stir well, and filter thru 30 mL coarse fritted glass gooch. Rinse beaker and gooch with 25 mL MeOH, applying vac. until all liq. is in filter flask. Place gooch and contents in original beaker, place 20 mL CH2 CI2 in gooch, and swirl to dissolve binder, letting solv. drip into beaker. Repeat with addnl 20 mL CH2CI2• Quant. transfer solv. to 50 mL g-s vol. flask and dil. to vol. with CH2CI2 • Scan soln as in detn and subtract A of binder soln (should be <0.005) from that of sample (=~A). % Aldicarb by wt = (~A/g sample) x (g std/A') x P, where P is % purity of ref. std. Amitrole (3-Amino-s-triazole) (B1)--Official Final Action (Caution: See 51.018 and 51.041.) 6.318 Preparation of Sample Solution (a) 50% Dry powder formulation.-Transfer 10.00 g sample to 100 mL g-s vol. flask, using powder funnel. Add 50 mL DMF. Shake 2-3 min to dissolve amitrole. (Undissolved amitrole is powder and can be differentiated visually from inerts which are usually crystals.) Let settle and carefully decant supernate into 100 mL vol. flask. Repeat extn of residue with three 15 mL portions DMF, letting settle each time before decanting into vol. flask. Dil. combined exts to vol. with DMF and shake well. Filter 40-50 mL thru fritted glass filter of medium porosity. Pipet 25 'TlL into 400 mL beaker contg 50 mL H20. (b) 90% Dry powder formulation.-Dissolve 1.0000 9 sample in 100 mL H2 0 in 400 mL beaker. (c) Aqueous amitrole.-Pipet 5 mL sample into 400 mL beaker contg 50 mL H2 0. 6.319 Determination Adjust sample soln or dild aliquot to pH 1.8 with 0.5N HCI. Stir mech. and titr. with 0.5 mL increments 0.5N NaOH to pH 3.5-4.0. (Use Beckman Model G pH meter, or equiv., equipped with glass-calomel electrode system, and stdzd at pH 4.0 and 7.0 with buffers, 50.007(c) and (d).) Add 0.5N NaOH rapidly to pH 6.5 and then dropwise to pH 7.5 (second inflection point). Plot pH against mL 0.5N NaOH and det. first inflection point (occurs at pH 2.5-2.9).
- 130. 108 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) % Amitrole by wt = (8 - C) x 0.5 x 8.408/F, where C = mL 0.5N NaOH required to titr. to first inflection point; 8 = mL 0.5N NaOH required to titr. to pH 7.5; and F = 2.5 for 50% dry powder formulation, (a). g sample for 90% dry powder formulation, (b), and 5.0 x sp gr sample for aq. amitrole, (c). Ib Amitrole in aq. amitrole/U.S. gal. = % amitrole x sp gr x 8.32/100. 6.320 Carbaryl (1-Naphthyl Methylcarbamate) (82) Official Final Action (Caution: See 51.018, 51.040, 51.041, and 51.056.) Apparatus (a) Centrifuge.-Glinical model, 8 place, or equiv. (b) Hypodermic syringe.-1 mL, glass barrel with rubber- tipped plastic plunger (1 mL B-D Glaspak Tuberculin disposable syringe supplied by Becton, Dickinson, and Co. is suitable). Disposable syringe may be used repeatedly. Wash with H20 and acetone or MeOH, air-dry, and lubricate rubber plunger tip with silicone stopcock grease. (c) Infrared spectrophotometer.-Perkin-Elmer Corp., Model 337, or equiv. Operator must adapt conditions to instrument. (d) Rotator.-Tube type, BBL, or equiv. (e) Shaking machine.-Wrist-action shaker (Burrell Corp., or equiv.). (f) Tubes.-Culture tubes, borosilicate glass, 16 x 150 mm with screw caps and Teflon liners (Corning Glass Works No. 9826, or equiv.). 6.321 Reagents (a) Methanol-chloroform soln.-10% (v/v) MeOH in CHCI3 • (b) Carbaryl std solns.-(T) 8 mg/mL.-Transfer 0.12±0.01 g carbaryl (anal. grade, available from Union Carbide Corp., Agricultural Products and Services). weighed to nearest 0.1 mg, to culture tube. Pipet 15 mL MeOH-CHCI3 soln into tube, cap securely, and rotate or shake mech. 30 min. (2) 2.5 mg/mL.- Transfer 0.25±0.01 g carbaryl, weighed to nearest 0.1 mg, to 250 mL g-s erlenmeyer. Pipet 100 mL CHCI3 into flask, stopper, and swirl to dissolve. 6.322 Preparation of Sample (a) Carbaryl dust and powder formulations.- Transfer weighed sample (,,;2.4 g) contg 0.12±0.01 g carbaryl to culture tube. Pipet 15 mL MeOH-CHCI3 soln into tube and cap securely. Rotate or shake mech. 30 min and centrf. 10 min. (b) Liquid suspensions.-Following steps must be performed in order described, as any deviation can cause erroneous results due to faulty sample transfer and incomplete extn: Place ca 20 g Na2S04 in 250 mL g-s erlenmeyer. Pipet 100 mL CHCI3 into flask. Vigorously shake sample bottle. Draw appropriate vol. sample into hypodermic syringe without needle. Use ca 0.5 mL sample for carbaryl 4 Ib/gal. and ca 1.0 mL for carbaryl 2 Ib/gal. Wipe outside of syringe with paper towel and weigh syringe and contents to nearest 0.1 mg. Add sample to erlenmeyer by slowly depressing syringe plunger. Do not let syringe or sample touch sides of flask. Sample must drop into CHCI3 • Reweigh syringe and calc. sample wt by difference. Stopper flask and shake vigorously 30 min on mech. shaker. 6.323 Determination (a) Carbaryl dust and powder formulations.-Using matched 0.2 mm NaCI cells, scan sample soln against MeOH-CHCI3 soln from 5.2 to 6.0 ~m (1900-1600 cm-'). Repeat scan with std soln. Measure A of carbaryl peak at 5.75 ~m (1740 cm-'). using A at 5.40 ~m (1850 cm-') as 0 point. A = ca 0.4 for both std and sample. % Carbaryl by wt = (A x 8' x PI/lA' x 8). where A and A' = absorbance of sample and std, resp., at 5.75 ~m; 8 and 8' = mg sample and mg std/mL, resp.; and P = % purity of carbaryl std. (b) Liquid s{Jspensions.-Proceed as in (a). except use matched 0.5 mm NaCI cells and scan sample soln against CHCI3 • 2,2-Diehlorovinyl Dimethyl Phosphate (DDVP) (83)-Official First Action Method I (Applicable to sand/sugar base fly bait contg ca 0.5% and 4 Ib/gal. DDVP emulsifiable concs. Caution: See 51.041.) 6.324 Apparatus and Reagent (a) Infrared spectrophotometer.-Capable of recording in re- gion 2-15 ~m. Slit width must be adjustable to give signal-to- noise ratio of ca 100: 1; with sealed liq. absorption cell, NaCI windows, and 0.2 mm path length. (b) Hypodermic syringe.-Luer type, glass, 1.0 mL. Use 18 gage (Stubbs). 2" slip-on needle. (e) 2,2-Dichlorovinyl dimethyl phosphate.-Use std DDVP of known purity. (Available from Shell Chemical Co.) 6.325 Calibration of Apparatus Into each offive 10 mL vol. flasks, weigh, to nearest 0.1 mg, 25, 75, 100, 150, and 200 mg DDVP std, and dil. to vol. with CHCI3 • Calibration solns contain ca 2.5, 7.5, 10, 15, and 20 g DDVP/L. Fill sealed liq. absorption cell with CHCI3, adjust spectrophtr to optimum settings, and scan over 10.7-9.9 ~m. Without changing settings, fill cell in turn with each of prepd calibration solns, starting with most dil., and scan each soln over 10.7-9.9 p,m. For each scan, construct baseline thru absorption min. at ca 10.0 p,m parallel to 0 radiation line. Draw perpendicular to 0 radiation line thru absorption max. of calibration soln at ca 10.2 ~m and measure radiant power Po (at 10.0 ~m) and P (at 10.2 p,m), in any convenient units but keeping same units thruout. Calc. A as log (Po/P). Repeat calcns, using absorption min. at ca 10.5 p,m as ref. point. Subtract A of cell and CHCI3 obtained above from A of cell and calibration solns. Plot LlA of DDVP as ordinate against gil DDVP as abscissa for each ref. point (10.0 and 10.5p,m). 6.326 Preparation of Sample Solution (a) Sand/sugar base fly baits.-Prep. 25 x 400 mm extn column by adding enough diat. earth (Hyflo Super-Cel) to make layer 5 cm high when gently packed. Place 250 mL vol. flask under outlet. Accurately weigh sample contg 0.2-1.0 g DDVP. Transfer sample to extn column with CHCI3, and rinse sample container with CHCI3• Working in well-ventilated hood, add 50 mL CHCI3 to column. Using stirring device, vigorously agitate sample and top half of adsorbent layer to form slurry with solv. Withdraw stirring device, and rinse it and column with addnl CHCI3 from wash bottle. Let solv. percolate thru column until level is few mm above diat. earth-sample layer. Add ca 50 mL CHCI3 to column, agitate sample and diat. earth with stirrer as above, and let solv. percolate thru column until upper level approaches sample layer. Repeat with two addnl 50
- 131. AOAC METHODS (1980) PARAQUAT 109 mL portions CHCI3• When solv. ht has diminished to 2-3 mm, rinse column with three 10 mL portions CHCI3 , letting each portion enter diat. earth layer before adding next. Let column drain and rinse outlet tip with CHCI3, collecting rinse in 250 mL vol. flask. Transfer CHCI3 eluate to evapg dish (125 mm diam.) marked at 40-50 mL. Evap. on steam bath to 40-50 mL. Remove dish and continue evapn at room temp. to 10-15 mL. Using CHCI3, quant. transfer to vol. flask of such size to give DDVP concn of 0.5-1.0 g/100 mL when soln is dild to vol. (b) Emulsifiable concentrates.-Weigh enough sample, to nearest 0.2 mg, to give ca 1 g DDVP/l00 mL CHCI3 when dild to vol. in 10,25, or 50 mL vol. flask. 6.327 Determination Oil. CHCI3 soln of DDVP to vol. with CHCI3, mix thoroly, and fill calibrated liq. absorption cell with sample soln. Using same instrument settings as for calibration, scan sample soln over 10.7-9.9 fLm. Examine spectra for possible interference and use appropriate absorption min. as ref. point. (If solvs or other ingredients interfere at one of ref. points, use alternative ref. point.) For example, (3-naphthol, often used as stabilizer in fly baits, exts with CHCI, and absorbs at ca 10.5 fLm, requiring use of 10.0 fLm ref. point. Calc. A of sample soln as in 6.325. From calcd A, read g DDVP/L from calibration curve. % DDVP by wt = [(g DDVP/L) x mL sample solnJl (10 x g sample). Method /I (84) (Applicable to ca 0.5% (w/w) spray soln and ca 1.0% (w/w) cattle spray in hydrocarbon solvs) 6.328 Apparatus and Reagent (a) Infrared spectrophotometer.-Double beam instrument with specifications as in 6.324(a). (b) 2,2-Dichlorovinvl dimethvl phosphate.-See 6.324(c). 6.329 Preparation of Compensating Solvent Transfer ca 30 mL sample to 125 mL separator and ext (2-3 min per extn) with 4 ca 30 mL portions 0.5N NaOH. Dry DDVP- free hydrocarbon phase by passing it thru 2-3 g anhyd. Na2S04 • Reserve dried solv. for prepn of DDVP std soln and as compen- sating solv. in ref. cell. 6.330 Determination Prep. std DDVP soln in compensating solv. that approximates (on wt basis) DDVP content of sample. Calc. DDVP content of std soln to nearest 0.01 % by wt. After detg optimum instrument parameters for compensation technic, scan std soln over 9.9-10.7 fLm (1010-935 cm-1 ) region with ref. cell contg compensating solv. in ref. beam of spec- trophtr. Scan sample against compensating solv. in same man- ner. From differential spectra, det. A of DDVP at 10.2 fLm (980 cm-1 ) of std, A', and sample, A, measured from baseline drawn between minima near 10.0 and 10.6 fLm. Calc. DDVP as follows: % DDVP by wt = % DDVP in std x A/A'. 6.331 Diazinon-Official First Action See 6.431-6.435. 6.332 Diquat (6,7-Dihydrodipyrido (1,2008:2',1 '-c) Pyrazinediium Ion) (85)-Official Final Action AOAC-CIPAC Method Reagents (a) Acetate buffer soln.-pH 4.05. Dissolve 10.88 g NaOAc .3H20 in H20, add 19 mL HOAc, dil. to 2 L with H20, and mix. (b) Diquat std solns.-(1) Stock soln.-0.2 mg diquat/mL. Prep. stock soln by dissolving 0.1968 g pure diquat dibromide monohydrate (C'2H'2N2Br2.H20, MW 362.1; 50.87% cation; avail- able from Chevron Chemical Co., 940 Hensley St, Richmond, CA 94804) in buffer soln, dil. to 500 mL with buffer soln, and mix. (2) Working soln.-O.02 mg diquat/mL. Oil. 10.0 mL stock soln to 100 mL with buffer soln. Prep. dild stds fresh as required. 6.333 Determination Using buret, transfer 10.0, 20.0, and 30.0 mL std diquat soln, contg 0.2, 0.4, and 0.6 mg diquat, resp., to three 100 mL vol. flasks, dil. each soln to vol. with buffer soln, and mix. Measure A of stds at 310 nm in 1 cm silica cell, with buffer soln as ref., and draw std curve relating A to mg diquat. Accurately weigh portion (w g) of well mixed sample contg ca 0.5 g diquat, transfer to 250 mL vol. flask, dil. to vol. with buffer soln, and mix (Soln 1). Transfer 10.0 mL Soln 1to 200 mL vol. flask, dil. to vol. with buffer soln, and mix (Soln 2). Transfer 5.0 mL Soln 2 to 100 mL vol. flask, dil. to vol. with buffer soln, and mix (Soln 3). Measure A of Soln 3 at 310 nm in 1 cm silica cell, with buffer soln as ref. Read diquat content of Soln 3 (V mg) directly from std curve or calc. diquat content by interpolation. 6.334 % Diquat, w/w = 100 V/w. Paraquat (1,1'·Dimethyl·4,4'·bipyridinium Ion) (85)-Official Final Action Reagents (a) Sodium dithionite.-l% soln in O.lN NaOH. (Sodium dithionite, Na2S20 4.2H20, is also called sodium hydrosulfite and sodium hyposulfite.) Do not keep soln >3 hr; solid is unstable in presence of moisture. Store solid in small air-tight bottles in vac. desiccator. (b) Paraquat std soln.-0.25 mg paraquat/mL. Dry anal. std (available from Chevron Chemical Co., 940 Hensley St, Rich- mond CA 94804) to const wt at 100-120° before weighing (para~uat salts are hygroscopic). Dissolve 0.1728 g 'paraquat dichloride (72.40% cation) in H2 0, dil. to 500 mL with H20, and mix. Prep. soln fresh as required. (c) Extracting soln.-Dissolve 11 g Na2S04.10H20 in 500 mL H20, add 500 mL alcohol, and mix. 6.335 Preparation of Standard Curve Pipet 50 mL std soln into 250 mL vol. flask, dil. to vol. with H20, and mix. Pipet 5,10,15, and 20 mL aliquots ofthis dild std soln into sep. 100 mL vol. flasks. (When dild to vol. these solns contain 2.5, 5.0, 7.5, and 10.0 fLg paraquat/mL, resp.) Proceed as in 6.337. Plot A against fLg paraquat/mL at final diln. 6.336 Preparation of Sample (Caution: Open aerosol can behind safety shield.) (a) Formulations not containing oil base.-Accurately weigh portion well mixed sample contg ca 0.25 g paraquat. Transfer to 500 mL vol. flask, dil. to vol. with H20, and mix well (Soln 1). Pipet 10 mL Soln 1 into 100 mL vol. flask, dil. to vol. with H20,
- 132. 110 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) and mix well (Soln 2). Pipet 10 mL Soln 2 into 100 mL vol. flask and proceed as in 6.337. (b) Aerosol formulations containing oil base.- Weigh aerosol can to nearest 0.1 g (C). Clamp can with bottom up and puncture smallest possible hole with punch and hammer. After hiss of escaping propellent is no longer heard, cut bottom Ya open with hand can opener. Push nearly detached lid into can. Immerse can 15 min in 50-70° H20 bath or in hot tap H20 running into 1 L beaker. Add 50 mL extg soln, (e), and 50 mL pentane to 250 mL separator. Remove can from H20 bath, dry well (especially inside cap and around valve), and weigh (D). Place pipet with capacity to deliver ca 20 mg paraquat in can, and weigh both (E). Withdraw liq., transfer contents to separator, replace pipet in can, and weigh (F). (Disregard material left in and on pipet.) Empty can, rinse completely with acetone, air dry, and weigh (G). Stopper separator and shake 30 sec, venting frequently. Let layers sep, and drain lower layer into 200 mL vol. flask. Add 25 mL extg soln to separator, repeat extn, and drain lower layer into same vol. flask. Dil. to vol. with extg soln and mix well. Pipet 5 mL into 100 mL vol. flask and proceed as in 6.337. 6.337 Determination (Complete analysis of one soln before adding dithionite to next soln.) Add 10 mL Na dithionite soln to one 100 mL vol. flask and dil. to vol. with H20. Mix by inverting end-over-end 3 times at such speed that air bubble travels from one end to other; do not shake flask vigorously, as this tends to cause fading of color due to oxidn. Immediately measure A of soln at 600 nm, using reagent blank (no paraquat) to set the 100% T orfor ref. side for dual beam instruments. Similarly, treat each flask in turn, completing color measurement without delay before adding dithionite to next soln. % Paraquat = (JLg/mL from std curve) x 5/g sample. % Paraquat (in aerosol formulations) = [(JLg/mL from std curve) x (D - G) x 0.4)/[(C - G) x (E - F)j. 6.338 Dithioearbamates (Ferbam, Maneb, Nabam, Zineb, and Ziram) (Caution: See 51.041.) Carbon Disulfide Evolution Method (86) Official Final Action (Applicable only to concs or formulations free from interfering substances) Principle Dithiocarbamates decompose on heating in acid medium. Evolved CS2 is passed thru Pb(OAc)2 soln traps to remove H2S and S02 formed from sample impurities. Washed CS2is reacted with methanolic KOH, and xanthate formed is titrd with I soln. 6.339 Apparatus Carbon disulfide evolution apparatus.-See Fig. 6:07. Avail- able from Scientific Glass Apparatus Co., No. JE-l000. 6.340 Reagent Methanolic potassium hydroxide.-2N. Dissolve 112 g KOH pellets in 500 mL anhyd. MeOH, filter thru cotton, and add addnl 500 mL anhyd. MeOH. 6.341 37MM O.D.BUlBS WITH 4 INDENTATIONS EACH - I ~ SPHERICAL JOINT AIR INlET -10"" RADIUS 20MM TUBING- LEAO ACETATE TRAPS- REACTION FLASK (2NECK-250ML) I -! 24/40 FIG. 6:07--<:arbon disulfide evolution apparatus II Determination Add 20 mL 10% Pb(OAc)2 soln to each Pb(OAc)2 trap and pipet 50 mL 2N MeOH-KOH soln into MeOH-KOH absorber (Fig. 6:07). (Absorber must be dry at time of addn and kept at 25±1°.) Add 50 mL H2S04 (1 +4) to reaction flask and heat acid to boiling. Adjust aspiration rate to "-'S:1 bubble/sec thru MeOH-KOH soln, using stopper in reaction flask. Weigh "-'S:5 g sample (contg 0.1-0.3 g dithiocarbamates) into small filter paper cone and fold cone to prevent sample loss. Remove stopper from reaction flask, insert wrapped sample, and immediately stopper flask. Adjust air flow if necessary and maintain steady, moderate boil. Do not let acid soln enter air inlet tube. Some dust formulations react vigorously and require special care to prevent ejection of hot acid. As reaction proceeds, adjust system so that rates of boiling and aspiration are almost in equilibrium, producing only very slow rate of bubbling thru MeOH-KOH soln. Continue boiling 1.5 hr. Disconnect MeOH- KOH absorber and rinse contents into 500 mL erlenmeyer, using ca 250 mL H20. (To remove absorber contents, apply slight air pressure to top of absorber and force soln thru side arm. Rinse with 4 ca 25 mL portions H20, forcing out rinse H20 in same manner with air pressure.) Add 3 drops phthln, and titr. with 30% HOAc until red just disappears. Immediately titr. with O.lN I; near end point, add 5 mL starch indicator soln, 6.005(f), and titr. to faint but definite color change. Det. blank (usually 0.1-0.2 mL 0.1N I) by dilg 50 mL MeOH- KOH soln with 250 mL H20, neutzg with 30% HOAc, and titrg as above. Calc. % dithiocarbamate = (Sample titrn - blank) x (I nor- mality) x (equiv. wt dithiocarbamate)/(g sample x 10). Equiv. wts (';2 MW) of zineb, maneb, ziram, nabam, and ('/3 MW) ferbam are 137.87, 132.65, 152.91,128.18, and 138.82, resp.
- 133. AOAC METHODS (1980) FORMALDEHYDE 111 Thiram (Bis(dimethylthiocarbamoyl)disulfide) (Tetramethylthiuram Disulfide) CIPAC Method (87)-Official Final Action 6.342 Principle Thiram is decomposed by boiling with HOAc and Zn(OAc)2 to Me2 NH, CS2, and carbonyl sulfide. The gaseous mixt. is carried by air stream thru CdSO. scrubber to remove H2S, and then into absorption system contg MeOH-KOH soln. Mixed xanthate- monothiocarbamate soln is neutzd and titrd with std aq. I. Method is not specific for thiram. Sep. characterization test, 29.171, must be made. 6.343 Apparatus Assembly and operating conditions.-Assemble app. as shown in Fig. 6:08 with 30 mL CdSO. soln in first absorber, 25 mL KOH soln in second absorber, and 5 mL in each bubbler. Turn on condenser H20 and maintain H20 bath surrounding CdSO. scrubber at 70-800 thruout test. Keep main KOH absorber at <250 by immersion in beaker of cold H,O. Absorber must be dry or rinsed with MeOH before adding KOH soln. Air bleed must reach nearly to bottom of digestion flask. Make all joints gas-tight, using small amts H3PO., petrolatum, or silicone grease. Check app. for absorber leaks and efficiency periodically, using pure Na diethyldithiocarbamate. Recoveries should be 99-101 %. Check purity of Na diethyldithiocarbamate by dis- solving ca 0.5 g, accurately weighed, in 100 mL H20 and titrg directly with 0.1N I, using ca 2% starch soln as indicator. 1 mL 0.1N I = 0.02253 g Na diethyldithiocarbamate. % Na diethyldi- thiocarbamate = 2.253 x mL 0.1N I/g sample. 6.344 Reagents (a) Acid mixture.-Dissolve 2.5 g ZnO in 100 mL HOAc (1+1). (b) Cadmium sulfate soln.-Dissolve 18.5 g 3CdSO•.8H20 in 100 mL H20. (c) Potassium hydroxide soln.-2N in MeOH and contg <1 ppm Cu or Fe. (d) Iodine std so/no -Q.1N. Stdze as in 50.019. 6.345 Determination Accurately weigh and transfer sample contg ca 0.3 g thiram to digestion flask, using small amt H20, if necessary. Assemble air bleed and dropping funnel, Fig. 6:08, and add 20 mL acid mixt. thru funnel. Connect app. to controlled aspiration (vac. or compressed air) so that ca 3 bubbles/sec pass thru absorbers. After sample is evenly dispersed, heat and reflux 30 min at moderate rate. Turn off cooling H20 and flush condenser and first absorber with steam from flask ,,;1 min. Remove burner 20ml. 50 ml. , I Shia!d--i I I and disconnect train. Wash contents of KOH absorber and bubblers into 600 mL beaker with 300-400 mL H20, add 1-2 drops phthln, just neutze with HOAc (1 +9) from buret, and add 3 drops excess. With continual stirring, titr. immediately (pref- erably within 1 min, as decomposition of mixed xanthate/ monothiocarbamate soln is extremely rapid under acidic con- ditions) with 0.1N I (t mL), using ca 2% starch soln as indicator. Det. blank in same manner, omitting sample (b mL). 1 mL 0.1N I = 0.01202 g thiram. % Thiram = 1.202 (t - b)/g sample. 6.346 Dodine (n-Dodecylguanidine acetate) (88) Official Final Action (Caution: See 51.022, 51.028(a) and (d). and 51.041.) Reagents (a) Perch/oric acid.-Q.05N. Dissolve 4.2 mL 72% HCIO. in HOAc and dil. to 1 L with HOAc. Stdze as follows: Accurately weigh 0.200 g KHC.H.O. into 250 mL erlenmeyer. Dissolve in 20 mL HOAc by gently heating flask on hot plate. Add 80 mL Ac20 and 8 drops metanil yellow indicator, (b). Place erlenmeyer contg bar on mag. stirrer and titr. with HCIO. to first definite red (magenta). Titr. reagent blank and correct sample titer. Normality = 0.200/(0.20422 x net mL HCIO.) (b) Metanil yel/ow.-0.20%. Dissolve 0.200 g metanil yellow powder in 100 mL MeOH. (e) Potassium acid phtha/ate.-NBS SRM KHC.H.O•. 6.347 Determination Accurately weigh sample contg ca 0.600 g dodine into 250 mL erlenmeyer. Add 10 mL HOAc followed by 90 mL Ac,O. Mix by swirling 5 min. Filter slurry with vac. thru large, medium porosity fritted glass buchner into 250 mL vac. flask. Wash erlenmeyer and residue in funnel with two 10 mL portions HOAc-Ac20 (10+90). Place vac. flask contg bar on mag. stirrer, add 8 drops metanil yellow indicator, and titr. with stdzd ca 0.05N HCIO. to first definite red (magenta). Titr. reagent blank and correct sample titer. % Dodecylguanidine acetate 6.348 = (net mL HCIO. x normality x 28.75)/g sample Formaldehyde in Solutions-Official Final Action Hydrogen Peroxide Method (89) Reagents (a) Sulfuric acid std so/n.-1N. Prep. and stdze as in 50.039-50.041. FIG. 6:08-Absorption system for thiram. Dimensions in em; N.S. = nonstandard; 810 = f10/30
- 134. 112 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) (b) Sodium hydroxide std soln.-1N. Stdze against (a), using litmus or bromothymol blue indicator. 1 mL = 30.03 mg HCHO. (c) Hydrogen peroxide soln.-Com., contg ca 3% H20 2. If acid, neutze with NaOH, (b), using litmus or bromothymol blue indicator. (d) Litmus indicator.-Soln. of purified litmus of such concn that 3 drops gives distinct blue color to 50 mL H20. (e) Bromothymol blue indicator.-Dissolve 1 g bromothymol blue in 500 mL alcohol, 50% by vol. 6.349 Determination Pipet 50 mL 1N NaOH soln into 500 mL erlenmeyer and add 50 mL H20 2, (c). Add weighed amt sample (ca 3 g) from weighing pipet, letting point of pipet reach nearly to liq. in flask. Place funnel in neck of flask and heat on steam bath 5 min, shaking occasionally. Remove from bath, wash funnel with H20, cool to room temp., and titr. excess NaOH with std acid, using bromothymol blue or litmus. (Cool flask before titrn to obtain sharp end point with litmus.) From mL 1N NaOH used and wt sample, calc. % HCHO according to following equation NaOH + HCHO + H20 2 = HCOONa + 2H20. If HCHO soln contains appreciable free acid, titr. sep. portion and calc. acidity as % HCOOH. Correct for this acidity in calcg % HCHO. 6.350 Cyanide Method (90) (Applicable only to dil. solns) Treat 15 mL 0.1N AgN03, 50.027-50.029, with 6 drops HN03 (1+1) in 50 mL vol. flask, add 10 mL KCN soln (3.1 gin 500 mL H20), dil. to vol., shake well, filter thru dry filter, and titr. 25 mL filtrate with 0.1N NH4SCN, 50.003-50.004, as in 3.074. Acidify another 15 mL portion 0.1 N AgN03 with 6 drops HN03 (1 +1) and treat with 10 mL of the KCN soln to which has been added measured amt of sample (wt calcd from sp gr) contg ,,;;25 mg HCHO. Dil. to 50 mL, filter, and titr. 25 mL aliquot with the 0.1N NH4SCN as before. Difference between mL NH4SCN used in these 2 titrns x 2 = mL 0.1N NH4SCN corresponding to KCN used by the HCHO. Calc. % HCHO present. 1 mL 0.1N NH4SCN = 3.003 mg HCHO. 6.351 Formaldehyde in Seed Disinfectants (97) Official Final Action (Applicable to detn of HCHO absorbed in inert carrier, e.g., bentonite, talc, charcoal, sawdust) Weigh ca 5 g sample contg 0.3-0.5 g HCHO in weighing bottle and transfer to SOO mL Kjeldahl flask. Add 25 mL H20 and 12 mL H2S04(1 +4). Steam distil rapidly, passing vapors thru condenser with delivery end dipping into 25 mL H20 in 500 mL vol. flask. Collect ca 450 mL distillate, keeping vol. in distg flask nearly const with aid of small flame. After distn, wash delivery tube, and dil. distillate to vol. with H20. Into each of two 200 mL vol. flasks measure 20 mL 0.1N AgN03• To each flask add 12 drops HN03 (1+1) and 30 mL H20. To one flask add slowly, with const shaking, 30 mL KCN soln (3.1 g in 1 L H20). Dil. to vol., shake well, and filter thru dry filter. To 100 mL filtrate add 3 mL HN03 and 5 mL ferric indicator, 6.019(e). and titr. with 0.1N KSCN. Pipet 25 mL HCHO distillate into small beaker contg 30 mL of the KCN soln, mix well, and add slowly, with const shaking, to second flask contg the acidified AgN03 soln. Dil. to vol. with H20, filter, acidify 100 mL filtrate with 3 mL HN03, and titr. with the KSCN soln, using FeNH4(S04)2 indicator. Difference between mL KSCN soln used in these 2 titrns x 2 = mL 0.1N KSCN equiv. to HCHO. Calc. % HCHO present. 1 mL 0.1N KSCN = 3.003 mg HCHO. Ethion (O,O,O',O'·Tetraethyl S,S'·methylene bis· phosphorodithioate) High Performance Liquid Chromatographic Method (92) Official First Action (Applicable to dry and liquid formulations contg ethion as only active ingredient.) 6.352 Apparatus (a) Liquid chromatograph.-Waters Associates with Model 6000A pump, or equiv., with 254 nm UV detector (Waters Associates, Inc). Typical operating conditions: eluant flow rate 1 mL/min (ca 1100 psi), chart speed 0.25 in./min, detector sensitivity 0.2 A unit full scale, ambient temp, injection vol. 10 ILL. Adjust operating conditions to elute ethion peak in 6±2 min. Column condition and H20 content of MeOH eluant can change retention times. Ethion peak must be sepd completely from internal std peak which normally elutes in ca 7 min (Waters C'8 column). (b) Liquid chromatographic column.-Either (1) Waters ILBondapakC,8, 300 x 3.9 mm id; or (2) DuPont ODS Permaphase, 0.5 m x 2.1 mm id. 6.353 Reagents (a) Eluant.-Either (1) degassed MeOH-H20 (90+ 10), UV cutoff <230 nm, or (2) degassed acetonitrile-H2 0 (40+60), UV cutoff <230 nm. (b) Light mineral oil.-USP, viscosity 3S.1 centistokes at 37.S0. (c) Internal std soln.-(T) For Waters column.-Accurately weigh ca 0.24 g pentachloronitrobenzene (PCNB), ref. grade, with no interfering peaks on HPLC, into 200 mL vol. flask. Dil. to vol. with MeOH and mix. (2) For DuPont column.-Using CH3CN as solv., vary amt PCNB in internal std to give peak ht approx. same as ethion peak. (d) Ethion std solns.-(1) For Waters column.-Stock soln.- Accurately weigh amt of std equiv. to 250 mg ethion, 95+% pure (available from Chemical and Biological Investigations, Environmental Protection Agency, Beltsville, MD 20705) into 25 mL vol. flask, dil. to vol. with MeOH, and mix. Working soln.- Pipet 10 mL stock soln into 50 mL vol flask, pipet 10 mL internal std soln, (c)(T), into flask, dil. to vol. with MeOH, and mix. Prep. std and samples daily. (2) For DuPont column.-Prep. as above, using CH3CN instead of MeOH. (3) For oil formulations.-Pipet 10 mL 1% stock soln (1) or (2) into 50 mL vol. flask contg ca same wt of light mineral oil as sample. Add 20 mL MeOH (or CH3CN) and proceed as in 6.354(c) beginning with "Stopper and agitate ..." 6.354 Preparation of Sample (a) Dry powder.-Accurately weigh sample contg ca 100 mg ethion into 250 mL g-s flask. Pipet in 40 mL MeOH (or CH3CN) and 10 mL internal std soln. Shake 30 min on mech. shaker and centrf. to sep. phases. (b) Liquid concentrates.-Prep. sample as in 6.353(d). (e) Oil formulations.-Accurately weigh sample contg ca 100 mg ethion into 50 mL vol. flask. Add 30 mL MeOH (or CH3CN). Stopper and agitate vigorously 1 min, with side to side action, keeping mixt. in main body offlask. Pipet in 4 mL H20 and repeat vigorous mixing 1 min. Dil. to approx. vol. with MeOH (or CH3CN). Cool to ambient temp and dil. to vol. Mix thoroly by inverting 10 times and swirling vigorously each time. Centrf. to sep. phases.
- 135. AOAC METHODS (1980) MALATHION 113 6.355 Determination Use high-pressure liq. syringe or sample injection loop to inject 10 ILL portions of std until 2 peak ht ratios agree within ±1%. Alternately inject two 10 JLL portions each of sample and std solns. Measure peak hts and calc. avo peak ht ratios for both std and sample. Adjust attenuation or amt injected for convenient size peaks (6D-80% full scale). Measure peak hts from baseline between ethion and internal std peaks. % Ethion = (R/R') x (W'/W) x P, where Rand R' = avo peak ht ratios for sample and std, resp.; W' = mg ethion in working std soln (ca 100 mg); W = mg sample in final diln; and P = % purity of std. 6.356 Formothion [S-[2-{Formyl methylamino}-2-oxoethyl) O,O-dimethyl phosphorodithioate; O,O-Dimethyl S-{N-formyl-2-mereapto-N-methylaeetamide} phosphorodithioate) {93}-Offieial Final Action CIPAC-AOAC Method Reagents (a) Solvent I.-Toluene contg 2% Ac20. (b) Solvent II.-Hexane-acetone (2+1) plus 2% Ac20. (e) Internal std soln.-Prep. soln contg ca 100 mg, accurately weighed, of ethion/mL solv. I. Ethion must be >95% pure and contain no impurities interfering at formothion retention time. (d) Reference std soln.-Accurately weigh ca 500 mg For- mothion Ref. Std (Sandoz Ltd, Agrochemical Division, CH4002 Basel, Switzerland) into 50 mL vol. flask, add 5.0 mL internal std soln, and dil. to vol. with solv. I. 6.357 Apparatus (a) Gas chromatograph.-(Varian Aerograph 1520, or equiv.} With flame photometric detector (Tracor, FPD 100AT, or equiv.), automatic injector (Hewlett-Packard 7600 A. or equiv.), integrator (lnfotronics CRS 104, or equiv.), and effluent splitter at column end with ratio 1:100-1:1000 in favor of outlet. Use glass spiral column, 1.0 m x 3.6 mm id, packed with 3% OV 225 on ao-100 mesh Chromosorb W-HP. Operating conditions: temps (0)_ oven 210, injector and detector 220; N carrier gas 60 mL/min; no. theoretical plates for ethion is ca 2000. Alternatively, flame ionization may be used. Conditions are same, except effluent splitter is not necessary. (b) Bottles.-50 mL with Mininert valve, or equiv. inert system for closure (Pierce Chemical Co.). 6.358 Determination Accurately weigh well mixed sample contg ca 500 mg for- mothion into bottle, (b). Add 5.0 g internal std soln, (e). and dil. to 50 mL with soIv. I. Close tightly and shake. Transfer 6 JLL soln to vial contg 1 mL solv. II. Seal vial with inert valve system. (For automatic injections with Hewlett-Packard sampler, dil. in AI foil-sealed vials and use Teflon rubber laminated disks as septa.) Keep tightly closed. Inject 1.0 JLL dild mixt. into column, by- passing solv. around detector by using splitter to avoid contam- ination and deterioration. Det. appropriate time for splitting by test chromatogram. Compds may be identified by retention times relative to ethion as 1.00 (ca 4.4 min): formothion 0.50, dimethoate (by-product) 0.36. Inject 1 JLL aliquots of reference std soln, (d), until ht or area ratio of formothion to ethion varies <2% for successive injec- tions. Precede and follow each sample by reference std soln and make 3 s~p. detns with all peak area ratios of reference std solns within ±2% of first accepted values. % Formothion = W' x H x f x P/W x H', where Wand W' = mg sample and internal std, resp.; Hand H' = peak hts or areas of formothion and internal std, resp.; P = % formothion in reference compd; f = correction factor = w x h'/w' x h; where wand w' = mg formothion ref. std and internal std, resp., and hand h' = peak hts or areas of formothion and internal std, resp. 6.359 Malathion {O,O-Dimethyl Dithiophosphate of Diethyl Mereaptosueeinate} (Caution: See 51.011 and 51.061.) Argentimetric Method {94}-Official First Action Principle Malathion is cleaved in alk. soln to dimethyl phosphorodi- thioate ion which forms insol. ppt with Ag ion. Pos. bias may be encountered. 6.360 Reagents (a) Potassium hydroxide soln.-1N. Dissolve 28 g KOH in 500 mL alcohol. (b) Silver nitrate soln.--o.1N. Prep. as in 50.027 and stdze against primary NaCI as in 6.363. (e) Cellulose powder.-Reeve Angel No. CF-ll. 6.361 Apparatus (a) Potentiometer.-Recording potentiometric titrator, oper- ated in derivative mode (Metrohm Models E336 thru E576. or equiv.; available from Brinkman Instruments). or pH meter with mv scale. (b) Glass reference electrode with Ag/ AgCI internal ele- ment.-Corning Glass Works, No. 476022. (e) Silver billet electrode.-Beckman Instruments, No. 39261. (d) Chromatographic tube.-30 cm x 13.5 mm id. 6.362 Preparation of Samples (a) Technical grade malathion or malathion emulsifiable con- centrates.-Place 2 g cellulose powder in 50 mL beaker, add 0.6 g H20 dropwise, and mix thoroly. Place plug of glass wool in bottom of chromatgc tube and pack with the wetted cellulose powder. Compress column to 5 cm with glass rod. Wet column with 5 mL hexane. Accurately weigh sample contg 0.5±0.1 g malathion into 250 mL beaker, add 50 mL hexane, and stir 10 min, using mag. stirrer. Transfer to column and collect eluate in another 250 mL beaker. Rinse original beaker with three 20 mL portions hexane and pour each thru column. Carefully evap. eluate just to dryness. Add 50 mL alcohol or isopropanol and stir mag.; add 20 mL IN KOH and stir 1 min more. Add 50 mL H20 and 20 mL 2N HN03 , and stir. (b) Powder formulations.-Accurately weigh sample contg 0.5±0.1 g malathion into 250 mL beaker, add 50 mL hexane, cover with watch glass, and stir mag. 20 min at high speed without splashing. Filter thru fiber glass paper in 4.25 cm buchner and Quant. recover filtrate. Thoroly wash beaker and funnel with two 20 mL portions hexane. Quant. transfer filtrate to 250 mL beaker and evap. hexane just to dryness. Add 50 mL alcohol or isopropanol and stir mag.; add 20 mL IN KOH and stir 1 min more. Add 50 mL H20 and 20 mL 2N HN03, and stir.
- 136. 114 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) 6.363 Determination (a) pH meter.-Immerse electrodes in soln and set pH meter to read absolute mv. Titr. with 0.1N AgN03 soln until meter reads ca 520 mv (within 1-2 mL of end point). Continue titrn by dropwise addn of titrant to end point, 425 mv. (b) Recording potentiometric titrator.-Set instrument in de- rivative mode and titr. at 2.0±0.2 mL 0.1N AgN03 soln/min to within ca 2 mL of expected end point. Decrease rate to 0.7±0.1 mL/min and continue titrn. Take first inflection in titrn curve as end point. % Malathion = (V x N x 33.04)/W, where V = mL AgN03 soln, N = normality of AgN03 soln, and W = g sample. Colorimetric Method (95)-Official First Action (Caution: See 51.018,51.040,51.041,51.043, and 51.051.) 6.364 Principle Malathion, S-(1,2-d icarbethoxyethyl) 0, O-dimethyl phospho- rodithioate, is decomposed by alkali in alcohol to Na 0,0- dimethyl phosphorodithioate (NaDMTA), Na fumarate, and al- cohol. NaDMTA is converted to Cu+2 complex sol. in cyclohexane with formation of intense yellow compd whose intensity is proportional to concn of O,O-dimethyl phosphorodithioic acid and which is measured colorimetrically at 420 nm. Pos. bias may be encountered. 6.365 Precautions Vol. of nonaq. solns is highly temp. dependent. Maintain all reagents at uniform temp. All glassware must be clean and dry. Rinse tubes and pipets with MeOH and oven dry before use. After use, rinse all glassware in contact with Cu reagent with acetone before washing to prevent contamination in future analyses. If diffi- culties are still encountered, use 1% HCI in MeOH as wash prior to oven drying. Det. A of blank against H20 whenever new reagents are prepd. Deviation from range 0.010--0.020 A units, using 1 cm cells, indicates either contamination of glassware or reagents, or reagents of improper concn. 6.366 Reagents (a) Cyclohexane.-Pass thru column of activated silica gel or A120 3, activity grade I. Accuracy of assay depends on stability of Cu complex which undergoes oxidn-reduction reaction cat- alyzed by strong proton donors. Net result is fading of developed color. Polar impurities in cyclohexane contribute to this problem and quality of solv. must be checked as follows before continuing with analysis: Perform detn, 6.368, with std soln (b)(1) or (2) and det. A at 2 min and again at 12 min. Calc. fade rate (FR) as follows: FR = [(A2 min - A'2 min) X 100l/(A2 min X 10) Fade rate should be ~0.5%/min for std or samples. If fade rate exceeds that limit, pass cyclohexane thru silica gel or AI20 3 column contg ca 100 g adsorbent/2 L cyclohexane to be treated. Ratio of ht of adsorbent bed to its diam. should be >5 and flow should be ~3 mL/min. Retest cyclohexane after treatment to det. its acceptability. Vol. of cyclohexane that can be treated in this manner will depend on extent of contamination. Check purity periodically. (b) Analytical stds.-Use either malathion or KDMTA, anal. grade of known purity (available from American Cyanamid Co.), for std soln. Use of malathion will provide assurance that quant. elimination reaction is taking place. Store malathion in refrig- erator, warming to room temp. before use. Store KDMTA in desiccator, avoiding elevated temp. (1) Malathion std solns.-Accurately weigh 130--170 mg mal- athion into tared 50 mL vol. flask. Dissolve in and dil. to vol. with cyclohexane. Store in refrigerator; use at room temp. (2) Potassium O,O-dimethyl phosphorodithioate (KDMTA) std solns.-Accurately weigh 80--120 mg KDMTA into tared 50 mL vol. flask. Dissolve in and dil. to vol. with acetone. Mix. Store in tightly stoppered flask. (e) Copper reagent.-Dissolve 410--430 mg cupric naphthen- ate (lCN-K&K Laboratories, Inc., No. 8172) or 8% Cu Nap-Allliq. (Mooney Chemicals Inc., 2301 Scranton Rd, Cleveland, OH 44113) in 100 mL cyclohexane. (d) Ethyl acetate.-Contg ~0.2% H20 and with acidity ~0.005% expressed as HOAc. (e) Sodium hydroxide soln.-1N. Dissolve 4 g carbonate-free NaOH in 100 mL absolute alcohol or absolute ethanol denatured with 0.5% benzene. (f) Acetonitrile.-Bp 80--82°. Pass thru column of silica gel. discarding yellow first portions of eluate and collecting colorless eluate. pH of 10% aq. soln should be 5-7. 6.367 Preparation of Sample (a) Technical materials and emulsifiable concentrates.-Ac- curately weigh sample contg 130--170 mg malathion into tared 50 mL vol. flask. Dissolve in and dil. to vol. with cyclohexane. Stopper flask and mix well. (b) Wettable powders and dusts.-Accurately weigh sample contg 130--170 mg malathion on tared weighing paper. Transfer to 8 oz narrow-mouth bottle fitted with Vinylite-lined screw caps. Add 100 mL cyclohexane or acetone, using vol. flask. Place sample 10 min on reciprocating shaker set for moderate agita- tion. Let solids settle or centrf.{ if necessary. Alternatively, use 150 mL g-s erlenmeyer and mag. stirrer. Cyclohexane will not quant. ext malathion from powders formulated with bentonite (montmorillonite, AI2(Si40 lO)(OH)2). and possibly with other carriers. In these cases, ext malathion with CH3CN or tetrahydrofuran. If CH3CN is used, evap. ext to dryness in rotary evaporator under vac. at ~50°. Similarly evap. appropriate vol. CH3CN for reagent blank. Carefully dissolve residue in cyclohexane and proceed with detn. Iftetrahydrofuran is used, proceed as directed, incorporating tetrahydrofuran reagent blank. Use alternative extn technics whenever it is not known whether method will give adequate extn. 6.368 Determination Pipet 5 mL aliquots from (a) or 10 mL aliquots from (b) and appropriate dild stds to sep. 50 mL vol. flasks. Prep. reagent blanks by transferring 5 or 10 mL aliquots cyclohexane to 50 mL vol. flask. Use acetone in reagent blank if KDMTA is std. At 1 min intervals, add 2 mL IN alc. NaOH to each flask in sequence, rinsing any sample adhering to neck or sides of flask into liq. in flask. Mix well by swirling. Stopper flasks and let stand 10 min. Do not have >9 flasks in a series. After 10 min, slowly dil. each to vol. with EtOAc, gently swirling flask during addn. (Voluminous ppt will form at this time.) Stopper flask and mix well. Complete this process for each flask before adding EtOAc to next. Let stand 10--20 min with occasional mixing. Remix contents of flask and pour portion into centrf. tube. Stopper tube and centrf. 5 min. (Supernate should be clear; recentrf. if necessary.) Transfer 5 mL to 50 mL vol. flask, taking care not to transfer any pptd NaOAc which may remain on surface of soln after centrfg.
- 137. AOAC METHODS (1980) MALATHION 115 Add 35--40 mL cyclohexane to reagent blank followed by 2 mL Cu reagent and immediately dil. to vol. with cyclohexane. Stopper flask and mix. Use this soln to zero spectrophtr, using 1 cm cells at max. A, ca 420 nm. Proceed as above for sample. Det. A against reagent blank exactly 2 min after adding Cu reagent. Slight haze may appear on initial diln with cyclohexane. If it does not clear after adding Cu reagent, check for presence of excessive H2 0 in analysis. Water can be introduced by EtOAc, acetone, CH3CN, alcohol, tetrahydrofuran, or contaminated glassware. Correct this con- dition before proceeding with analysis. 6.369 Calculations (a) Absorptivity of samples (a).- (1) Tech. malathion and emulsifiable concs a = (A x 50 x 50 x 50)/(mg sample x 5 x 5) = (A x 5000)/mg sample (2) Wettable powders and dusts a = (A x 100 x 50 x 50)/(mg sample x 10 x 5) = (A x 5000)/mg sample (b) Absorptivity of stds (a').- (1) Malathion: Use formula in (a)( 1). (2) Potassium O,O-dimethyl phosphorodithioate a' =[(A x 50 x 50 x 50)/(mg sample x 5 x 5)] x 0.5939 = (A x 2969)/mg sample, where 0.5939 is factor to convert K salt to equiv. wt malathion. (In general, a of stds are in range 17.2-17.6, corrected for quality of std, at concn 1 mg/mL in 1 cm cell.) (3) % Malathion = (a x % purity of std)/a' 6.370 Preparation of Standard Curve (Optional) Transfer 3, 4,5, and 6 mL aliquots centrfgd EtOAc soln of std, 6.368, par. 3, into sep. 50 mL vol. flasks. Proceed with color development, and plot A against wt malathion in aliquot. Use std curve with considerable caution, since fade rate may vary from day to day, depending on solvs used for analysis. Std curve is not appropriate where fluctuations in temp. occur in laboratory environment. 6.371 Interferences Test for free O,O-dimethyl phosphorodithioic acid or other interfering components in sample ext as follows: Transfer 2 drops ext to test tube, dil. with ca 10 mL cyclohexane, add 8 drops Cu reagent, and mix. Development of discernible yellow in soln indicates necessity of obtaining sample-blank correction. Obtain sample-blank correction as follows: Transfer 5 or 10 mL aliquot sample to 50 mL vol. flask and dil. to vol. with EtOAc. (Do not add alc. NaOH soln.) For color development, transfer 5 mL dild sample soln and continue as in 6.368. Correct A for base-treated sample and proceed with calcn. Gas Chromatographic Method (96)-Official First Action 6.372 Apparatus (a) Gas chromatograph.-With glass column, on-column in- jection system, flame ionization detector, and electrometer with sensitivity of ~1O-11 amp driving 1 mv recorder. Drift should be <1%/hr. Totally solid state amplifier with FET input is recom- mended. Electronic digital integrator or computer calcd area measurements must be used. Integrator should have independ- ent controls for selection of up and down slope sensitivities so that start and stop integration points can be selected. Automated sample injection system contributes significantly to precision. Hewlett-Packard Model 7600 is suitable when equipped as described. Equiv. instrumentation may be used but may require modification of operating conditions to obtain good peak shape, adequate resolution, and appropriate retention times. Typical conditions for Hewlett-Packard Model 7600 (instru- ment may have to be adjusted to give complete resolution of well shaped peaks): Cycle timers (min): analysis and stop integrate, 16; range, 103; temps (0): oven 180, injection port 200, flame detector 300; gas flow rotameters (mL/min): H 35, air 425, He carrier gas 30; integrator settings (adjusted so that deflections on slope meter do not exceed ±50% before injec- tion): noise suppression max., slope sensitivity up and down 0.1, BL reset delay 0.15, area threshold 1000; retention times (min): malathion 10, internal std 6, min. time between malathion and internal std 3.5. (b) Column.-Borosilicate glass tube 1.22 m (6') x 4 mm id, 6 mm od, bent to fit chromatograph and packed with 5% SP- 2401 or OV-210 on Gas-Chrom Q or Supelcoport (100/120 mesh). Can be purchased as prepd packing from Supelco, Inc., (specify "Pesticide Grade"); Alltech Associates, 202 Campus Dr, Arling- ton Heights, IL 60006; and Applied Science Laboratories, Inc. Use exclusively for malathion analysis. (e) Glass wool.-Silane treated. (No. 14502, Applied Science Laboratories, Inc.) (d) Syringes.-10 ILL, Series 700, Hamilton Co. (e) HI-EFF Fluidizer.-Applied Science Laboratories, Inc. 6.373 Reagents (a) Internal std soln.-1.2% m-Diphenoxybenzene in CHCI3 • Must not contain any impurities which elute at or near malathion peak. Bring soln to consistent temp. above ambient (e.g. 25°) before taking aliquots. (b) Malathion std solns.-Accurately weigh ca 170, 200, and 230 mg malathion std (anal. grade, available from American Cyanamid Co.) into sep. preweighed 25 mL vol. flasks. Add by pipet 5 mL internal std soln and dil. to vol. with CHCI3 • Label A, B, and C. Soln B is working std soln for detn; solns A and C are used for linearity check and to guard against weighing error in prepn of working std soln. Solns are stable ca 4 weeks if kept tightly sealed in refrigerator. Warm to room temp. before use. Soln B can be prepd independently of solns A and C, if conditions of linearity check are met. 6.374 Preparation and Conditioning of Column Weigh 6.25 g of trifluoropropylsilicone (SP-2401 or' OV-210) in 250 mL beaker and dissolve in 125 mL EtOAc. Stir to obtain vortex and add 25 g solid support (Gas-Chrom Q or Supelcoport, 100/120 mesh) with continued agitation. Filter slurry thru What- man No.1 paper, or equiv., on buchner, using gentle vac. to minimize evapn of soIv. Continue filtration until drop rate is ca 1/sec. Transfer packing to HI-EFF Fluidizer, connect source of N thru pressure reducer to base, and place fluidizer on controlled temp. hot plate set for 75°. Continue gas flow until soIv. vapors can no longer be detected by odor, taking care that packing is not blown out top of fluidizer. To pack column, attach 75 mm funnel to exit end of prebent glass tube. Tap tube with pencil or small wooden rod, and add prepd packing in small amts until exit end is filled to ca 15 mm from end. Move funnel to entrance end of column. Insert pledget of silane-treated glass wool in exit end and attach source of moderate vac. to this end. Continue to add packing slowly with tapping until tube is filled to ca 20 mm from entrance end. Insert pledget of silane-treated glass wool in entrance end, compress- ing it only enough to hold it in place. Condition column with He carrier gas flowing at 30 mL/min
- 138. 116 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) :315 hr (overnight) at 255° or ca 20° below max. temp. recom- mended for liq. phase. Exit end of column should not be connected to detector during this conditioning. Connect exit end of column to detector, adjust controls to conditions given in 6.372(a). and let instrument come to equi- librium. Inject 3 ILL aliquots std soln C until :33 consecutive injections give response ratios agreeing within 2%. 6.375 Linearity Check Check gas chromatograph for linearity at least weekly, when- ever new std solns are prepd, and whenever column, new or used, is newly installed in instrument. Using digital integration for peak area measurements, det. appropriate attenuation setting and injection aliquot (2-4 ILL) of std soln B to give area count of :3100,000 counts (optimum electrometer output with acceptable noise level). Use conditions so detd for all samples and stds in series. Inject triplicate aliquots of detd vol. of std solns A, B, and C into chromatograph, det. response ratio for each, and aver- age ratios for each soln. Divide av. ratio for each soln by corresponding malathion content in mg. Ratio/mg should agree within 2%. Failure to meet this specification indicates either weighing error in prepn of a std soln or instrumental difficulties which must be corrected before proceeding with analysis of samples. 6.376 Preparation of Sample (Analyze samples at least in duplicate) (a) Liquid formulations and technical materials.-Accurately weigh sample contg ca 200 mg malathion into preweighed 25 mL vol. flask. Pipet in 5 mL internal std soln, dil. to vol. with CHCI3, and mix well. (b) Solid formulations containing 10% or more ofmalathion.- Accurately weigh sample contg ca 1.0 g malathion and transfer to 200-250 mL (8 oz) bottle. Pipet in 50 mL CHCI3, stopper tightly, and shake on reciprocating shaker 30 min. Let settle ca 15 min; if not clear, centrf. Layer of solids will float at interface. Avoid entrainment of particles by exerting pos. pressure from bulb on pipet while it is carefully inserted into soln for removal of aliquot. Particles in final soln can clog syringe needle. Transfer 10 mL aliquot clear soln to 25 mL vol. flask, pipet in 5 mL internal std soln, dil. to vol. with CHCI3, and mix well. (c) Solid formulations containing less than 10% malathion.- Accurately weigh sample contg ca 400 mg malathion and transfer to 500 mL (16 oz) bottle. Add exactly 200 mL CHCI3 and shake 30 min on reciprocal shaker. Let settle, observing precau- tions given in (b). Pipet 100 mL aliquot to 500 mL r-b flask and evap. to dryness. Pipet in 5 mL internal std soln and 20 mL CHCI3, swirl to dissolve residue, and mix well. 6.377 Determination Inject duplicate aliquots of appropriate vol. of std soln B as detd in linearity check, 6.375. Response ratios should agree within 2%; if not, repeat with 2 more injections. Failure to meet specification with second pair of injections indicates instru- mental difficulties which must be resolved before proceeding with analysis. Inject duplicate aliquots of each sample soln of same vol. as std soln. Average response ratios for each sample. Precision considerations stated for std soln also apply to sample soln injection response. Inject duplicate aliquots std soln B after every 2 sample solns. Average response ratios of stds immediately before and after sample solns. Use this av. to calc. malathion content of the 2 sample solns. Each detn of av. response ratio for std soln B should yield value within 2% of previously detd value. Failure to meet this specification indicates instrumental drift which must either be corrected or compensated for by more frequent measurements of response of std soln B. In extreme cases, follow each sample injection with std injection but this would indicate an instability which should be corrected at once. 6.378 Calculations For each sample injection, calc. response ratio: R = area of malathion peak/area of internal std peak % Malathion = (R/R') x (W'/W) x P x D where R' and R = av. response ratio for std soln B and sample soln, resp; W' and W = g malathion std and sample, resp.; P = % purity of malathion std; and D = diln factor (1 for liqs; (50/10)(25/25) 5 for solids :310% malathion; and (200/100)(25/25) = 2 for solids <10% malathion). Parathion Gas Chromatographic Method (97)-Official First Action (Not applicable to dusts and powders) 6.379 Standard Solutions (a) Dipentyl phthalate internal std soln.-Dissolve 2.0±0.1 g dipentyl phthalate (Eastman Kodak Co., No. P2473, or equiv.) in CS2 and dil. to 500 mL with CS2• (b) Parathion stdsoln.-Accurately weigh ca 125 mg parathion (Monsanto Chemical Co., or equiv.) into 50 mL g-s erlenmeyer, pipet in 25 mL internal std soln, and mix thoroly. 6.380 Preparation of Sample Accurately weigh sample contg ca 125 mg parathion into 50 mL g-s erlenmeyer. Pipet in 25 mL internal std soln and mix thoroly. 6.381 Gas Chromatograph See 6.402. Column should have :31200 theoretical plates for parathion. Vary attenuation and injection vol. (1-2 ILL) so that peak hts of parathion and dipentyl phthalate are 60-80% full scale on 1 mv recorder. Retention times for parathion and dipentyl phthalate are 6-8 and 8-10.5 min, resp. 6.382 Determination Proceed as in 6.403, except substitute parathion for Me parathion and dipentyl phthalate for p,p'-DDE. 6.383 Calculations Proceed as in 6.404, except substitute parathion for Me parathion and delete F from equation. 6.384 High Pressure Liquid Chromatographic Method (97) Official First Action (Not applicable to dusts and powders) Apparatus (a) Liquid chromatograph.-5ee 6.405(a), except use eluant flow rate of 1.5 mL/min (ca 800 psi). (b) Liquid chromatographic column.-5ee 6.294(b).
- 139. AOAC METHODS (1980) PARATHION 117 6.385 Reagents (a) Choroform.-See 6.406(a). (b) Eluant.-Stir 500 mL CHCI3 on mag. stirrer 3-4 min under moderate vac. (ca 350 mm Hg). (c) Internal std soln.-Accurately weigh ca 110 mg benzo- phenone (MC/B Manufacturing Chemists, No. BX0410, or equiv.) into 250 mL vol. flask, and dissolve and dil. to vol. with CHCI3 • (d) Parathion std solns.-(1) Stock soln.-1500 ~g/mL. Ac- curately weigh ca 75 mg anal. grade parathion (Monsanto Chemical Co., or equiv.) into 50 mL vol. flask, and dissolve and dil. to vol. with CHCI3 • (2) Working soln.-(150 ~g parathion + 44 ~g benzophenone)/mL. Pipet 5 mL stock soln and 5 mL internal std soln into 50 mL vol. flask, and dil. to vol. with CHCI3 • 6.386 Preparation of Sample Accurately weigh sample contg ca 75 mg parathion into 50 mL vol. flask, and dissolve and dil. to vol. with CHCI3• Pipet 5 mL sample soln and 5 mL internal std soln into 50 mL vol. flask, and dil. to vol. with CHCI3 • 6.387 Determination Proceed as in 6.408, except substitute parathion for Me parathion and benzophenone for acetophenone, and delete F from equation. Retention times for parathion and benzophenone are 4.0-5.5 and 7-9 min, resp. Volumetric Method (98)-Official First Action (Applicable to dusts and powders only. Caution: See 51.041.) 6.388 Apparatus (a) Photoelectric colorimeter.-With filter to give max. T be- tween 400 and 450 nm. Spectrophtr set at 405 nm may also be used. (b) Potentiometer.-With adapter for outside Pt and calomel electrodes. Dead-stop end point equipment may also be used. 6.389 Reagents (a) Zinc dust.-Low in Fe. (b) Sulfanilic acid.-Anhyd. recrystd material. Check purity by N detn. (c) p-Nitrophenol.-Mp 112-113°. (d) Sodium nitrite std soln.-O.1N. Stdze weekly. Accurately weigh 0.4--0.45 g of the sulfanilic acid into 400 mL tall beaker. Add 80 mL H20, 10 mL HCI, 30 mL HOAc, and 5 g NaBr. Place electrodes and mech. stirrer in reaction mixt. and titr. with the 0.1N NaN02• Add in 5 mL portions until within 1 mL of calcd end point; then add NaN02 soln in 0.1 mL portions until max. rise in potential is obtained. At first, 3-5 min is required for potential to become const; as end point approaches, espe- cially after 0.1 mL addns, reaction should be complete within 1 min. As alternative, dead-stop end point technic may be used (98), or following spot test, adding NaN02 soln in 4 drop portions near end point: Dip glass rod into soln being titrd and touch rod quickly to piece of KI-starch paper, (e). End point is reached when intense blue-black color appears immediately and can be obtained repeatedly during 1 min period without further addn of NaN02• Normality NaN02 soln = g sulfanilic acid x 1000/(mL NaN02 x 173.2). (e) Starch iodide paper.-Triturate 10 parts starch with 200 parts H2 0, bring to bp, and add 1 part KI. Impregnate strips of filter paper with this soln, dry, and preserve in g-s bottles. 6.390 Preparation of Standard Curve of p-Nitrophenol Accurately weigh 100 mg p-nitrophenol, transfer to 1 L vol. flask, and dil. to vol. with 0.1N NaOH. Transfer 2, 4, 6, 8,10, and 20 mL aliquots of this soln to 100 mL vol. flasks and dil. each soln to vol. with 0.1N NaOH. Read A of each soln in photoelec. colorimeter (400-450 nm) or spectrophtr (405 nm) against H20 as ref. Plot A against concn in mg/mL. 6.391 Preparation of Sample Dust preparations and wettable powders.-Transfer weighed sample to thimble and ext with 150 mL ether in Soxhlet app. 1 hr. Transfer ether ext to 250 mL separator, and sep. p-nitrophenol and parathion as in 6.392. Det. sample size by parathion concn as follows: 10%,6.75 g; 15% 4-5 g; 25%,2.5-3.5 g. 6.392 Separation of Parathion and p-Nitrophenol Ext ether soln with four (or until ext is colorless) 20 mL portions chilled 1% Na2C03 soln, collecting combined aq. layers in 200 mL vol. flask. Transfer ether layer to 400 mL tall beaker, rinsing separator with small portions ether. 6.393 Determination of p-Nitrophenol Add 20 mL 1N NaOH to combined aq. exts and dil. to vol. with H20. MeasureA of soln as in 6.390 and read concnp-nitrophenol in mg/mL from std curve. % p-Nitrophenol = (mg/mL) x 200 x 100/(1000 x g sample). 6.394 Determination of Parathion (Caution: See 51.011, 51.039, and 51.054.) Add 35 mL HOAc-HCI mixt. (9+ 1) to ether soln, 6.392. Add 2 g Zn dust, cover beaker with watch glass, and gently heat soln on steam bath 45 min, or until most of ether evaps and soln is colorless. Add 30 mL HCI and heat 10 min longer to complete soln of Zn dust. Wash down beaker and watch glass with H2 0. Filter reduced mixt. thru paper and rinse beaker thoroly with H20. Oil. to 125 mL and cool to room temp. Add 5 g NaBr (or KBr) and titr. with 0.1N NaN02 as in 6.389(d). % Parathion = mL NaN02 x normality x 29.13/g sample. 6.395 Colorimetric Method (99)-Official First Action (Applicable to dusts and powders only) Principle Parathion is extd with alcohol and hydrolyzed with KOH to form K p-nitrophenate, which is detd calorimetrically. 6.396 Preparation of Standard Curve Weigh 60 mg reagent grade p-nitrophenol into 100 mL vol. flask, dissolve in alcohol, and dil. to vol. with alcohol. Pipet 10 mL into 100 mL vol. flask and dil. to vol. with alcohol. Prep. p- nitrophenol stds contg 0.3, 0.18, and 0.06 mg/100 mL by pipetting 5, 3, and 1 mL aliquots, resp., of second diln into sep. 100 mL vol. flasks, adding from pipet 5 mL 1N KOH in 50% alcohol, and dilg to vol. with 50% alcohol. MeasureA at 405 nm in 1 cm Corex cells against 50% alcohol and plot A against concn. 6.397 Preparation of Sample Weigh sample contg ca 10 mg parathion into 250 mL g-s flask. Pipet in 100 mL alcohol and shake occasionally during 10 min. Filter ca 25 mL into g-s container.
- 140. 118 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) 6.398 Determination of Free p-Nitrophenol Pipet 10 mL aliquot of above soln into 100 mL vol. flask and dil. to vol. with 50% alcohol. Add 5 drops IN KOH in 50% alcohol, and measure A at 405 nm within 2 min against 50% alcohol. Calc. free p-nitrophenol. 6.399 Determination of Parathion Pipet 5 mL filtered soln into 125 mL g-s flask, pipet in 5 mL IN KOH in 50% alcohol, and add glass beads to prevent bumping. Reflux ;.30 min. Cool, and transfer to 100 mL vol. flask with 50% alcohol. Dil. to vol. with 50% alcohol and measure A as in 6.396. Calc. parathion, using std curve, dilns, and factor: Parathion =p-nitrophenoI/0.478. Correct for freep-nitrophenol. Methyl Parathion Gas Chromatographic Method (tOOl-Official First Action 6.400 Standard Solutions (a) p,p'-DDE internal std soln.-Dissolve 5.0±0.1 g 2,2-bis(p- chlorophenyl)-l,l-dichloroethylene (p,p'-DDE, No. 12.389-7, Ald- rich Chemical Co., Inc., or equiv.) in CS2 and dil. to 1 L with CS2• (b) Methyl parathion std soln.-Accurately weigh ca 125 mg Me parathion (Monsanto Chemical Co., 800 N Lindbergh Blvd, St. Louis, MO 63166, or equiv.) into 50 mL g-s erlenmeyer, pipet in 25 mL internal std soln, and mix thoroly. 6.401 Preparation of Sample (a) Liquid.-Accurately weigh into 50 mL g-s erlenmeyer sample contg ca 125 mg Me parathion. Pipet in 25 mL internal std soln and mix thoroly. (b) Wettable powder.-Accurately weigh into 100-150 mL (4 oz) round bottle sample contg ca 625 mg Me parathion. Pipet in 50 mL CHCl3-acetone (9+1), cap, and shake mech. 30 min. Let settle and pipet 10 mL supernate into 50 mL g-s erlenmeyer. Place erlenmeyer in 550 H2 0 bath and evap. solv. under stream of dry air or N. Pipet in 25 mL internal std soln and mix thoroly. 6.402 Gas Chromatograph Use instrument equipped with flame ionization detector and 1.2 m x 4 (id) mm glass column packed with 1.5% SE-30 plus 1.5% OV-210 on 80-100 mesh Gas-Chrom Q. Prep. column by accurately weighing ca 0.12 g SE-30 and ca 0.12 g OV-210 into 250 mL beaker. Add 50 mL CHCl3-acetone (3+2), cover with watch glass, and heat on steam bath until stationary phases are dissolved. Speed dissoln of SE-30 by spreading material on walls of beaker with small spatula or stirring rod. Add enough 80-100 mesh Gas-Chrom Q to yield 1.5% of each phase on sol id support. Heat on steam bath, stirring frequently until all solv. is removed. Air dry 2-3 hr. Pack in column and condition 24 hr at 2450 with N or He at 30 mLjmin. Column should have ;.1200 theoretical plates for p,p'-DDE. Typical operating conditions: temps (D)-inlet 210, column 180±10, detector 250; N or He carrier gas, 55--75 mLjmin; air and H as specified by manufacturer; attenuation and injection vol. (1-2 p,L) varied so that peak hts of Me parathion and p,p'- DDE are 60-80% full scale on 1 mv recorder. Retention times for Me parathion and p,p'-DDE are 3.5--5.5 and 6-8 min, resp. 6.403 Determination Inject aliquots of std soln until peak ht ratio of Me parathion: p,p'-DDE varies ~1% for successive injections. Then make duplicate injections of sample followed by duplicate injections of std. Peak ht ratios of stds must be within ± 1% offirst accepted std values or repeat series of injections. Repeat for addnl samples. 6.404 Calculations Calc. peak ht ratios for both duplicate std injections preceding and following samples. Average the 4 values (R'). Calc. and average peak ht ratios of the 2 samples (R). % Me parathion = (RjR') x (W'jW) x F x P, where Wand W' = mg sample and std, resp., F = 1 for liq. and 5 for wettable powder samples; and P = % purity of std. High Pressure Liquid Chromatographic Method (101) Official First Action 6.405 Apparatus (a) Liquid chromatograph.-Waters Model ALC 202jGPC 204 (Waters Associates, Inc.), or equiv., with 254 nm UV detector and 10 mv recorder. Typical operating conditions: eluant flow rate, 1.2 mLjmin (ca 700 psi); detector sensitivity, 0.16 A unit full scale; temp., ambient; valve injection vol., 10 p,L. (b) Liquid chromatographic column.-See 6.294(b). (c) Chromatographic tubes.-Glass, 900 x 25 (id) mm, with coarse porosity frit in bottom (SGA Scientific, Inc., No. JC-2650, or equiv.). 6.406 Reagents (a) Chloroform.-Alcohol-free with <0.01 % H20 (Burdick & Jackson Laboratories, Inc., distd in glass, or equiv.). (b) Silicic acid-water.-75% (wjv). Add 25 mL H20 to 75 g silicic acid (Mallinckrodt Chemical Works, Code 2847, or equiv.), and shake until lumps disappear. (c) Water-saturated chloroform.-Shake 700 mL CHCI3 with 150 mL H20 2-3 min, and pass thru 900 x 25 mm glass tube packed with 100 g silicic acid-H20. (d) Eluant.-Blend 200 mL H20-satd CHCI3 with 300 mL CHCI3 on mag. stirrer 2-3 min under moderate vac. (ca 350 mm Hg). (e) Internal std soln.-Accurately weigh ca 115 mg aceto- phenone (MCjB Manufacturing Chemists, No. AX0164, or equiv.) into 250 mL vol. flask, and dissolve and dil. to vol. with CHCI3 • (f) Methyl parathion std solns.-(1) Stock soln.-700 p,gjmL. Accurately weigh ca 70 mg anal. grade Me parathion (Monsanto Chemical Co., or equiv.) into 100 mL vol. flask, and dissolve and dil. to vol. with CHCI3• (2) Working soln.-(70 ttg Me parathion + 46 p,g acetophenone)jmL. Pipet 5 mL stock soln and 5 mL internal std soln into 50 mL vol. flask, and dil. to vol. with CHCI3 • 6.407 Preparation of Sample Accurately weigh ca 95 mg tech. Me parathion into 100 mL vol. flask, or accurately weigh emulsifiable sample contg ca 35 mg Me parathion into 50 mL vol. flask, and dil. to vol. with CHCI3 • Pipet 5 mL sample soln and 5 mL internal std soln into 50 mL vol. flask, and dil. to vol. with CHCI3• 6.408 Determination Pump sufficient eluant thru column to equilibrate system. Inject 10 ttL working std soln onto column thru sampling valve, and adjust operating conditions to give peak hts 60-80% full scale and retention times of 3.5--5.0 and 5.5--8.0 min for Me parathion and acetophenone, resp. Repeat injections until ratio of Me parathion to acetophenone peak hts is within ± 1% of previous injection. Without changing conditions, alternately inject 10 ttL aliquots of working std soln and duplicate 10 p,L aliquots of sample soln until peak ht ratios for sample soln vary
- 141. AOAC METHODS (1980) PHORATE 119 ~1% for successive injections. Average last 2 peak ht ratios for sample and for std, resp., and calc. % Me parathion. % Me parathion = (R/R') x (W'/W) x (p/n where Rand R' = avo peak ht ratios of Me parathion to acetophenone for sample and std, resp.; Wand W' = mg sample and std, resp.; P = % purity of std; and F = 1 for tech. and 2 for emulsifiable samples. Methyl Parathion in Water-based Microencapsulated Formulations Gas Chromatographic Method (t02)-Official First Action 6.409 Principle Me parathion is released from microcapsules by grinding, and is extd into CH3CN. Dimethoate is added as internal std and concn of Me parathion is detd by flame ionization GLC. 6.410 Apparatus (a) Gas chromatograph.-Perkin-Elmer Model 900, or equiv., with flame ionization detector, glass lined injection port, 1 mv strip chart recorder, and 1.8 m x 2 (id) mm glass column packed with 3% OV-17 on 80-100 mesh Supelcoport (Supelco, Inc.). Typical operating conditions: temps (0): column 200, injection port 225, detector 250; flow rates (mL/min): He carrier gas 35, air 400, H optimize for max. sensitivity; sample: 1 ~L CH3CN contg dimethoate and Me parathion with retention times of ca 3 min and 4 min, resp. Injection vol. may be varied to give peak hts 50-90% of full scale. (b) Sample grinder.-Spex Industries Mixer/Mill No. 8000 (Spex Industries, Inc., PO Box 798, Metuchen, NJ 08840) or 40 mL Corning 7726 glass tissue grinder (No. 441969, Corning Glass Works). (c) Weighing dishes.-With natural AI surface towhich sample does not stick (Fisher Scientific Co., No. 8-732, or equiv.). 6.411 Reagents (a) Dimethoate.-Cygon® insecticide, anal. grade (obtainable from American Cyanamid Co.). (b) Methyl parathion.-Anal. grade (obtainable from Mon- santo Chemical Co.). 6.412 Determination of Correction Factor Prep. 2 duplicate std solns by accurately weighing ca 0.1 g Me parathion and ca 0.1 g dimethoate directly into same 50 mL vol. flask, and dilg to vol. with CH3CN. Shake thoroly to dissolve. Inject ca 1 ~L each soln into gas chromatograph. Repeat injections until ratio of peak hts is reproducible; then record peak hts and attenuations for dimethoate and Me parathion. CF = (Pd X Wmp)/(Pmp x Wd). where CF = correction factor, Pd = peak ht x attenuation for dimethoate, Wmp = g Me parathion, Pmp = peak ht x attenuation for Me parathion, and Wd = g dimethoate. Average results for the 2 solns. 6.413 Preparation of Sample Prep. duplicate samples as follows: Thoroly shake sample container to assure that slurry of microcapsules is homogene- ous. Withdraw ca 1 g sample using medicine dropper while stirring. Immediately discharge contents into tared AI weighing dish, and record exact wt. Transfer to Mixer/Mill or glass tissue grinder, using small amt of CH3CN. (If anal. balance can accom- modate grinder, sample may be weighed directly in it.) Add ca 30 mL CH3CN and grind ca 4 min. Quant. transfer ground sample to 100 mL vol. flask, using CH3CN. Accurately weigh ca 0.2 g dimethoate in tared AI weighing dish, transfer quant. to vol. flask, and dil. to vol. with CH3CN. 6.414 Determination Inject ca 1 ~L soln contg sample and internal std into gas chromatograph. Record peak hts and attenuations for dime- thoate and Me parathion. wt % Me parathion = (Pmp x Wd x CF X 100)/(Pd x Wl. where Pmp = peak ht x attenuation for Me parathion, Wd = g dimethoate, CF = correction factor, Pd = peak ht x attenuation for dimethoate, and W = g sample. Analyze duplicate samples and avo results. Phorate (Thimet®) (O,O-Diethyl S-(Ethylthio)methyl Phosphorodithioate) (t03)-Official Final Action (Applicable to analysis of 5 and 10% granules. Presence of other pesticides and extractable org. materials such as dispersing agents, emulsifiers, and solvs requires testing for interference.) 6.415 Apparatus (a) Infrared spectrophotometer.-Capable of measurement in 7.9--8.6 ~m range; with 0.5 mm cell. (b) Chromatographic tube.-15 x 450 mm with stopcock or Ultramax valve (Fischer & Porter Co., Lab Crest Scientific Div., Cat. No. 274-019 or 274-100). 6.416 Reagents (a) Phorate reference std.-Purified (obtainable from Ameri- can Cyanamid Co.). (b) Phorate std soln.-Accurately weigh by difference from Smith or Lunge pipet 1.0--1.1 g Phorate Ref. Std into 250 mL beaker contg 45 mL CH3CN. (c) Cyclohexane.-Practical grade. (d) Acetonitrile.-Practical grade, bp 82-84'. 6.417 Preparation of Sample Solution (Caution: See 51.011, 51.040, and 51.043.) Accurately weigh 20±0.01 g sample of 5% granular material (10±0.01 g for 10%). Place small glass wool plug in bottom of chromatgc tube, transfer sample to tube, and gently tap sides with spatula or rod to settle contents. Place 250 mL beaker under column. Add 50 mL CH3CN to column and let percolate thru at rate of 40--50 drops/min until flow stops. Place beakers contg std (from 6.416(b)) and samplesolns in shallow H20 bath at 30--35° and evap. under gentle stream of air until odor of CH3CN is no longer detectable. (Sample solns on evapn will change from clear to cloudy and then to residue of 2 layers.) Treat residue with four 5 mL portions and one 4 mL portion cyclo- hexane, quant. transferring cyclohexane layers to 25 mL vol. flask. (Keep cyclohexane-immiscible layer in beaker during each extn.) Dil. to vol. with cyclohexane. 6.418 Determination Using hypodermic syringe, fill 0.5 mm cell with prepd std soln, and obtain IR spectrum from 7.9 to 8.6 ~m. (With single beam instrument, adjust to give 75% Tat 8.2 ~m with cell contg std soln in position.) Using same instrument settings, treat prepd sample solns similarly. Draw baseline from inflection points 8.10 to 8.48 ~m. Draw perpendicular from 0 radiation line thru absorption peak, and measure distance from 0 to baseline (Y) and from 0 to absorption
- 142. 120 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) peak (X) in same units. Calc. A = log (YIX) for sample (A) and std (A 'I. % Phorate = (A/A ') x (wt std/wt sample) x % purity of std. 6.419 * Sulfoxide (n-Octyl Sulfoxide of * Isosafrole) (104)-Offieial First Action Sulfoxide is sepd from solvs, emulsifiers, pyrethrins, and other insecticides by silicic acid column chromatgy with suc- cessive eluting solns: CHCI3, 2% acetone in CHCI3, and 10% acetone in CHCI3 • Sulfoxide is removed in last eluate and is detd by UV spectrophotometry. See 6.296-6.302, 11th ed. 6.420 Tetraethylpyrophosphate (TEPP) (105)-Offieial Final Action (Caution: See 51.041.) Reagents (a) Indicator.-O.l % aq. soln Me red or chlorophenol red. (b) Amberlite IR-4B(OH) (free base form) resin.-Anal. grade. Amberlite IR-45, Dowex 3, or equiv., are satisfactory. 6.421 Preparation of Resin Column Screen resin to remove particles <30 mesh. Slurry 30 g screened resin with H20, and pour into 100 mL buret contg small plug of glass wool at bottom. Wash resin column with 150 mL 3% NaOH soln at flow rate of ca 5 mL/min and then rinse with H20 until effluent is acid to phthln, adjusting stopcock of buret so flow rate is ca 25 mL/min. Wash with aq. acetone (1+3) to displace H20. Column is now ready for use. Note.s: Because channeling may result if column runs dry, keep Ilq. level ca 2.5 cm above resin bed at all times. Because resin tends to pack in column as it adsorbs acidic material expand resin bed after each detn before adding new sample by back-wash.ing with ac~tone (1 +3) as follows: Connect large funnel to tiP of buret with rubber hose, and add the dil. acetone from funnel until liq. level reaches top of buret; let resin settle, and then let soln flow from buret until surface is 2.5 cm above resin bed. Column is now ready to receive next sample. After 8-1 0 samples have passed thru column, regenerate resin by repeating original treatment with 3% NaOH soln, H20, and acetone (1 +3). Washing with dil. acetone must be continued until effluent is colorless. 6.422 Determination (a) In purified or technical grades of tetraethylpyrophosphate not mixed with solvent emulsifying agent etc.-From 5-10 mL weighing buret, weigh by difference, to nearest mg, 2.5 g sample (1.0 g if tetraethylpyrophosphate content is >50%) into 50 mL acetone (1 +3) in 125 mL separator. Mix by swirling, and let soln stand 15 min at 25±2°. Let soln flow thru resin column by gravity at ca 25 mL/min, and catch effluent in 250 mL vol. flask. Wash separator and column with three 50 mL portions acetone (1 +3), collecting washings in same flask. Dil. combined effluent to vol. with H20, mix, and transfer 100 mL aliquot to 250 mL beaker. Add 50 mL O.lN NaOH to beaker, stir well, let stand 30 min at room temp., and back-titr. with O.lN HCI, using pH meter (or indicator, 6.420(a), if pH meter is not available). Calc. % tetra- ethyl pyrophosphate = net mL O.lN NaOH x 3.67/wt sample taken. (b) In formulations of tetraethylpyrophosphate containing organic solvent and emulsifying agent.-Proceed as in (a), except filter acetone soln thru 25 mm cotton plug in cylindrical * Surplus method-see inside front cover. funnel (25 mm diam., 75 mm long) before adding it to column if oil seps from soln. Pass acetone washings successively thru separator, cylindrical funnel, and resin column as in (a). (Cotton plug absorbs oil.) 6.423 Organic Thioeyanates Thiocyanate Nitrogen in Livestock or Fly Sprays (106)-Official Final Action (Caution: See 51.041.) Reagents (a) Strong potassium polysulfide soln.-Dissolve 180 g KOH in 120 mL H20. Sat. 100 mL of this soln with H2S (ca 42 g) (Caution: See 51.059) while cooling. Add remaining 100 mL KOH soln and 80 g S. Shake until dissolved. (b) Mixed sulfide soln.-To 100 mL (a) add 50 g Na2S.9H20, 30 g KOH, and 200 mL H20. (c) Sodium bisulfite.-Na2S20 s or NaHS03 • (d) Copper sulfate soln.-20% aq. soln CuS04.5H20. (e) Wash soln.-To 300 mL H20 add 1 mL H2S04 (1+4), 1 g (c), 10 mL (d), and 12 g Na2S04, and pass S02 into soln 10 min. 6.424 Preparation of Sample Weigh sample preferably contg ca 0.03 g thiocyanate N into 250 mL g-s erlenmeyer. (If SCN content is very low, do not unduly increase amt sample without correspondingly increasing amt mixed sulfide soln used; 2~25 g fly spray is usually enough.) Add 35 mL mixed sulfide soln and shake vigorously at room temp. 10 min, during which time reaction is nearly completed. Heat to 70° on steam bath, carefully releasing pressure resulting from heating, shake 15 min at 70°, and cool. Removal of petroleum oil.-Transfer mixt. to separator with ca 200 mL H20. Add 50 mL pet ether, shake, and drain aq. layer into 600 mL beaker. Wash pet ether layer with two 10 mL portions H20, adding washings to main soln. (If emulsions form during washing, break by acidifying with H2S04 (1 +4).) Drain aq. layer and wash pet ether layer with H20 as above. Discard pet ether layer. 6.425 Determination Dil. combined aq. soln to ca 300 mL and neutze with H2S04 (1 +4), using litmus paper as outside indicator. Add 2 mL H2S04 (1+4), quickly bring mixt. to bp, and boil 8 min to remove H2S. Cool. If fatty acids or oils are present, transfer to separator, ext with pet ether, and return aq. phase to original beaker. Filter thru small buchner and transfer filtrate to beaker. Neutze to litmus paper with 10% KOH soln and add 1 mL H2S04 (1+4). Add 1 g Na bisulfite and stir until dissolved. Add excess (ca 15 mL) CuS04 soln and pass S02 into soln 10 min. Let pptd CuSCN settle 2 hr, and filter with suction thru 56 mm buchner coated with layer of asbestos (Caution: See 51.086), upon which is placed No. 42 Whatman paper, or equiv., second layer of asbestos, layer of diatomite, and finally third layer of asbestos. If filtrate is not clear, centrf. soln at 2000 rpm 10-15 min, and pour thru filter again. Wash filter and ppt once or twice with wash soln, continue suction until filter pad is dry, and transfer to 800 mL Kjeldahl flask. (Filter pad may be folded in filter paper together with bits of moist filter paper used to wipe out buchner, and whole placed in Kjeldahl flask.) Add few glass beads, 35 mL H2S04, 10 g K2S04, and ca 0.7 g HgO or 0.65 g Hg. (Caution: See 51.030 and 51.065.) Digest until colorless; then 15 min more. Det. N as in 2.057, second par. Perform blank analysis on paper, filter pad, and reagents.
- 143. AOAC METHODS (1980) TRIAZINES 121 Thiocarbamate Herbicides (1071-0fficial First Action (S-Ethyl Dipropylthiocarbamate (EPTC, Eptam®I, S-Ethyl Hexahydro-1H-azepine-1-carbothioate (Molinate, Ordram®1. S-Ethyl Cyclohexylethylthiocarbamate (Cycloate, Ro-Neet®I, S-Ethyl Diisobutylthiocarbamate (Butylate, Sutan®), S-Propyl Butylethylthiocarbamate (Pebulate, Tillam®I, S-Propyl Dipropylthiocarbamate 6.426 (Vernolate, Vernam®)) Gas Chromatographic Method (Applicable to liq. and granular formulations. Caution: See 51.041.) Apparatus (a) Gas chromatograph.-With flame ionization detector. Op- erating conditions: temps (O)-injection port 225, column 130 (EPTC and butylate). 170 (molinate), 140 (cycloate, vernolate), 150 (pebulate). detector 250; gas flows (mL/min)-N carrier 30--35, H 25-30, air 200--300 (or as specified by manufacturer). (b) Recorder.-1 mv full scale sensitivity and 1 sec response. (c) Columns.--6' (1.8 m) x 0.25" od, Pyrex, AI, or stainless steel, packed with 3% OV-17 on 60--80 Gas-Chrom Q, or equiv. (for mol inate). and 3% SE-30 or OV-1 on 60--80 mesh Gas-Chrom Q, or equiv. (for other 5 compds). Condition columns 12 hr at 250° under N flow of 30 mL/min. 6.427 Preparation of Standards (a) Internal std solns.-Accurately weigh ca 400 mg each ref. grade thiocarbamate (EPTC, cycloate, butylate, or pebulate; Stauffer Chemical Co., 1200 S 47th St, Richmond, CA 94804) and transfer to sep. 100 mL vol. flasks. Dil. to vol. with CS2- CHCI3-MeOH (80+15+5). and mix thoroly. (b) Std solns.-Accurately weigh ca 100 mg each ref. grade thiocarbamate into sep. 2 oz (50 mL) polyethylene-lined screw- cap, conical bottles. Add 25 mL internal std soln indicated below, and mix thoroly. Approx. Approx. retention retention time, Internal 5td time, Std 50ln min 50ln added min EPTC 2.0 Butylate 2.4 Molinate 4.3 Cycloate 4.8 Cycloate 5.4 Pebulate 2.6 Butylate 2.4 EPTC 2.0 Pebulate 4.0 Cycloate 8.0 Vernolate 3.5 Cycloate 5.5 6.428 Preparation of Sample Accurately weigh sample contg ca 100 mg thiocarbamate into 2 oz (50 mL) polyethylene-lined screw-cap, conical bottle. Add 25 mL appropriate internal std soln, (a), as indicated in (b), and shake thoroly. Vigorously shake granular formulations 30 min on wrist-action shaker. 6.429 Determination Inject 2 J.tL clear supernate or soln into chromatograph pread- justed to appropriate conditions. Make triplicate injections of sample and appropriate std soln in random order. Det. peak areas, preferably with digital integrator. Adjust sensitivity of gas chromatograph so that larger com- ponent or internal std peak is ca '% full scale. 6.430 Calculations Response Factor (RF) = (I' x g compd in std soln x % purity)/S' Wt % compd = (RF x S)/I x g sample). where I and I' = areas of internal std peak in sample and std solns, resp.; and Sand S' = areas of compd peak in sample and std solns, resp. Triazines and Other Pesticides (lOBI-Official Final Action (See Table 6:03 for applicability to and official status of specific compds.) AOAC-CIPAC Method 6.431 Standard Solutions (Caution: See 51.041.) (a) Dieldrin internal std soln.-Std should be ~90% pure and contain no impurities eluting at retention time for pesticide being detd. (1) For propazine.-Weigh 14.0±0.1 g tech. dieldrin, dissolve in ca 300 mL CHCI3 , and dil. to 1 L with CHCI3 . (2) For other compounds.-Weigh 2.00±0.02 g tech. dieldrin, dissolve in ca 200 mL CHCI3 , and dil. to 250 mL with CHCI3 • (b) Aldrin internal std soln.-(For Diazinon®.) Weigh 4.0±0.1 g tech. aldrin into 600 mL beaker. Slurry with 400 mL acetone to dissolve, filter thru paper into 1 L vol. flask, washing with several 100 mL portions acetone, and dil. to vol. Std should be ~90% pure and contain no impurities eluting at retention time of Diazinon. (e) Dibenzyl succinate internal std soln.-(For chlorobenzilate and chloropropylate.) Weigh 5.0±0.1 g dibenzyl succinate, dis- solve in ca 300 mL acetone, and dil. to 1 L with acetone. Std should be >98% pure and contain no impurities eluting at retention time for pesticide being detd. (d) Pesticide std solns.-Accurately weigh 250 mg (125 mg for Diazinon and 150 mg for simazine) of ref. std of pesticide being detd (available from Ciba-Geigy Corp., PO Box 11422, Greensboro, NC 27409) into 4 oz (125 mL) round bottle with AI- lined screw cap. Pipet in 50 mL internal std soln (see Table 6:03) and shake mech. 30 min. (e) Dioctyl phthalate internal std so/n.-(For simazine.) Weigh 3.0±0.1 g tech. dioctyl phthalate, dissolve in ca 200 mL DMF, and dil. to 1 L with DMF. (Caution: See 51.053.) Std should be >98% pure and contain no impurities eluting at retention time of simazine. 6.432 Preparation of Sample Accurately weigh amt sample specified in Table 6:03 into 4 oz (125 mL) round bottle with AI-lined screw cap. Pipet in same vol. internal std used for prepn of std soln, (d), and shake mech. 30 min. Let insol. materials settle or centrf. portion of ext to obtain clear soln. 6.433 Gas Chromatography Use instrument equipped with flame ionization detector and 4 mm id glass column (length specified in Table 6:03) packed with 3% Carbowax 20M (Applied Science Laboratories, Inc.) on 80--100 mesh Gas-Chrom Q. (For Diazinon, use 10% silicone DC- 200 viscosity 12500.) Condition 24 hr at 240° with N or He at ca 40 mL/min. Column should have ~2000 (~1500 for chloroben- zilate, chloropropylate, propazine, and simazine) theoretical plates (see 6.156(a)). Operate at following conditions: temps-as specified in Table 6:03; N or He carrier gas, 80-100 mL/min; air and H, 80-100 mL/min; attenuation varied so that peak hts of pesticide and internal std are 60--80% ful.l scale. Retention times are specified
- 144. 122 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) Table 6:03 Chemical and Gas Chromatographic Parameters for Triazines and Other Pesticides CA Internal Common or Registry Std Soln Chemical Name Trade Name No. 6.431 2-(Ethylamino)-4-(isopropylamino)- Ametryn 834-12-8 (a)(2) 6-(methylthio)-s-triazine 2-Chloro-4-(ethylamino)-6- Atrazine 1912-24-9 (a)(2) (isopropylamino)-s-triazine Ethyl-4,4'-dichlorobenzilate Chlorobenzilate 510-15-6 (e) Isopropyl-4,4'-dichlorobenzilate Chloropropylate 5836-10-2 (e) O,O-Diethyl-O-(2-isopropyl-6- Diazinon 333-41-5 (b) methyl-4-pyrimidinyl) phosphorothioate 2,4-Bis(isopropylamino)-6- Prometon 1610-18-0 (a)(2) methoxy-s-triazine 2,4-Bis(isopropylamino)-6- Prometryn 7287-19-6 (a)(2) (methylthio)-s-triazine 2-Chloro-4,6-bis(isopropylamino)- Propazine 139-40-2 (a)(1) s-triazine 2-Chloro-4,6-bis(ethylamino)-s- Simazine 122-34-9 (e) triazine 2-(tert-Butylamino)-4-(ethylamino)- Terbutryn 886-50-0 (a)(2) 6-(methylthio)-s-triazine in Table 6:03. (Ametryn and dieldrin peaks must be resolved. Prep. new column if variation of flow rate or temp. does not resolve peaks. Resolution may be improved by increasing column temp.) 6.434 Determination Inject 3 ILL aliquots std soln until peak ht ratio of pesticide: internal std varies ~1% for successive injections. Then make duplicate injections of sample followed by duplicate injections of std. Peak ht ratios of stds must be within ± 1% offirst accepted std values or repeat series of injections. Repeat for addnl samples. 6.435 Calculations Calc. peak ht ratios for both duplicate std injections preceding and following samples. Average the 4 values (R'). Calc. and average peak ht ratios of the 2 samples (R). % Pesticide = (R/W) x (W' x P/R'), where Wand W' = mg sample and std, resp.; and P = % purity of std. Triphenyltin Compounds (Fentin) Potentiometric Titration Method I109)-Official First Action lelPAC Method) 6.436 Principle Org. Sn compds are extd with acetone, diphenyltin compds are quant. converted to insol. oxide with alk. alumina and filtered, and acetone soln is titrd potentiometrically. 6_437 Apparatus (a) Filtration apparatus.-Glass bell with neck and removable plate to permit glass buchner with fine porosity fritted disk and long stem to drain into beaker under vac. (Fig. 6:09). Length Temperature (0) Retention Times (min) Column Wt Sample m Inlet Column Detector Pesticide Internal Std 300 mg 80% 1.8 240 215±15 240 8-12 9-15 wettable powder 300 mg 80% 1.8 240 200±10 240 5-7 9-15 wettable powder 500 mg liq. 1.2 260 230±10 260 5-8 8-10 formulation 1 9 liq. 1.2 260 230±10 260 4-6 8-10 formulation Sample 1.8 240 190±10 240 5-6 10-12 contg 110 mg 1 9 liq. 1.8 240 200±20 240 3-5 9-15 formulation 300 mg 80% 1.8 240 200±10 240 6-8 9-15 wettable powder 300 mg 80% 1.2 250 210±10 240 3-5 7-9 wettable powder 190 mg 80% 1.8 250 210±5 250 6-8 10-14 wettable powder 300 mg 80% 1.8 240 200±20 240 8-10 9-15 wettable powder (b) Potentiometric titration apparatus.-pH meter with glass and satd calomel electrodes is satisfactory. 6.438 Reagents (a) Alkaline alumina.-Mix 150 g neutral AI20 3 (Woelm 4649, or equiv.) with 150 mL alcohol contg 15 g KOH in 1 L r-b flask. Reflux 30 min, cool, and filter with suction thru buchner. Dry powder in vac. 1 hr at 100° and 3-3.5 hr at 130°. Pour warm powder into bottle and stopper tightly. Com. alk. AI20 3 is not satisfactory. (b) Cellulose powder.-Whatman CF 11, or equiv. FIG. 6:09--Filtration apparatus
- 145. AOAC METHODS (1980) SELECTED REFERENCES 123 6.439 Determination Accurately weigh into 100 mL glass beaker sample contg ca 0.30 9 triphenyltin com pd. Add 2 9 alk. AI20 3 and 25 mL acetone and stir with mag. stirrer 10 min. Prep. and process blank of 2 9 alk. AI20 3 and 25 mL acetone in same manner. Place 1 9 cellulose powder and 1 9 alk. AI20 3 in funnel and mix thoroly. Assemble filtration app. contg 250 mL beaker and filter suspen- sion thru funnel. Wash beaker and funnel with four 20 mL portions acetone. Titr. filtrate potentiometrically with stdzd 0.1N HCI. % Triphenyltin compd = (S - B) x N x (M/W) x 10 where S = mL HCI used for sample, B = mL HCI used for blank, N = normality of HCI, M = mol. wt of compd (367.0 for triphenyltin hydroxide and 409.0 for the acetate), and W = 9 sample. QUATERNARY AMMONIUM COMPOUNDS Chloride (110)-Official Final Action 6.440 Potentiometric Titration Method Transfer sample contg 30-35 mg CI to 600 mL beaker, dil. to 200 mL with H20, and add 5 mL HN03 (1+1). Add just enough acetone to dissolve ppt that forms and titr. with O.lN AgN03 , using app. for potentiometric titrn. Calc. % CI (1 mL 0.1N AgN03 = 3.545 mg CI) and equiv. % quaternary NH. salt. Adsorption Indicator Method 6.441 Reagents (a) Bromothymol blue indicator.-Dissolve 1 9 indicator in 500 mL 50% alcohol. (b) Dichlorofluorescein soln.-0.1%. Dissolve 100 mg indi- cator in 100 mL 70% alcohol. 6.442 Determination Transfer sample contg 30-140 mg CI (usually ca 1 9 quaternary NH. salt) into 300 mL erlenmeyer, dil. to 75 mL with H2 0, and add 25 mL isopropanol. Neutze if necessary with HOAc (1 +9), using 1 drop bromothymol blue (pH 4-6). Add 10 drops dich- lorofluorescein, and titr. with 0.1N AgN03, avoiding direct sun- light. Ppt becomes red at end point and may flocculate just before end point. Calc. % CI and equiv. % quaternary NH. salt. SELECTED REFERENCES (1) JAOAC 62, 494(1979) (2) Ind. Eng. Chem. 14,207(1922); JAOAC 5, 33, 402(1922); 6,313(1923). (3) JAOAC 7, 313(1924). (4) Anal. Chem. 22, 1066(1950); JAOAC 46, 672 (1963). (5) JAOAC 5, 398(1922). (6) J. Am. Chem. Soc. 40, 1036(1918); JAOAC 5, 398(1922). (7) J. Res. Natl. Bur. Standards 3, 581 (1929); JAOAC 25,670 (1942); 27,74(1944); 28,72(1945). (8) Compt. rend. 173, 714, 836(1921); JAOAC 14, 253(1931). (9) J. Am. Chem. Soc. 55, 1741(1933); Ind. Eng. Chem., Anal. Ed. 5,7(1933); 9, 551(1937); 11, 21(1939); JAOAC 21, 459(1938). (10) JAOAC 55, 851 (1972). (11) JAOAC 43,367(1960). (12) Ind. Eng. Chem. 1,208(1909); JAOAC 3, 158(1917). (13) JAOAC 5, 34(1922). (14) J. Am. Chem. Soc. 24, 1082(1902). (15) USDA Bur. Chem. Bull. 105, p. 167. (16) JAOAC 3, 332(1920). (17) JAOAC 3,333(1920). (18) USDA Bur. Chem. Bull. 137, p. 40; 152, p. 68. (19) JAOAC 5, 33(1921); 392(1922). (20) JAOAC 47,253(1964). (21) Fresenius, "Quantitative Chemical Analysis," Trans. 6th German Ed., 1906, amplified and revised, Vol. 2, 1180; U.S. Geol. Survey Bull. 700, p. 218. (22) JAOAC 15, 289(1932); 17,62(1934). (23) JAOAC 10, 29(1927). (24) JAOAC 35,377(1952). (25) JAOAC 10, 27(1927) (26) JAOAC 10, 28(1927). (27) JAOAC 3, 353(1920). (28) JAOAC 18, 63, 65(1935); 43, 346(1960). (29) JAOAC 43,346(1960). (30) JAOAC 10, 30, 124(1927); 11,35(1928). (31) USDA Bull. 898, p. 48. (32) JAOAC 9,27(1926). (33) USDA Bur. Chem. Circ. 10, p. 7. (34) JAOAC 9,28(1926). (35) USDA Bur. Chem. Bull. 105, p. 165. (36) JAOAC 9,29(1926). (37) Whitmore, "Organic Compounds of Mercury," p. 365; JAOAC 13, 156(1930). (38) JAOAC 54, 685(1971). (39) JAOAC 56, 572(1973). (40) JAOAC 31, 366(1948). (41) JAOAC 22, 411(1939); 25,79(1942); 28,72(1945). (42) JAOAC 43,365(1960). (43) JAOAC 48,576(1965); 49, 207(1966). (44) Anal. Chem. 25, 1207(1953); JAOAC 40, 732 (1957). (45) JAOAC 55, 907(1972). (46) Ind. Eng. Chem., Anal. Ed. 10, 19(1938); JAOAC 21, 148(1938); 22,408(1939); 24,70(1941); 43,376(1960). (47) JAOAC 44,580(1961); 46, 668(1963). (48) JAOAC 35, 771(1952); 43, 350(1960). (49) Contrib. Boyce Thompson Inst. 8, No.3, 175(1936); Ind. Eng. Chem., Anal. Ed. 10,5(1938); JAOAC 43,358(1960). (50) Soap 10, No.5, 89(1934); JAOAC 43, 354(1960); 46, 664(1963). (51) JAOAC 43, 374(1960). (52) USDA Bur. Animal Ind. Bull. 133. (53) JAOAC 44,595(1961). (54) JAOAC 53, 1155(1970). (55) JAOAC 32,684(1949); 39,373(1956). (56) Ind. Eng. Chem. 8, 312(1936). (57) Anal. Chem.19, 779(1947); Report No. 4760, May 15,1949, Phys. Chem. Lab., Hooker Electrochemical Co., Niagara Falls, NY. (58) Anal. Chem. 25,1661(1953); JAOAC 40, 737(1957). (59) JAOAC 56, 567(1973). (60) JAOAC 54, 688(1971). (61) JAOAC 45,513(1962). (62) JAOAC 48,573(1965). (63) JAOAC 49,254(1966). (64) JAOAC 55,942(1972). (65) JAOAC 59, 716(1976); 61,1499(1978). (66) JAOAC 62, 8(1979). (67) JAOAC 30,319(1947); 31,368(1948). (68) JAOAC 40,286(1957); 43, 342(1960). (69) JAOAC 52,1284(1969). (70) JAOAC 51,1301(1968).
- 146. 124 6. PESTICIDE FORMULATIONS AOAC METHODS (1980) (71) JAOAC 54, 706(1971). (72) JAOAC 61, 1163(1978). (73) Anal. Chem. 31,418(1959); JAOAC 43, 382 (1960); 45, 522(1962). (74) JAOAC 59,1109(1976). (75) JAOAC 60,716(1977). (76) JAOAC 60, 1157(1977). (77) JAOAC 51, 565(1968). (78) JAOAC 59, 748(1976). (79) Anal. Chem. 27, 1774(1955); JAOAC 43, 382(1960); 45, 522(1962). (80) JAOAC 57, 642(1974). (81) JAOAC 50, 568(1967). (82) JAOAC 50, 566(1967); 56,576(1973); 59,753, 1196(1976). (83) JAOAC 47,268(1964). (84) JAOAC 49,251(1966). (85) Analyst 92, 375(1967); JAOAC 51,1304,1306 (1968); 55, 857(1972). (86) JAOAC 48, 562(1965). (87) J. Sci. Food Agric. 15,509(1964); JAOAC 49, 40(1966); 51, 447(1968). (88) JAOAC 52, 1292(1969). (89) Ber. 31, 2979(1898); J. Am. Chem. Soc. 27, 1183(1905); USDA Bur. Chem. Bull. 99, p. 30; 132, p. 49; 137, p. 47. (90) Z. anal. Chem. 36, 18(1897); USDA Bur. Chem. Bull. 132, p.49. (91) Ind. Eng. Chem., Anal. Ed. 3, 357(1931); JAOAC 25, 80, 668(1942). (92) JAOAC 62,11(1979). (93) JAOAC 57, 771(1974). (94) JAOAC 55,1133(1972). (95) JAOAC 5~, 926(1972). (96) JAOAC 62,272(1979). (97) JAOAC 61; 495(1978). (98) Anal. Chem. 23, 1167(1951); JAOAC 35, 381(1952); 36, 384(1953). (99) JAOAC 43,344(1960); 47, 242(1964). (100) JAOAC 60, 720(1977). (101) JAOAC 60, 724(1977). (102) JAOAC 60, 862(1977). (103) JAOAC 47,245(1964). (104) JAOAC 51,562(1968). (105) Anal. Chem. 21, 808(1949). (106) JAOAC 34, 677(1951). (107) JAOAC 57, 53(1974). (108) JAOAC 56, 586(1973); 58, 513, 516(1975); 59, 758(1976). (109) JAOAC 61, 1504(1978). (110) JAOAC 43, 352( 1960).
- 147. 7. Animal Feed 7.001 Sampling (1)-Procedure Use slotted single or double tube, or slotted tube and rod, all with pointed ends. Take ~500 g sample, 1 kg preferred, as follows: Lay bag horizontally and remove core diagonally from end to end. Det. number of cores as follows: From lots of 1-10 bags, sample all bags; from lot of ~11, sample 10 bags. Take 1 core from each bag sampled, except that for lots of 1-4 bags take enough diagonal cores from each bag to total ~5 cores. For bulk feeds draw ~10 cores from different regions; in sampling small containers (",,10 Ib) 1 package is enough. Reduce composite sample to amt required, preferably by riffling, or by mixing thoroly on clean oil-cloth or paper and quartering. Place sample in air-tight container. A sample from less than these numbers of bags may be declared an official sample if guarantor agrees. For samples that cannot be representatively taken with probe described, use other sampling means. 7.002 Preparation of Sample-Official Final Action Grind sample to pass sieve with circular openings 1 mm (1/25") diam. and mix thoroly. If sample cannot be ground, reduce to as fine condition as possible. Do not grind molasses feeds. 7.003 Moisture-Official Final Action I. Drying in Vacuo at 95-100" (2) Determination Dry amt sample contg ca 2 g dry material to const wt at 95---100° under pressure ",,100 mm Hg (ca 5 hr). For feeds with high molasses content, use temp. ",,70° and pressure ",,50 mm Hg. Use covered AI dish ~50 mm diam. and ",,40 mm deep. Report loss in wt as moisture. II. By Distillation with Toluene (3) 7.004 Apparatus Connect 250 mL flask of Pyrex or other resistant glass by means of Bidwell-Sterling moisture receiver to 500 mm Liebig condenser. Calibrate receiver, 5 mL capacity, by distg known amts H20 into graduated column, and estg column of H20 to 0.01 mL. Clean tube and condenser with chromic acid cleaning mixt., rinse thoroly with H20, then alcohol, and dry in oven to prevent undue amt H20 from adhering to inner surfaces during detn. 7.005 Determination If sample is likely to bump, add dry sand to cover bottom of flask. Add enough toluene to cover sample completely (ca 75 mL). Weigh and introduce enough sample into toluene to give 2-5 mL H20 and connect app. Fill receiving tube with toluene, pouring it thru top of condenser. Bring to boil and distil slowly, ca 2 drops/sec, until most ofthe H20 passes over; then increase rate of distn to ca 4 drops/sec. When all H20 is apparently over, wash down condenser by pouring toluene in at top, continuing distn short time to see whether any more H20 distils over; if it does, repeat washing- down process. If any H2 0 remains in condenser, remove by 125 brushing down with tube brush attached to Cu wire and satd with toluene, washing down condenser at same time. (Entire process is usually completed within 1 hr.) Let receiving tube come to room temp. If any drops adhere to sides of tube, force them down, using Cu wire with end wrapped with rubber band. Read vol. H20 and calc. to %. 7.006 * III. Drying without Heat over * Sulfuric Acid (4) See 7.006-7.007, 12th ed. 7.007 IV. Drying at 135° (5) (Not to be used when fat detn is to be made on same sample) Regulate air oven to 135±2°. Using low, covered AI dishes, 7.003, weigh ca 2 g sample into each dish and shake until contents are evenly distributed. With covers removed, place dishes and covers in oven as quickly as possible and dry samples 2 hr. Place covers on dishes and transfer to desiccator to cool. Weigh, and calc. loss in wt as H20. 7.008 V. In Highly Acid Milk By-products (6) Add ca 2 g ZnO, freshly ignited or oven dried, to flat-bottom dish ~5 cm diam. and weigh. Add ca 1 g sample and weigh quickly. Add ca 5 mL H20 and distribute sample evenly on bottom of dish. Heat on steam bath, exposing max. surface of dish bottom to live steam until apparently dry. Heat at 98---100° in air oven 3 hr or to const wt. Cool in desiccator; then weigh quickly. Det. wt residue. Titr. acidity of sample and calc. as lactic acid, 16.023. To compensate for H20 formed when acid is neutzd by ZnO, add 0.1 g to residue wt for each g acid (as lactic) in weighed sample. Report % residue (corrected) as total solids. 7.009 Ash (l)-Official Final Action Weigh 2 g sample into porcelain crucible and place in temp. controlled furnace preheated to 600'. Hold at this temp. 2 hr. Transfer crucible directly to desiccator, cool, and weigh imme- diately, reporting % ash to first decimal place. NITROGEN Qualitative Tests for Proteins (S)-Official Final Action 7.010 * Biuret Test * See 22.012-22.013, 10th ed. 7.011 * Millon Test * See 22.014-22.015, 10th ed. (Caution: See 51.018, 51.026, and 51.065.) 7.012 * Glyoxylic Acid Test (Hopkins-Cole) * See 22.016-22.017, 10th ed. (Caution: Wear face shield and heavy rubber gloves as protection against reagent bump. See also 51.018 and 51.063.) *Surplus method-see inside front cover.
- 148. 126 7. ANIMAL FEED AOAC METHODS (1980) 7.013 * Adamkiewicz Test * See 22.018, 10th ed. 7.014 * Xanthoproteic Test * See 22.019, 10th ed. Crude Protein-Official Final Action 7.015 Kjeldahl Method (9) Det. N as in 2.057. Multiply result by 6.25, or in case of wheat grains by 5.70. Dumas Method (10) 7.016 Principle N, freed by pyrolysis and subsequent combustions, is swept by CO2 carrier into nitrometer. CO2 is absorbed in KOH and vol. residual N is measured and converted to equiv. protein by numerical factor. 7.017 Apparatus and Reagents (a) Nitrogen analyzer and accessories.-Consists of combus- tion and collection and measuring systems. Suitable instrument, Model 29A. with following accessories and reagents is available from Coleman Instruments Div., 2000 York Rd, Oak Brook, IL 60521: AI combustion boats, No. 29-412; Vycor combustion tubes, No. 29-328; CuO-Pt catalyst (CuO wire form with 2.5% Pt reforming catalyst), No. 29-160; reduced Cu wire, No. 29-120; C030 4, No. 29-170; CuO powder, fines, No. 29-140; 45% KOH, No. 29-110. (b) Balance.-Accurate to 0.01 mg. (c) Barometer.-Hg type, readable to 0.1 mm. 7.018 Preparation of Samples Grind to pass No. 30 sieve. Store in capped bottles. 7.019 Determination Operate instrument in accordance with instructions of man- ufacturer. (Following directions apply to Coleman Model 29A Nitrogen Analyzer. Consult Operating Directions D-360B, Cole- man Cat. No. 29-904, for addnl details.) After combustion furnaces have come to thermal equilibrium, turn combustion cycle control to START and let proceed normally thru cycle. Observe indicated temp. on pyrometer of both upper and lower combustion furnaces at end of combustion portion of cycle. Furnace temps should be 850-900°. If not, adjust. Prep. combustion tube by inserting stainless steel screen in lower end of combustion tube (end farthest from trademark). In upper end, place enough glass wool to form 6 mm plug when packed. With 11 mm glass rod, drive glass wool down to stainless steel plug. Holding tube vertically, pour CuO-Pt catalyst directly from dispenser bottle into combustion tube until it reaches upper end of trademark. Tap or vibrate tube on bench until reagent settles to approx. center of trademark. Weigh and record wt of empty AI combustion boat. Place sample in boat. Weigh and record wt of sample and combustion boat. Difference between wts is sample wt. Use following sample wts (mg) as guides to suitable sample sizes: bermuda grass 150-300; rice bran, wheat shorts, dehydrated alfalfa 150-250; range feed 100-200; cottonseed meal 75-150; edible soy protein 50-150. Weigh sample to nearest 0.01 mg. To avoid wt changes, *Surplus method-see inside front cover. Table 7:01 Volume correction for temperature correction factor (e,) (J.LL/"K)a Final Counter Reading (J.'L) o 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 aVol. correction, Vt = C,(t, - ttl (C,) (Nitrometers with Check Value) 12 29 45 62 79 95 112 129 145 162 179 record wt within 1 min after sample and boat are placed on balance. If this is impossible, weigh sample inside weighing bottle, such as Kimble No. 15165 or 15166. Turn combustion tube to horizontal, and carefully insert loaded sample boat into open end of tube. Slide or push boat, without spilling contents, until it reaches trademark. Raise open end until tube forms 60-70° angle to horizontal. Tap or vibrate combustion tube on bench top while rotating tube between thumb and forefinger. Raise open end of tube and add vol. C030 4 and vol. CuO fines equal to vol. sample. For convenient means of adding above reagents to samples, place vol. CuO fines and vol. C030 4, each equal to vol. sample, in addnl combustion boat; add contents of boat, but not boat itself, to combustion tube; and rotate partially filled combustion tube between thumb and forefinger while varying angle of tube 20-45° from horizontal. Continue rotating, tapping, and vibrating until sample is dispelled from boat and is thoroly mixed with oxidizing agents. Raise open end until tube forms 60-70° angle to horizontal; add CuO-Pt catalyst ca 12 mm above sample boat. Tap or vibrate gently to eliminate voids. Add CuO-Pt catalyst to within 20 mm of top of tube, again tapping or vibrating gently to eliminate voids. Install prepd combustion tube in N analyzer. Adjust 45% KOH soln meniscus to calibrating mark in nitrometer with digital readout meter. Record counter reading, R,. (Counter reading should preferably lie between 500 and 1000 J.LL at this point. Vent control may be used to assist in arriving at this counter setting, if necessary.) Record syringe temp., t" indicated on special scale thermometer. Add 2 min more to combustion portion of cycle by turning auxiliary timer to setting 3. (Once this is done, addnl 2 min will be automatically programmed into each subsequent cycle.) Turn combustion cycle control to START. Let analyzer proceed thru its cycle. After cycle is complete and combustion cycle control has entered STAND-BY section, readjust KOH meniscus to calibration mark with digital readout counter. Record new counter reading, R2, and syringe temp., t2 • Det. blank for instrument under same conditions as actual analysis except omit sample. Table 7:02 Barometric temperature correction (Pb) Po (mm Hg) Temperature,OC 700-749 750-780 10 15 20 25 30 35 1.2 1.8 2.3 2.9 3.5 4.1 1.3 1.9 2.5 3.1 3.7 4.3
- 149. AOAC METHODS (1980) NITROGEN 127 7.020 Calculations (a) Record observed N vol., Vo = R2 - R" where Vo = observed N vol. (ttL). R, = initial counter reading, and R2 = final counter reading. (b) Det. corrected N vol. (in ttL), Vc = Vo - (Vb + Vt), where Vb = vol. blank (ttL), Vt = vol. correction for temp. (ttL) = C,(t2 - t,). Cf is obtained from Table 7:01 (based on final counter reading); t2 and t, are in OK. (c) Det. corrected barometric pressure, Pc = Po - (Pb + Pv). where Po = observed barometric pressure (mm Hg), Pb = barometric temp. correction (from Table 7:02), and Pv = pressure correction for vapor pressure of KOH soln (from Table 7:03). (Note: Empirical approximation of (Pb + Pv ) = 11.0 will be satisfactorily accurate for Po between 740 and 780 mm Hg and syringe temp. between 298 and 305°K.) (d) Calc. % N = (Pc x Vc x 0.0449)/(T x W). where T = final syringe temp. in oK and W = sample wt in mg. Example: Po = 750.1 mm Hg at 25°C; W = 148.91 mg Start Finish Counter readings, blank 500 ILL 524 ILL Counter readings, sample 524 6955 t, = 302.rK, t2 = 303.00 K, Vo = 6955 - 524 = 6431 ILL Vc = 6431 - [24 + C,(t2- t,)] = 6431 - (24 + 35 x 0.3) = 6396 ttL Pc = 750.1 - (3.1 x 9.6) = 737.4 % N = (737.4 x 6396 x 0.04493)/(303.0 x 148.91) = 4.69% (e) Calc. % protein = % N x 6.25, or % N x 5.70 in case of wheat grains. Table 7:03 Pressure correction (Pv) for vapor pressure of KOH (for practical purposes, temp. of KOH is same as syringe) 7.021 Temperature, OK Pv (mm Hg) 288 293 298 303 308 313 4.1 5.7 7.4 9.6 12.5 16.5 Automated Method (tt)-Official Final Action Principle Automation of macro Kjeldahl method is in 6 steps: sample and reagent addn, initial and final digestion, cooling and diln, NaOH addn, steam distn and titrn, and automatic pumping of flask contents to waste. Chemistry is carried out in macro Kjeldahl flasks equipped with side arms which are rotated at 3 min intervals thru each successive step. 7.022 Apparatus (a) Kjeldahl (protein/nitrogen) analyzer. -Kjel-Foss Auto- matic, Model 16210 (Foss America, Inc., PO Box 504, Rt 82, Fishkill, NY 12524). or equiv. (b) Weighing papers.-120 x 120 mm N-free tissues, Foss America, Inc., or equiv. 7.023 Reagents (a) Kjel-tabs.-Contg 5 g K2S04and 0.25 g HgO (Foss America, Inc.). (b) Kjeldahl (protein/nitrogen) analyzer reagents.-Prep. fol- lowing according to manufacturer's instructions: (1) Sulfuric acid.-96--98%. (2) Hydrogen peroxide.-30-35%. (3) Ammo- nium sulfate std solns.-(a) Std soln I.-Dissolve 30.000±0.030 g (NH4)2S04 in H20 and dil. to 1 L with H20. (b) Std soln 11.- Dissolve 0.750±0.001 g (NH4)2S04 in H20 and dil. to 1 L with H20. (4) Mixed indicator soln.-Dissolve 1.000 g Me red and 0.250 g methylene blue in alcohol and dil. to 1 L with alcohol. Dil. 10 mL this soln to 1 L with H20. (5) Sodium hydroxide- sodium thiosulfate soln.-40% NaOH-8% Na2S20 3.5H20. (6) Di- lute sulfuric acid soln.--fJ.6%. Dil. 30 mL 96--98% H2S04to 5 L with H20. 7.024 Determination (Caution: See 51.019, 51.030, 51.065, and 51.070.) Place 3 Kjel-tabs in special flask (500 mL of design compatible to Foss instrument) in position 1. Shift dispenser arm over flask and depress H2S04 lever, initiating simultaneous addn of 10 mL 30-35% H20 2 and 12-15 mL 96--98% H2S04 (depending on fat content of sample). To flask, add accurately weighed sample (ca 1.0 g if <45% protein, and ca 0.5 g if >45% protein) wrapped in weighing paper and close lid. Flask automatically rotates to position 2 where sample digests 3 min, and then to position 3 for 3 min addnl digestion. In position 4, flask is cooled by centrifugal blower, lid opens automatically, and 140 mL H20 is added automatically. Flask rotates to position 5, where NaOH- Na2S20 3 soln is automatically introduced in excess. Released NH3 is steam distd Quant. into 200 mL tall-form titrn beaker contg 50 mL mixed indicator soln, and is simultaneously titrd automatically with dil. H2S04soln delivered by photometrically regulated syringe. Final position of syringe is measured by potentiometer, output of which feeds electronic circuitry for conversion to visual display and/or printout in % Nor % protein with appropriate conversion factors. In position 6, flask is emptied. Calibrate instrument initially each day with aliquots of (NH4)2S04 std solns and check periodically as stated in operating manual. Semiautomated Method (12)-Official Final Action 7.025 Principle Samples are digested in 250 mL calibrated tubes, using block digestor. A of NH3-salicylate complex is read in flowcell at 660 nm, or NH3, is distd into std acid and back-titrd with std alkali. 7.026 Apparatus (a) Block digestor.-Model BD-20 (Technicon Instruments Corp.) or Model DS-20 (Tecator, Inc., 1898 S Flatiron Ct, Boulder, CO 80301). Capable of maintaining 410° and digesting 20 samples at a time in 250 mL calibrated volumetric tubes constricted at top. Block must be equipped with removable shields to enclose exposed area of tubes comfJ:etely at or above ht of constriction. (b) Automatic analyzer.-AutoAnalyzer with following mod- ules (Technicon Instruments Corp.), or equiv.: Sampler II or IV with 40/hr (2: 1) cam (higher ratio cams result in carry-over and poorer peak sepn); proportioning pump III; NH3 anal. cartridge No. 116-D531-01 (or construct equiv. manifold from flow dia- gram); AAII single channel colorimeter with 15 x 1.5-2.0 mm id tubular flowcell, matched 660 nm interference filters, and voltage stabilizer; and recorder of appropriate span. (See Fig. 7:01.) 7.027 Reagents (a) Phosphate-tartrate buffer soln.-pH 14.0. Dissolve 50 g NaK tartrate and 26.8 g Na2HP04.7H20 in 600 mL H20. Add 54 g NaOH and dissolve. Add 1 mL Brij-35 (Technicon Instru- ments Corp.), dil. to 1 L with H20, and mix.
- 150. 128 7. ANIMAL FEED AOAC METHODS (1980) (b) Sodium chloride-sulfuric acid soln.-Dissolve 200 g NaCI in H20 in 2 L vol. flask. Add 15 mL H2S04 and 2 mL Brij-35. Dil. to vol. with H20 and mix. (e) Sodium hypochlorite soln.-Dil. 6 mL com. bleach soln contg 5.25% available CI (Clorox, or equiv.) to 100 mL with H20 and mix. Prep. fresh daily. (d) Sodium nitroprusside-sodium salicylate soln.- Dissolve 150 g NaC7Hs03 and 0.3 g Na2Fe(CN)s.NO.2H20 in 600 mL H20. Add 1 mL Brij-35, dil. to 1 L with H20, and mix. (e) Nitrogen std solns.-Prep. 6 stds by accurately weighing (±10 mg) 59,118,177,236,295, and 354 mg (NH4)2S04 primary std (Fisher Scientific Co. No. A-938, or equiv.; dry 2 hr at 105° before use and assume theoretical value of 21.20% N after drying) into individual 250 mL digestion tubes. Proceed as in 7.031, beginning "Add 9 g K2S04, 0.42 g HgO, and 15 mL H2S04 ..." Stds may be stored and reused until exhausted. (f) Sodium hydroxide-potassium sulfide soln:-Dissolve 400 g NaOH in H20. While still warm, dissolve 30 g K2S in soln, and dil. to 1 L. 7.028 Analytical System If manifold is to be constructed, use clear std pump tubes for all air and soln flows. All fittings, coils, and glass transmission lines are AAII type and size. Use glass transmission tubing for all connections after pump to colorimeter. Construct modified AO fitting on sample diln loop using AO fitting, N13 stainless steel nipple connector, and '12" length of 0.035" id Tygon tubing. Insert N13 nipple approx. halfway into 0.035" Tygon tubing. Insert tubing into side arm of AO fitting far enough so resample line will not pump any air. Space pump tubes equally across pump rollers. Cut 0.16 mL/min resample pump tube ,,;1" at entrance before connecting to side arm of AO fitting. In opera- tion, add buffer and hypochlorite solns thru metal side arms of A10 type fittings; add salicylate soln, (d), thru metal insert to 20T coil. Air, reagents, and sample are combined immediately after pump thru injection fittings. 37" e "G"Coil COLORIMETER 660 nm 15 mm Fie D RECORDER 7.029 Start-Up Start automatic system and place all lines except salicylate line in resp. solns. After :35 min, place salicylate line in resp. soln and let system equilibrate. If ppt forms after addn of salicylate, pH is too low. Immediately stop proportioning pump and flush coils with H20, using syringe. Before restarting system, check concns of NaCI-H2S04 soln and phosphate-tartrate buffer soln. Pump lowest concn N std soln continuously thru system :35 min and adjust baseline control on colorimeter to read 10% full scale. Pump highest concn N std soln continuously thru system until no drift exists (usually :310 min) and adjust "std. caL" control to read 85% full scale. Recorder tracings must be stable and show <0.3 division noise. If noisy conditions exist, replace dialyzer membrane. When recorder tracing indicates stable condition, immediately start sampling. 7.030 Shut-Down Place reagent lines in H20, removing salicylate line first. Let system wash out :320 min. 7.031 Colorimetric Determination (Caution: See 51.065.) Weigh samples (See Table 7:04) into dry digestion tubes. Add 9 g K2S04, 0.42 g HgO, and 15 mL H2S04to each tube. (Calibrated metal scoops may be used for solids.) Insert tubes into digestor block preheated to 410°, place shields around tubes, and digest 45 min. After digestion, remove rack of tubes from block, place in hood, and let cool 8-10 min. (Time depends upon air flow around tubes.) Direct rapid spray of H20 (kitchen sink dish rinsing sprayer works well) to bottom of each tube to dissolve acid digest completely. If ppt forms, place tube in ultrasonic bath to aid in redissolving salt. Let cool, dil. to vol., and mix thoroly. Transfer portion of each sample soln to AutoAnalyzer beaker. SAMPLER mlimin. 0.42 Buffe ic 0.16 C orite waste _.l..1..>,l20l--t.!Frl!!0ml:!...tF../C'----- FIG. 7:01-Flow diagram for semiautomated analysis for crude protein
- 151. AOAC METHODS (1980) NITROGEN 129 Table 7:04 Sample Weight Protein, % 6-24 25-40 41-50 51-60 61-90 >90 Sample, 9 1.5±0.1 1.0±0.1 0.B±0.1 0.HO.1 0.5±0.01 Weigh sample equiv. to 50 mg N Place stds in tray in increasing order of concn, followed by group of samples. Analyze lowest concn std in duplicate, dis- carding first peak. Precede and follow each group of samples with std ref. curve to correct for possible drift. Analyze stds and samples at rate of 40!hr, 2!1 sample-to-wash ratio. Prep. std curve by averaging peak hts of first and second set of stds. Plot avo peak ht stds against N concn contained in each 250 mL tube. 7.032 % Protein = [(mg N!250 mL from graph) x 6.25 x 100l/mg sample Titrimetric Determination Digest as in 7.031. Cool 5 min and add only enough H20 to dissolve salts (70-75 mL). Cool and attach digestion tube to distn head according to manufacturer's directions. Place receiver flask contg 25 mL std acid, 2.055(j}, and 5--7 drops Me red indicator on platform. Condenser tip must be below surface of std acid soln. Add 50 mL NaOH-K2S soln to tube and steam distil vigorously until 125 mL distillate collects. Titr. excess acid with std 0.1N NaOH soln, 2.055(k). Correct for reagent blank. % N = [(mL std acid x normality acid) - (mL std NaOH x normality NaOH)] x 1.4007!g sample % crude protein = % N x 6.25 7.033 Urea and Ammoniacal Nitrogen (13) Official Final Action Reagents (a) Defoaming so/n.-Dow Corning Corp. Antifoam B Emul- sion. (b) Urease so/n.-Prep. fresh soln by dissolving stdzd urease in H20 so that each 10 mL neutzd soln will convert N of ~0.1 g pure urea. Standardization.-To det. alky of com. urease prepn dissolve 0.1 gin 50 mL H20 and titr. with 0.1N HCI, using Me red, 2.055(0. Add same vol. 0.1N HCI to each 0.1 g urease in prepg urease soln. To det. enzyme activity, prep. ca 50 mL neutzd 1% soln. Add different amts of soln to 0.1 g samples pure urea and follow with enzyme digestion and distn as in detn. Calc. activity of urease prepn from amt of this urease soln that completely converted urea, as detd by complete recovery of N by distn. (c) Calcium chloride so/n.-Dissolve 25 g CaCI2 in 100 mL H20. 7.034 Determination Place 2 g sample in Kjeldahl flask with ca 250 mL H20. Add 10 mL urease soln, stopper tightly, and let stand 1 hr at room temp. or 20 min at 40°. Cool to room temp. if necessary. Use addnl urease soln if feed contains >5% urea (ca 12% protein equiv.). Rinse stopper and neck with few mL H20. Add ~2 g MgO (heavy type). 5 mL CaCI2 soln, and 3 mL defoaming soln, and connect flask with condenser by Kjeldahl connecting bulb. Distil 100 mL into measured vol. std acid, 2.055(j), and titr. with std alkali, 2.055(k). using Me red, 2.055(0. *Surplus method---see inside front cover. Urea (14)--Official Final Action (Applicable to animal feeds and their ingredients) 7.035 Apparatus Spectrophotometer.-Instrument with max. band width 2.4 nm at 420 nm, with 1 em cells. 7.036 Reagents (a) p-Dimethy/aminobenza/dehyde (DMAB) so/n.-Dissolve 16.00 g (Eastman Kodak Co. No. 95 only) in 1 L alcohol and add 100 mL HCI. Stable 1 month. Prep. new std curve with each new batch of reagent. (b) Zinc acetate soln.-Dissolve 22.0 g Zn(OAc)2.2H,O in H20, add 3 mL HOAc, and dil. to 100 mL. (c) Potassium ferrocyanide so/n.-Dissolve 10.6 g K4 Fe(CN)6 .3H20 in H20 and dil. to 100 mL. (d) Vegetable charcoa/.-Darco G-60. (e) Phosphate buffer so/n.-pH 7.0. Dissolve 3.403 g anhyd. KH2P04 and 4.355 g anhyd. K2HP04 sep. in ca 100 mL portions freshly distd H20. Combine solns and dil. to 1 L with H20. (f) Urea std so/ns.-(1) Stock so/n.-5 mg!mL. Dissolve 5.000±0.001 g reagent grade urea in H20 and dil. to 1 L with H20. (2) Working so/ns.--0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, and 2.0 mg urea!5 mL. Pipet 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 mL stock soln into 250 mL vol. flasks and dil. to vol. with phosphate buffer. (3) Reference so/n.-Use std soln contg 1.0 mg urea!5 mL as ref. std. Store at <24°. Stable 1 week. 7.037 Preparation of Standard Curve Pipet 5 mL aliquots of working std solns into 20 x 150 mm (25 mL) test tubes and add 5 mL DMAB soln to each. Prep. reagent blank of 5 mL buffer soln and 5 mL DMAB soln. Shake tubes thoroly and let stand 10 min in H2 0 bath at 25°. Read A in 1 em cell at 420 nm with reagent blank at zero A. Plot A against concn urea. Plot should be straight line; if not, repeat, using new lot of DMAB. 7.038 Determination Weigh 1.00 g ground sample into 500 mL vol. flask. Add 1 g charcoal, ca 250 mL H20, 5 mL Zn(OAc)2 soln, and 5 mL K4 Fe(CN)6 soln. Shake mech. 30 min and dil. to vol. with H20. Let stand until ppt settles. Decant thru Whatman No. 40 paper and collect clear filtrate. Pipet 5 mL filtrate into test tube, add 5 mL DMAB soln, and shake thoroly. Include reference std (5 mL soln (f)(3) and 5 mL DMAB soln) and reagent blank with each 'group of samples. Let stand 10 min in H20 bath at 250 • Read A at 420 nm against reagent blank. % Urea = (1.0 x AsamPle x 100)!(Astd X mg sample in aliquot). 7.039 * Albuminoid Nitrogen-Official Final Action * Pptn with Cu(OH)2 and N detn. See 22.020-22.021, 10th ed. 7.040 * Amido Nitrogen-Official Final Action * % total N - % albuminoid N = % amido N. Nitrate and Nitrite Nitrogen (15}-Official Final Action 7.041 Princ;ple Nitrate and nitrite are extd with Cd and Ba chloride soln. Bulk of sol. proteins are pptd in alk. soln and clarified soln is passed thru metallic Cd column, reducing nitrate to nitrite. Nitrite is measured colorimetrically. (Caution: Cd salts are toxic. See 51.084.)
- 152. 130 7. ANIMAL FEED AOAC METHODS (1980) 7.042 Reagents and Apparatus (a) Nitrate nitrogen std solns.-(1) Stock soln.-12 ILg nitrate N/mL. Dissolve 0.867 g KN03 in 1 L H20. Oil. 25 mL to 250 mL with H20. (2) Working solns.-O.6, 1.2, 1.8,2.4,3.0 ILg N/mL. Oil. 5, 10, 15, 20, and 25 mL stock soln to 100 mL with H20. (b) Extracting soln.-Dissolve and dil. 50 g CdCI2 and 50 g BaCI2 to 1 L with H20. Adjust to pH 1 with HCI. (c) Ammonium chloride buffer soln.-pH 9.6. Dissolve 50 g NH4CI in 500 mL H20 and adjust pH with NH4 0H. Oil. to 1 L with H20. (d) Sodium hydroxide soln.-2.5N. Dissolve 50 g NaOH in 500 mL H2 0. (e) Sulfanilamide soln.-O.5%. Dissolve 1.25 g sulfanilamide in 250 mL HCI (1+1). Soln is stable 1-2 months. (f) Coupling reagent.-Dissolve 0.5 g N(1-naphthyl)ethylene- diamine.HCI in 100 mL H20. Store in g-s dark bottle in refrigerator. Soln is stable several weeks. (g) Salt soln .-Dissolve 100 g NaCI in 500 mL H20. Add 50 mL buffer soln, (c), and dil. to 1 L with H20. (h) Reduction tube.-25 mL buret or equiv. id chromatgc tube with stopcock and reservoir (Kontes Glass Co. Cat. No. K-420280 or SGA Scientific Inc. Cat. No. JC-1506). 7.043 Preparation of Columns Prep. supply of metallic Cd by placing Zn rods into 500 mL 20% CdS04 soln. After reaction for 3 hr, discard soln and scrape moss-like Cd growth from Zn rods. Place Cd in high-speed blender, add 500 mL H20, and blend 2 sec. Wash fine metal particles with H20 onto sieves, collecting only 20-40 mesh size. Fill reduction tube with H20 and add 2 cm plug of glass wool. Press any trapped air from glass wool as it is pushed to bottom of column with glass rod. Add Cd to depth of 10 cm, using min. of very gentle tapping. Wash column with 25 mL 0.10N HCI, two 25 mL portions H20, and finally 25 mL buffer, (c), dild 1 + 9. Keep column covered with salt soln, (g), when not in use. Normally columns can be used repeatedly if kept under salt soln between analyses. When succession of highly proteina- ceous or other sol. org. contg samples are treated, flow rate may decrease gradually. Repeating 25 mL 0.10N HCI treatment may restore original flow rate; if not, prep. new column. Reproducible flow rate is important. Actual rate can be 3-5 mL/min but once established, it must be identical (:to.1 mL) for samples and stds. 7.044 Preparation of Standard Curve Prep. std curve of 3, 6, 9, 12, and 15 ILg nitrate-nitrite N by pipetting 5.0 mL aliquots of working std solns into 30 mL beakers. Add 5 mL buffer soln, (c), and 15 mL H20, mix well, and transfer quant. to reduction column, using min. H20. Adjust flow rate thru column to 3-5 mL/min. Just as reservoir empties, add 15 mL salt soln, (g). Collect eluate, including salt wash, in 50 mL vol. flask (total vol. of eluate should be ca 40 mL). Add 5 mL sulfanilamide soln, (e), mix, and let stand 3 min. Add 2 mL coupling reagent, (f), mix, dil. to vol. with H20, mix, and let stand 20 min for max. color development. Color is stable ~2 hr. Det. A in 1 cm cells at 540 nm against reagent blank. Plot A against ILg nitrate-nitrite N. 7.045 Extraction (a) Low level nitrate samples (grains, meals, supplements, etc.).-Wash 5.0 g finely ground sample into 250 mL vol. flask. Add 100 mL extg soln, (b), and 100 mL H20, and mix. Let stand 1 hr with occasional swirling. Add 20 mL 2.5N NaOH, dil. to vol. with H2 0, mix, and filter immediately thru rapid paper. Pipet 10 mL buffer soln, (c), into 100 mL vol. flask, dil. to vol. with clear filtrate, and mix. (b) Dry, high level nitrate products (dried plants, hays, meals, etc.).-Weigh 5.0 g finely ground sample into 500 mL vol. flask. Add 100 mL extg soln, (b), and 300 mL H20, and mix. Let stand 1 hr with occasional swirling, add 40 mL 2.5N NaOH, dil. to vol. with H20, mix, and filter immediately thru rapid paper. Pipet 10 mL buffer soln, (c), into 100 mL vol. flask, dil. to vol. with clear filtrate, and mix. (c) Grasses, silages, and other wet materials.- Weigh 100 g sample into 1 gal. capacity high-speed blender. Add 100 mL extg soln, (b), and 800 mL H20, including vol. H20 contributed by sample as detd in 7.003 or 7.005. Homogenize 1 min, pour into 2 L beaker, and let stand 1 hr. Add 100 mL buffer soln, (c) (total vol. 1 L), mix well, and filter thru Whatman No. 42 paper, collecting portion of clear filtrate. 7.046 Determination (a) Nitrate plus nitrite nitrogen.-Pipet 25 mL buffered sample exts, 7.045(a) or (b), or 5 mL ext, (c), into reduction column and treat as in 7.044, beginning, "Adjust flow rate thru column . .." Rinse column with 30 mL H20 between samples to remove NaCI. Use portion of buffered sample exts with equiv. diln and pH as ref. soln in detg A at 540 nm. Also det. nitrate-nitrite in reagents and correct for this blank value. Calc. total nitrate- nitrite N from std curve. (b) Nitrite nitrogen.-Pipet aliquot clear sample filtrate (contg <15 p,g nitrite) into 50 mL vol. flask and dil. with H20 to ca 40 mL. Mix well, add 5 mL sulfanilamide soln, (e), mix, and let stand 3 min. Add 2 mL coupling reagent, (f), and dil. to vol. with H20. Mix well and let stand 20 min for max. color development. Measure A in 1 em cells against sample ext with equiv. diln at 540 nm. Correct for nitrite reagent blank. (c) Nitrate nitrogen.-Calc. by difference between (a) and (b) above. 7.047 Calculation ppm N02 and/or N03-N = ILg N03-N found x diln factor/g sample. Diln factors for exts: 7.045(a), 11.1; (b), 22.2; (c), 200. Pepsin Digestibility of Animal Protein Feeds (16) Official Final Action 7.048 Principle Defatted sample is digested 16 hr with warm soln of pepsin under const agitation. Insol. residue is isolated by filtering, washed, dried, and weighed to det. % residue. Residue is examined microscopically and analyzed for protein. Filtration method is applicable to all animal proteins. Methods are not applicable to vegetable proteins or mixed feeds because of presence of complex carbohydrates and other compds not digested by pepsin. 7.049 Apparatus (a) Agitator.-5ee Fig. 7:02. Continuous, slow speed (15 rpm), end-over-end type, to operate inside incubator at 45:t2° and carry 8 oz screw-cap prescription bottles, or equiv. Agitator and bottles available from D. E. Sims, 716 Forrest Ave, Quincy, IL 62301. Stirring or reciprocating (shaking) type agitator cannot be used because solid particles collect on sides of bottle and do not contact pepsin soln. If heat from agitator motor raises incubator temp. to >45°, mount motor outside incubator by drilling hole thru side of incubator and connecting motor to
- 153. AOAC METHODS (1980) NITROGEN 131 FIG.7:02-Agitator agitator with extension shaft and coupling (available from agitator supplier). (Caution: See 51.012.) (b) Settling rack.-Wood or metal to hold digestion bottles at 45° angle. May be made from 2 boards nailed horizontally into "V" cut into vertical end pieces. Also available from agitator supplier, (a). (e) Filtering device.-Modified California buchner, 7.063(d), available from Labconco Corp., 8811 Prospect Ave, Kansas City, MO 64132, No. 55100. (If edge of screen is rough, smooth with small-tip soldering iron.) Use with retainer sleeve, 2 x 2.75" od stainless steel tube, available from agitator supplier, (a). (d) Glass fiber filter.-7 cm, Reeve Angel No. 934-AH, or equiv. (e) Moisture dishes.-AI, 78 mm od x 20 mm, with outside cover and vertical sides (Curtin Matheson Scientific, Inc., No. 19370-30, or equiv.). 7.050 Reagent Pepsin soln.-O.2% pepsin (activity 1:10,000) in 0.075N HCI; do not use pepsin of activity other than 1: 10,000. Prep. just before use by dilg 6.1 mL HCI to 1 L and heating to 42-45°. Add pepsin and stir gently until dissolved. Do not heat pepsin soln on hot plate or overheat. 7.051 Preparation of Sample Sieve sample, 7.001, thru No. 20 sieve. Grind portion retained on sieve to pass No. 20 sieve. Combine both portions and blend by stirring and shaking in pt (500 mL) jar. Thoro blending is essential. Because of high fat content of many animal products, grinding without sieving may cause sticking in mill, loss of moisture or fat, or poorly blended sample. 7.052 Extraction (Caution: See 51.011, 51.039, and 51.054.) Prep. extn thimble from 11 cm Whatman No.2 paper, or equiv., as follows: Fold paper in half; straighten paper and refold at right angles to first fold; turn paper over and repeat process with folds at 45° to original fold; while holding creased paper in one hand, place short test tube (6-8 mm smaller in diam. than extractor sample holder or cup in which thimble is to be used) at its center; fold along natural crease lines to form 4-pointed star around tube; and wrap points in same direction around tube to complete thimble. Weigh 1.000 g ground sample (0.500 g of poultry byproducts or hydrolyzed feathers because of gummy nature and amt of residue) into thimble and ext 1 hr with ether at condensation rate of 3-4 drops/sec. (If Soxhlet is used, top of thimble should extend above siphon tube to avoid loss of solid particles. If paper contg sample is totally submerged in siphon cup, sample must be c.ompletely wrapped in paper.) Observe ether ext to det. that no solid particles were carried into solv. For approx. fat content detn, evap. ether, and dry and weigh residue. Remove paper from sample container or cup and let dry at room temp. Unfold, and quant. brush defatted sample into digestion bottle, avoiding contamination by brush bristles or filter paper fibers. Use of powder funnel is helpful to avoid loss. 7.053 Pepsin Digestion To defatted sample in agitator bottle add 150 mL freshly prepd pepsin soln prewarmed to 42-45°. Be sure sample is completely wetted by pepsin soln. Stopper bottle, clamp in agitator, and incubate with const agitation 16 hr at 45°. 7.054 Treatment of Residue Dry individual sheets of glass fiber filter, (d). 30 min at 110° in moisture dishes with cover open. Cool in desiccator 30 min with cover closed, and weigh (W,). Remove bottles from agitator. Place in 45° angle settling rack and loosen caps. Let residue settle ;;.15 min. Place weighed filter in California buchner, (e), apply suction, and moisten with H20. Place retainer sleeve on filter and press down gently. Rinse particles of residue on cap onto filter with small amt H20. Carry bottle from rack to filter at same angle as settled and slowly pour contents thru filter as continuous small stream, avoiding all unnecessary agitation. Liq. passes thru paper as rapidly as poured, with residue spreading over surface of filter but not covering it completely until all or pratically all of liq. has passed thru. If filtration rate becomes slow, it may be accelerated by adding acetone washes described below, but only if no signif- icant amt of digestion mixt. remains on funnel when acetone is added. (Filtration (passage of aq. mixt. thru filter) should be complete within 1 min with most proteins.) After supernate has passed thru filter, quant. transfer residue onto filter as follows: Add 15 mL acetone to bottle. Hold thumb over bottle neck and shake vigorously. Release pressure, replace thumb over bottle neck, and shake bottle in inverted position over filter. Remove thumb, letting acetone and residue discharge onto filter. Repeat rinse with second 15 mL portion acetone, shaking and releasing pressure as above. Inspect bottle, and rinse further with acetone, using policeman, if necessary. If >3 mm liq. remains on paper when acetone washes are started, it may be necessary to use three 15 mL acetone washes instead of 2 to increase filtration rate. After all liq. passes thru funnel, wash residue and inside surface of retainer sleeve with 2 small portions acetone from wash bottle or hypodermic syringe, and suck dry. Remove retainer sleeve from funnel. Transfer filter to original moisture dish. Scrape or brush any residue particles or filter clinging to retainer sleeve or funnel onto filter in moisture dish. Dry in oven, cool, and weigh as before (W2 ). Calc. % indigestible residue = (W2 - W,) x 100/g sample. Det. indigestible protein by transferring filter contg residue directly to Kjeldahl flask. Proceed as in 7.015. (Caution: Violent reaction may take place when NaOH is mixed with dild digestion mixt., caused by large excess H2S04 due to small amt org. material from residue and none from glass filter. Avoid by
- 154. 132 7. ANIMAL FEED AOAC METHODS (1980) thoroly mixing and cooling digestion mixt. before addn of NaOH or by using 20 mL H2S04 in Kjeldahl digestion instead of 25 mL.) Make blank detn on 1 sheet of glass filter and subtract from each sample detn, if necessary. Calc. % protein based on original sample wt. Result represents % indigestible protein in sample. Convert to % crude protein content of sample not digested, "protein indigestible" = % indigestible protein in sample x 100/% total crude protein in sample. OTHER CONSTITUENTS Crude Fat or Ether Extract' Use method 7.056 or 7.057 for mixed feeds other than (1) entirely baked and/or expanded, (2) entirely dried milk products, or (3) contg urea. Direct Method-Official Final Action 7.055 Reagent Anhydrous ether.-Wash com. ether with 2 or 3 portions H2 0, add solid NaOH or KOH, and let stand until most of H20 is abstracted from the ether. Decant into dry bottle, add small pieces of carefully cleaned metallic Na, and let stand until H evolution ceases. Keep ether, thus dehydrated, over metallic Na in loosely stoppered bottles. (Caution: See 51.034 and 51.054.) 7.056 Determination (Large amts H2 0-sol. components such as carbohydrates, urea, lactic acid, glycerol, and others may interfere with extn of fat; if present, ext 2 g sample on small paper in funnel with five 20 mL portions H20 prior to drying for ether extn. Caution: See 51.009,51.011, and 51.054.) Ext ca 2 g sample, dried as in 7.003 or 7.006*, with anhyd. ether. Use thimble with porosity permitting rapid passage of ether. Extn period may vary from 4 hr at condensation rate of 5-6 drops/sec to 16 hr at 2-3 drops/sec. Dry ext 30 min at 100°, cool, and weigh. 7.057 Indirect Method-Official Final Action Det. moisture as in 7.003 or 7.006*; then ext dried substance as in 7.056, and dry again. Report loss in wt as ether ext. 7.058 In Baked or Expanded and Intermediate Moisture (Semimoist or Soft-Moist) Pet Foods (171 Official Final Action (To be used only on products which have been baked and/or expanded, and on intermediate moisture pet foods. Not appli- cable to canned, fresh, or frozen pet food. Such products should be dried at 70-110°, then ground, and drying completed by 7.003 or 7.006* followed by 7.056 or 7.057. Caution: See 51.011, 51.054, and 51.073.) Place ca 2 g, accurately weighed, ground, well mixed sample in Mojonnier fat-extn tube, add 2 mL alcohol to prevent lumping on addn of acid, and shake to moisten all particles. Add 10 mL HCI (25+ 11). mix well. and set tube 30-40 min in H20 bath at 70-BO°, shaking frequently. Cool to room temp. and add alcohol until liq. level rises into constricted portion of Mojonnier tube. Add 25 mL ether, stopper with glass, Neoprene, or good quality rubber stopper thoroly cleaned with alcohol, and shake vigorously 1 min. Carefully release pressure so that no solv. is lost. Wash adhering solv. and fat from stopper back into extn tube with few mL redistd pet ether (bp <60°). Add 25 mL redistd pet ether, stopper, and shake vigorously 1 min. Let stand until upper liq. is practically clear or centrf. 20 min at ca 600 rpm. Pour as much of ether-fat soln as possible thru filter consisting of cotton pledget packed just firmly enough in funnel stem to let ether pass freely into 150 mL beaker contg several glass beads. Rinse lip of tube with few mL pet ether. Re-ext liq. remaining in tube twice, each time with only 15 mL of each ether, shaking 1 min after addn of each ether. Pour clear ether soln thru filter into same beaker as before, and wash tip of tube, stopper, funnel, and end of funnel stem with few mL of mixt. of 2 ethers (1 +1). Evap. slowly on steam bath under gentle stream of air or N. Continue heating on steam bath 15 min after solv. has evapd; then cool to room temp. Redissolve dried fat residue in four 10 mL portions Et ether, filtering each portion thru small pledget of cotton into 100 mL beaker, contg few glass beads, that has been predried 30 min at 100°, cooled to room temp. in desiccator, and weighed immediately. Use fifth 10 mL portion ether for rinsing cotton and funnel. Evap. ether on steam bath, dry 90 min at 1000 , cool to room temp. in desiccator, and weigh immediately. Correct this wt by blank detn on reagents used. 7.059 In Dried Milk Products (TB}-Official Final Action Proceed as in 16.199(b) and 16.200, using B.5 mL H20 and 1.5 mL NH40H. 7.060 In Fish Meal See 18.046 and 18.047-18.049. 7.061 Crude Fiber (T9)-Official Final Action AOCS-AOAC Method (Caution: See 51.086.) Principle Crude fiber is loss on ignition of dried residue remaining after digestion of sample with 1.25% H2S04 and 1.25% NaOH solns under specific conditions. Method is applicable to grains, meals, flours, feeds, and fiber-bearing material from which fat can be extd to leave workable residue. 7.062 Reagents (a) Sulfuric acid soln.-D.255±0.005N. 1.25 g H2S04/100 mL. Concn must be checked by titrn. (b) Sodium hydroxide soln.-D.313±0.005N. 1.25 g NaOH/l 00 mL, free, or nearly so, from Na2C03• Concn must be checked by titrn. (c) Prepared asbestos.-Spread thin layer acid-washed, me- dium or long fiber asbestos in evapg dish and heat 16 hr at 600° in furnace. Boil 30 min with 1.25% H2S04, filter, wash thoroly with H2 0, and boil 30 min with 1.25% NaOH. Filter, wash once with 1.25% H2S04 , wash thoroly with H20, dry, and ignite 2 hr at 600°. Det. blank by treating 1.0 g prepd asbestos with acid and alkali as in detn. Correct crude fiber results for any blank, which should be negligible (ca 1 mg). Asbestos recovered from detn may be used in subsequent detns. (d) Alcohol.-95% or reagent alcohol, MeOH, or isopropanol. (e) Antifoam.-Dow Corning Corp. Antifoam A compd dild 1+4 with mineral spirits or pet ether, or H2 0-dild Antifoam B Emulsion (1 +4). Do not use Antifoam Spray. (f) Bumping chips or granules.-Broken Alundum crucibles or equiv. granules (RR Alundum 90 mesh, Norton Co., 1 New Bond St, Worcester, MA 01606) are satisfactory. 7.063 Apparatus (a) Digestion apparatus.-With condenser to fit 600 mL beaker, and hot plate adjustable to temp. that will bring 200 mL
- 155. AOAC METHODS (1980) ....-,, , : : I : FIG. 7:03---{)klahoma State filter screen H2 0 at 25° to rolling boil in 15±2 min. (Available from Labconco Corp., 8811 Prospect Ave, Kansas City, MO 64132.) (b) Ashing dishes.-Silica, Vitreosil70 x 15 mm; or porcelain, Coors, No. 450, size 1, or equiv. (e) Desiccator.-With efficient desiccant such as 4-8 mesh Drierite (CaCI2 is not satisfactory). (d) Filtering device.-With No. 200 type 304 or 316 stainless steel screen (W. S. Tyler Co., 8200 Tyler Blvd, Mentor, OH 44060), easily washed free of digested residue. Either Oklahoma State filter screen (see Fig. 7:03; available from Labconco Corp.) or modified California plastic buchner (see Fig. 7:04; consists of 2 piece polypropylene plastic funnel manufactured by Nalge Co., 75 Panorama Creek Drive, Rochester, NY 14602, Cat. No. 4280, 70 mm (without No. 200 screen), or equiv. (also available from Labconco Corp.). Seal screen to filtering surface of funnel, using small-tip soldering iron). (e) Suction filter.-To accommodate filtering devices. Attach suction flask to trap in line with aspirator or other source of vac. with valve to break vac. (f) Liquid preheater.-For preheating H20, 1.25% H2S04 , and 1.25% NaOH solns to bp of H2 0. Convenient system, shown in Fig. 7:05, consists of sheet Cu tank with 3 coils of %" (10 mm) od Cu tubing, 12.5' (3.8 m) long. Solder inlets and outlets where tubing passes thru tank walls. Connect to reflux condenser and fill with H20. Keep H20 boiling with two 750 watt thermostatically controlled hot plates. Use Tygon for inlet leads to reservoirs of H20, acid, and alkali; use gum rubber tubing for outlets. Capacity of preheater is adequate for 60 analyses in 8 hr. 7.064 Preparation of Sample Reduce sample (riffle is suitable) to 100 g and place portion in sealed container for H20 detn. Immediately det. H20. Grind remainder to uniform fineness. (Weber mill (Sargent-Welch Scientific Co. S-60870) with screen 0.033-0.040" (No. 18 or 20), Micro mill (Pulverizing Machinery, Div. Mikropul Corp., 10 Chatham Rd, Summit, NJ 07901) with screen 1/25-1/16" (No. 18--No. 12), and Wiley mill with 1 mm (No. 18) screen give comparable fineness.) Since most materials lose moisture during grinding, det. H2 0 on ground sample at same time sample is taken for crude fiber detn. FIBER 133 II II CJJ----3 1/8""---1 FIG. 7:04--Modified California State buchner funnel, 2-piece polypro- pylene plastic, covered with 200-mesh screen, A, heat-sealed to edge of filtering surface 7.065 Determination Ext 2 g ground material with ether or pet ether, 14.088. If fat is <1 %, extn may be omitted. Transfer to 600 mL beaker, avoiding fiber contamination from paper or brush. Add ca 1 g prepd asbestos, 200 mL boiling 1.25% H2S04, and 1 drop dild antifoam. (Excess antifoam may give high results; use only if necessary to control foaming.) Bumping chips or granules may also be added. Place beaker on digestion app. with preadjusted hot plate and boil exactly 30 min, rotating beaker periodically to keep solids from adhering to sides. Remove beaker, and filter as in (a) or (b). (a) Using Oklahoma filter screen.-Turn on suction and insert screen (precoated with asbestos if extremely fine materials are analyzed) into beaker, keeping face of screen just under surface of liq. until all liq. is removed. Without breaking suction or raising filter, add 50--75 mL boiling H20. After wash is removed, , : ~¢J~~ff,;;~;f~ta (-':: ,> FIG. 7:0S-Continuous heater for distilled water, 1.25% alkali, and 1.25% acid
- 156. 134 7. ANIMAL FEED AOAC METHODS (1980) repeat with three 50 mL washings. (Work rapidly to keep mat from becoming dry.) Remove filter from beaker and drain all H20 from line by raising above trap level. Return mat and residue to beaker by breaking suction and blowing back. Add 200 mL boiling 1.25% NaOH and boil exactly 30 min. Remove beaker, and filter as above. Without breaking suction, wash with 25 mL boiling 1.25% H2S04 and three 50 mL portions boiling H20. Drain free of excess H2 0 by raising filter. Lower filter into beaker and wash with 25 mL alcohol. Drain line, break suction, and remove mat by blowing back thru filter screen into ashing dish. Proceed as in (c). (b) Using California buchner.-Filter contents of beaker thru buchner (precoated with asbestos if extremely fine materials are being analyzed). rinse beaker with 50-75 mL boiling H20, and wash thru buchner. Repeat with three 50 mL portions H20, and suck dry. Remove mat and residue by snapping bottom of buchner against top while covering stem with thumb or forefin- ger and replace in beaker. Add 200 mL boiling 1.25% NaOH and boil exactly 30 min. Remove beaker, and filter as above. Wash with 25 mL boiling 1.25% H2S04, three 50 mL portions H20, and 25 mL alcohol. Remove mat and residue; transfer to ashing dish. (c) Treatment ofresidue.-Dry mat and residue 2 hr at 130±2°. Cool in desiccator and weigh. Ignite 30 min at 600± 15°. Cool in desiccator and reweigh. % Crude fiber in ground sample = C = (Loss in wt on ignition - loss in wt of asbestos blank) x 100/wt sample. % Crude fiber on desired moisture basis = C x (100 - % moisture desired)/(100 - % moisture in ground sample). Report results to 0.1 %. 7.066 Asbestos-Free (AF) Method (20) Official Final Action Principle Principle is same as in 7.061, except sample is exposed to min. vac. needed to regulate filtration, and heating of sample solns prevents gelling or pptn of possible satd solns. 7.067 Apparatus and Reagents See reagents 7.062(a), (b). and (f); app. 7.063(a). (e). (d). and (f). and 14.088; and in addn: (a) Filtration apparatus.-System to permit application of min. vac. necessary for filtration and washing of each sample within 3-5 min. Each unit consists of reservoir manifold connected to (1) H20 aspirator thru 120° stopcock, (2) atm. thru second stopcock with metering device, and (3) receptacle contg cone- shaped hard rubber gasket which provides vac. seal with cru- cible. Vac. gage attached to manifold indicates vac. applied to crucible. Crucible can be heated before and during filtration by flow of hot H2 0 in surrounding jacket. (For photograph of app., see JAOAC 56, 1353(1973). Filtration unit is available as Model 150 from Analytical BioChemistry Laboratories, Inc., PO Box 1097, Columbia, MO 65201.) (b) Crucible.-Fritted glass, 50 mL, coarse porosity. Clean as follows: Brush, and flow hot tap H2 0 into crucible to remove as much ash as possible. Submerge crucible in base soln, (c)(2), ~5 min, remove, and rinse with hot tap H20. Submerge in HCI (1+1). (e) (T), ~5 min, remove, and rinse thoroly with hot tap H20 followed by distd H20. After 3-4 uses, back wash by inverting crucible on hard rubber gasket in filtration app., and flowing near-boiling H20 thru crucible under partial vac. (c) Cleaning solns.-( 1) Acid soln.-HCI (1 +1). (2) Base soln.- Dissolve 5 9 Na2H2EDTA, 50 g Na2HP04 (tech. grade). and 200 g KOH in H20, and dil. to 1 L. Storage in sep. wide mouth containers holding 2-3 L soln into which crucibles can be placed is convenient. 7.068 Determination (Caution: See 51.011 and 51.073.) Ext 2 g ground material with ether or pet ether, 14.088. If fat is <1 %, extn may be omitted. Transfer to 600 mL reflux beaker, avoiding fiber contamination from paper or brush. Add 0.25-0.5 g bumping granules, followed by 200 mL near-boiling 1.25% H2S04 soln in small stream directly on sample to aid in complete wetting of sample. Place beakers on digestion app. at 5 min intervals and boil exactly 30 min, rotating beakers periodically to keep solids from adhering to sides. Near end of refluxing place California buchner, 7.063(d). previously fitted with No.9 rubber stopper to provide vac. seal, into filtration app., and adjust vac. to ca 25 mm Hg (735 mm pressure). At end of refluxing, flow near-boiling H20 thru funnel to warm it; then decant liq. thru funnel, washing solids into funnel with min. of near-boiling H20. Filter to dryness, using 25 mm vac., and wash residue with four 40-50 mL portions near-boiling H20, filtering after each washing. Do not add wash to funnel under vac.; lift funnel from app. when adding wash. Wash residue from funnel into reflux beaker with near-boiling 1.25% NaOH soin. Place beakers on reflux app. at 5 min intervals and reflux 30 min. Near end of refluxing, turn on filtration app., place crucible, (b). in app., and adjust vac. to ca 25 mm. Flow near-boiling H20 thru crucible to warm it. (Keep near-boiling H20 flowing thru jacket during filtration and washing.) At end of refluxing, decant liq. thru crucible and wash solids into crucible with min. of near-boiling H20. Increase vac. as needed to maintain filtration rate. Wash residue once with 25-30 mL near-boiling 1.25% H2S04 soln, and then with two 25-30 mL portions near-boiling H20, filtering after each washing. (Filtering and washing takes ca 3-5 min/sample.) Do not add wash to crucible under vac. Dry crucible with residue 2 hr at 130±2° or overnight at 110°, cool in desiccator, and weigh. Ash 2 hr at 550±10°, cool in desiccator, and weigh. Do not remove crucibles from furnace until temp. is ",250°, as fritted disk may be damaged if cooled too rapidly. % Crude fiber = Loss in wt on ignition x 100/wt sample. 7.069 Acid-Detergent Fiber and Lignin (21) Official Final Action (Caution: See 51.086.) Reagents (a) Sulfuric acid.-72% by wt. Stdze reagent grade H2 S04 to sp gr 1.634 at 20° or 24.00N: Add 1200 g H2S04 to 440 mL H20 in 1 L MCA vol. flask with cooling. Stdze to 1634 gil at 20° by removing soln and adding H20 or H2S04 as required. (Caution: See 51.030.) (b) Acid-detergent soln.-Add 20 g cetyl trimethylammonium bromide (tech. grade) to 1 L 1.00N H2S04 , previously stdzd. Agitate to aid soln. (e) Asbestos.-Place 100 g asbestos in 3 L flask contg 850 mL H20. Add 1.4 L H2S04 (tech. grade). mix, and let cool 2 hr at room temp. Filter on large buchner and wash with H20. Resus- pend mat in H20 and pour into bag sewn from rectangle of fiberglass window screening, 14 x 18 mesh (bag should be ~45 cm wide x 30 cm deep). Wash by immersion and agitation in partly filled sink to remove fine particles. Ash recovered asbestos 16 hr in 800° furnace. Store in dry form until use. Used asbestos may be rewashed, reashed, and reused. Com. prepd acid-washed asbestos is unsatisfactory unless treated with 72% H2S04 and ashed at 800°.
- 157. AOAC METHODS (1980) CARBOHYDRATES 135 7.070 Apparatus (a) Refluxing apparatus.-Any conventional app. suitable for crude fiber detns. Berzelius beakers (600 mL) and condensers made from 500 mL r-b flasks are also satisfactory. (b) Fritted glass crucibles.-Use coarse porosity, 40-50 mL Pyrex crucible. Wash new crucibles and ash at 500°. Remove while still hot and place in 100° forced-draft oven ",,1 hr. Cool 15 min in desiccator over P20 S or Mg(CIO')2 and weigh in same order samples are to be weighed. Check balance 0 after each weighing if crucibles are still warm. Hold length of time from oven to balance pan as const as possible and always weigh crucibles in same order. 7.071 Determination of Acid-Detergent Fiber Weigh 1 g air-dried sample ground to pass 1 mm screen, or approx. equiv. amt wet material, into refluxing container. Add 100 mL acid-detergent soln at room temp. Heat to boiling in 5-10 min; reduce heat to avoid foaming as boiling begins. Reflux 60 min from onset of boiling, adjusting boiling to slow, even level. Remove container, swirl, and filter thru weighed (W,) fritted glass crucible, using min. suction. Increase vac. only as needed. Shut off vac. Break up filtered mat with rod and fill crucible % full with hot (90-100°) H20. Stir and let soak 15-30 sec. Dry with vac. and repeat H20 washing, rinsing sides of crucible. Wash twice similarly with acetone. Repeat acetone washings until no more color is removed, breaking up all lumps so that solv. wets all particles of fiber. Remove residual acetone with vac. Dry 3 hr or overnight in 100° forced-draft oven and weigh (W2 ). Calc. % acid-detergent fiber = 100 (W2 - W,)/S, where S = g sample x g oven-dried matter/g air-dried or wet matter, detd on sep. sample. 7.072 Determination of Ugnin To crucible contg fiber, 7.071, add 1 g asbestos. Place crucible in 50 mL beaker for support or arrange crucibles in shallow enamel pan. Cover contents of crucible with cooled (15°) 72% H2SO. and stir with glass rod to smooth paste, breaking all lumps. Fill crucible about half-way with acid and stir. Leave glass rod in crucible; refill with 72% H2SO. and stir hourly as acid drains, keeping crucible at 20-23° (cool if necessary). After 3 hr, filter as completely as possible with vac., and wash with hot H20 until acid-free to pH paper. Rinse sides of crucible and remove stirring rod. Dry crucible in 100° forced-draft oven, cool in desiccator over P20 S or Mg(CIO.)" and weigh (W3 ). Ignite crucible in 500° furnace 2 hr or until C-free. Place crucible while still hot into 100° forced-draft oven 1 hr. Transfer to desiccator, cool, and weigh (W.). Det. asbestos blank by weighing 1 g asbestos into tared crucible. Proceed as above, beginning "Cover contents of cru- cible ..." Record any loss in wt on ashing (Ws). Discontinue detn of blank if asbestos blank is <0.0020 gig asbestos. Calc. % acid-insol. lignin = (W3 - W. - Ws)/S. Total Sugars (22J-Official Final Action 7.073 Reagents (a) Soxhlet modification ofFehling soln.-Prep. as in 31.034(a) and (b). (b) Invert sugar std soln.-1.0%. Prep. as in 31.034(c). but do not neutze. Oil. to 0.5% just before use for analysis of most products. (c) Lactose std soln.-1.0%. Dissolve 5.000 g lactose in H20 and dil. to 500 mL. Prep. daily. 7.074 Apparatus (a) Lamp.-Fluorescent desk lamp or 150 watt reflector spot lamp, to illuminate boiling soln. (b) Heater.-Glas-Col mantle, 250 mL, placed over mag. stir- rer. Adjust heat so that 50 mL H20 contg stirring bar will boil in 3 min. Mag. stirring hot plate is also satisfactory. 7.075 Preparation of Sample and Inversion (a) Feeds containing molasses.-Weigh appropriate size sam- ple, prepd as in 7.002 but not ground, to provide final soln ca 0.5% invert sugar but ""5 g, into 250 mL P flask (Corning Glass Works No. 5840, or equiv.). Add 150 mL H20, swirl to wet and mix, and heat just to bp. Let stand to cool, dil. to vol., mix, and let stand to settle coarse particles. Transfer 50 mL supernate to 100 mL vol. flask and add 2.5 mL HCI (sp gr 1.18 at 20/4°). Let stand overnight at ",,25°, dil. to vol., and mix. (If aliquot to be used in detn is >25 mL, it is necessary to neutze inverted soln.) (b) Feeds containing milk products.-Weigh appropriate size sample to provide final soln ca 1% lactose into 250 mL vol. flask. Thoroly moisten sample with H20, swirl to dissolve lactose, dil. to vol., mix, and let stand to settle coarse particles. Proceed as in 7.077(b). 7.076 Standardization Fill 50 mL buret, with offset tip, with std sugar soln (invert sugar for use with 7.077(a) and lactose with 7.077(b)). Proceed as in 31.080, par. 2, except use same type flask as used in 7.077, do not add H20, and start stirring after addn of indicator. 7.077 Determination (a) Difference method.-Add reagents and stirring bar to 250 mL extn flask (Corning Glass Works No. 5160, or equiv.) or to erlenmeyer, as in 7.076. Transfer aliquot inverted soln, (a). to flask so that >1 but <5 mL std soln will be required to reach end point, place on preheated mantle or hot plate, heat to bp, boil 2 min, add ca 1 mL indicator, and begin stirring. Complete detn by titrg with std sugar soln to same end point used in stdzn. Color change is not so sharp as in stdzn, but under suitable light it is definite, discernible, and repeatable. (b) Alternative method.-Fill buret with sample soln, (b), or inverted sample soln, (a). As in 7.076, place reagents in flask, place on heater, add sample soln to within 2 mL of final titrn (detd by trial), bring to bp, boil 2 min, and complete titrn as in (a). 7.078 Calculations % Total sugar (as invert or lactose) =[(F - M) x I x 100)/[V x (W/250) x DlJ, where F = mL std sugar required to reduce mixed Soxhlet reagent in stdzn; M = mL std soln required to complete detn (omit in alternative method); 1= concn std soln; V = mL sample soln in aliquot used; W = g sample; and D = diln factor. Report total sugars, expressed as invert or as lactose. 7.079 Sucrose (23)-Official Final Action Place 10 g sample in 250 mL vol. flask. If material is acid, neutze by adding 1-3 g CaC03• Add 125 mL 50% alcohol by vol., mix thoroly, and boil on steam bath or by partially immersing flask in H20 bath 1 hr at 83-8r, using small funnel in neck of flask to condense vapor. Cool and let mixt. stand several hr, preferably overnight. Oil. to vol. with neut. 95% alcohol, mix thoroly, let settle or centrf. 15 min at 1500 rpm, and decant closely. Pipet 200 mL supernate into beaker and evap. on steam
- 158. 136 7. ANIMAL FEED AOAC METHODS (1980) bath to 20-30 mL. Do not evap. to dryness. Little alcohol in residue does no harm. Transfer to 100 mL vol. flask and rinse beaker thoroly with H20, adding rinsings to flask. Add enough satd neut. Pb(OAc)2 soln (ca 2 mL) to produce flocculent ppt, shake thoroly, and let stand 15 min. Oil. to vol. with H20, mix thoroly, and filter thru dry paper. Add enough anhyd. Na2C03 or K oxalate to filtrate to ppt all Pb, again filter thru dry paper, and test filtrate with little anhyd. Na2C03 or K oxalate to make sure that all Pb has been removed. Place 50 mL prepd soln in 100 mL vol. flask, add piece of litmus paper, neutze with HCI, add 5 mL HCI, and let inversion proceed at room temp. as in 31.026(c). When inversion is complete, transfer soln to beaker, neutze with Na2C03, return soln to 100 mL flask, dil. to vol. with H20, filter if necessary, and det. reducing sugars in 50 mL soln (representing 2 g sample) as in 31.038. Calc. results as invert sugar. % Sucrose = [% total sugar after inversion - % reducing sugars before inversion (both calcd as invert sugar)) x 0.95. Because insol. material of grain or cattle food occupies some space in flask as originally made up, correct by mUltiplying all results by factor 0.97, as results of large number of detns on various materials show avo vol. of 10 g material to be 7.5 mL. * Starch-Official Final Action * 7.080 Direct Acid Hydrolysis See 8.019. Use sample contg 2.5-3 g dry material. 7.081 Diastase Method with Subsequent Acid Hydrolysis See 7.067, 12th ed. 7.082 Extraction with Subsequent Enzyme Hydrolysis See 14.075-14.080. 7.083 In Presence of Interfering Polysaccharides (24) See 22.048, 10th ed. 7.084 In Condensed or Dried Milk Products- Qualitative Test (25) See 22.049, 10th ed. 7.085 * Pentosans (26)-Official Final Action * See 22.050-22.051, 10th ed. 7.086 * Galactan-Official Final Action * See 22.052, 10th ed. (Caution: See 51.011 and 51.026.) 7.087 * Water-Soluble Acidity (27) * Official Final Action See 22.053, 10th ed. 7.088 * Ferrous Salts (28)-Official Final Action * K3Fe(CN)6 spot test. See 7.074, 12th ed. 7.089 * Copper Salts (28)-Official Final Action * K.Fe(CN)6 spot test. See 7.075, 12th ed. * Surplus method-see inside front cover. 7.090 * Potassium Iodide (28)-Official Final Action * Starch-iodine spot test. See 7.076, 12th ed. 7.091 Minerals in Feeds by Atomic Absorption Spectrophotometry (29)-Official Final Action (Caution: See 51.006.) Apparatus Atomic absorption spectrophotometer.--See 2.109. 7.092 Operating Parameters See Table 2:04, except use fuel-rich air-C2H2 flame for Ca and Mg, and ranges of operation for /Lg element/mL soln are: Ca 5--20, Cu 2-20, Fe 5--20, Mg 0.5--2.5, Mn 5--20, and Zn 1-5. 7.093 Reagents (See introduction to 2.110. Com. prepd std solns may be used.) (a) Calcium std solns.-Prep. as in 2.110(a). (b) Copper, iron, magnesium, manganese, and zinc std solns.-Prep. stock solns as in 2.110(b). (c), (e). (f), and (g). and dil. aliquots with 0.1-Q.5N HCI to make ;;,4 std solns of each element within range of detn. 7.094 Preparation of Sample Solution (a) Dry ashing (not applicable to mineral-mix feeds).-Ash 2-10 g sample in well-glazed porcelain dish. Start in cold furnace, bring to 550°, and hold 4 hr. Cool, add 10 mL 3N HCI, cover with watch glass, and boil gently 10 min. Cool, filter into 100 mL vol. flask, and dil. to vol. with H20. Subsequent dilns with 0.1-0.5N HCI may be necessary to bring sample solns into anal. range, except for Ca. Final Ca diln must contain enough La soln, 2.110(d), to provide 1% La concn after diln to vol. with H20. (b) Wet digestion.-Proceed as in 7.097(a), adding 25 mL HN03 for each 2.5 g sample and dilg to 100 mL with H20. Digestion can be made at low heat on hot plate, using 600 mL beaker covered with watch glass. Subsequent dilns with 0.1-0.5N HCI may be necessary to bring sample solns into anal. range, as in (a). 7.095 Determination and Calculation See 2.112-2.113. Calcium-Official Final Action 7.096 Method 1(30) (Applicable to mineral feeds only) Weigh 2 g finely ground sample into Si02 or porcelain dish and ignite in furnace to C-free ash, but avoid fusing. Boil residue in 40 mL HCI (1 +3) and few drops HN03 • Transfer to 250 mL vol. flask, cool, dil. to vol., and mix thoroly. Pipet 25 mL clear liq. into beaker, dil. to ca 100 mL, and add 2 drops Me red, 2.055(i). Add NH.OH (1 + 1) dropwise to pH 5.6, as shown by intermediate brownish-orange. If overstepped, add HCI (1 +3) with dropper to orange. Add 2 more drops HCI (1 +3). Color should now be pink (pH 2.5--3.0). not orange. Oil. to ca 150 mL, bring to boil, and slowly add, with const stirring, 10 mL hot satd (4.2%) soln of (NH4)2C204. If red changes to orange or yellow, add HCI (1 +3) dropwise until color again changes to pink. Let stand overnight for ppt to settle. Filter supernate thru quant. paper, gooch, or fritted glass filter (fine Pyrex is preferable), and wash ppt thoroly with NH.OH (1 +50). Place paper or crucible with ppt in original beaker, and add mixt. of 125 mL H20 and 5 mL H2S04.
- 159. AOAC METHODS (1980) MINERALS 137 Heat to ~70° and titr. with 0.1N KMnO., 50.025-50.026, to first slight pink. Presence of paper may cause color to fade in few sec. Correct for blank and calc. % Ca. 7.097 Method II (31) Preparation of Solution (Caution: See 51.026 and 51.028.) (a) Weigh 2.5 g sample into 500 or 800 mL Kjeldahl flask. Add 20-30 mL HN03 and boil gently 30-45 min to oxidize all easily oxidizable matter. Cool soln somewhat and add 10 mL 70-72% HCIO•. Boil very gently, adjusting flame as necessary, until soln is colorless or nearly so and dense white fumes appear. Use particular care not to boil to dryness (Danger!) at any time. Cool slightly, add 50 mL H20, and boil to drive out any remaining N02 fumes. Cool, dil., filter into 250 mL vol. flask, dil. to vol., and mix thoroly. (b) Weigh 2.5 g finely ground sample into Si02 or porcelain dish and ignite as in 7.009. Add 40 mL HCI (1 +3) and few drops HN03to residue, boil, transfer to 250 mL vol. flask, cool, dil. to vol., and mix thoroly. 7.098 Determination Pipet suitable aliquot of clear soln, 7.097(a) or (b), into beaker, dil. to 100 mL, and add 2 drops Me red, 2.055(0. Continue as in 7.096, beginning "Add NH.OH (1+1) dropwise ..." except use 0.05N KMnO. for titrn. (100 mL is suitable aliquot of sample soln for grain feeds; for mineral feeds, 25 mL aliquot may be taken and titrd with 0.1N KMnO•. For suitable precision, size of sample, aliquot, and concn of KMnO. must be so adjusted that ~20 mL std KMnO. soln is used.) Soluble Chlorine Titration Method (32)-Official Final Action 7.099 Reagents (a) Potassium chloride std soln.-o.001 g CI/mL. Recrystallize reagent KCI 3 times from H20, dry at 110°, and heat at ca 500° to canst wt. Dissolve 2.1028 g in H20 and dil. to 1 L. (b) Silver nitrate soln.-Dissolve 5 g AgN03 in 1 L H20 and adjust soln so that 1 mL = 1 mL std KCI soln. (c) Potassium thiocyanate soln.-Dissolve 2.5 g KSCN in 1 L H20 and adjust so that 1 mL = 1 mL std AgN03 soln. Stdze as in 50.004. (d) Ferric sulfate soln.-Dissolve 60 g Fe2(SO')3 + Aq. in H20 and dil. to 1 L. (e) Ferric sulfate indicator.-To filtered 25% soln of Fe2(SO.), + Aq. add equal vol. HNO,. 7.100 Determination Transfer 3 g sample to 300 mL erlenmeyer. Add 50 mL Fe2(SO')3 soln (accurately measured), swirling flask to prevent caking of sample and to facilitate soln of CI. Add 100 mL (also accurately measured) NH.OH (1 +19). Swirl flask just enough to ensure soln of CI and thoro mixing of soln. (Very little swirling is necessary. If soln is agitated by vigorous vertical shaking, filtration will be difficult.) Let mixt. settle 10 min. Filter thru dry 11 cm Whatman No. 41 paper, or equiv. Use 50 mL aliquots ('h of total) on samples low in CI (0-2% CI) and 25 mL aliquots (1/6 of total) on samples high in CI (>2%). For mineral and other feeds contg ~10% CI, weigh 1 g and use 15 mL (1/10 of total). If approx. % CI in sample is not known, take 10 mL aliquot for trial titrn. To this add 10 mL HNO, and 10 mL Fe2(SO.), indicator. Dil. to ca 50 mL. Add 0.5 mL KSCN soln and immediately add, with stirring, enough AgNO, soln to entirely eliminate any reddish color. From this titrn calc. vol. AgNO, soln necessary to ppt all CI in aliquot to be used, adding excess equal to ca 10% total vol. necessary, altho somewhat greater excess will not affect results. Use min. total of 10 mL. To sample aliquot in 250 mL beaker add 10 mL HNO, and 10 mL Fe2(SO.), indicator (or 20 mL soln contg equal vols of these solns). Add, with stirring, calcd vol. AgN03soln. Heat to boiling and cool to room temp., stirring enough to coagulate ppt. (Cooling may be hastened by immersion of beakers in cold H20.) Titr. excess AgN03 with KSCN. End point is indicated by first appearance of reddish tint that persists 15 sec. For accurate work, use ref. soln contg all ingredients except KSCN. End point is first change in color. 7.101 Potentiometric Method (33) Official Final Action Apparatus Potentiometer.-With Ag-AgCI reference electrode and Ag- indicating electrode (Fisher Scientific Co. No. 9-313-216 and 13-639-122, or equiv.). 7.102 Standardization Weigh 125 mg dry NaCI into 400 mL beaker. Add 200 mL H20 and 1 mL HNO,. Null potentiometer and titr. NaCI soln with 0.1N AgN03 soln. Plot mL AgNO, soln against mv or scale readings. Add titrant in small enough increments so that voltage end point is obvious. Use same end point for samples. 7.103 Determination (a) Samples containing less than 5% sodium chloride.- Weigh 5.844 g sample into 400 mL beaker. Add ca 200 mL H20 and 1 mL HN03. Swirl mixt. gently and let stand 10 min for complete soln of chlorides. Titr., while stirring, to same voltage end point as in stdzn. % NaCI = mL 0.1N AgNO,/10. (b) Samples containing more than 5% sodium chloride.- Weigh 5.844 g sample into 200 mL vol. flask. Add ca 190 mL H20 and 1 mL HNO" dil. to vol. with H20, mix, and let stand 10 min. Transfer aliquot contg equiv. of ca 125 mg NaCI to 400 mL beaker, dil. to ca 200 mL, add 1 mL HN03, and titr. as in (a). % NaCI = diln factor x mL 0.1N AgN03/10. Cobalt (34)-Offieial Final Action 7.104 Reagents (a) Cobalt std soln.-O.05 mg Co/mL. Dissolve 0.2385 g CoSO•.7H20 (do not dry; use as received) in H20 and dil. to 1 L. Dil., if necessary, to suitable concn to prep. std curve. (b) Nitroso-R salt soln.-Dissolve 1 g C1oH.OH.NO(SO,Na)2 in H20 and dil. to 500 mL. (c) Spekker acid.-Mix 150 mL 85% H3PO. and 150 mL H2SO., and dil. to 1 L with H20. (d) Sodium acetatesoln.-Dissolve 500 g NaOAc.3H20 in H20 and dil. to 1 L with H20. 7.105 Preparation of Standard Curve To 1, 2, etc., up to 11 mL portions std Co soln in 100 mL vol. flasks add 2 mL Spekker acid, 10 mL nitroso-R salt soln, and 10 mL NaOAc soln. Prep. blank by using 2 mL Spekker acid and 10 mL NaOAc soln, but omitting nitroso-R salt soln. Bring solns to bp on hot plate. Add 5 mL HNO, and boil ~1, but,,;;2 min. Cool, and dil. to 100 mL.
- 160. 138 7. ANIMAL FEED AOAC METHODS (1980) 7.106 Determination (Caution: See 51.026 and 51.059.) Ash 2 g sample 2 hr at 600°, transfer to 200 mL vol. flask with 20 mL HCI and 50 mL H20, boil 5 min, cool, and dil. to vol. Let soln settle. Pipet suitable aliquot into small flask. For samples contg 0.01-0.2% Co use equiv. of 0.25 g sample. Adjust amt to ,;;0.5 mg Co. Soln no longer appears to follow Beer's law above this amt. Pass brisk current of H2S thru soln 10 min. Filter directly into 100 mL vol. flask thru Whatman No. 40 paper. Wash with ca 50 mL 1% H2SO. satd with H2S. Add 2 small glass beads and boil off H2S. (Flasks must be given individual attention, as violent bumping may occur.) Shake flasks often. Add 5 mL HN03 and boil until nitrous fumes no longer appear. (Take care, as vol. of soln will be low and bumping and spattering may occur. At first indication of this, remove immediately from hot plate.) Small amt HN03 remaining will not affect result. Cool, add 2 drops phthln, and adjust to first faint pink with ca 30% NaOH soln. Immediately add 2 mL Spekker acid followed by 10 mL nitroso- R salt soln and 10 mL NaOAc soln. Bring to vigorous boil, carefully add 5 mL HN03, and boil ~1 but ,;;2 min. Cool, and dil. to vol. Compare color with std Co solns in photoelec. colorimeter,· using green or No. 54 filter, or in spectrophtr at 540 nm. Read color within 2 hr. Report % Co to third decimal place. Copper (35)-Official Final Action 7.107 Preparation of Standard Curve Dissolve 1.9645 g CuSO•.5H20 in H20 and dil. to 500 mL. (1 mL = 1 mg Cu.) Use from 1 to 10 mL ofthis soln to prep. set of stds in 100 mL Pyrex g-s vol. flasks. Add 4 mL HCI, dil. to 50 mL, add 5 mL tetraethylenepentamine, dil. to vol. with H20, stopper, and mix thoroly. Prep. blank, using all reagents except Cu. Filter blank and stds before reading color as in 7.108. 7.108 Determination Prep. sample soln as in 7.106, using 8 g sample. Pipet 50 mL aliquot into 100 mL Pyrex g-s vol. flask, add 5 mL tetraethyle- nepentamine, dil. to vol. with H20, and mix thoroly. Filter, and compare colors within 30 min in photoelec. colorimeter (red or No. 66 filter) or read in spectrophtr at 620 nm. Report % Cu to third decimal place. Fluorine-Official Final Action 7.109 Colorimetric Method See 25.049-25.055, especially 25.053. Ion Selective Electrode Method (36) 7.110 Apparatus (a) Electrodes.-Fluoride ion selective electrode (Model 94- 09, Orion Research Inc., or equiv.) and single junction calomel ref. electrode, plastic sleeve-type (Model 90-01, Orion Research Inc., or equiv.). (b) Magnetic stirrer.-With 4 cm (1 '12") Teflon-coated stirring bar. Use mat to insulate sample from motor heat. (c) pH meter.--Corning digital Model 112 (Corning Scientific Instruments, Medfield, MA 02052, or equiv.). 7.111 Reagents (Deionized H20 may be used.) (a) Sodium acetate soln.-3M. Dissolve 408 g NaOAc.3H20 with H20 in 1 L vol. flask. When soln warms to room temp., dil. to vol. with H20. Adjust to pH 7.0 with few drops HOAc. (b) Sodium citrate soln.-l.32M. Dissolve 222 g Na citrate.2H20 with ca 250 mL H20 in 1 L vol. flask. Add 28 mL HCIO., dil. to vol., and mix. (c) Fluoride std solns.-(1) Stock soln.-500 ppm. Accurately weigh 1.105 g NaF (reagent grade, dried 4 hr at 100°) into 1 L vol. flask. Dissolve and dil. to vol. with H20, and mix. Store in plastic bottle. (2) Intermediate soln 1.-100 ppm. Pipet 20 mL stock soln into 100 mL vol. flask, dil. to vol. with H20, and mix. (3) Intermediate soln 11.-10 ppm. Pipet 2 mL stock soln into 100 mL vol. flask, dil. to vol. with H20, and mix. (4) Working solns.- Pipet 3, 5, and 10 mL intermediate soln II and 5 and 10 mL intermediate soln I into five 100 mL vol. flasks to prep. 0.3,0.5, 1.0, 5.0, and 10 ppm F working solns, resp. To each add 10.0 mL IN HCI, 25.0 mL NaOAc.3H20 soln, (a), and 25.0 mL Na citrate soln, (b). Dil. to vol. with H20 and mix. 7.112 Preparation of Sample Accurately weigh well mixed sample contg ca 400 JLg F into 200 mL vol. flask. Pipet in 20 mL IN HCI and stir 20 min at high speed on mag. stirrer. Add 50.0 mL NaOAc soln, (a), and 50.0 mL Na citrate soln, (b). to dissolved sample. Dil. to vol. with H20 and mix. 7.113 Determination Connect F and ref. electrodes to pH meter, place electrodes in low concn F soln, and warm up pH meter. Pour 50-70 mL std and corresponding sample solns into sep. 100 mL beakers. Place electrodes in each soln and while stirring with mag. stirrer at const rate, read mv of std and unknown solns. Rinse and blot off electrodes and stirring bar between solns. Construct std curve on 3 cycle semilogarithmic paper. Read ppm F of sample soln from std curve. % F = ppm F x mL sample soln x 10-6 x 100/g sample Iodine in Mineral Mixed Feeds-Official Final Action 7.114 * Knapheide-Lamb Method (37) * See 22.084-22.086, 10th ed. 7.115 Elmslie-Caldwell Method (38) (Not applicable to iodized mineral feeds contg little or no org. matter. Caution: See 51.047.) Place sample contg 3-4 mg I in 200-300 mL Ni dish. Add ca 5 g Na2C03, 5 mL NaOH soln (1 +1). and 10 mL alcohol, taking care that entire sample is moist. Dry at ca 100° to prevent spattering upon subsequent heating (30 min is usually enough). Place dish and contents in furnace heated to 500° and keep at that temp. 15 min. (Ignition of sample at 500° appears to be necessary only to carbonize any sol. org. matter that would be oxidized by Br-H20 if not so treated. Temp. >500° may be used if necessary.) Cool, add 25 mL H20, cover dish with watch glass, and boil gently 10 min. Filter thru 18 cm paper and wash with boiling H20, catching filtrate and washings in 600 mL beaker (soln should total ca 300 mL). Neutze to Me orange with 85% H3PO. and add 1 mL excess. *Surplus method-see inside front cover.
- 161. AOAC METHODS (1980) MICROSCOPY 139 Add excess Br-H20 and boil soln gently until colorless, and then 5 min longer. Add few crystals salicylic acid and cool soln to ca 20°. Add 1 mL 85% H3P04 and ca 0.5 g KI, and titr. I with 0.005N Na2S20 3, adding starch soln when liberated I color is nearly gone. 1 mL 0.005N Na2S20 3 = 0.1058 mg I. 7.116 Acid-Soluble Manganese (39) Official Final Action Reagent Potassium permanganate std soln.-500 ppm Mn. Prep. and stdze as in 50.025-50.026, except use 1.4383 g KMn04 and 0.12 g Na oxalate. Transfer aliquot contg 20 mg Mn to beaker. Add 100 mL H20, 15 mL H3P04, and 0.3 g KIO., and heat to bp. Cool, and dil. to 1 L. Protect from light. Oil. this soln contg 20 ppm Mn with H20 (previously boiled with 0.3 g KI04/L) to make convenient working stds in range of concns to be compared. 7.117 Determination (Caution: See 51.026 and 51.030.) Ash weighed sample, 5--15 g, at dull red heat (ca 600°) in porcelain dish. Cool, and add 5 mL H2S04 and 5 mL HN03 to ash in dish or to ash transferred to beaker with 20-30 mL H20. Evap. to white fumes. If C is not completely destroyed, add addnl portions HN03 , boiling after each addn. Cool slightly, transfer to 50 or 100 mL vol. flask, and add vol. dil. H3P04 soln (8+92) equal to 'h vol. of flask (25 or 50 mL). Cool, dil. to vol., mix, and filter or let stand until clear. If 50 mL flask was used, pipet 25 mL clear soln into beaker or 50 or 100 mL vol. flask and add 15 mL H20. If 100 mL flask was used, pipet 50 mL into beaker or 100 mL flask and add 30 mL H2 0. Heat nearly to bp, and with stirring or swirling add 0.3 g KIO. for each 15 mg Mn present. Keep 30-60 min at 90-100°, or until color development is complete. Cool, dil. to measured vol. of 50 or 100 mL, and mix. Compare with std KMn04 soln in photoelec. colorimeter or in spectrophtr at 530 nm. Calc. ppm Mn. Phosphorus Alkalimetric Ammonium Molybdophosphate Method (40)-Official Final Action 7.118 Reagents (e) Molybdate soln.-Dissolve 100 g Mo03 in mixt. of 144 mL NH40H and 271 mL H20. Cool, and slowly pour soln, stirring constantly, into cool mixt. of 489 mL HN03 and 1148 mL H20. Keep final mixt. in warm place several days or until portion heated to 40° deposits no yellow ppt. Decant soln from any sediment and keep in g-s vessels. (b) Acidified molybdate soln.-To 100 mL molybdate soln, (a), add 5 mL HN03• Filter immediately before use. (c) Sodium hydroxide std soln.-Dil. 324.03 mL IN alkali, carbonate-free, 50.032-50.036, to 1 L. (100 mL of this soln should neutze 32.40 mL IN acid; 1 mL = 1 mg or 1% P20 S on basis of 0.1 g sample.) (Since burets in const use may become so corroded as to increase their capacity, test them at least an- nually.) (d) Std acid soln.-Prep. soln of HCI or of HN03, corresponding to concn of (c) or to 'h this concn, and stdze by titrn against (c), using phthln. 7.119 Determination Prep. sample soln as in 7.097(a). Pipet, into beaker or flask, aliquot corresponding to 0.4 g sample for P20 S content of sample <5%; 0.2 g for 5-20%; 0.1 g for >20%. Add 5-10 mL HN03, depending on method of soln (or equiv. in NH4 N03 ); then add NH4 0H until ppt that forms dissolves only slowly on vigorous stirring, dil. to 75-100 mL, and adjust to 25-30°. If sample does not give ppt with NH.OH as test of neutzn, make soln slightly alk. to litmus paper with NH40H and then slightly acid with HN03 (1 +3). Add 20-25 mL acidified molybdate soln for P20s content <5%; 30-35 mL for 5-20%; and enough acidified molybdate soln to ensure complete pptn for >20%. Shake or stir mech. 30 min at room temp.; decant at once thru filter and wash ppt twice by decanting with 25-30 mL portions H2 0, agitating thoroly and allowing to settle. Transfer ppt to filter and wash with cold H2 0 until filtrate from 2 fillings of filter yields pink color on adding phthln and 1 drop of the std alkali. Transfer ppt and filter to beaker or pptg vessel, dissolve ppt in small excess of the std alkali, add few drops of phthln, and titr. with std acid. Report as % P. 7.120 Photometric Method (41)-Official Final Action Apparatus Spectrophotometer.-Capable of isolating 400 nm band and accepting ~15 mm diam. cells. 7.121 Reagents (a) Molybdovanadate reagent.-Prep. as in 2.022(a), except add only 250 mL 70% HCI04 to NH.V03 soln. (b) Phosphorus std solns.-(1) Stock soln.-2 mg P/mL. Dis- solve 8.788 g KH2P04 in H20 and dil. to 1 L. (2) Working soln.- 0.1 mg P/ mL. Oil. 50 mL stock soln to 1 L. 7.122 Preparation of Standard Curve Transfer aliquots of working std soln contg 0.5, 0.8, 1.0, and 1.5 mg P to 100 mL vol. flasks. Treat as in 7.123, beginning "Add 20 mL molybdovanadate reagent, ..." Prep. std curve by plotting mg P against %T on semilog paper. 7.123 Determination Ash 2 g sample, in 150 mL beaker, 4 hr at 600°. Cool, add 40 mL HCI (1 +3) and several drops HN03, and bring to bp. Cool, transfer to 200 mL vol. flask, and dil. to vol. with H20. Filter, and place aliquot contg 0.5-1.5 mg P in 100 mL vol. flask. Add 20 mL molybdovanadate reagent, dil. to vol. with H20, and mix well. Let stand 10 min; then read %T at 400 nm against 0.5 mg std set at 100% T. (Use ~15 mm diam. cells.) Det. mg P from std curve. % P = mg P in aliquot/(g sample in aliquot x 10). 7.124 Basic Feed Microscopy (42)--()fficial Final Action Apparatus (a) Magnifier-fluorescent illuminator with desk base, 3 x, or reading glass. (b) Microscopes and illuminator.--5ee 44.002(n) and (q). Fol- lowing are preferred: (1) Widefield stereoscopic microscope.-With arm rests, flat stage (remove spring holders), optional substage illumination, inclined eyepiece, and lenses to magnify ca 7-30x, 15x opti- mum. (2) Compound microscope.-With mech. stage, substage con- denser, incl ined binocular eyepiece, 3 position rotating nose- piece, lenses to magnify ca 36-400x, 120x optimum.
- 162. 140 7. ANIMAL FEED AOAC METHODS (1980) (3) Microscope i/luminator.-With iris diaphragm; movable stand holder with rod to permit adjusting light source as to ht and angle for substage or direct over-stage lighting; able to hold 2 blue glass filters or 1 blue and 1 ground glass; 60-100 watt bulb. (c) Sieves.-Nest of 5" No. 10,20,40,60,80, and bottom pan. (d) Stages.-Dark Co glass plates 4 x 4" (Fisher Scientific Co. No. 13-735); or blue paper and microscope slides. (e) Spot plates.-Black and white. (f) Forceps.-Fine pointed, curved. If necessary, bend and grind on emery wheel for good contact of points. (9) Dropping bottles.-Amber, 30 mL, as reagent dispensers. (h) Micro-spatula; micro-stirring rods made by drawing out glass rods; spoon. 7.125 Reagents (a) Chloroform.-Tech. Recover by filtration and distn. (b) Acetone.-Tech. (c) Acetone, dilute.-Dil. 75 mL acetone with 25 mL H20. (d) Dilute hydrochloric acid.-Dil. 1 vol. HCI with 1 vol. H20. (e) Dilute sulfuric acid.-Dil. 1 vol. H2S04 with 1 vol. H20. (f) Iodine soln.-Dissolve 0.75 g KI and 0.1 g I in 30 mL H20 and add 0.5 mL HCI. Store in amber dropping bottle. (9) Millon reagent.-Dissolve, by gently warming, 1 part by wt Hg in 2 parts by wt HN03• Dil. with 2 vols H20. Let mixt. stand overnight and decant supernate. Soln contains Hg(N03)2, HgN03, HN03, and some HN02 • Store in g-s bottle. (Caution: See 51.079.) (h) Molybdate soln.-Add 100 mL 10% NH4N03 soln to 400 mL molybdate soln, 7.118(a). Use only clear supernate to fill 30 mL amber dropping bottle. Discard and refill when crystn occurs. (i) Mountant I.-Dissolve 10 g chloral hydrate in 10 mL H2 0 and add 10 mL glycerol. Store in amber dropping bottle. (j) Mountant II.-Dissolve 160 g chloral hydrate in 100 mL H2 0 and add 10 mL HCI. (k) Silver nitrate soln.-l0%. Dissolve 10 g AgN03 in 100 mL H20. 7.126 Standards (a) Feed ingredients.-Collect ingredients used in grain and stock feeds known to conform to definitions of Association of American Feed Control Officials as stds. Store in 4 oz bottles. To control insects, add ca 1 mL CS2, and stopper. Become thoroly familiar with structural appearance of stds before and after treatment with org. solvs. (b) Weed seeds.-Collect common weed seeds occurring in grains. Most may be found in foreign material obtained after sieving com. whole grains with U.S. Grain Testing Sieve having %4" (2.5 mm) triangular holes. Identify from illustration in "Identification of Crop and Weed Seeds" (USDA Handbook 219 (1963), Government Printing Office, Washington, DC 20402). Store in numbered vials. Become familiar with those weed seeds designated as prohibited and restricted noxious under state laws of individual concern. (See "State Noxious-Weed Seed Requirements Recognized in the Administration of the Federal Seed Act" (USDA, Agricultural Marketing Service, Grain Div., Hyattsville, MD 20782).) Identification of Vegetable Tissues 7.127 Principle Feeds are fractionated according to particle size and cleared where necessary for clear observation; conglomerates are dis- integrated into constituents and fractions arranged on stage suitable for microscopic examination at lowest magnification that permits identification of components when compared to std feed ingredients. 7.128 General Methods (a) Scratch feeds.-Spread representative portion of sample on white paper and examine under magnifier-fluorescent illu- minator at 3x or with reading glass. Identify grains and weed seeds; note other foreign material, heat- and insect-damaged particles, live insects, and rodent excreta; examine for smut, ergot, and mold ("Grain Inspection Manual," USDA). (b) Mashes comparatively free from adhering fine particles.- (1) Low power microscopy.-Arrange in nest form 3 sieves that will adequately fractionate feed according to particle size. Gen- erally, for cattle feeds use No. 10,20, and 40; for poultry feeds, No. 20, 40, and 60. Include bottom pan. Add ca 10 g unground feed (plastic tablespoon makes convenient scoop) to nest, and sieve thoroly. With spatula, spread portion from each sieve on 4 x 4" Co glass stage and place under stereoscopic microscope. (Blue paper may also be used as stage.) Arrange illuminator above and near stage so light strikes sample at angle of ca 45° for shadow contrast. Adjust magnification (ca 15x optimum), illumination, and light filters to individual preference for clear observation. Blue light or northern daylight is preferred. Examine each fraction on stage sep. and systematically. Observe feed particles, continually probing, turning, and testing resistance to pressure with forceps. Note particle size, shape, color, resistance to pressure, texture, odor, and major structural features. Com- pare with stds. If desired, transfer individual particles with forceps to second glass plate for direct comparison with cor- responding tissues from stds. Likewise transfer and break up conglomerates by gentle pressure with flat end of forceps. Make list of observed ingredients. Neglect trace grains which may be normal inpurities in major grains. (Consult "Official Grain Stand- ards of the United States," USDA, for amts of "other grains" permissible as impurities in whole grains.) (2) High power microscopy.-Lower illuminator and select filters so adequate blue light is reflected thru substage condenser of high power microscope. With microspatula, transfer little of fine sievings from bottom sieve and pan to slide, add 2 drops mountant I, stir, and disperse with microstirring rod. Examine microscopically (120x optimum). Compare histologically with stds. Remove slide, add 1 drop I soln, stir, and re-examine. Starch cells are stained pale blue to black; yeasts and other protein cells, pale yellow to brown. If further tissue clarification is desired, boil little of same fine sievings 1 min with ca 5 mL mountant II. Cool, transfer drop or 2 of bottom settlings to slide, cover, and examine microscopically. (c) Oily feeds or those containing large particles obscured by adhering fine particles.-(Most pOUltry feeds and unknowns are best examined by this technic.) Place ca 10 g unground feed in 100 mL tall-form beaker and nearly fill with CHCI3 (hood). Stir briefly and let settle ca 1 min. With spoon, transfer floating (org.) material to 3.5" (9 cm) cover glass, drain, and dry on steam bath. Sieve, and proceed as in (b). If desired, filter, dry, suspend fine particles in CHCI3, and examine microscopically (rarely necessary). (d) Feeds in which molasses has caused lumpiness and otherwise obscured vision.-Place ca 10 g unground feed in 100 mL tall-form beaker. Add 75 mL 75% acetone, stir few min to dissolve molasses, and let settle. Carefully decant and repeat extn. Wash residue twice with acetone by decantation, dry on steam bath, sieve, and proceed as in (b). (e) Pellets or crumbles.-Gently grind few pellets at time in mortar with pestle with enough pressure to sep. pellet into its constituents, but not to break up constituents themselves. Sieve first grind thru No. 20 sieve and return particles remaining on
- 163. AOAC METHODS (1980) PRESERVATIVES 141 sieve to mortar for further grinding. Depending on nature of pellet, proceed with ground material as in (b), (c), or (d). 7.129 Identification of Animal Tissues and Major Mineral Constituents Principle Feeds contg animal tissues and minerals when suspended in CHCI3 readily sep. into 2 fractions: (1) Org. fraction which floats, consisting of muscle fibers, connective tissue, dried ground organs, feather remains, hoof and horn particles, etc. from either animal or marine products, plus all vegetable tissues. (2) Mineral fraction which sinks, consisting of bones, fish scales, teeth, and minerals. 7.130 Preparation of Sample Perform CHCI3 flotation sepn as in 7.128(c). Collect floating material and dry on steam bath. Decant CHCI3, collect mineral fraction, and dry on steam bath. 7.131 Identification of Animal Tissue Examine dried floating material as in 7.128(b). 7.132 Identification 01 Major Mineral Constituents Place dried mineral fraction on nest of No. 40, 60, and 80 sieves and bottom pan. Sieve and place the 4 fractions in sep. groups on same Co glass plate or blue paper stage. Examine under stereoscopic microscope at ca 15x. Animal and fish bones, fish scales, and mollusc shells are generally recogniz- able. Salt usually occurs in cubes which may be dyed. Calcite form of limestone occurs as rhombohedrons. 7.133 Confirmatory Tests With forceps, place unknown particle on glass plate and break up by applying gentle pressure with flat surface. Working under stereoscopic microscope, sep. particles ca 2.5 cm and place beside each a fractional drop of reagent solns listed by touching end of dropper to plate. Push particle into Iiq. with microstirring rod and observe what occurs at interface. Follow order given until pos. identification is obtained. If preferred, perform tests in black spot plate. (a) Silver nitrate soln.-(1) Crystal immediately turns chalk white and slowly expands: chloride, probably salt. (2) Crystal turns yellow and yellow needles begin to grow: mono- or dibasic phosphate, generally dicalcium phosphate. (3) Sparingly sol. white needles form (A92S04): sulfate, Mn-MgS04' (4) Particles slowly darken: bone. (b) Dilute hydrochloric acid.-( 1) Vigorous effervescence: CaC03 • (2) Mild effervescence or none: make following tests. (c) Molybdate soln.-Formation of minute yellow crystals at some distance from particle: tricalcium phosphate, either bone or rock phosphate. (All phosphates react, but mono- and dibasic phosphates have been identified with AgN03 .) (d) Millon reagent.-( 1) Disintegrated particles mostly float, turn pink to red (protein), and fade in ca 5 min: bone phosphate. (2) Particles appear to swell and disintegrate but remain on bottom: defluorinated rock phosphate. (3) Particles merely dis- integrate slowly: rock phosphate. (e) Dilute sulfuric acid.-Long, thin white needles slowly form on addn of drop of H2S04 (1+1) to HCI (1+1) soln of particle: confirms-Ca. 7.134 Identification 01 Furazolidone, Tylosin, and Zoalene See 42.001-42.004. PRESERVATIVES Ethoxyquin (1 ,2-Dihydro-6-ethoxy-2,2,4- trimethylquinoline) (43)-Official Final Action 7.135 Reagents and Apparatus (a) Quinine sulfate reference soln.-1 j..tg/mL 0.1N H2S04, Dissolve 0.100 g quinine sulfate USP in 1 L 0.1N H2S04, Oil. 10 mL aliquot of this soln to 1 L with 0.1N H2S04, Use to calibrate photofluorometer. (b) Ethoxyquin std solns.-Add 100.0 mg liq. ethoxyquin to 100 mL vol. flask and dil. to vol. with pet ether (Soln A). Oil. 5 mL Soln A to 100 mL with pet ether (Soln B, 50 j..tg/mL). Oil. 5 mL Soln B to 100 mL with pet ether (Soln C, 2.5 j..tg/mL). Oil. 10 mL Soln C to 20 mL with pet ether (1.25 j..tg/mL) and 5 mL to 25 mL (0.50 j..tg/mL). (c) Photof/uorometer.-Equipped with primary filter passing 365 nm Hg line (Corning Glass Works No. 5874 (CS7-39), or equiv.) and secondary filter passing 42G-500 nm (Corning Glass Works 3389 + 5543 + 4784, half stock thickness, or equiv.). 7.136 Preparation of Standard Curve Adjust photofluorometer to read 0 with pet ether and 100 with quinine sulfate ref. soln. Obtain fluorescence readings for ethoxyquin std solns contg G-2.5 j..tg/mL. Plot readings against j..tg ethoxyquin/mL on linear paper. 7.137 Determination Place 10±0.1 g finely ground sample in 100 mL beaker and slurry with 50 mL MeOH. Stir and let stand 10 min. Decant thru plug of glass wool into 250 mL vol. flask. Reslurry residue with two 50 mL portions MeOH, decant, and filter, combining all filtrates. Oil. to vol. with MeOH. Transfer 25 mL aliquot to 250 mL separator, add 100 mL H20, and mix well. Add 50 mL pet ether, stopper, and shake moderately 1 min. Let stand few min to sep. (If emulsion forms, add ca 100 mg NaCI crystals. After emulsion breaks, drain aq. lower layer into 250 mL beaker.) Transfer pet ether layer to second 250 mL separator, return aq. layer to first separator, and re-ext with two 25 mL portions pet ether. Add 50 mL H20 to combined pet ether exts in separator, stopper, and shake moderately. Let sep., drain lower aq. layer, and discard. Transfer pet ether layer to 100 mL vol. flask, and dil. to vol. with pet ether. Adjust photofluorometer as above and det. fluorescence readings. Obtain j..tg ethoxyquin/mL from std curve. Ppm ethoxyquin = 100 x j..tg/mL. - If untreated feed is available, prep. std curve from series of samples contg G-250 j..tg ethoxyquin/10 g and carried thru detn. 7.138 Drugs in Feeds See Chapter 42. 7.139 Molasses and Molasses Products See Chapter 31. 7.140 Cyanogenetic Glucosides See 26.134. 7.141 Hydrocyanic Acid Formed by Hydrolysis of Glucosides in Beans See 26.135-26.136.
- 164. 142 7. ANIMAL FEED AOAC METHODS (1980) SELECTED REFERENCES (1) JAOAC 33,424(1950); 41, 223(1958); 48, 658(1965). (2) JAOAC 17, 68(1934). (3) JAOAC 8,295(1925); 9,30(1926). (4) USDA Bur. Chern. Bull. 122, p. 219; 132, p. 150. (5) JAOAC 13, 173(1930); 14, 152(1931); 17, 178(1934); 18, 80(1935). (6) JAOAC 36,213(1953); 37, 253(1954). (7) JAOAC 25, 857(1942); 26, 220(1943). (8) JAOAC 18,81,369(1935). (9) JAOAC 37, 241(1954); 38, 56(1955). (10) JAOAC 51, 766(1968). (11) JAOAC 59, 141 (1976). (12) JAOAC 59,134(1976); 62,290(1979). (13) JAOAC 24, 867(1941); 25,874(1942); 27,494(1944). (14) JAOAC 50, 56(1967). (15) JAOAC 51, 763(1968). (16) J. Agric. Food Chern. 3, 159(1955); JAOAC 40,606(1957); 41,233(1958); 42, 231(1959); 43, 320(1960); 54, 669(1971); 55,702(1972). (17) JAOAC 37, 250(1954); 38, 225(1955); 59, 1218(1976). (18) JAOAC 15, 524(1932); 17, 190(1934); 18, 351(1935); 28, 80(1945). (19) JAOAC 42, 222(1959); 43, 335(1960); 44, 567(1961); 45, 578(1962). (20) JAOAC 61,154(1978). (21) JAOAC 46, 829( 1963); 56, 781 (1973). (22) JAOAC 57, 382(1974). (23) USDA Bur. Chern. Circ. 71; JAOAC 41, 276(1958); 42, 39(1959). (24) J. Agr. Research 23, 995(1923); JAOAC 9, 31 (1926). (25) JAOAC 21, 595(1938); 23,656(1940). (26) J. Landw. 48, 357(1900); 49, 7(1901). (27) USDA Bur. Chern. Bull. 137, p. 152; JAOAC 30,594(1947). (28) JAOAC 14, 142(1931); 15, 77(1932); 23, 86(1940); 28, 80(1945). (29) JAOAC 51, 776(1968); 59, 937(1976). (30) JAOAC 10, 177(1927); 19,93,574(1936); 28,80(1945). (31) Ind. Eng. Chern., Anal. Ed. 7, 116, 167(1935); JAOAC 30, 606(1947); 31, 98(1948); 32, 650(1949); 33, 162(1950); 34, 563(1951). (32) JAOAC 26,87(1943); 28, 80(1945). (33) JAOAC 52, 607(1969) (34) JAOAC 35,559(1952). (35) Anal. Chern. 19, 325(1947); JAOAC 37, 246 (1954); 38, 222(1955). (36) JAOAC 58, 477(1975). (37) JAOAC 17,67, 173(1934); 18,335(1935); 38, 96(1955). (38) JAOAC 18, 338(1935); 21, 596(1938); 23, 688(1940); 33, 83(1950). (39) J. Am. Chern. Soc. 39, 2366(1917); G. Frederick Smith Chemical Co. Pub. 209, 5th ed. (1950); JAOAC 22, 78, 673(1939); 24, 865(1941); 25,892(1942). (40) USDA Div. Chern. Bull. 56,36(1898); JAOAC 47,420(1964). (41) JAOAC 48,654(1965). (42) JAOAC 47,504(1964). (43) JAOAC 44, 560(1961); 46, 306(1963); 47, 512 (1964).
- 165. 8. Baking Powders and Baking Chemicals 8.001 Preparation of Sample-Official Final Action Remove entire sample from package, pass thru No. 20 sieve, and mix thoroly. 8.002 Total Carbon Dioxide (1)-Official Final Action (Applicable to baking powders contg added CaC03) Reagent Displacement soln.-Dissolve 100 g NaCI or Na2S04.10H20 in 350 mL H20. Add ca 1 g NaHC03 and 2 mL Me orange, 5.014(e), and then enough H2S04 (1+5) or HCI (1+2) to make just acid (decided pink). Stir until all CO2 is removed. This soln is used in gas-measuring tube and leveling bulb and seldom needs replacement. 8.003 Apparatus Chittick apparatus.-Fig. 8:01. Connect decomposition flask, A, by glass T-tube, B, provided with stopcock, C, to graduated gas-measuring tube, D, connected in turn with leveling bulb, E. For A always use 250 mL wide-mouth extn flask of Pyrex or FIG. 8:01~hittick apparatus for gasometric determination of carbon dioxide 143 other resistant glass fitted with 2-hole rubber stopper, thru one hole of which passes extended tip of 25 mL buret, F, and thru other, glass tube of same diam. as connecting T-tube. Use buret graduated in mL at 20°, numbered at 5 mL intervals, and fitted with extra-long tip bent to pass thru rubber stopper. Connect glass tube leading from decomposition flask to T-tube with rubber tubing to permit rotation of flask. Use gas-measuring tube graduated in mL at 20° with 0 mark at point 25 mL below top marking to allow for graduating upward from 0 to 25 mL and downward from 0 to 200 mL. Connect gas-measuring tube to ca 300 mL leveling bulb with long rubber tube. (Available from Sargent-Welch Scientific Co.) 8.004 Determination (2) Weigh 1.7 g prepd sample, 8.001, into flask A, and connect flask with app., Fig. 8:01. Open stopcock C, and using leveling bulb E, bring displacement soln to 10 mL graduation above 0 mark. (This 10 mL is practically equal to vol. of acid to be used in decomposition.) Let app. stand 1-2 min fortemp. and pressure within app. to come to room conditions. Close stopcock, lower leveling bulb somewhat to reduce pressure within app., and slowly add 10 mL H2S04 (1 +5) or HCI (1 +2) to decomposition flask from buret F. To prevent escape of liberated CO2 thru acid buret into air, at all times during decomposition keep displacement soln at level lower in leveling bulb than that in gas-measuring tube. Rotate and then vigorously agitate flask to mix contents intimately. Let stand 5 min to secure equilibrium. Equalize pressure in measuring tube, using leveling bulb, and read vol. of gas in tube. Observe temp. of air surrounding app. and also barometric pressure, and multiply mL gas evolved by factor given in table, 52.007, for this temp. and pressure. % CO2 by wt = corrected reading/ 1O. 8.005 Residual Carbon Dioxide (3)-Official Final Action (a) After drying on water bath.-Place 1.7 g baking powder in clean, dry, 250 mL wide-mouth Soxhlet extn flask, A, Fig. 8:01. Add 20 mL H20. Put flask on cover of H20 bath (single or mUltiple) in which boiling H2 0 is kept at const level of 5 cm below top of bath. (H2 0 in bath must boil vigorously all thru detn. Opening in cover of bath must be 7.5 cm diam. to prevent flask from touching H20.) Evap. contents of flask until no moisture is visible in residue or inside surface of flask. (Sample should be completely dry in 1.5-2 hr.) Leave flask on H2 0 bath 2 hr more. Add 10 mL H2 0, and let stand until flask is at room temp. (ca 1 hr). Det. CO2 with Chittick app. as in 8.004, using correction factors in 52.007. Shake flask vigorously until further shaking produces no increase in reading. (b) After drying in oven.-Place 1.7 g sample in clean, dry, 250 mL wide-mouth Soxhlet extn flask, A, Fig. 8:01. Tap flask to spread sample evenly on bottom. Add 10 mL H2 0 with pipet. Stir with glass rod to break up powder that may have caked on bottom of flask. Wash down stirring rod and sides of flask with 10 mL H20. Place flask on shelf near center of air oven set at 100±2°, and evap. to dryness. After 5 hr, remove from oven, add 10 mL H20, and cool to same temp. as air surrounding Chittick app., 8.003. Det. CO2 in residue with Chittick app., using correction factors in 52.007. Shake flask vigorously until further shaking produces no increase in reading.
- 166. 144 8. BAKING POWDERS AND BAKING CHEMICALS AOAC METHODS (1980) 8.006 Available Carbon Dioxide-Official Final Action (Applicable to baking powders contg added CaC03 ) Subtract residual CO2, 8.005, from total CO2, 8.004. Neutralizing Value-Official Final Action 8.007 Of Acid-Reacting Materials Other Than Phosphates Dissolve 1 g sample in hot H20 and titr. with 0.2N NaOH, using phthln. Express result as parts NaHC03 equiv. to 100 parts of the acid-reacting material. 8.008 Of Monocalcium Phosphate (4) Weigh 0.84 g sample into 375 mL casserole. Add 24 mL cold H2 0 and, after stirring for moment, add 90.0 mL 0.1N NaOH. Bring suspension to bp in exactly 2 min, and boil 1 min. While soln is still boiling hot, add 1 drop phthln, and back-titr. with 0.2N HCI until all pink disappears. Boil soln 1 min, and again add 0.2N HCI until pink just disappears. 90 - (mL 0.2N HCI x 2) = neutzg value, parts NaHC03 equiv. to 100 parts of the phosphate. 8.009 Of Anhydrous Monocalcium Phosphate (4) Use 100 mL 0.1N NaOH and stir intermittently 5 min before bringing to bp. Proceed as in 8.008. 8.010 Of Sodium Acid Pyrophosphate (4) Weigh 0.84 g sample and 20 g NaCI into 375 mL casserole, and slowly add 25 mL H20 while stirring. Stir and crush with flat-end rod 3-5 min. Add 90.0 mL 0.1N NaOH and 1 drop phthln, and titr. with 0.2N HCI until pink disappears. If "starch filled" or 50% neutzg strength pyrophosphate is being titrd, use 70.0 mL 0.1N NaOH. Vol. (mL) 0.1N NaOH - (mL 0.2N HCI x 2) = neutzg value, parts NaHC03 equiv. to 100 parts Na acid pyrophosphate. 8.011 Of Sodium Aluminum Phosphate (5) AOAC-Food Chemicals Codex Method Apparatus Magnetic stirrer-hot plate.-Must be capable of bringing assay soln to bp within 5 min. Alternatively, use ordinary hot plate and manual stirring. 8.012 Determination Accurately weigh ca 0.84 g sample, transfer to 250 mL beaker, and add 20 g NaCI, 5 mL 10% Na citrate.2H20 soln, and 25 mL H2 0. Pipet (or deliver from buret) 120 mL stdzd 0.1N NaOH, 50.032-50.034, swirling during addn. Stir on mag. stirrer-hot plate at slow to medium speed; avoid spattering. Bring to bp in 3-5 min, and boil exactly 5 min. Remove from hot plate, and immediately cool to 25°. Titr. immediately with mag. stirring to pH 8.5 with stdzd 0.2N HCI, 50.011-50.012, using pH meter previously stdzd with pH 7.0 buffer. Stir addnl 5 min and add 0.2N HCI to obtain pH 8.5. Neutzg value = (V,N, - V2N2 ) x (0.84 x 10)jW, where V, and N, = vol. and normality of NaOH, resp.; V2and N2 = vol. and normality of HCI, resp.; and W = g sample. 8.013 Tartaric Acid, Free or Combined (Qualitative Test) (6)-Official Final Action (Applicable in presence of phosphates) Shake ca 5 g sample repeatedly with ca 250 mL cold H20 in flask, and let insol. portion settle. Decant soln thru filter, and evap. filtrate to dryness. Powder residue, add few drops 1% resorcinol soln, 31.146, and ca 3 mL H2S04, and heat slowly. Tartaric acid is indicated by rose-red, discharged on diln with H20. 8.014 Cream of Tartar and Free Tartaric Acid in Tartrate Powders (7)-Official Final Action Total, Combined, and Free Tartaric Acid Determination To 2.5 g sample in 250 mL vol. flask add 100 mL H20 at ca 50°, and hold at room temp. ca 30 min, shaking occasionally. Cool, dil. to vol. with H2 0, shake vigorously, and filter thru large fluted paper. Pipet 2 portions of 100 mL each of clear filtrate into 250 mL beakers, and evap. to ca 20 mL. To 1 portion add 3.5 mL ca 1N KOH. Mix well, and add 2 mL HOAc. Again mix well and add 100 mL alcohol, stirring constantly. Treat other portion similarly, but use 1N NaOH instead of KOH. Then treat each mixt. sep. as follows: Cool to ca 15°, stir vigorously ca 1 min, and leave in refrigerator overnight. Collect ppt in gooch on thin, tightly tamped pad of asbestos. Rinse beaker with ca 75 mL ice-cold 80% alcohol, carefully washing down sides of beaker. Finally wash sides of crucible with 25 mL alcohol and suck dry. Transfer contents of crucible to original beaker with ca 100 mL hot H20, and titr. with 0.1N alkali, using phthln. Designate titer of portion treated with KOH as "x" and that treated with NaOH as "y." 8.015 Calculations % Total tartaric acid = 1.5(x + 0.6). % Cream of tartar = 1.88(y + 0.6). % Free tartaric acid = 1.5(x - y). In above formulas "0.6" represents solubility of cream of tartar in reaction mixt. in terms of 0.1N alkali. Free Tartaric Acid (Direct Determination) 8.016 Reagent Saturated alcohol.-To ca 50 g finely powd pure cream of tartar in erlenmeyer add ca 100 mL alcohol and 100 mL H2 0, shake vigorously several min, and let stand 15 min, shaking occasionally. Filter on paper in buchner; wash with ca 200 mL alcohol (1 +1), then with alcohol, and finally with ether. Dry at temp. of boiling H20. To 500 mL absolute alcohol add ca 5 g of the purified cream of tartar and let stand 2 hr, shaking occa- sionally. Properly purified cream of tartar requires ,,;0.15 mL 0.1N alkal ito neutze 100 mL of mixt. of 50 mL CHCI3 and 150 mL of the satd alcohol. 8.017 Determination Weigh 1.25 g sample into absolutely dry 200 mL vol. flask, add 50 mL CHCI3, and let stand ca 5 min, shaking occasionally. (Discard detn if upon addn of CHCI3 , powder sticks to bottom of flask, indicating moisture.) Add 100 mL satd alcohol, shake ca 5 min, and let stand 30 min, shaking at frequent intervals. (It is not necessary to filter the alcohol reagent.) Oil. to vol. with the satd alcohol, shake few min, and filter thru large fluted paper. Titr. 100 mL clear filtrate with 0.1N alkali, using phthln. Vol. (mL) alkali used x 1.2 = % free tartaric acid.
- 167. AOAC METHODS (1980) ALUMINUM 145 8.018 Free Tartaric Acid (Qualitative Test) Ext 5 g sample with absolute alcohol and evap. alcohol from ext. Dissolve residue in NH4 0H (1 + 10), transfer to test tube, add good-size crystal of AgN03, and heat gently. Tartaric acid is indicated by formation of Ag mirror. (If desired, alc. ext may be tested as in 8.013.) Starch-Official Final Action 8.019 Direct Acid Hydrolysis Method (For baking powders and baking chemicals free from Cal Stir 5 g sample 1 hr in 250 mL beaker with 50 mL cold H2 0. Transfer to filter and wash with 250 mL cold H2 0. Heat insol. residue 2.5 hr with 200 mL H20 and 20 mL HCI (sp gr 1.125, 52.002(c)) in flask provided with reflux condenser. Cool, and nearly neutze with NaOH. Transfer to 250 mL vol. flask, dil. to vol., filter, and det. glucose in aliquot of filtrate as in 31.038. Wt glucose obtained x 0.925 = wt starch. 8.020 Indirect Method (8) (For baking powders and baking chemicals contg Cal Mix 5 g sample with 200 mL HCI (1+ 11) in 500 mL vol. flask and let mixt. stand 1 hr, shaking frequently. Filter on 11 cm hardened paper, taking care to obtain clear filtrate. Rinse flask once without attempting to remove all starch, and wash paper twice with cold H20. Carefully wash starch from paper back into flask with 200 mL H2 0. Add 20 mL HCI (sp gr 1.125) and proceed as in 8.019. (Treatment with HCI, without dissolving starch, effectively removes Ca, which otherwise would be pptd as tartrate by alk. Cu soln.) 8.021 Aluminum Qualitative Test (9)-Official Final Action (In presence of phosphates) Reagents (a) Ammonium acetate soln.-50%. Dissolve 50 g NH4 0Ac in 50 mL H20. (b) Aurintricarboxylic acid soln.-O.l %. Dissolve 0.1 g aurin- tricarboxylic acid in H2 0 and dil. to 100 mL. 8.022 Detection Place 1 g sample in 250 mL beaker, add 5 mL ea 1N HCI and 20 mL H20, and heat until starch hydrolyzes. Add 100 mL cold H2 0, 5 mL 10% NaNH4 HP04 .4H20 soln, and 3 drops Me orange. Add NH40H dropwise until ppt forms or color changes; then add 1N HCI dropwise until ppt dissolves or color changes plus 2 or 3 drops excess. Add 5 mL aurintricarboxylic acid soln and let stand 1 min. Add 50% NH4 0Ac soln dropwise until ppt forms or color changes and then 1 mL excess. Let stand 5 min, stirring occasionally, and filter. Bright red ppt on filter paper indicates presence of AI. 8.023 Atomic Absorption Spectrophotometric Method (10)-Official First Action Apparatus Atomic absorption spectrophotometer.-Perkin-Elmer Model 303, or equiv. Typical operating parameters for this app. are given in Table 8:01. Operator must become familiar with opti- mum settings for his own app. and use table only as guide. (Caution: See 51.006.) Table 8:01 Operating Parameters Wavelength, nm Slit width, mm Source, ma N20, aspirating N20, auxiliary and aspirating C2H2 fuel Flame Ht, burner to light path, in. Sample uptake, mL/min Optimum concn range, ILg/mL 309.3 1 30 4.5 (scale divisions) 5.5 (scale divisions) 6 (metal ball scale division) reducing 'I. 4 50-150 8.024 Reagents (Do not use <2 mL pipets or <25 mL vol. flasks. Stock solns and (1+10) dilns may be stored indefinitely.) (a) Diluting soln.-To 500 mL H20 add 20 mL H2S04 and 2.5 g NaCI; dil. to 1 L with H20. (b) Aluminum std solns.-(1) Stock soln.-l mg Al/mL. Dis- solve 1.000 g pure AI wire in min. amt HCI. Evap. almost to dryness, add 500 mL H20, 20 mL H2S04, and 2.5 g NaCI, and dil. to 1 L with H20. (2) Working solns.-Dil. aliquots of stock soln with dilg soln, (al. to make ",,4 std solns within conen range of instrument. 8.025 Preparation of Sample (Caution: See 51.019.) Accurately weigh ca 1 g sample into 250 mL Kjeldahl flask, add 2.0 mL H2S04, and then slowly add 3 mL 30% H20 2• When initial vigorous reaction subsides, apply heat from Bunsen flame until sample begins to char. Add addnl 1 mL increments H20 2 and heat until soln no longer chars; finally heat to fumes of S03' Cool, add 50 mL H2 0 and 1 Pyrex glass chip, and boil 3-5 min. Cool and filter, if necessary, thru Whatman No.2 paper into 100 mL vol. flask, washing paper thoroly with H20, and dil. to vol. Prep. reagent blank of 2.0 mL H2S04 and same total amt 30% H20 2 used for sample. Measure A directly or dil. with dilg soln, (a), within range of instrument. 8.026 Determination Set up app. as in Table 8:01, or use previously established optimum settings for app. Zero app. while aspirating dilg soln, (a). Det. A of ""4 std solns within anal. range, alternating with sample soln readings. Flush burner with dilg soln, (a), and check a point between readings. Correct for reagent blank reading if significant, and det. AI content from std curve of A against I1-g Al/mL: % AI = (l1-g Al/mL) x (Fig sample) x 10-4 , where F = 100 or 100 x mL final diln/mL aliquot, if original 100 mL is dild. 8.027 Insoluble Ash and Preparation of Solution (11) Official Final Action Char 5 g sample in Pt dish at heat below redness (ca 5000 ). Boil carbonaceous mass with HCI (1 +2.5), filter into 500 mL vol. flask, and wash with hot H20. Return residue, together with paper, to Pt dish, and burn to white ash. Boil again with the dil. HCI, filter, wash, combine filtrates, and dil. to 500 mL. Incinerate residue after last filtration and weigh ash insol. in acid. 8.028 Iron and Aluminum (71)-Official Final Action Sep. Si02, if necessary, from 100 mL aliquot prepd soln, 8.027. Mix soln with excess 10% Na2 HP04 .12H2 0 soln. Add NH4 0H until permanent ppt is obtained, then HCI dropwise until ppt
- 168. 146 8. BAKING POWDERS AND BAKING CHEMICALS AOAC METHODS (1980) dissolves. Bring soln to bp and boil 2-3 min; mix with consid- erable excess 50% NH40Ac soln, 8.021 (a), and 4 mL HOAc (4+1). As soon as ppt of AIP04, mixed with FeP04, settles, collect on filter, wash with hot H20, ignite, and weigh. Fuse mixed phos- phates with 10 parts Na,C03, dissolve in H2S04 (1 +6); reduce with Zn, and det. Fe by titrn with std KMn04soln (1 mL = 1 mg Fe). Det. P20 S in aliquot from 8.027 as in 8.033 or 7.119. Wt AI20 3 = wt mixed phosphates - wt (Fe20 3 + P20 S )' 8.029 Calcium (71)-Official Final Action Heat combined filtrate and washings obtained in 8.028 to 50°, and add excess satd NH4 axalate saln. Let stand in warm place until ppt settles, filter, wash ppt with hot H20, dry, and ignite over Bunsen burner and finally over blast lamp at ",,950°. Cool in desiccator and weigh as CaO. 8.030 Potassium and Sodium (71)-Official Final Action Evap. aliquot prepd soIn, 8.027, nearly to dryness to remove excess HCI, dil., and heat to bp. While soln is still boiling add 10% BaC/2.2H20 saln as long as ppt forms, and then enough satd Ba(OH)2 saln to make liq. strongly alk. After ppt settles, filter, and wash with hot H20; heat filtrate to bp, add enough (NH4)2C03 saln (1 part (NH4)2C03 in 5 parts NH40H soln (1 + 12» to ppt all the Ba, filter, and wash with hot H2 0. Evap. filtrate to dryness and ignite residue below redness to remove NH4 salts. Add little H20 and few drops (NH4)2C03 soln to residue. Filter into weighed Pt dish, evap., ignite below redness, and weigh mixed K and Na chlorides. Digest residue with hot H20, filter thru small filter, and dil. filtrate, if necessary, to provide ",,20 mL liq. for each 100 mg K20. Acidify with few drops HCI and add excess Pt soln (10.5 g H2PtC16/100 mL). Evap. on H20 bath to thick paste; treat residue repeatedly with 80% alcohol, decanting thru weighed gooch or other filter; transfer ppt to filter, and wash thoroly with 80% alcohol. Dry 30 min at 100° and weigh. Calc. K found to its equiv. of KCI and subtract result from wt mixed chlorides to obtain wt NaCI. Phosphorus-Official Final Action 8.031 Reagents (a) Ammanium nitrate saln.-Dissolve 100 g P-free NH4N03 in H20 and dil. to 1 L. (b) Magnesia mixture.-(1) Dissolve 55 g crystd MgCI,.6H,O in H20, add 140 g NH4CI and 130.5 mL NH.OH, and dil. to 1 L. Or, (2) dissolve 55 g crystd MgCI2.6H20 in H20, add 140 g NH4CI, dil. to 870 mL, and add NH40H to each required portion of soln just before using, at rate of 15 mL/l00 mL soln. (c) Ammanium hydraxide saln far washing.-(l +9). Should contain ""2.5% NH3 by wt. 8.032 Preparation of Solution Mix 5 g sample with little Mg(N03)2 soln, 2.019, dry, ignite, dissolve in HCI (1 +2.5), and dil. to definite vol. In aliquot of soln det. P as in 8.033 or 7.119. 8.033 Determination Pipet aliquot of prepd soln into 250 mL beaker; add NH.OH in slight excess and barely dissolve ppt formed with few drops HN03, stirring vigorously. If HCI or H2SO. has been used as solv., add ca 15 g cryst. NH4N03 or soln contg that amt. To hot soln add 70 mL molybdate soln, 7.118(a), for every 100 mg P20 S present. Digest 1 hr at ca 65° and test for complete pptn of P20 S by adding more molybdate soln to clear supernate. Filter, and wash with cold H20 or preferably with the NH4N03soln. Dissolve ppt on filter with NH.OH (1 +1) and hot H20, and wash into beaker to vol. ,,;100 mL. Neutze with HCI, using litmus paper or bramathymal blue as indicator; cool, and from buret slowly add (ca 1 drop/sec), stirring vigorously, 15 mL magnesia mixt./l00 mg P20 S present. After 15 min add 12 mL NH.OH and let stand until supernate is clear (usually 2 hr); filter, wash ppt with NH.OH (1 +9) until washings are practically CI-free, dry, burn at low heat, and ignite to const wt, preferably in furnace at 950-1000°; cool in desiccator, and weigh as Mg2P20,. Report as % P2 0 S' 8.034 Qualitative Test-Official Final Action Add 10 mL H20 to 1-2 g sample in 150 mL beaker. Make just acid with HN03, filter, take equal vols filtrate and molybdate soln, 7.118(a), and warm at 40-50°. Yellow ppt indicates presence of phosphate. 8.035 Sulfate (12)-Official Final Action Boil 5 g sample 1.5 hr with mixt. of 300 mL H20 and 15 mL HCI. Filter, wash filter thoroly with hot H20, cool combined filtrate and washings, and dil. to 500 mL with H20. Det. sulfate in 100 mL aliquot as in 3.062. 8.036 Ammonia-Official Final Action To 2 g sample in distn flask add 300-400 mL H20 and excess of NaOH soln (1+1), connect with condenser, and distil into measured vol. std acid. Titr. excess acid in distillate with std alkali, using Me red. 8.037 Arseni~fficial Final Action Place 5 g sample directly in generator, 25.007(a); add 10 mL H20, little at time to prevent foaming over, and then 15 mL As- free HCI, adding it dropwise until foaming ceases. Heat on steam bath until drop of mixt., when dild and treated with I soln, does not show blue. Then dil. to ca 30 mL with H20 and continue as in 25.010 or 25.012, beginning with addn of KI reagent. Prep. blank and stds for comparison, using As-free HCI of same concn as that used in detn. 8.038 Fluorine-Official Final Action See 25.049-25.055. 8.039 lead-Official Final Action See 25.061-25.062 and 25.095-25.105. SELECTED REFERENCES (1) JAOAC 6, 453(1923). (2) JAOAC 10, 36(1927). (3) JAOAC 31, 278(1948); 32,83,269(1949); 33,77(1950). (4) JAOAC 33, 77(1950); 34,296(1951). (5) JAOAC 59, 26(1976). (6) Ann. chim. anal. 4, 263(1899). (7) JAOAC 13,385(1930); 22, 599(1939). (8) Conn. Agr. Expt. Sta. Rpt. 1900 (II), p. 174. (9) J. Am. Chern. Soc. 47,142(1925); JAOAC 34, 61(1951); 35, 57(1952). (10) JAOAC 55, 684(1972). (11) Conn. Agr. Expt. Sta. Rpt. 1900, p. 178. (12) USDA Bur. Chem. Bull. 13 (V), p. 596; Conn. Agr. Exp. Sta. Rpt. 1900, p. 179.
- 169. 9. Beverages: Distilled liquors SPIRITS 9.001 Physical Examination-Procedure Note and record following: (a) Color and depth of color; (b) odor-whisky, brandy, rum, etc., or foreign; (c) taste-whisky, brandy, rum, etc., or foreign. Color (7)-Official Final Action 9.002 Definition Whisky color units are defined as lOx A at 430 nm, measured in monochromatic light, of sample '12" thick which has spectral color characteristics of an avo whisky free of turbidity. This definition applies only toA values obtained with precise spectrophtr with band width of ~1 nm at 430 nm, and whose wavelength and photometer scales have been checked and corrected by methods recommended by NBS, in LC-l017, Jan. 1967, and in SP260-41. 9.003 Potassium Dichromate Calibration Method Preparation of Standard Curve Prep. solns of K2Cr20 7 in O.OlN H2S04 as follows: Color Color Unit giL Unit giL 1 0.0500 6 0.3000 2 0.1000 7 0.3500 3 0.1500 8 0.4000 4 0.2000 9 0.4500 0.2500 10 0.5000 Read A of these solns in spectrophtr at 430 nm against H2 0, using same size cell as used in detns. If other than '12" cell is used, convert reading to this size. Plot color units against A or calc. avo factor for converting instrument reading to color units if straight line is obtained. 9.004 Determination Place sample, or sample dild with 50% alcohol, in cell and det. A against H20. Calc. color units, using factor or std curve. 9.005 Natural and Artificial Coloring Matter (Organic and Water-Soluble Color) (2)-Official Final Action Spectrophotometric Method Apparatus (a) Spectrophotometer.-See 9.002. (b) Graduated cylinder.-Cylindrical type of uniform diam., with pressed or molded base and l stopper. Distance from base to top is 285-295 mm. To contain 50 mL at 20°, graduated in 0.2 mL with each fifth mark distinguished by longer line; numbered from bottom upward at 2 mL intervals; error of graduations ~0.2 mL at any point. (Available from SGA Scientific Inc., No. JC-9675.) 9.006 Reagents (a) n-Methyl propyl ketone.-2-Pentanone, practical. (b) Saturated sodium chloride soln.-Use USP or ACS NaCI. (c) Alcohol.-MeOH, reagent grade, or alcohol, USP. 147 9.007 Determination Pipet 20 mL whisky into cylinder, 9.005(b). Add by pipet, in order, 10 mL satd NaCI soln, 0.5 mL HCI, and 10 mL Me Pr ketone. Immediately invert 10-15 times and let layers sep. Color in lower layer indicates presence of caramel, vegetable extrac- tives, or synthetic dye. Read vol. of org. layer within 1 hr and det. its A at 430 nm. If A is too great or if solv. layer is cloudy, dil. aliquot to known vol. with either 50% MeOH or 50% alcohol and read A. 9.008 Calculation Example: If from 20 mL sample, org. layer of 16.1 mL was obtained which had A of 0.420 in 1 cm cell after diln 1+ 1 with 50% alcohol: (16.1 x 0.420 x 12.7 x 2)/20 = 8.59 color units (Lovibond number), where 12.7 is conversion factor to color units. 9.009 Specific Gravity (Apparentl-Officiai Fina! Action Apparatus (a) Constant temperature water bath. (b) Pycnometers.-l00 and 50 mL (Fig. 9:01). 9.010 Calibration Fill thoroly cleaned pycnometer with recently distd H20, stopper, and immerse in const temp. H2 0 bath with bath level above graduation mark on pycnometer. After 30 min, remove stopper and with capillary tube adjust until bottom of meniscus is tangent to graduation mark. With small roll of filter paper, dry inside neck of pycnometer, stopper, and immerse in H20 at room temp. 15 min. Remove pycnometer, dry, let stand 15 min, and weigh. Empty pycnometer, rinse with acetone, and dry thoroly in air with suction. Let empty flask come to room temp., stopper, and weigh. Wt in air of contained H2 0 = wt filled pycnometer - wt empty pycnometer. 9.011 Determination Obtain wt sample as in 9.010. Sp gr in air = S/W, where S = wt sample, and W = wt H20. 9.012 Alcohol by Volume From Specific Gravity by Pycnometer (3) Official Final Action Apparatus Distillation apparatus.-500 mL flask, connected thru bulb (Iowa State type is convenient) to vertically assembled Liebig condenser with jacket ~400 mm long, inner tube 9±1 mm id, with adapter. Joints may be live rubber or $. Heat with elec.- or gas-operated unit. 9.013 Determination (Some samples do not require distn prior to detn, e.g., straight bourbon whisky; alcohol-H2 0 mixts contg traces of volatile ingredients.) (a) Samples containing 60% or less alcohol by volume.- Calibrate 100 mL pycnometer, Fig. 9:01, as in 9.010, at one of
- 170. 148 9. BEVERAGES: DISTILLED LIQUORS AOAC METHODS (1980) Ground in Stopper No no. contains 100 Ml 20°_ C ~::::~ no. contains 50 Ml 20°- C ~:;::::::~ FIG. 9:01-100 mL and 50 mL pycnometers temps specified in 52.003. Fill clean, dry pycnometer with sample and adjust to vol. at calibration temp. as in 9.010. Transfer contents of pycnometer to distg flask, just previously rinsed with cold H20 and contg few glass beads, or equiv. Rinse pycnometer 3 times, using total of 25 mL cold H20 (40 mL for cordials or wines), and add rinse H20 to flask. Place wet pycnometer so that adapter extends just into bulb. Surround pycnometer with ice or ice-H2 0. Complete connections and pass thru condenser rapid stream of H20 kept at ".,;25° at outlet. Distil ca 96 mL at uniform rate in ~30 but ".,;60 min, using longer times for higher percentages of alcohol. Remove and stopper pycnometer, mix distillate by swirling, and wash down with H2 0 any drops that may be above graduation mark. Immerse in const temp. bath at calibration temp. and after 30 min carefully dil. to vol., with aid of capillary tube, by adding H20 previously boiled and cooled to same temp. Det. sp gr of distillate as in 9.011. Obtain corresponding % alcohol by vol. from 52.003. (This result is % alcohol by vol. at 15.56° (60°F).) (b) Samples containing more than 60% alcohol by volume.- Proceed as in (a) with following changes: Calibrate 100 mL and 50 mL pycnometers, Fig. 9:01, at 15.56°, fill 50 mL pycnometer with sample, and adjust to vol. at 15.56°. Add 50 mL cold H2 0 to distg flask before transfer of sample and collect distillate in 100 mL pycnometer. Adjust to vol. at 15.56°. Obtain sp gr of distillate, and from table, 52.003, obtain % alcohol by vol. in distillate. Calc. as follows: % alcohol by vol. in sample at 15.56° = 0 x W/W'; where 0 = % alcohol by vol. in distillate at 15.56°; W = wt H2 0 at 15.56° in 100 mL pycnometer; and W' = wt H2 0 at 15.56° in 50 mL pycnometer. 9.014 From Specific Gravity by Hydrometer (4) Official Final Action (Applicable to spirits contg ".,;600 mg ext/100 mL) Apparatus (a) Hydrometer.-Graduated to 0.1 or 0.2° proof, with calibra- tion corrections. (b) Thermometer.-Graduated to 0.25 or OSF, with calibra- tion corrections. (e) Cylinder.-Clear glass, 2.5" diam., 14" high. (d) Metal c/ips.-To hold thermometer in cylinder. 9.015 Determination Clean and dry hydrometer before use. Let hydrometer, ther- mometer, cylinder, and sample come to room temp. Rinse cylinder, contg thermometer held in place by spring frame clip, 2 or 3 times with portion of sample. Fill cylinder to desired level with sample, holding cylinder at ca 45° angle to reduce agitation and air bubbles. (After hydrometer is inserted, liq. level should be slightly below rim of cylinder.) Place palm of hand over top of cylinder and slowly invert 3 or 4 times to equalize temps of liq. and cylinder. Wipe off any liq. on outside of cylinder. (Do not place hands on cylinder in such way as to warm liq. inside.) Insert hydrometer in liq.; then raise and lower hydrometer bulb from top to bottom 5 or 6 times to temper and distribute slight temp. changes thruout liq. Keep hydrometer bulb in liq., dry stem, and let hydrometer come to rest without wetting more than few tenths degrees of exposed stem. Read hydrometer, then thermometer. To read hydrometer
- 171. AOAC METHODS (1980) ALCOHOL 149 scale, place eye slightly below plane of surface of liq., and then slowly raise head, keeping eye perpendicular to hydrometer, until surface flattens from ellipse into straight line. Take point where this line intersects hydrometer scale as reading of hy- drometer. Raise hydrometer slightly above its point of rest and again let it come to rest in liq. Read hydrometer and thermometer again to verify original readings. Read hydrometer to nearest 0.02° and thermometer to nearest 0.1°. Remove and dry hydrometer. Reinvert cylinder and contents several times (with thermometer left in place) to thermally equilibrate system. Retemper hydrom- eter, dry stem, and again read hydrometer and thermometer. Apply calibration corrections for both hydrometer and ther- mometer. Calc. true % of proof from Table No. 1 of the U.S. Treasury Department Gauging Manual, 1970. Average calcd values if they agree within 0.1 ° proof; otherwise take addnl readings and average. Det. ext as in 9.022, and for every 100 mg ext/1 00 mL add 0.4° proof to apparent proof. 9.016 From Refraction-Official Final Action Measure 25 mL sample into distn flask, noting temp.; dil. with 100 mL H20, distil nearly 100 mL, dil. to vol. at same temp., and det. immersion refractometer reading. Obtain corresponding % alcohol from 52.004. When vol. measurements are made at temp. other than 15.56°, multiply % alcohol from 52.004 by appropriate factor from Table 9:01. Williams Field Test (51-Procedure 9.017 Apparatus Williams tube.-See Fig. 9:02. Available from Kontes Glass Co., No. K-899400. Clean frequently and dry. 9.018 Reagents (a) Dilute hydrochloric acid.-Dil. 10 mL HCI to 100 mL with H20. (b) Solvent.-Mix 70 mL Pentasol or isoamyl alcohol, 28 mL toluene, and 2 mL dil. Hel. Shake well until acid completely dissolves. 9.019 Determination Place sample in tube, accurately adjusting bottom of meniscus to coincide with 7.5 mL mark. Remove any excess sample on sides of tube above 7.5 mL mark with swab or roll offilter paper. Add soIv. to 15 mL mark. Stopper tube and invert number of 15ml 8·3/8" '!. 7.5ml 10 20 25 30 "40 45 - 50 = " 60 FIG. 9:02-Williams tube times to mix intimately. Stand tube upright and let sep. When sepn is complete, rotate tube to shake down globules of lower soln that adhere to sides, and stopper. When settling and drainage are complete, read % alcohol (by vol.) where meniscus between the 2 layers falls on calibration mark. Repeat mixing and settling, and read again. 9.020 Temperature Correction Correct for temp. and alcohol concn according to Table 9:02. Example: % alcohol from tube: 48.0% at 90°F. Correction factor from table: -1.1; 48.0 - 1.1 = 46.9%. 9.021 Alcohol by Weight-Official Final Action Accurately weigh 40-50 g sample in clean, dry 50 mL pyc- nometer, Fig. 9:01, or other closed vessel. (If alcohol is ,,;60% by vol., the 100 mL sample of 9.013(a) may be weighed and used.) Transfer to 500 mL distn flask contg 50 mL H20 and few Table 9:01 Factors for calculating original alcohol content for 25 and 50 mL samples distilled to 100 mL when refractometer measurements are made at 20, 25, 30, or 35° 25 mL Sample 50 ml Sample Temp. of Measurement Temp. of Measurement Alc. % by Vol. 20° in Distillate 25° 30° 35° 20° 25° 30° 35° at 15.56° Multiply Alcohol in Distillate by: Multiply Alcohol in Distillate by: 0-3.99 4.001 4.002 4.003 4.004 2.000 2.000 2.000 2.000 4-5.99 4.003 4.006 4.009 4.013 2.000 2.001 2.001 2.001 6-7.99 4.005 4.011 4.016 4.021 2.001 2.001 2.002 2.002 8-9.99 4.007 4.015 4.023 4.030 2.001 2.002 2.003 2.004 10-11.99 4.009 4.019 4.028 4.037 2.001 2.003 2.004 2.006 12-13.99 4.010 4.021 4.031 4.041 2.002 2.004 2.006 2.007 14-15.99 4.011 4.022 4.032 4.043 2.002 2.005 2.007 2.009 16-19.99 4.011 4.023 4.034 4.045 2.003 2.006 2.008 2.011 20-more 4.011 4.024 4.036 4.047 2.003 2.006 2.009 2.012
- 172. 150 9. BEVERAGES: DISTILLED LIQUORS AOAC METHODS (1980) Table 9:02. Temperature (OF) correction factors % 60' 62' 64' 66' 43 +0.5 +0.3 +0.2 0.0 43.4 +0.5 +0.4 +0.2 +0.1 44 +0.6 +0.4 +0.3 +0.2 45 +0.7 +0.5 +0.4 +0.3 46 +0.8 +0.7 +0.5 +0.4 47 +0.9 +0.8 +0.6 +0.5 47.5 +1.0 +0.8 +0.7 +0.5 48 +1.1 +0.9 +0.7 +0.6 49 +1.1 +1.0 +O.B +0.7 50 +1.2 +1.1 +0.9 +0.8 % 80' 82' 84' 86' 43 -0.9 -1.1 -1.2 -1.4 43.4 -0.9 -1.0 -1.2 -1.3 44 -0.8 -0.9 -1.1 -1.2 45 -0.7 -0.8 -1.0 -1.1 46 -0.6 -0.7 -0.9 -1.0 47 -0.5 -0.6 -0.8 -0.9 47.5 -0.4 -0.6 -0.7 -0.8 48 -0.4 -0.5 -0.6 -0.8 49 -0.3 -0.4 -0.5 -0.7 50 -0.2 -0.3 -0.4 -0.6 clean glass beads, or equiv. Rinse pycnometer 3 times, bringing contents of distn flask to ca 125 mL. Distil, and det. % alcohol by vol. in distillate as in 9.013(a). Det. corresponding % alcohol by wt in distillate from table, 52.005. Multiply result by wt distillate and divide by wt sample. 9.022 Extract-Official Final Action Weigh, or measure at 20°, 25-100 mL sample, evap. to dryness on steam bath, dry 30 min at 100°, cool in desiccator 30 min, and weigh. 9.023 Ash-Official Final Action Proceed as in 31.012 or 31.013, using residue from 9.022. Potassium Flame Photometric Method (6)-Official Final Action 9.024 Reagent and Apparatus (a) Std solns.-Prep. as in 11.024(a). except make final dilns, i.e., 1-10 ppm K, with 50% alcohol. (b) Flame spectrophotometer.-5ee 11.024(b). 9.025 Determination (Caution: See 51.007.) Proceed as in 11.025, except burn sample undild, or, if necessary, dild with 50% alcohol (usually 2.5-5.0 times). Calc. ppm K as in 11.025. Sodium Flame Photometric Method (6)-Official Final Action 9.026 Reagent and Apparatus (a) Std solns.-Prep. as in 11.026, except make final dilns, i.e., 1-10 ppm Na, with 50% alcohol. (b) Flame spectrophotometer.-5ee 11.024(b). 68' -0.1 0.0 0.0 +0.1 +0.2 +0.3 +0.4 +0.4 +0.6 +0.7 8B' -1.5 -1.4 -1.4 -1.3 -1.2 -1.0 -1.0 -0.9 -0.8 -0.7 70' 72' 74' 76' 78' -0.2 -0.4 -0.5 -0.7 -0.8 -0.2 -0.3 -0.5 -0.6 -0.7 -0.1 -0.3 -0.4 -0.5 -0.7 0.0 -0.2 -0.3 -0.4 -0.6 +0.1 0.0 -0.2 -0.3 -0.5 +0.2 +0.1 -0.1 -0.2 -0.4 +0.3 +0.1 0.0 -0.2 -0.3 +0.3 +0.2 0.0 -0.1 -0.2 +0.4 +0.3 +0.1 0.0 -0.1 +0.5 +0.4 +0.3 +0.1 0.0 90' 92' 94' 96' 98' -1.6 -1.8 -1.9 -2.0 -2.2 -1.6 -1.7 -1.9 -2.0 -2.2 -1.5 -1.6 -1.8 -1.9 -2.1 -1.4 -1.5 -1.7 -1.8 -2.0 -1.3 -1.4 -1.6 -1.7 -1.8 -1.2 -1.3 -1.5 -1.6 -1.7 -1.1 -1.3 -1.4 -1.5 -1.7 -1.1 -1.2 -1.3 -1.5 -1.6 -1.0 -1.1 -1.2 -1.4 -1.5 -0.9 -1.0 -1.1 -1.3 -1.4 9.027 Determination (Caution: See 51.007.) Proceed as in 11.025, except burn sample undild, or, if necessary, dild with 50% alcohol until %T falls within %T range of stds. Calc. ppm Na as in 11.025. 9.028 Phosphorus-Official Final Action See 11.034. 9.029 Copper-Official Final Action Atomic Absorption Method (7) (Distil H20 and alcohol from all-Pyrex stills into Cu-free receiver.) Reagent Copper std solns.-( 1) Stock soln.-0.2 mg/ mL. Dissolve 0.393 g CuS04.5H20 (free from any whitish deposit) in 500 mL vol. flask contg H20 and 2 mL H2S04, Dil. to vol. and mix. (2) Working soln.-o.004 mg/mL. Prep. daily by dilg 2.00 mL stock soln to 100 mL. 9.030 Preparation of Standard Curve To series of 50 mL vol. flasks each contg 25 mL alcohol, add 0,2,4,6, 10, and 12 mL Cu working std soln. Dil. nearly to mark with H20, mix, and cool to room temp. before dilg to vol. Stds contain 0.0, 0.16, 0.32, 0.48, 0.80, and 0.96 ppm Cu (lLg/mL), resp. 9.031 Determination (Caution: See 51.006.) Follow operating instructions supplied by manufacturer or previously established optimum settings for AA spectrophtr used. Adjust instrument to 0 A while aspirating blank. Read at 324.7 nm ~4 std solns within anal. range before and after each 6-10 samples. Prep. calibration curve from avo of each std before and after sample group. Use std curve to convert A values for samples to ppm Cu.
- 173. AOAC METHODS (1980) ZDST Colorimetric Method (8) 9.032 Reagents Prep. H20, alcohol, and Cu stds as in 9.029, and in addn: Zinc dibenzyldithiocarbamate (ZDBT)-carbon tetrachloride soln.-0.2%. Dissolve 2 9 ZDBT (Uniroyal Chemical or ICN- K&K Laboratories) in 1 L CCI. by warming in H20 bath at <77°. Filter thru Whatman No. 41, or equiv. acid-washed paper, into dark bottle. Store in refrigerator. 9.033 Apparatus Separators .-60 or 125 mL pear-shaped separators with Teflon stopcocks. Clean separators with hot chromic acid cleaning soln and rinse with H20. Before each analysis, shake mixt. of 10 mL H20, 0.5 mL 6N H2SO., and 10 mL ZDBT-CCI. soln in each separator 1 min. Clean inside of stems with cotton swab soaked in ZDBT-CCI. soln. Drain and rinse separators with H20. 9.034 Preparation of Standard Curves (a) 80-135° proof alcoholic samples. - To separators contg 5 mL alcohol, add 0, 0.50, 1.00, 2.00, and 3.00 mL Cu working std soln and 5, 4.5, 4, 3, and 2 mL H20, resp. Solns in separators contain 0.0, 0.20, 0.40, 0.80, and 1.20 JLg Cu/ mL (ppm), resp. Treat as in 9.035. Plot ppm Cu againstA. (b) Aqueous samples, wines, and other low proof samples.- Prep. as in (a) except use 5 mL H20 instead of alcohol. 9.035 Determination To separator contg 10 mL sample (dil. sample >135° proof to 80-135° proof) or std, add 0.5 mL 6N H2S04 and 10.0 mL ZDBT- CCI. soln. Stopper and shake briefly; release pressure by re- moving stopper. Replace stopper and shake vigorously 100 times. If funnel stems are not dry, remove drops of liq. with small roll acid-washed paper (e.g., Whatman 41) to prevent draining H20 drops into cell. Insert plug of fibre glass (Corning Glass Works No. 3950) or cotton into each stem to filter out possible haze materials. Within 10--60 min, det. A of CCI. layer at 438 nm. Let few mL CCI4 layer pass thru filtering medium before collecting sample in cell. Use CCI. layer from appropriate o ppm Cu soln (prepd as for std curve (a) or (b)) as ref. Det. Cu concn from appropriate std curve. Multiply by diln factor if sample was dild. Iron (9)-Official Final Action Atomic Absorption Method 9.036 Apparatus Spectrophotometer.-Perkin-Elmer Corp. 303 (or later double beam model), or equiv., with 3-slot Boling burner head, or equiv. 9.037 Reagent (Use Fe-free H20 and reagents; rinse glassware with HCI and H20 before use.) Iron std solns.-(T) Stock soln.-l0 p.g/mL. Dissolve 0.0684 g ferrous ethylenediammonium sulfate (FeC2H4(NH3)2S0•.4H20, G. Frederick Smith Chemical Co.) in H20, add 2.5 mL H2S04, dil. to 1 L with H20, and mix thoroly. (2) Working solns.-0.0, 0.1, 0.2, 0.3, and 0.4 ppm. To 100 mL vol. flask contg 50 mL 43% alcohol, add 0, 1, 2,3, and 4 mL stock soln and dil. each soln nearly to vol. with 43% alcohol. Mix thoroly, let cool to room temp., and adjust to 100 mL with 43% alcohol. IRON 151 9.038 Determination (Caution: See 51.006.) Follow manufacturer's operating instructions, using lean air- C2H2flame and single element Fe lamp. Set wavelength at 248.3 nm and adjust spectrophtr to 0 A while aspirating blank (0.0 ppm Fe). Analyze stds before and after duplicate series of samples. Det. avo A values. Aspirate H20 between each detn to flush burner, and reset A to 0 with blank. Plot std curve of A against ppm Fe. Read ppm Fe in sample from this curve. 9.039 TPTZ Colorimetric Method (Not applicable to brandy) Apparatus (a) Spectrophotometer.-Beckman Instruments Model DU, or equiv., or photoelec. colorimeter with suitable filter, e.g., Klett colorimeter with No. 60 filter. (b) Water bath.-To maintain const temp. at ca 60°. 9.040 Reagents (Use Fe-free H20 and reagents; rinse glassware with HCI and H20 before use.) (a) Hydroxylamine hydrochloride soln.-40%. Dissolve 10 g NH20H.HCI in 25 mL H20. (b) Ammonium perchlorate soln.-10%. Dissolve 10 g NH.CIO. in 100 mL H20. (c) 1,2-Propanediol cyclic carbonate (propylene carbonate ).- Reagent grade (MC/B Manufacturing Chemists). (d) 2,4,6-Tripyridyl-s-triazine (TPTZ) soln.-0.001M. Add 0.0781 g TPTZ (G. Frederick Smith Chemical Co.) to 100 mL H20 contg 5 drops HCI in 250 mL vol. flask. Dil. to vol. with H20 and filter thru coarse fritted glass funnel. Store in Fe-free glassware. (e) Sodium acetatesoln.-40%. Dissolve 48.2 g anhyd. NaOAc in 75.7 mL H20 and 24.3 mL HCI. Add 5 mL 0.001M TPTZ, 5 mL 40% NH20H.HCI, 2 mL 10% NH4CI04, and 10 mL propylene carbonate. Add 5 mL CHCI3, shake, and discard lower layer. After 2 CHCI3 extns, add 5 mL 0.001M TPTZ and repeat CHCI3 extn. (Four extns are necessary to obtain colorless soln.) Store in Fe- free glassware. (f) Ascorbic acid solns.-( 1) 5% .-Dissolve 2.5 g ascorbic acid in 25 mL H20 and dil. to 50 mL with alcohol. Prep. fresh daily. (2) 5% Ascorbic acid in sodium acetate buffer.-Dissolve 2.5 g ascorbic acid in 20 mL H20, add 5 mL 40% NaOAc, and dil. to 50 mL with alcohol. Prep. fresh daily. (g) Iron std solns .-Prep. same concns as in 9.037, but prep. 200 mL each working soln. 9.041 Preparation of Standard Curve Pipet 50 mL 43% alcohol contg known amts Fe (i.e., °(reagent color blank), 0.1, 0.2, 0.3, and 0.4 ppm) into 5 sep. 100 mL vol. flasks. Add 2 mL 5% ascorbic acid in NaOAc buffer and 2 mL TPTZ, and heat 15 min in ca 60° H20 bath. Cool solns to room temp. and det. A on spectrophtr at 593 nm or photoelec. colorimeter with appropriate filter. Use 43% alcohol to zero colorimeter. Subtract A of reagent color blank from A of samples. Plot corrected A readings against ppm Fe to obtain std curve. (Straight line should be obtained with Fe concns used.) 9.042 Determination Pipet 50 mL sample into each of 2 sep. 100 mL vol. flasks and add 2 mL 5% ascorbic acid to each. Add 2 mL TPTZ to one flask
- 174. 152 9. BEVERAGES: DISTILLED LIQUORS AOAC METHODS (1980) and 2 mL H20 to other (product blank). Heat 15 min in ca 60° H20 bath. Cool solns and measure A of each soln as for std curve. Subtract A of reagent color blank, 9.041, and also A of product color blank from A of samples. Det. Fe concn from std curve. Chloride (to)-Official Final Action 9.043 Apparatus See 11.029. 9.044 Reagents See 11.030 and in addn: (a) Dilute chloride std soln.-Dil. 50 mL chloride std soln, 11.030(e)' to 500 mL with H20. Alternatively, dissolve 0.2103 9 KCI in H20 and dil. to 1 L. 1 mL = 0.1 mg CI. (b) Dilute silver nitrate stdsoln .-Dil. 50 mL std soln, 11.030(d), to 500 mL with H20. Alternatively, dissolve 0.4791 9 AgN03 in H20 and dil. to 1 L. 1 mL = 0.1 mg CI. (c) Alcohol soln.-Place 500 mL alcohol in 1 L vol. flask, add ca 475 mL H20, mix, cool to room temp., and dil. to 1 L with H20. 9.045 Determination Det. equivalence point voltage as in 11.031 except use alcohol soln instead of H2 0 to adjust vol. to 100 mL. Use std solns 11.030(c) and (d). Pipet 5 mL dil. std CI soln into 250 mL beaker, and add 95 mL alcohol soln and 1.0 mL HN03• Titr. with dil. std AgN03 soln to predetd equivalence voltage as in 11.031. Pipet 100 mL distd spirits sample into 250 mL beaker, add 1.0 mL HN03, and titr. with dil. std AgN03soln as above. Ppm CI in sample = (Vw!Vs) x C x 10, where Vw = mL std AgN03 used by sample, Vs = mL std AgN03 used by std CI soln, and C = 0.5 mg CI in 5 mL std CI soln used. 9.046 Total Acids-Official Final Action Neutze ca 250 mL boiled H20 in porcelain evapg dish (185 mm dish is convenient). Add 25 mL sample and titr. with O.lN NaOH, using ca 2 mL phthln. 9.047 Fixed Acids-Official Final Action Evap. 25-50 mL sample to dryness in Pt dish on steam bath and dry 30 min in oven at 100°. Dissolve and transfer residue with several portions of neut. alcohol of ca same proof as sample, using 25-50 mL in all, to porcelain dish contg ca 250 mL neutzd boiled H20. Titr. with O.lN NaOH, using 10 mL buret graduated in 0.05 mL, and ca 2 mL phthln. 9.048 Volatile Acids--Official Final Action Volatile acids = total acids from 9.046 - fixed acids from 9.047. Esters and Aldehydes--Offieial Final Action 9.049 Reagents (a) Sodium thiosulfate std soln.--O.05N. Prep. by dilg O.lN soln, 50.037. (b) Iodine soln.-Approx. 0.05N. (c) Sodium bisulfite soln.-Approx. 0.05N. (Deterioration is retarded if soln contains ca 10% alcohol; do not use after ca 1 week.) 9.050 Preparation of Sample To 200 mL sample in 500 mL erlenmeyer, add ca 35 mL H2 0 and few grains SiC (Carborundum). Distil slowly into 200 mL vol. flask until distillate is nearly at mark. Dil. to vol. and mix. 9.051 Determination of Esters (11) Transfer 100 mL distillate to 500 mL flask, neutze free acid, add measured excess O.lN NaOH, connect flask with air-cooled condenser ca 60 cm long, heat 2 hr on steam bath, let cool, and titr. excess alkali. Reject detns in which excess O.lN alkali is <2 mL, or is >10 mL. Calc. esters as EtOAc. Correct for blank detn performed on 100 mL 50% alcohol (absolute alcohol-H20, 1+1). 9.052 Determination of Aldehyde (Indirect Method) Place remainder of distillate from 9.050 in 500 mL flask, add ca 100 mL H20 and excess NaHS03 soln, and let stand ca 30 min, shaking occasionally. (Excess NaHS03 should be equiv. of ca 25 mL I soln.) Add excess I soln, and titr. this excess with std Na2S20 3 soln. Run blank contg same vols of I soln and bisulfite soln as used in sample. Difference between titrns in mL Na2S20 3 soln x 1.1 = mg acetaldehyde in sample. Esters Spectrophotometric Method (t2)---Official Final Action 9.053 Principle Esters react quant. with H2 NOH in alk. soln to form a hydrox- amic acid which, after acidification, forms colored complex with ferric ions. Ester concn is proportional to A at 525 nm at canst alcohol concn and can be calcd from either of 2 std curves: (1) At constant proof.-Plot A against known EtOAc concn in 100° proof spirits (or any other convenient const proof); or (2) At any actual proof.-Plot A of EtOAc!g against proof and calc. concn from this proof factor. 9.054 Reagents (a) Hydrochloric acid.-4N. Oil. 333 mL HCI to 1 L with H20. (b) Ferric chloride soln.-0.37M. Dissolve 50 9 FeCI3.6H20 in ca 400 mL H2 0 in 500 mL vol. flask. Add 12.5 mL 4NHCI and dil. to vol. with H20. (e) Hydroxylamine hydrochloride soln.-2M. Dissolve 69.6 g H2 NOH.HCI in H20 in 500 mL vol. flask and dil. to vol. with H20. Store in refrigerator. (d) Sodium hydroxide soln.-3.5N. Dissolve 70 g NaOH in ca 400 mL H20 in 500 mL vol. flask. Cool, and dil. to vol. with H20. 9.055 Preparation of Ethyl Acetate Standard Solutions (a) Stock soln No.7 for std curve.--O.333 9 EtOAc!L in 100° proof spirits. Weigh 0.1667 g EtOAc in weighing bottle and transfer quant. to 500 mL vol. flask with 100° proof spirits. Oil. to vol. with 100° proof spirits at room temp. (b) Std solns for std curve.-To series of five 100 mL vol. flasks add, from pipet or buret, 0.0, 15.0, 30.0,45.0, and 60.0 mL stock soln No.1. Oil. to vol. with 100° proof spirits and mix. Stds contain 0.0, 5.0, 10.0, 15.0, and 20.0 9 EtOAc!100 L, resp. (c) Stock soln No.2 for proof factor determination.-0.500 g EtOAc/L H,O. Wei~h 0.2500 9 EtOAc in weighing bottle and
- 175. AOAC METHODS (1980) ALDEHYDES 153 transfer quant. to 500 mL vol. flask with H20. Dil. to vol. with H20 and mix. (d) Stock soln No.3 for proof factor determination.---fJ.500 g EtOAc/L 192° proof spirits. Prep. as in (c). using 192° proof spirits for transfer and diln. (e) Proof factor std solns.-(1) 0-50° stds.-To 6 sep. 250 mL vol. flasks add 50.0 mL EtOAc stock soln No.2. Using graduate, add 0, 13, 20, 39, 53, and 66 mL 192° proof spirits. Dil. each nearly to vol. with H2 0, mix, and cool to room temp. before dilg to vol. with H20. Std solns contain 10 g EtOAc/100 L in ca 0,10, 20,30,40, and 50° proof spirits. (2) 60-192° stds.-To 9 sep. 250 mL vol. flasks, add 50.0 mL EtOAc stock soln No.3. Using appropriate graduate, add 29, 55,81, 108, 134, 161, 174, 187, and 200 mL 192° proof spirits, resp. Dil. nearly to vol. with H20, mix, and cool to room temp. before dilg to vol. with H2 0. Std solns contain 10 g EtOAc/100 L in ca 60, 80,100,120,140,160, 170, 180, and 192° proof spirits, resp. Det. exact proof of solns by std method, e.g. hydrometer. 9.056 Preparation of Samples Analyze whisky distillates, spirits, and colorless gin samples directly. Distil colored or turbid samples as in 9.050. If sample contains >20 g ester/100 L, dil. with H20 to ester concn of 5--20 g/100 L. 9.057 Determination (Mix all solns by swirling to avoid formation of bubbles.) Just before use, prep. stocksoln of reaction mixt. by combining 5.0 mL H2NOH.HCI and 5.0 mL 3.5N NaOH for each std and sample soln. Discard after 6 hr. Prep. ref. soln by pipetting 4 mL reaction mixt. and 2 mL 4N HCI into 25 x 200 mm test tube. Mix and add 2.0 mL sample. Same ref. soln may be used for series of samples of different ester content, but they must have same proof. Pipet 2 mL sample and 4 mL reaction mixt. into another 25 x 200 mm test tube. Mix and let react 1-20 min. Pipet in 2 mL 4N HCI and mix. To ref. soln, pipet in 2 mL FeCI3 soln. Rinse ref. cell twice with this soln, fill cell, and place in cell holder. This ref. soln may be used for 1 day if tightly capped; otherwise refill periodically to avoid evapn error. To sample soln, pipet in 2 mL FeCI3 soln and mix. Complete reading of each sample before proceeding to next. Rinse sample cell twice, fill cell, and place in cell holder. Read A at 525 nm immediately, since color of sample fades rapidly. If instrument has single cell or tube, use same cell or tube for both ref. and sample. Calc. or obtain ilA = AsamPle - A,ef' 9.058 Preparation of Standard Curve Analyze std solns, 9.055(b). as in 9.057. Plot ilA against EtOAc concn (g/100 L at 100° proof). (Note: Std curve need not be repeated for each analysis. Check periodically and repeat if new instrument or reagents are used.) 9.059 Preparation of Proof Factor Curve Analyze std solns, 9.055(e). as in 9.057. Plot ilA/g EtOAc against proof in the 15 solns (0-192° proof). See Note, 9.058. To calc. ester content of samples, read A/g value from proof factor curve at sample proof. If sample was dild, use dild proof in calcn. Divide observed A by A/g to obtain g EtOAc/100 L. Correct for sample diln, if necessary. To express as g/100 L at 100° proof, multiply above ester value by ratio: 100/sample proof. Aldehydes-Official Final Action Method 1(13) (Applicable to ext-free spirits-brandy and wine spirits) 9.060 Reagents (a) Potassium metabisulfite soln.-Dissolve 15 g K2 S2 0 S in H20, add 70 mL HCI, and dil. to 1 L with H20. Bisulfite titer of 10 mL soln should be ~24 mL 0.1N I soln. (b) Phosphate-EDTA soln .-Dissolve 200 g Na3PO•. 12H20 (or 188 g Na2HPO•. 12H20 + 21 g NaOH; or 72.6 g NaH2PO•.H20 + 42 g NaOH; or 71.7 g KH2PO. + 42 g NaOH) and 4.5 g Na2H2EDTA in H20 and dil. to 1 L. (c) Dilute hydrochloric acid.-Dil. 250 mL HCI to 1 L with H2 0. (d) Sodium borate soln.-Mix 100 g H3B03 with 170 g NaOH and dil. to 1 L with H20. 9.061 Total Aldehydes Pipet 50 mL sample (contg ~30 mg acetaldehyde), reduced to ca 1000 proof, or 25 mL high proof sample and 25 mL H20, into 750 mL or 1 L erlenmeyer contg 300 mL boiled or deaerated H20 and 10 mL K2S20 S soln. Stopper flask, swirl to mix, and let stand 15 min. Add 10 mL phosphate-EDTA soln. (pH should be 7.0-7.2. If not, adjust pH by adding HCI or NaOH soln to K2S20 S soln and start with new sample.) Stopper flask, swirl, and let stand addnl 15 min. Add 10 mL HCI, (c) (when analyzing series, make complete detn on first sample before adding acid to next), and ca 10 mL fresh 0.2% starch indicator. Swirl to mix. Add enough ca 0.1N I soln to just destroy excess bisulfite and bring soln to light blue end point. Add 10 mL Na borate soln, and rapidly titr. liberated bisulfite with 0.05N I soln from 10 mL buret (or 0.02N I soln from 25 mL buret) to same light blue end point as above, swirling gently and continuously, avoiding direct sunlight. (pH should be 8.8-9.5. If necessary, adjust by adding HCI or NaOH soln to Na borate soln and start with fresh sample.) mg CH3CHO/100 mL = mL I soln x normality I soln x 22.0 x 100/mL sample. 9.062 Free Aldehydes Pipet identical sample as in 9.061 into 750 mL or 1 L erlen- meyer contg 300 mL boiled or deaerated H20 and 10 mL each K2S20 S and phosphate-EDTA solns. Stopper flask, swirl, and let stand 15 min. Proceed as in 9.061, beginning "Add 10 mL HCI, (c) ..." Method /I (Applicable to spirits contg ext-aged in wood) 9.063 Free Aldehydes Pipet 50 mL sample (contg ~30 mg acetaldehyde), reduced to 80-1000 proof, if necessary, into 500 mL distg flask, add 50 mL satd borax soln, and distil ca 50 mL into 750 mL or 1 L erlenmeyer contg 300 mL H20 and 10 mL each K2S20 S and phosphate-EDTA solns. (pH should be 7.0-7.2. If necessary, adjust by adding HCI or NaOH soln to K2S20 S soln and start with fresh sample.) Proceed as in 9.061, beginning "Add 10 mL Hel, (c) ..." 9.064 Aldehydes as Acetal Transfer 200 mL sample measured at std temp. in vol. flask to 500 mL distg flask, and rinse vol. flask 2-3 times with small amts H2 0 into distg flask. Add 50 mL satd borax soln and distil,
- 176. 154 9. BEVERAGES: DISTILLED liQUORS AOAC METHODS (1980) slowly at first, nearly 200 mL into same vol. flask contg 2-3 mL H20 and immersed in ice bath. Bring distillate to vol. at same temp. used for measuring sample. Det. total aldehydes (including acetal) as in 9.061. Det. free aldehydes as in 9.062. Total aldehydes - free aldehydes = combined aldehydes equiv. to acetal as mg CH3CHO/l00 mL. Alternatively, combined aldehydes as acetal/l00 mL = (com- bined aldehydes equiv. to acetal as mg CH3CHO/l00 mL) x 2.68. 9.065 Total Aldehydes Report total aldehydes as sum of free aldehydes, 9.063, and combined aldehydes equiv. to acetal, 9.064. Fusel Oil-Otficial Final Action Method I (74) 9.066 Reagents (a) p-Dimethylaminobenzaldehyde (DMAB) soln.-In 100 mL vol. flask dissolve 1 g DMAB in mixt. of 5 mL H2S04 and 90 mL H20, and dil. to vol. with H20. (b) Isobutyl alcohol.-Eastman Kodak Co. 303 (highest purity for fusel oil assay). (c) Isoamyl alcohol.-Eastman Kodak Co. X-18, isopentyl al- cohol (highest purity for fusel oil assay). (d) Ethyl alcohol.-Redistd middle 50% fraction. (e) Synthetic std fusel oil.-Weigh 2 g std isobutyl alcohol and 8 g std isoamyl alcohol into 1 L vol. flask and dil. to vol. with H20. Pipet two 10 mL portions into 100 mL vol. flasks and dil. to vol., one with H2 0 and other with alcohol. Prep. working stds for products in range of 0--170 proof contg 1.0--6.0 g synthetic fusel oil/l00 L by dilg 1.0--6.0 mL aliquots of aq. std soln to 100 mL with alc. soln of proof expected for dild sample when pipetted into analysis tube. Prep. similar working stds for products in range of 170--190 proof by dilg 1.0--6.0 mL aliquots of alc. std soln to 100 mL with alc. soln of proof of sample or its diln. When 6 mL synthetic std dild with 190 proof alcohol is analyzed, A should be 0.83±0.03 at 530 nm. 9.067 Preparation of Samples (Aged, blended and rectified products, whiskies, brandies, rums, vodka, and liqueurs require distn prior to analysis.) Determination of true proof of sample.-Det. alcohol as in 9.013. For samples contg >6 g fusel oil/ 100 L, dil. distd sample with H20 to concn of 2.0--5.0 g fusel oil/l00 L. Oil. 5 mL brandy, rum, or blended whisky to 100 mL; dil. 5 mL heavy brandy, rum, or straight whisky to 250 mL. 9.068 Determination Pipet 2 mL aliquots of sample or dild sample, distd, if necessary, 2 mL H20 (for reagent blank), and 2 mL aliquots of stds into 15 x 150 mm g-s or covered test tubes. Stopper or cover tubes, and place in rack, then in ice bath. Pipet 1 mL DMAB soln into each tube, shake, and replace in ice bath for 3 min. With tubes still in ice bath, add 10 mL chilled H2S04 from buret down side of tubes. Shake tubes individually and replace in ice bath for 3 min. Transfer rack of tubes from ice bath to boiling H20 bath and boil 20 min. Transfer tubes to ice bath for 3-5 min, then to room temp. bath. Read %T of developed color of samples and stds on spectrophtr at 538-543 nm against reagent blank as ref. (Use same wavelength for both stds and unknowns.) Plot g fusel oil in std samples/l00 L on linear scale as abscissa against % T as ordinate on log scale of semilog paper. Convert %T of samples to g fusel oil/l00 L from std curve. If diln was used, multiply g fusel oil/l00 L found by diln factor to obtain g fusel oil/l00 L in original sample. Analyze 2 levels of stds with each series of unknowns. Precision expected: Whisky and brandy, ±5%; rum, ±8%; gin, vodka, and spirits, ±0.4 g/100 L. Method II (75) 9.069 Reagents (a) Color reagent.-Dissolve 1 g Na salt of 4-hydroxybenzal- dehyde-3-sulfonic acid in H2 0, dil. to vol. in 25 mL vol. flask with H2 0, and filter. (b) Fusel oil std solns.-Weigh 2 g isobutyl alcohol, 9.066(b)' and 8 g isoamyl alcohol, 9.066(c), into 1 L vol. flask and dil. to vol. with 50% alcohol. Dil. 0, 1, 2, 3,5, 10, and 15 mL portions to vol. with 50% alcohol in 100 mL vol. flasks (0.0, 0.1, 0.2, 0.3, 0.5,1.0, and 1.5 g fusel oil/L). Std soln contg 1.0 g fusel oil/L should give A of ca 0.4 at 445 nm in 9.071. (c) Alcohol.-50%. Free of fusel oil (ACS). 9.070 Preliminary Distillation Add 20 mL H2 0 to 50 mL sample and distil, slowly at first, collecting ca 50 mL in 50 mL vol. flask. Dil. to vol. with H2 0. (For samples known to contain >150 g fusel oil/l00 L, use 25 mL sample plus 45 mL H20.) 9.071 Determination To dry 10 mL vol. flask, add 0.1 mL distillate from serological blow-out pipet. (Pipet should have pointed tip and should be thoroly cleaned (chromic acid plus H20 rinse) before use. Rinse pipet several times with distillate, and wipe end dry. Bring liq. to line while holding tip to outside surface of vol. flask. Then insert end to bottom of vol. flask and release sample. After draining pipet, hold flask at 45° angle and blowout pipet.) Add 0.1 mL color reagent from 1 mL buret graduated in 0.01 mL; then add 2 mL H2S04 from 50 mL buret. Mix, and place unstoppered flask in H20 bath at room temp. (250 mL beaker is convenient). Bring to bp and boil 30 min. Let cool, dil. to vol. with H2S04, and det. A at 445 nm against H20 on Beckman DU spectrophtr, or equiv. instrument. 9.072 Blank and Standards Develop color of 0.1 mL 50% alcohol and 0.1 mL portions of std solns. Use same 0.1 mL pipet for blank, stds, and sample. Subtract A of 50% alcohol blank from A of sample and stds. 9.073 Calibration Curve or Factor Plot corrected A of stds against concn. (Straight line is obtained up to ca 150 g fusel oil/l00 L; above this value, curve flattens. Concn of color reagent may be increased, but it is best to dil. distd sample so that A is <0.6.) Calc. or obtain A' (A of 1.0 g fusel oil/L) from curve. 9.074 Calculations g fusel oil/l00 L = 100A x D/A' where 100 = diln of 0.1 mL sample to 10 mL in color development, A = corrected A of sample, A' = factor, 9.073, and 0 = diln of sample before distn.
- 177. AOAC METHODS (1980) HIGHER ALCOHOLS 155 Higher Alcohols (n-Propyl Alcohol, Isobutyl Alcohol, and Isoamyl Alcohol) and Ethyl Acetate-Official Final Action Gas Chromatographic Method (16) 9.075 Apparatus (a) Gas chromatograph.-Equipped with flame ionization de- tector. (1) Column.-23% Carbowax 1500 (w/w) on Chromosorb W (60-80 mesh, acid-washed). Weigh 9 g Carbowax 1500 into 250 mL beaker and mix with H20 on steam bath. Weigh 30 g Chromosorb W in 250 mL beaker and combine with Carbowax soln in large flat-bottom Pyrex glass baking dish or flat-bottom polyethylene container (ca 20 x 25 cm). Add H2 0 to just cover solid support and mix thoroly. Evap. H20 with frequent stirring in hood. (Gentle steam may be applied to hasten evapn.) After evapn of H20, heat coated support ca 2 hr in 100° oven. Pack 2.4 m (8') x 1.4" od Cu tubing tightly and evenly by repeated tapping, and condition in column oven at 150° with He flow rate of 150 mL/min until steady baseline is observed at attenuation 1x at operating parameters (ca 24 hr). (2) Approximate parameters.-Column temp. 70° (isother- mal); detector and inlet temp. 150°; He carrier flow 150 mL/min. Optimum operating conditions vary with column and instru- ment, and must be detd by using std solns. Adjust parameters for max. peak sharpness and optimum sepn. With high level std, n-PrOH should give almost complete baseline sepn from EtOH. (b) Syringe.-10 ILL, Hamilton Co. No. 701, or equiv. 9.076 (a) Isobutyl alcohol.-see 9.066(b). (b) Isoamyl alcohol.-See 9.066(c). (c) n-Propyl alcohol.-Redistd, reagent grade. (d) Ethyl acetate.-Redistd, reagent grade. (e) n-Butyl alcohol.-Redistd, reagent grade. Reagents (f) n-Butyl alcohol internal std solns.-(T) High level.-Oil. 10 mL n-BuOH to 100 mL with 40% alcohol. (1 mL added to 100 mL sample or std is equiv. to ca 81 g n-BuOH/100 L.) (2) Low level.-Oil. 1 mL n-BuOH to 200 mL with 95% alcohol. (1 mL added to 100 mL sample or std is equiv. to ca 4.1 g/ 100 L.) (g) n-Propyl alcohol, isobutyl alcohol, isoamyl alcohol, and ethyl acetate high level std solns.-(T) Stock soln.-Accurately weigh 1 mL n-PrOH, 1 mL isobutyl alcohol, 2 mL isoamyl alco- hol, and 1 mL EtOAc into 100 mL vol. flask and dil. to vol. with 40% alcohol. (2) Intermediate soln.-Oil. 10 mL stock soln to 200 mL with 40% alcohol. (3) Working soln.- (Approx. 40.2, 41.1, 81.2, and 45.1 g/ 100 L n-PrOH, isobutyl alcohol, isoamyl alcohol, and EtOAc, resp.) Oil. 5 mL stock soln to 100 mL with 40% alcohol. Add 1 mL high level n-BuOH internal std soln, (f)(1), and mix. Prep. fresh weekly. (h) n-Propyl alcohol, isobutyl alcohol, isoamyl alcohol, and ethyl acetate low level working solns.- (Approx. 2.0, 2.1, 4.1, and 2.3 g/100 L n-PrOH,isobutyl alcohol, isoamyl alcohol. and EtOAc, resp.) Oil. 5 mL high level intermediate soln, (g)(2). to 100 mL with 95% alcohol. Add 1 mL low level internal std soln, (f)(2), and mix. Prep. fresh weekly. Prep. std soln of ca same concn as sample if latter differs grossly from appropriate (high or low level) std. 9.077 Determination Make preliminary injection of 10 ILL sample to det. absence of n-BuOH. (If present, subtract its amt from total n-BuOH (original and internal std) content.) Add 1 mL internal std soln, (f) (high or low level, depending on higher alcohol and EtOAc concn), to 100 mL sample in vol. flask, and chromatograph 10 f.LL aliquots of sample and std solns in triplicate. Measure peak hts ofn-PrOH, isobutyl alcohol, isoamyl alcohol, and EtOAc to nearest 0.05 cm and calc. peak ht ratio of each to n-BuOH (internal std) in sample and std solns. (For more accurate detn of isoamyl alcohol, use peak areas.) X = H x S/H', where X = concn of higher alcohol or EtOAc in sample (g/100 L); H = peak ht (or area for isoamyl alcohol) ratio of higher alcohol or EtOAc to n-BuOH in sample; H' = peak ht (or area for isoamyl alcohol) ratio of higher alcohol or EtOAc to n-BuOH in std; S = concn of higher alcohol or EtOAc in std (g/100 L). Sum of isoamyl and isobutyl alcohol concns is ca equiv. to fusel oil concn as detd in 9.069-9.074. Alternative Method (17) 9.078 Apparatus Gas chromatograph.-Equipped with flame ionization detec- tor. (1) Column.-2% glycerol and 2% 1,2,6-hexanetriol (Aldrich Chemical Co.) (w/w) on Gas-Chrom R (100-120 mesh, non-acid- washed) (Applied Science Laboratories, Inc.). Weigh 0.2 g each of glycerol and 1,2,6-hexanetriol into 50 or 100 mL beaker and dissolve by stirring with MeOH. Weigh 9.6 g Gas-Chrom R in evapg dish (top diam. ca 100-125 mm). moisten with MeOH, and add soln of stationary phases. Mix thoroly, and gently heat mixt. on steam bath, stirring continuously. When material appears to be dry (light pink). continue heating and occasional stirring to ensure complete removal of solv. Store in capped jar. Pack 3 m (10') x 'Is" od Cu or stainless steel tube (0.030" wall; if steel is used, rinse inside several times with acetone to remove manufacturing oils, and air dry) with prepd support, using vibrator to ensure complete packing of column. Condition overnight in 80° column oven with He flow rate of 10-25 mL/min and detector end of column disconnected. (2) Approximate parameters.--Column, injector, and detector temps (°)-80, 100, and 125, resp.; gas flows (mL/min)-He carrier and H 25, air 250-400; attenuation 64x. Optimum operating conditions vary with column and instru- ment and must be detd by using std solns. Adjust parameters for max. peak sharpness and optimum sepn. Analysis is com- plete in ca 11 min. 9.079 Reagents (Use absolute alcohol thruout when alcohol is specified.) (a) n-Propyl alcohol.-Reagent grade. (b) Isobutyl alcohol.-(Fisher Scientific Co., Certified Reagent No. A-379.) (c) Amyl alcohol.-(Gallard-Schlesinger Chemical Manufac- turing Co., 584 Mineola Ave., Carle Place, NY 11514, AnalaR No. 10038.) Mixt. of active-amyl and isoamyl alcohols, ca 22 and 78%, resp. Concn of 2 isomers varies from batch to batch. Oet. composition of reagent by 9.080. Measure areas of 2 peaks by triangulation (ht x width at half-ht). and obtain concn of each by dividing area of each peak by sum of both peak areas. (d) 3-Pentanol internal std soln.-40.76 mg/mL (Aldrich Chemical Co., Inc., No. P802-5). Prep. soln contg 10 mL reagent in 200 mL alcohol-H2 0 (1 + 1). (e) Ethyl acefate.-(Fisher Scientific Co., Certified Reagent No. E-145.) (f) n-Propyl alcohol, isobutyl alcohol, and amyl alcohol std solns.-Prep. 3 or 4 std solns contg varying amts alcohols as follows: Into tared 100 mL vol. flasks contg alcohol-H20 (1 +1). pipet fusel alcohols and weigh after addn of each component.
- 178. 156 9. BEVERAGES: DISTILLED LIQUORS AOAC METHODS (1980) Proportions of fusel alcohols in each std soln should vary so that desired concn range of each is represented in random manner in series of std solns. Suggested amts: 0.25-1.5 mL n- PrOH, 1.0-2.5 mL isobutyl alcohol, and 2.0-5.0 mL amyl alcohol. Oil. each to vol. with alcohol-H20 (1 +1). (g) n-Propyl alcohol, isobutyl alcohol, and amyl alcohol work- ing std solns.-Dil. 10 mL each std soln and 2.0 mL 3-pentanol internal std soln to 200 mL with alcohol-H20 (1 +1) (1 :20 diln). (h) Ethyl acetate std solns.-Prep. 3 or 4 std solns contg 0-0.5 g/L (0-50 g/ 100 L) in H20 or alcohol-H2 0 (1+ 1). Use for prepg direct std curve by plotting peak ht (mm) against concn in g/100 L. 9.080 Determination Pipet 10 mL sample into convenient vessel (e.g., 1 oz French sq glass bottle with screw cap). add, by pipet (0.2 mL pipet graduated in 0.01 mL), 0.1 mL 3-pentanol internal std soln, and mix. Inject 2 JLL sample and working std solns. Measure peak ht of each component in working std soln and calc. peak ht ratio of each to internal std. Calc. concn ratio of each by dividing wt of component by that of internal std. (Proportion of active-amyl and isoamyl alcohols in mixt. must be taken into consideration in calcns of actual wts of each isomer in working std solns.) Plot concn ratios (horizontal axis) against peak ht ratios (vertical axis) for each higher alcohol in all working stds to obtain family of curves. For EtOAc, plot peak ht directly against concn. Similarly, measure peak ht of each component in sample and calc. peak ht ratios. Read concn ratios of all alcohols, using proper std curve. Multiply concn ratio of each fusel alcohol in sample by 40.76 to obtain g/100 L. New std curves need be prepd only when new instruments, parameters, or stds are used. Furfural (18)-Official Final Action 9.081 Reagent Furfural std soln.-Redistil furfural thru short packed fraction- ating column at atm. pressure, and collect fraction boiling at 161.2° (uncorrected). Weigh 1 mL redistd furfural into 100 mL vol. flask and dil. to vol. with alcohol. Pipet 5 mL ofthis soln into 500 mL vol. flask and dil. to vol. with 50% alcohol (concn, ca 116 mg/L). Concd soln retains strength, but dil. soln does not. 9.082 Determination Pipet 25 mL distd spirits into volatile acid distn flask, Fig. 9:03, with$" joints and steam distil until 200 mL collects. If haze is present in distillate, dil. with known vol. alcohol. Det. A at 277 nm. Det. A of std solns of furfural contg 0, 1, 2, 3, 4, and 5 mg furfural/L. Plot std curve or calc. avo A of 1 mg furfural/L, A' (ca 0.15). mg Furfural/L = (A/A') x F, where F is diln factor (vol. final soln on which A is detd/vol. sample). 9.083 Detection of Acetone, Other Ketones, Isopropanol, and Tertiary Butyl Alcohol Official Final Action Reagent Mercuric sulfate soln.-Mix 5 g yellow HgO with 40 mL H20 and add, with stirring, 20 mL H2S04 and 40 mL H20. Stir until completely dissolved. (Caution: See 51.079.) FIG. 9:03-Steam distillation flask 9.084 Determination To 2 mL distillate, 9.050, add 3 mL H20 and 10 mL HgS04 soln. Heat on boiling H20 bath 3 min. White or yellow ppt forming within 3 min indicates presence of acetone, other ketones, or tert-BuOH. Disregard any ppt forming after 3 min on boiling H20 bath. If no ppt forms, test for isopropanol as follows: Place 8 g Cr03 in 100 mL Kohlrausch flask, and add 15 mL H20 and 2 mL H2S04, Connect flask with reflux condenser and add 5 mL sample very slowly thru condenser. Reflux 30 min; then cool and distil 2 mL, collecting distillate in 10 mL graduate. Add 3 mL H20 and 10 mL HgS04 soln, and proceed as above. 9.085 Sugars-Official Final Action See Chapter 31. 9.086 Methanol-Official Final Action Chromotropic Acid Colorimetric Method (19) Reagents (a) Potassium permanganate soln.-Dissolve 3.0 g KMn04 and 15.0 mL H3P04 in 100 mL H20. Prep. monthly. (b) Sodium salt of chromotropic acid (sodium 1,8-dihy- droxynaphthalene-3,6-disulfonate) soln.-5% aq. soln. Filter if not clear. Prep. weekly. Either acid or salt may be used. 9.087 Purification of Chromotropic Acid If A of blank is >ca 0.05, purify reagent as follows: Dissolve 10 g chromotropic acid or its salt in 25 mL H20. (Add 2 mL H2S04to aq. soln of salt to convert it to free acid.) Add 50 mL MeOH, heat just to bp, and filter. Add 100 mL isopropanol to ppt free chromotropic acid. (Add more isopropanol to increase yield of purified acid.) 9.088 Preparation of Sample Dil. or adjust sample to total alc. concn of 5-6%. Using 50 mL sample, distil thru simple still, collecting 40 mL distillate. Oil. to 50 mL with H2 0. (If alcohol has been detd previously, distillate may be adjusted to 5-6% alc. concn and used for this' test.) If
- 179. AOAC METHODS (1980) METHANOL 157 >0.05% MeOH by vol. is present, dil. to ca that concn with 5.5% alcohol. For samples contg <0.05% MeOH, measure 200 mL into efficient fractionating still, place system under total reflux 15 min, and then slowly distil at high rate of reflux (;,20: 1). Collect 10 mL distillate and dil. to 160 mL with H20. 9.089 Determination Pipet 2 mL KMn04 soln into 50 mL vol. flask. Chill in ice bath, add 1 mL chilled dild sample, and let stand 30 min in ice bath. Decolorize with little dry NaHS03 and add 1 mL chromotropic acid soln. Add 15 mL H2S04 slowly with swirling and place in hot (60-75°) H2 0 bath 15 min. Cool, add enough H20 to bring approx. to 50 mL mark, mix, and dil. to vol. with H20 at room temp. Read A at 575 nm against reagent blank of 5.5% alcohol treated similarly. Treat std MeOH soln contg 0.025% by vol. MeOH in 5.5% alcohol simultaneously in same manner, and read A'. (Temp. of std and sample should be within 1° since temp. affects A.) % MeOH in sample = (A/A') x 0.025 x F, where F = diln factor of sample. Example: Sample was dild 25x; A of sample = 0.421; A of std MeOH = 0.368. Then (0.421/0.368) x 0.025 x 25 = 0.715% (If color of sample is too intense, dil. with H2S04-alcohol blank prepd as above. Not more than 3-fold diln is permitted, as ratio of chromotropic acid to HCHO is too low if diln is greater.) 9.090 Immersion Refractometer Method (20) Det. Zeiss immersion refractometer reading at 17S of distillate obtained in detn of alcohol. If, on ref. to table, 9:03, refractometer reading shows sp gr agreeing with that obtained in alcohol detn, 9.013, it may be assumed that MeOH is absent. Low refractom- eter reading indicates presence of appreciable amt of MeOH. If absence from the soln of refractive substances other than H20 and the alcohols is assured, this difference in refraction is conclusive evidence of presence of MeOH. Addn of MeOH to alcohol decreases refractive index in direct proportion to amt added; hence, quant. calcn is made by interpolation in Table 9:03 of figures for pure alcohol and MeOH of same sp gr as sample. Example.-Distillate has sp gr at 15.56° of 0.9625 and refrac- tometer reading at 17.5° of 43.1. By interpolation in Table 9:03, readings for alcohol and MeOH at this gravity are 65.2 and 31.7, resp., and difference is 33.5; 65.2 - 43.1 = 22.1; (22.1 -7 33.5) x 100 = 66.0, showing 66.0% of total alcohol present is MeOH. Gas Chromatographic Method (21) 9.091 Apparatus See 9.075. 9.092 Reagents (a) Alcohol.-USP, MeOH-free. (b) Methanol stock soln.-Dil. 10 mL MeOH, 99.9 mol % (Fisher Scientific Co., A-936, or equiv.) to 100 mL with 40% alcohol. (c) n-Butyl alcohol internal std stock soln.-Dil. 10 mL n- BuOH, 99.9 mol % (Fisher Scientific Co., A-384, or equiv.) to 100 mL with 40% alcohol. (d) Methanol std soln.-0.050% MeOH plus 0.030% n-BuOH internal std. Fill 100 mL vol. flask to ca 99 mL with 40% alcohol and add, by syringe, 500 J.LL MeOH stock soln, (b). and 300 J.LL n-BuOH stock soln, (c). Mix and dil. to vol. with 40% alcohol. Mix again. Table 9:03 Scale readings on Zeiss immersion refractometer at 17.5°, corresponding to specific gravities of ethyl and methyl alcohol solutions Sp. Gr. Scale Readings Sp. Gr. Scale Readings 15.56° Ethyl Methyl 15.56° Ethyl Methyl 15.56° Alcohol Alcohol Differences 15.56 Alcohol Alcohol Differences 1.0000 15.0 15.0 0.0 0.9720 51.5 27.0 24.5 .9990 15.8 15.3 0.5 .9710 53.0 27.5 25.5 .9980 16.6 15.6 1.0 .9700 54.6 28.1 26.5 .9970 17.5 15.9 1.6 .9690 56.1 28.7 27.4 .9960 18.5 16.2 2.3 .9680 57.6 29.2 28.4 .9950 19.4 16.5 2.9 .9670 59.1 29.6 29.5 .9940 20.4 16.9 3.5 .9660 60.6 30.1 30.5 .9930 21.4 17.2 4.2 .9650 62.0 30.6 31.4 .9920 22.5 17.5 5.0 .9640 63.3 31.0 32.3 .9910 23.6 17.9 5.7 .9630 64.6 31.5 33.1 .9900 24.7 18.2 6.5 .9620 65.8 31.9 33.9 .9890 25.9 18.6 7.3 .9610 67.0 32.4 34.6 .9880 27.1 19.0 8.1 .9600 68.1 32.8 35.3 .9870 28.4 19.5 8.9 .9590 69.2 33.3 35.9 .9860 29.6 19.9 9.7 .9580 70.2 33.7 36.5 .9850 31.0 20.4 10.6 .9570 71.2 34.1 37.1 .9840 32.4 20.8 11.6 .9560 72.1 34.5 37.6 .9830 33.8 21.3 12.5 .9550 73.0 34.9 38.1 .9820 35.2 21.8 13.4 .9540 73.8 35.3 38.5 .9810 36.7 22.3 14.4 .9530 74.6 35.6 39.0 .9800 38.3 22.8 15.5 .9520 75.4 35.9 39.5 .9790 39.9 23.4 16.5 .9510 76.2 36.2 40.0 .9780 41.5 24.0 17.5 .9500 76.9 36.5 40.4 .9770 43.1 24.5 18.6 .9490 77.6 36.8 40.8 .9760 44.8 25.0 19.8 .9480 78.3 37.0 41.3 .9750 46.5 25.5 21.0 .9470 79.0 37.3 41.7 .9740 48.2 26.0 22.2 .9460 79.7 37.6 42.1 .9730 49.8 26.5 23.3 Scale readings are applicable only to instruments calibrated in arbitrary scale units proposed by Pulfrich, Z. angew. Chem., 1899, p. 1168. According to this scale, 14.5 = 1.33300,50.0 = 1.34650, and 100.0 = 1.36464. If instrument used is calibrated in other arbitrary units, refractive index corresponding to observed reading can be converted into equivalent Zeiss reading by referring to 52.004-
- 180. 158 9. BEVERAGES: DISTILLED LIQUORS AOAC METHODS (1980) 9.093 Determination Inject 10 JLL MeOH std soln. Adjust operating parameters and attenuation to obtain measurable peak ht (ca v.. full scale deflection). Det. retention time of MeOH and n-BuOH (ca 3 and 12 min, resp.). Inject 10 JLL sample to est. MeOH, using 'atten- uation if necessary, and to check for absence of n-BuOH. On basis of presence or absence of n-BuOH in sample, det. MeOH content from std curve prepd according to (a) or (b): (a) n-Butyl alcohol absent.-On basis of est. of MeOH, prep. series of stds (4 or 5) in which range of concn includes MeOH concn in sample. Add internal std to both sample and std solns at concn similar to that of MeOH in sample. Calc. peak ht ratios of MeOH:n-butyl alcohol, using avo of duplicate injections, and plot ratios against MeOH concn. (b) n-Butyl alcohol present.-Prep. series of MeOH stds as in (a), but do not add n-BuOH to sample or stds. Plot actual peak ht of MeOH against concn. Artificial Colors (See also 9.00S-9.008) 9.094 Marsh Test-Official First Action To 10 mL sample in 20 mL test tube add enough freshly shaken Marsh reagent (100 mL amyl alcohol, 3 mL H3P04, and 3 mL H2 0) to nearly fill tube, and shake several times. Let layers sep. Color in lower layer indicates that sample has been colored with caramel, synthetic dye, or extractive material from un- charred white oak chips. In absence of any color, test 10 mL in same manner, using enough fusel oil, amyl alcohol, or Pentasol to nearly fill tube and shaking several times. Deeply colored lower layer indicates synthetic dye. Det. its identity as in Chapter 34. To confirm caramel apply one or more of following tests: 9.095 Mathers Test-Official Final Action See 11.0SS. 9.096 * Cyclohexanol Test-Official Final Action * See 9.0S9, 10th ed. 9.097 Coal-Tar Colors See Chapter 34. Tannin (22)-Official Final Action 9.098 Reagents (a) Folin-Denis reagent.-To 750 mL H20 add 100 g Na2W04 .2H20, 20 g phosphomolybdic acid, and 50 mL H3 P04 • Reflux 2 hr, cool, and dil. to 1 L. (b) Sodium carbonate saturated soln.-To each 100 mL H20 add 35 g anhyd. Na2C03, dissolve at 70-800 , and let cool overnight. Seed supersatd soln with crystal of Na2C03.10H20, and after crystn filter thru glass wool. (c) Tannic acid std soln.-O.l mg/mL. Dissolve 100 mg tannic acid in 1 L H2 0. Prep. fresh soln for each detn. 9.099 Preparation of Standard Curve Pipet 0-10 mL aliquots std tannic acid soln into 100 mL vol. flasks contg 75 mL H2 0. Add 5 mL Folin-Denis reagent and 10 * Surplus method-see inside front cover. mL Na2C03 soln, and dil. to vol. with H2 0. Mix well and det. A after 30 min at 760 nm. Plot A against mg tannic acid/l00 mL. 9.100 Determination Using 1 mL sample, det. A as in 9.099 and obtain mg tannic acid/l00 mL from std curve. If A is too great, repeat detn on 1+4 diln of sample. Samples treated as above may be compared in Nessler tubes against freshly prepd tannic acid stds treated in same manner. Cyanide (23)-Official First Action 9.101 Apparatus (Letters refer to Fig. 9:04.) (a) Distilling flask.-500 mL, r-b, 3-neck with "$ joints, angle- type (Fisher Scientific Co., No. 10-165B, or equiv.) (A). (b) Thermometer.-Range -10 to 1100 , with "$ screw-cap adapter to fit one "$ side neck of distg flask (B). (c) Air inlet tube.-With "$ cone to fit side neck of distg flask (C). (d) Condenser.-Graham coil-type, with inner and outer "$ joints, "$ cone to fit center neck of distg flask (Fisher Scientific Co., No. 7-728B, 400 mm jacket length, or equiv.) (D). (e) Flange a;sembly.-Consisting of 2 sep. ground-glass flanges at one end of sep. glass tubing, 7 mm diam. Lower tube (E) is ca 14 mm long and is also fitted with "$ cone to fit "$ socket at top of condenser; "$ cone has 2 glass hooks. Upper tube (F) is ca 5 mm long. (f) Clip.-To connect 2 ground-glass flanges together (G). (9) Springs.-To connect (D) and (F) securely together (H). ~-'-ij'r--IE_G_~Jb?N lJ--E- - ',;:,' ,4'COlUMN PLUG .. ___ LEAD .... f DIOXIDE ( H-! FIG. 9:04--Apparatus for determining hydrogen cyanide in distilled spirits.
- 181. AOAC METHODS (1980) CORDIALS AND LIQUEURS 159 9.102 Reagents (a) Potassium ferrocyanide soln.-( 1) Stock soln.- 0.01 % K4Fe(CN)6.3H20 in 0.2% Na2C03. Store in amber bottle. Na2C03 stabilizes this soln. (2) Working soln.-o.OOOl % K4Fe(CN)6.3H20; 27 mL equiv. to 10 J.Lg CN. Prep. fresh daily by dilg 1.0 mL stock soln to 100 mL with H20. (b) 4A'-Methylenebis (N,N-dimethylaniline) (tetrabase ).- Eastman Kodak Co., No. 244; or equiv. (c) Bis(ethyl acetoacetato) copper (copper ethyl acetoace- tate).-Fisher Scientific Co., No. 10057; or equiv. (d) Color reagent.-Dissolve 50 mg tetra base and 50 mg Cu Et acetoacetate in 10 mL CHCI3. Store in closed bottle; prep. fresh weekly. (e) Test papers.-Whatman No. 44 paper, or equiv., cut to size in strips to fit between flanges of ground-glass flange assembly. Impregnate with several drops color reagent imme~ diately before use; let CHCI3 evap. completely before inserting test paper between flanges of assembly. (f) Lead dioxide.-Dissolve ca 40 g Pb(OAc)2 in 200 mL H20 by heating on H20 bath in hood. Add NaOCI (13% available CI) until there appears to be no further darkening in color (ca 100 mL). Ppt should be dark brown at this stage. Let stand ca 15 min. Filter thru double thickness Whatman No. 42 papers in buchner. Wash with H20, discard filtrate, and wash ppt with ca 200 mL HN03 (1 +9). Finally wash with H20 and dry overnight in 105° oven. Pb02 produced by this method is granular and should be broken into pieces ca 20 mesh, but must not be ground any finer. Check each batch by applying control test for ferrocyanide std, 11.057(a), on 100 mL aliquots of H20 contg 10 J.Lg CN and 500 ppm 502 (as NaHS03) and comparing stain produced with std stain. Anal. grade Pb02is not suitable because it is too fine and prevents free flow of N to test papers. 9.103 Distilled Spirits Free of Sulfur Dioxide (For products contg 502, see 11.057.) (a) Control test.-Add 27 mL K4Fe(CN)6.3H20 working soln (equiv. to 10 J.Lg CN) to 100 mL alcohol and 100 mL H20 in 500 mL 3-neck flask with stopper in 1 side neck and gas inlet tube connected to N (or CO2) in other. (Use of gas to flush HCN thru test papers is preferred, but vac. may be used.) Connect center neck of flask to condenser and place ground-glass flange as~ sembly in top of condenser; then connect hooks with springs. Insert test paper between 2 glass flanges and clip together. Remove stopper momentarily, add 10 mL H2S04 (1 +9), and immediately replace stopper. Assure rapid flow of cold H20 thru condenser and then heat contents of flask just to bp. When boiling temp. is reached, pass N thru liq. at rate such that individual bubbles just cease to be visible as sep. bubbles, but form continuous stream. Check that all joints are tight. Let distn proceed 15 min; then remove test paper and examine for blue stain which indicates cyanide. (Alcohol vapors must not reach test paper.) Intense, well defined blue circle should be obtained with 10 J.Lg CN. Blank detn performed concurrently must show no color. (1 I-'g CN, equiv. to 0.01 ppm CN in 100 mL sample, can be readily detected.) (b) Samples.-Add 100 mL sample to 100 mL H20 in 500 mL 3-neck distg flask, and test as in (a). For pos. results, confirm test papers showing faint or questionable stains by 2 addnl analyses. Blue stains are stable ca 1 week if kept out of direct light. (c) Confirmation.-To confirm that pos. reactions from sam- ples of unknown origin are due to CN, expose blue test paper to NH3 vapors. Colorless carbinol base is formed. With addn of HOAc, blue reappears. CORDIALS AND LIQUEURS 9.104 Physical Examination-Procedure Note and record following: (a) Appearance, whether bright or turbid and presence of sediment; (b) color and depth of color; (c) odor; (d) taste. 9.105 Specific Gravity-Official Final Action See 9.011. 9.106 Alcohol-Official Final Action (a) By weight.-See 9.021. (b) By volume.-See 9.013. Use pycnometer calibrated at 15.56°. Methanol-Official Final Action 9.107 Preparation of Sample Measure sample contg 20-25 mL absolute alcohol into distg flask, add enough H20 to make total ca 100 mL, and distil, collecting ca 50 mL distillate. To distillate add 4 g NaCI for each 10 mL H20 and let stand several hr for complete satn. Transfer to separator, using ca 10 mL satd NaCI soln to wash out container, and shake with 25 mL pet ether. When sepn is complete, transfer aq. soln to second separator contg 25 mL pet ether; shake, and transfer aq. soln to third separator, also contg 25 mL pet ether; shake, and when sepn is complete, drain aq. soln into 200 mL distg flask. Meanwhile add 25 mL satd NaCI soln to first separator and follow sample thru with this soln, finally adding washings to sample soln in distg flask. Repeat this operation with second 25 mL portion satd NaCI soln, finally adding this also to distg flask. Distil mixt. into 50 mL vol. flask, using suitable adapter. After 48-49 mL distils, disconnect app., fill flask to mark with H20, mix, and det. MeOH as in 9.089 or 9.090. 9.108 Aldehydes-Official Final Action Measure 100-200 mL sample into distn flask. If solid content is ,-s25 g/100 mL, add 12.5-25 mL H20; if >25 g/100 mL, add 5 mL H20 for each 10 g solids present; distil slowly, collecting vol. distillate equal to that of sample, and proceed as in 9.052. 9.109 Fusel Oil-Official Final Action Using 50 mL prepd distillate, 9.108, proceed as in 9.068, 9.071-9.074, or 9.077. 9.110 Total Solids-Official Final Action (a) From specific gravity of dealcoholized sample.-Transfer residue from alcohol detn, 9.106(b), to original pycnometer with H20, dil. to mark with H20 at 15.56°, and mix. Adjust temp. of pycnometer and contents to 20°; adjust meniscus to mark, using capillary tube or narrow strips of filter paper to remove any excess liq. while in 20° bath. Weigh, and calc. sp gr of liq. From 52.008 det. % dry substance and corresponding sp gr at 20°/4°. Sp gr at 20°/4° x % dry substance = total solids (g/100 mL). (b) By evaporation.-Fill 25 mL vol. flask with sample at 20°, and adjust meniscus, using capillary tube or narrow strips of filter paper, while flask is immersed in bath held at same temp. ca 30 min. Quant. transfer contents of flask to 100 mL vol. flask with H20 and dil. to vol. with H20 at convenient temp. At same temp., pipet 10 mL dild sample into dish contg sand and dryas in 31.008. Wt residue x 40 = total solids (g/100 mL).
- 182. 160 9. BEVERAGES: DISTILLED LIQUORS AOAC METHODS (1980) (c) From refractive index of dealcoholized sample.-Restore residue from alcohol detn to original vol. by evapg or dilg as necessary. Det. refractometer reading of soln at 20° and obtain corresponding % dry substance. From 52.008 det. sp gr corre- sponding to % dry substance found and multiply by % dry substance to obtain g total solids/100 mL sample. To obtain % total solids, divide total solids/100 mL by sp gr, 9.011. 9.111 * Glycerol-Official Final Action * By direct weighing, or by oxidn with K2Cr20 7 soln. See 9.111,12th ed. 9.112 Sucrose-Official Final Action (a) By polarization.-Pipet, into evapg dish, vol. sample equiv. to 52 g as calcd from sp gr, 9.011, and exactly neutze with 1N NaOH, calcg amt required from acidity, 9.119. Evap. on steam bath to remove alcohol, transfer to 200 mL vol. flask, and proceed as in 31.025 or 31.026, beginning " ... add necessary clarifying agent, ..." in 31.025(a). (b) By reducing sugars before and after inversion.-Approx- imate sugar content of sample from total sol ids, 9.110, and pipet sample contg 5-7 g sugars into porcelain dish; exactly neutze with 1N NaOH soln, calcg amt required from acidity, 9.119, and evap. on steam bath to remove alcohol. Transfer to 200 mL vol. flask, clarify with neut. Pb(OAc)2 soln, 31.021(d), remove excess Pb with K oxalate, and proceed as in 31.031, using 31.038 for detn of reducing sugars. 9.113 Ash-Official Final Action Proceed as in 31.012 or 31.013, using 25 mL sample. 9.114 Soluble and Insoluble Ash-Official Final Action Using ash from 9.113, proceed as in 31.015. 9.115 Alkalinity of Soluble Ash-Official Final Action Using sol. ash from 9.114, proceed as in 31.016. 9.116 Alkalinity of Insoluble Ash-Official Final Action Using insol. ash from 9.114, proceed as in 31.017. 9.117 Phosphorus-Official Final Action Using ash obtained in 9.113, det. P20 5 as in 11.034. 9.118 Caramel-Official Final Action See 11.055. 9.119 Total Acidity-Official First Action Place ca 600 mL H20 in 800 mL beaker, add ca 1 mL phthln, and titr. to pink soln with 0.1N NaOH. Add 10-20 mL sample (unless this vol. gives soln such deep color that it will obscure end point, in which case 5 mL may be used) and titr. to pink comparable to that of soln before sample was added. Calc. acidity as g/100 mL sample in terms of predominating acid present in sample. 9.120 Characteristic Acids-Preparation of Sample-Procedure Use sample contg ~30 g solids and ~200 mg acid to be detd, as calcd from acidity; evap. to ca 30 mL and treat as in 9.121-9.124. *Surplus method-,see inside front cover. 9.121 * Tartaric Acid-Official Final Action * See 9.121, 12th ed. 9.122 * Citric Acid-Official Final Action * See 9.122, 12th ed. 9.123 * Total Malic Acid (Laevo and Inactive) * Official First Action See 9.123, 12th ed. 9.124 * Laevo-Malic Acid-Official First Action * See 9.124, 12th ed. 9.125 Volatile Esters-Official Final Action Measure 100-500 mL sample into distg flask and steam distil as in 12.026, collecting vol. distillate at least twice as great as vol. alcohol contained in sample. (If detn 9.126 is to be made, use 500 mL sample.) Disconnect app. and wash out condenser with little H20. Add ca 1 mL phthln, and titr. to pink that persists >1 min, using 0.1N NaOH or KOH. Add measured excess of 25-50 mL 0.1N alkali to soln, reflux 1 hr, cool, and titr. excess alkali with 0.1N H2S04 , Calc. number of mL 0.1N alkali used in saponification of esters as EtOAc. 1 mL 0.1N alkali = 8.8 mg EtOAc. 9.126 * Gamma Undecalactone * (Qualitative Test) (24) Official Final Action (Peach and apricot cordials) See 9.087, 10th ed. 9.127 * Optical-Crystallographic Properties * of Hydrazino-y-Undecalactone Official Final Action See 9.088, 10th ed. 9.128 Benzaldehyde-Official Final Action See 19.100-19.101. 9.129 Thujone (25)-Official First Action To 500 mL sample add 1 mL freshly distd aniline and 1 mL H3P04, and reflux 30 min on steam bath. Distil two 100 mL portions; reject first and test second for thujone as follows: Add 0.5 g semicarbazide hydrochloride and 0.6 g anhyd. NaOAc (or 1.0 g crystd salt) and let mixt. stand overnight. Distil off alcohol at min. pressure. Steam distil to remove essential oils and other volatile material; collect and reject first ca 15 mL distillate. Wash down condenser with little alcohol and with H20. Cool sample, add 1 mL H2S04 (1 +1), and again steam distil, collecting 20 mL distillate in cylinder. Pour distillate into small separator, and add 20 mL ether, using receiver as measure. Shake and sep. ether soln. Add 10 mL 65% alcohol and let ether evap. spontaneously. After all ether evaps, note odor of residue. Odor of thujone will be apparent if ;,2 mg is present in soln, provided it is not masked by presence of other odoriferous substances. Make modified Legal test as follows: To soln obtained as above, add 1 mL 10% ZnS04 soln and 0.25 mL freshly prepd aq. Na nitroprusside soln (0.1 g/mL). Slowly, with const stirring, add 2 mL 5% NaOH soln. Let stand 1-2 min. Add 1.5 mL HOAc and mix. Ppt of raspberry red color (resembling alcohol ppt of red fruit juice) shows presence of thujone. Neg. test is shown by similar ppt having appearance
- 183. AOAC METHODS (1980) SELECTED REFERENCES 161 similar to that of alcohol ppt from apple jelly or other light colored fruit. SELECTED REFERENCES (1) JAOAC 39, 723(1956); 41,118(1958). (2) JAOAC 38, 821(1955); 39,730(1956); 40, 440(1957). (3) Ind. Eng. Chern., Anal. Ed. 14, 237(1942); JAOAC 28, 88(1945); 41, 118(1958); 42, 329(1959). (4) JAOAC 40, 436(1957); 42, 327(1959); 43, 657(1960). (5) Ind. Eng. Chern. 18,841(1926); JAOAC 35, 239(1952). (6) JAOAC 37, 945(1954); 46, 299(1963); 47. 720(1964). (7) JAOAC 50. 338(1967). (8) JAOAC 50, 334(1967). (9) JAOAC 53,12(1970). (10) JAOAC 49,498(1966). (11) JAOAC 37,921(1954). (12) JAOAC 55, 559(1972). (13) JAOAC 55, 566(1972). (14) JAOAC 42,331(1959); 43,655(1960); 44, 383(1961). (15) JAOAC 46,285(1963). (16) JAOAC 51, 915(1968). (17) JAOAC 55, 549(1972). (18) JAOAC 43,659(1960); 44,392(1961). (19) JAOAC 41,121(1958); 42, 336(1959). (20) J. Am. Chern. Soc. 27, 964(1905); Ind. Eng. Chern. 19, 844(1927); JAOAC 28, 800(1945). (21) JAOAC 55,564(1972). (22) JAOAC 35,255(1952); 37, 665(1954). (23) JAOAC 53, 777(1970). (24) JAOAC 16,420(1933); 19, 75, 183(1936). (25) Ann. chim. anal. 13, 227(1908); Schweiz. Wochschr. 49, 337,507(1911); JAOAC 19, 120(1936); 20,69(1937).
- 184. 10. Beverages: Malt Beverages and Brewing Materials* BEER (Unless otherwise directed, express results as % by wt.) 10.001 Preparation of Sample Official Final Action Remove CO2 by transferring sample to large flask and shaking, gently at first and then vigorously, keeping temp. of beer at 20-25°. If necessary, remove suspended material by passing the CO2-free beer thru dry filter paper. 10.002 Color Spectrophotometric Method (Standard Reference Color Method) (11 Official Final Action Apparatus Spectrophotometer.-Capable of isolating band width of ,;:;1 nm at 430 nm with wavelength and photometer scales checked and corrected for inaccuracies in accordance with instructions contained in NBS Letter Circular LC-1017 of Jan. 1967. 10.003 Preparation of Sample Partially degas sample by opening bottle at room temp., pouring contents into 1 L erlenmeyer, and swirling gently. Avoid formation of turbidity, and conduct partial degassing and read- ings as rapidly as possible. 10.004 Determination Place prepd sample in suitable cell and det. A at 430 nm and at 700 nm. 10.005 Calculations Calc. A from thickness at which read to 1,1," (1.27 em) (Av.). If (A y, at 430 nm) X 0.039 > (A v. at 700 nm), sample is assumed "free of turbidity" and color is calcd as follows: Beer color intensity = 10 x (A y, at 430 nm)' If (A v. a1700 nm) > 0.039 X (A v. at 430 nm), clarify sample by centrfg or filtering, and redet. A. Report color intensity values to nearest 0.1 unit. 10.006 Photometric Method-Official Final Action Apparatus Use any com. available filter photometer or abridged spec- trophtr utilizing moderately broad spectral band and having adequate sensitivity. Use light filter with peak T in range 420-450 nm (blue-violet) for max. sensitivity and precision. (Filters for wavelengths in blue or blue-green range may also be used, but result in reduced precision.) Cell should be of such size, if possible, as to give A values between 0.187 and 0.699 (20-65% T), where max. precision is achieved. Use same size cell for both color measurement and calibration. 10.007 Calibration of Photometers Beer calibration method.-For each color intensity value for which measurements are to be made, obtain 6-8 replicate bottles of beer which are low in air content and have been pasteurized. Det. color intensity value of the beer by averaging readings obtained for ?o2 bottles by Standard Reference Color (SRC) Method, 10.004. If these values must be obtained from another laboratory, ship bottles of beer by the fastest available method, marked to avoid rough handling. Det. photometer reading of the beer by averaging readings obtained for ?o2 bottles with wavelength and cell as in 10.006. Calc. calibration factor in accordance with photometer instruc- tions or prep. calibration curve by plotting A or photometer scale reading against the SRC value for sample, assuming that curve passes thru origin. This calibration will be accurate only for readings in immediate vicinity of calibration point. If it is desired to accurately measure color intensity of >1 sample or colors over range of values, calibrate photometer for each sample or use beers having colors which cover desired range. Calc. avo calibration factor or prep. avo calibration curve. 10.008 Preparation of Sample See 10.003. 10.009 Determination Place sample in cell and det. photometer reading. Calc. color intensity value, using calibration factor or calibration curve. Report color to nearest 0.1 unit. Total Haze after Chilling (2)-Official Final Action 10.010 Reagents (Use turbidity-free distd H2 0 thruout.) (a) Hydrazine sulfate soln.-l%. Dissolve 1.000 g H4 N2 .H2S04 in H20 (may require 4-6 hr) and dil. to 100 mL. (b) Stock formazin suspension.-Dissolve 2.500 g hexame- thylenetetramine (formin) in 25 mL H20 in 125 mL erlenmeyer, pipet in 25 mL 1% hydrazine sulfate soln, and stopper flask. Formazin begins to ppt in 6-8 hr and pptn is complete within 24 hr. Prep. every 3 months. (c) 1000 Turbidity std.-Oil. 14.5 mL well mixed stock sus- pension, (b), to 1 L with H2 0 in vol. flask. Prep. weekly. (1000 formazin turbidity units (FTU) on empirical formazin turbidity scale represents reflectance of insol. reaction products of 0.0725 g hydrazine sulfate with 0.7250 g hexamethylenetetramine dild to 1 L.) (d) Working stds for visual method.-Prep. suitable dilns, daily, of 1000 turbidity std with H20. FTU stds <100 are suitable for fresh beers; higher stds may be required for older samples. Use increments of 10 FTU for stds <100 FTU; in 20 FTU increments for 100-200; and in 50 FTU increments for >200 FTU. Visual Method 10.011 Apparatus (a) Clark Turbidimeter, Model CL T.-Code 648. Available from Cargille Scientific Inc., 55 Commerce Rd, Cedar Grove, NJ 07009. * Many methods in this chapter have been tested by both American Society of Brewing Chemists and Association of Official Analytical Chemists and have been adopted by both Associations. See "Methods of Analysis, A.S.B.C.," 7th rev. ed., 1976. 162
- 185. AOAC METHODS (1980) Viewing box of same dimensions and lighting is also suitable. (b) Red Plexiglas sheet.-'/.." thick, ca 1 sq ft (930 sq em). (c) Constant temperature bath.-D±0.2°. (d) Ice-water bath.-Contg few drops wetting agent. (e) Flint glass bottles.-Of same dimensions as flint glass bottles contg beer test samples; or clear drinking glasses (shells), 10 oz (300 mL), od ca 66 mm at bottom and 67 mm at top. 10.012 Determination (Make comparisons with samples at 0°. Keep test samples in 0° bath when not matching turbidities.) Place container of beer to be tested in upright position in 0° bath and hold 24 hr. Prep. series of formazin turbidity working stds covering range of expected turbidities of test samples. Fill into flint glass bottles of same dimensions as those holding beer test samples. If beer is in flint glass bottles, carefully remove bottle from const temp. bath without disturbing sediment. Dip bottle into ice-H,O bath contg few drops wetting agent to prevent fogging or accumulation of H,O droplets on bottle while in viewing box. Place bottle of beer in viewing box between 2 bottles of formazin turbidity working stds. Compare turbidities by viewing thru red Plexiglas sheet placed 5 cm (2") in front of bottles. Change formazin stds until that working std is found which most closely matches turbidity of test sample. If beer is not in flint glass bottles, carefully remove container from const temp. bath and, without disturbing sediment, pour beer into clear 10 oz drinking glass (shell) which has been prechilled by standing (external contact only) in ice-H,O bath contg wetting agent. Degassing is not necessary. Use formazin turbidity working stds in identical 10 oz glasses to match turbidities as above for bottles. Report as total haze of the beer after chill ing, formazin turbidity units (FTU) of working std giving closest match. In range 0-100 FTU, report to nearest 10 FTU; 100-200,20; >200,50. Nephelometric Method 10.013 Calibration Nephelometer.-Prep. calibration curve at 580 nm or other suitable wavelength for instrument employed by use of series of working stds or dilns of 1000 turbidity std. If readout device of nephelometer is 0-100 scale of arbitrary units, set needle to indicate 0 units when cell is filled with turbidity-free distd H,O and 100 units when it is filled with selected formazin turbidity std. 10.014 Determination Place containers of beer to be tested in upright position in 0° bath and hold 24 hr. Prechill nephelometer cell in small ice-H20 bath contg wetting agent (external contact only). Carefully remove container of beer from bath and, without disturbing sediment, rinse and fill cell with test sample. Place cell in ice-H,O bath contg wetting agent, and degas beer by stirring with thermometer. When beer temp. is 0°, place cell in sample chamber of nephelometer and det. reading. (Beer must be at 0° when taking reading.) 10.015 Calculations Calc. FTU total haze from calibration curve or by formula: FTU = R x S/100, where R = nephelometer (galvanometer scale) reading; S = FTU of formazin turbidity std used for calibration of nephelometer. BEER 163 10.016 Specific Gravity-Official Final Action Det. sp gr of prepd sample, 10.001, at 20/20° (in air) as in 9.011, but use pycnometers described in 10.l06(i) and 10.107(b) or (c). Viscosity of Beer (3)-Official Final Action 10.017 Apparatus (a) Constant temperature bath.-Adjusted to 20.00±0.05°. (b) Viscometer.-Ostwald or Cannon-Fenske; H20 time range 50-150 sec. 10.018 Determination Prep. sample as in 10.001, ensuring complete decarbonation but avoiding loss of surface active components. Det. sp. gr. as in 10.016. Clean viscometer with chromic acid cleaning soln, rinse with H,O, and drain. Attemperate viscometer, H,O, and beer to 20° in H,O bath. Add appropriate vol. H,O (usually 5 or 10 mL) at 20° to viscometer. Using suction, draw H20 above upper mark. Let liq. level fall, and start timing with stopwatch as meniscus passes upper mark. Stop timing when meniscus passes lower mark. Rinse viscometer with beer sample, and det. time required for passage of beer between menisci as above. 10.019 Calculations (a) Absolute or dynamic viscosity (Centipoise).-(1) Viscosity (cP) = flow time of beer at 20° x sp gr of beer x 1.002/flow time of H,O at 20°, where 1.002 = viscosity of H20 in cP at 20°. Report to second decimal place. (2) Example.-Flow time of H,O = 89.8 sec; flow time of beer = 130.2 sec; sp gr of beer = 1.03425; Viscosity, cP = 130.2 x 1.03425 x 1.002/89.8 = 1.503 = 1.50 cP (b) Kinematic viscosity (Centistoke).-( 1) Viscosity (cS) = flow time of beer at 20° x 1.0038/flow time of H,O at 20°, where 1.0038 is viscosity of H,O at 20° in cS. Report to second decimal place. (2) Example.-Flow time of H,O = 89.8 sec; flow time of beer = 130.2 sec; Viscosity, cS = 130.2 x 1.0038/89.8 = 1.455 = 1.46 cS (c) SI system.-( 1) SI unit for dynamic viscosity is Pascal- second (Pa' sec). 1 cP = 0.001 Pa' sec = 1 mPa' sec. Example.-1.503 cP = 0.001503 Pa'sec, or 1.503 milliPascal- sec, or 1.50 mPa' sec. (2) SI unit for kinetic viscosity is meter/second (m2/sec). 1cS = 0.000001 m'/sec = 1 IL'/sec. 10.020 Apparent Extract-Official Final Action Find apparent ext corresponding to sp gr detd at 20/20° from 52.009, reporting to second decimal place. 10.021 Real Extract-Official Final Action (a) Evap. 75-100 mL sample (accurately weighed to 0.1 g) on H,O bath or asbestos plate, at temp. ~80°, to ca '13 original vol. Cool, dil. to original wt with H20, and det. sp gr with pycnometer at 20/20°. Det. real ext directly from 52.009. (b) If no antifoam material was used in detn of alcohol, 10.023, quant. transfer residue with hot H2 0 to 100 mL vol. flask. Cool, and dil. to 100 mL at 20°. Det. sp gr at 20/20°, 10.016, and find
- 186. 164 10. BEVERAGES: MALT BEVERAGES AND BREWING MATERIALS AOAC METHODS (1980) ext directly from 52.009. If 100 mL beer was taken, correct as follows: Ext found x sp gr of dealcoholized beer /sp gr of beer = g ext/l00 g beer. 10.022 Extract of Original Wort-Official Final Action Calc. from following formula and report to first decimal place: 0= [(P x 2.0665) + E] x 100/[100 + (P x 1.0665)], where 0 = ext of original wort; P = % alcohol by wt (g/100 g beer); and E = % real ext, 10.021(a) or (b). 10.023 Alcohol by Volume-Official Final Action See 11.005, but use pycnometers described in 10.106(i) and 10.107(b) or (c). Alcohol by Weight 10.024 Specific Gravity Method-Official Final Action See 9.021, but use pycnometers described in 10.106(i) and 10.107(b) or (c). 10.025 Refractometer Method (4) Official Final Action Apparatus (a) Immersion refractometer.-Bausch & Lomb, Carl Zeiss, Inc., 444 Fifth Ave, New York, NY 10018, or equiv., with prisms covering range 1.32-1.37 n. (b) Water bath.-5ee 10.106(k). (c) Pycnometer.-5ee 10.106(i) and 10.107(b) or (c). 10.026 Calibration Adjust refractometer light to give max. contrast between light and dark fields. Adjust color compensator and focus for sharp, color-free dividing line. H20, double-distd from glass, should read ca 14.50 at 20°. Read H20 before each sample series. Rinse prism with H20 after each sample and dry with soft tissue. Prep. calibration curve to convert refractometer readings and sp gr detns to % alcohol by wt by analyzing beers covering alcohol range of interest by 10.024 and 10.027. Plot results, using ordinates (R - N), where R = refractometer reading (Rbee, - R wate,), N = 1000 x (sp gr - 1.00000), and abscissa = % alcohol by wt, 10.024. Fit least squares line to adequate number of points to get accuracy desired. Det. equation of line and slope. (See Definitions of Terms and Explanatory Notes, Item (24).) Calc. % alcohol by wt by formula or read from calibration curve. % alcohol by wt = F x (R - N) + C, where F = slope of calibration line, and C = const of calibration curve equation. 10.027 Determination Det. sp gr of decarbonated beer by 10.016. Place refractometer cuvet contg distd H20 and clear decarbonated beer samples in 20° H20 bath. Place prism of refractometer in H20 cuvet and check temp. after 15 min. Make 5 readings to nearest 0.1 scale division and average results (Rwate,). Transfer dry prism to beer sample cuvet, wait ~1 min, make 5 readings to nearest 0.1 scale division, and average results (Rbee,). Calc. % alcohol by wt by formula or read from calibration curve. 10.028 Real Degree of Fermentation or Real Attenuation-Official Final Action Calc. as follows and report to first decimal place: (orig. ext - real ext) x 100/orig. ext. 10.029 Apparent Degree of Fermentation or Apparent Attenuation-Official Final Action Calc. as follows and report to first decimal place: (orig. ext - apparent ext) x 100/orig. ext. 10.030 End Fermentation (Yeast Fermentable Extract) (Fermentable Sugars)-Official Final Action Det. real ext, 10.021, or apparent ext, 10.020. Ferment 250 mL beer with 1 g active compressed brewers yeast 24-48 hr at 15-25°, or until fermentation is complete, providing fermentation flask with H20 or Hg seal. Filter; det. real ext, 10.021, or apparent ext, 10.020. Fermentable sugars = difference in real ext before and after fermentation; or fermentable sugars = 0.82 x differ- ence in apparent ext before and after fermentation. 10.031 Caloric Content-Official First Action Det. sp gr (= density), 10.016, % alcohol by wt, 10.024 or 10.027, real ext, 10.021, and ash, 10.047, of beer sample. Calories/l00 g = (g real ext - % ash) x 4 + (% EtOH by wt x 6.9); Calories in vol. = (Calories/l00g) x (beer vol.20o x density)/100, where vol. is expressed in units required by test. 10.032 * Glycerol-Official Final Action * Dichromate oxdn method. See 11.010(b), 12th ed. Total Acidity (5)-Official Final Action 10.033 Indicator Titration Method Bring 250 mL H20 to bp and continue boiling 2 min. From fast-flowing pipet add 25 mL beer previously decarbonated by shaking and filtering, 10.001. After emptying pipet, continue heating 60 sec, regulating heat so that soln resumes boiling during final 30 sec. Remove from heat, stir 5 sec, and cool rapidly to room temp. Add 0.5 mL 0.5% phthln. Titr. with O.lN NaOH against white background. Make frequent color comparisons with sample of equal vol. and diln to which has been added approx. anticipated amt of alkali but no indicator. Titr. to first appearance of faint pink. Read buret. Add 0.2 mL more alkali; color should then be permanent, definite pinkish red, indicative of overtitrn. Take first buret reading as end point. Observe strictly all details of method. However, 100 mL H20, 10 mL beer, and 0.2 mL indicator may be used in place of amts specified above. (Use potentiometric titrn method, 10.034, for beers of dark color which, even when dild, may not permit judging phthln end point with necessary precision.) Report results: (a) as lactic acid, to nearest 0.01% (1 mL O.lN alkali = 0.0090 g lactic acid); or (b) as mL IN alkali, to nearest 0.1 mL, necessary for neutzn of 100 g beer. 10.034 Potentiometric Titration Method Use glass-calomel electrode system. Decarbonate beer com- pletely by shaking, 10.001. Using 50 mL undild sample (or such amt as best suits titrn assembly)' titr. potentiometrically with O.lN NaOH to pH 8.2. Add alkali in 1.5 mL portions to ca pH 7.6, and in 0.15 mL portions from there to pH 8.2. Make sure that complete equilibrium and good convergence are attained before reading buret at exactly pH 8.2. Report results as in 10.033. *Surplus method-see inside front cover.
- 187. AOAC METHODS (1980) Precautions: Observe all details of good potentiometric tech- nic, including following: Stdze potentiometer against fresh 0.05M K acid phthalate, 50.007(c). before and after any set of titrns; read potentiometer to nearest 0.02 unit; use flexible shielding around electrode leads and motor cords; ground motor and motor cords, preferably to H20 pipes; avoid contact between electrodes and glass beaker; use proper stirring speed to ensure quick mixing but to avoid foaming which may tem- porarily trap some of alkali added; stop titrn at ,,;pH 8.6 to minimize alkali contamination of glass electrode; check batteries frequently. Follow manufacturer's instructions for potentiometer used. Hydrogen-Ion Activity (pH) Official Final Action 10.035 Potentiometric Method Det. pH of undild sample, 10.001, using glass-calomel elec- trode system. Follow manufacturer's instructions for potentiom- eter used. Check pH meter before and after use against std K acid phthalate buffer, 50.007(c). Observe precautions in 10.034. Report results to nearest 0.05 pH. 10.036 Volatile Acids-Official Final Action Using 100 mL beer, proceed as in 11.040. Express result as HOAc, g/100 mL. 1 mL 0.1N alkali = 0.0060 g HOAc. 10.037 Carbohydrate Content-Official First Action Det. sp gr (=density). 10.016, real ext, 10.021, ash, 10.047, and protein, 10.045, of beer sample. g Carbohydrate/100 g = (g real ext/100g) - (% protein + % ash) g Carbohydrate in vol. = (g carbohydrate/100 g) x (beer vol.20o x density)/100, where vol. is expressed in units required by test. 10.038 Reducing Sugars-Official Final Action Dil. 25 mL prepd sample, 10.001, measured at 20°, to 100 mL with H20 at same temp. Det. reducing sugars in 25 mL of this soln by Munson-Walker method, 31.060, or dil. 50 mL beer with H20 to 100 mL and use Lane-Eynon method, 31.059. Express result as g maltose/100 mL beer. For conversion to % by wt, divide results by sp gr of beer. 10.039 Dextrin-Official Final Action To 25 mL prepd sample, 10.001, measured at 20° in 500 mL boiling flask, add 15 mL HCI (sp gr 1.125) and dil. to 200 mL. Attach flask to reflux condenser, and keep in boiling H20 bath 2 hr. Cool. nearly neutze with NaOH soln (1 + 1), dil. to 250 mL in vol. flask, filter, and det. glucose as in 31.051. g Dextrin/100 mL beer = [glucose (g/100 mL) - (1.053 x maltose, 10.038)] x 0.9. 10.040 Diacetyl (6)-Official Final Action ASBC Colorimetric Method Apparatus (a) Compressed carbon dioxide gas.-In steel cylinder with reducing valve connected thru 1-hole rubber stopper into 1 neck of boiling flask, (b), ending high up in flask. (b) Glass distillation apparatus.-With following parts: boiling flask, 2-neck, 500 mL; distg tube, mounted vertically on boiling flask; condenser, H20-cooled, connected by 75° adapter, if necessary, to distg tube so that condenser slopes downward; BEER 165 curved, tapered tube adapter, connected to condenser delivery tip to dip below liq. level in receiver; and receiver, such as 50 mL beaker, marked at 15 and 35 mL. (c) Heating mantle.-For boiling flask, (b). or burner. (d) Spectrophotometer or colorimeter.-Capable of measur- ing A at 520 nm; with matched 1 cm cells. 10.041 Reagents (a) Dimethylglyoxime std soln.-O.0674 mg/mL. Dissolve 0.1348 g dimethylglyoxime in 3-5 mL alcohol, dil. to 2 L with H20, and mix well. (b) Dipotassium hydrogen phosphate in aqueous acetone soln.-O.827M. Dissolve 14.4 g K2HP04 (or 19.9 g K2HP04.3H20) in H20, add 20 mL acetone, dil. to 100 mL with H20, and mix well. Store in refrigerator. (c) Ferrous sulfate soln.-5%. Dissolve 5 g FeS04.7H20 in 100 mL H2S04 (1 +99). and mix well. Discard when soln turns yellow due to oxidn of Fe. (d) Hydroxylamine hydrochloride soln.-6% aq. soln of NH20H.HCI. (e) Potassium dihydrogen phosphate alkaline soln.-0.30M. Dissolve 1.0 g KH2P04 in 25 mL 0.1N NaOH, and mix well. (f) Potassium sodium tartrate, saturated soln.-Shake 90 g K Na tartrate well with 50 mL H20, and let settle. (g) Silicone antifoam.-Dow Corning Antifoam A, or equiv. 10.042 Preparation of Standard Curve In 20 mL vol. flasks, prep. series of dimethylglyoxime std solns contg, in increments of 0.025 mg, 0.025--0.200 mg diacetyl by mixing appropriate vols dimethylglyoxime std soln, (a) (0.741 x mg dimethylglyoxime/mL = mg diacetyl/mL). with 0.75 mL 6% NH20H.HCI soln and sufficient H20 to yield 15 mL. Heat each soln 15 min in ca 80° H20 bath, cool to room temp., add 1.0 mL 0.827M K2HP04, and let stand 5 min. Successively add 0.6 mL NH4 0H, 2.5 mL satd K Na tartrate soln, and 0.2 mL 5% FeS04 soln. Dil. to 20 mL with H20, and mix by inversion. Prep. blank contg all reagents, except dimethylglyoxime, in sufficient H20 to yield 20 mL. Set spectrophtr to read 0 A at 520 nm with blank in cell. Obtain A readings at 520 nm for dimethylglyoxime std solns contg 0.025--0.200 mg diacetyl equiv. Plot A at 520 nm against mg diacetyl/20 mL. Std curve should be straight line passing thru origin. 10.043 Determination Place 25 mL 0.30M KH2P04 in 500 mL boiling flask. Add 250 mL cold beer, not decarbonated, and 1 drop antifoam. Fit 1 neck of boiling flask to lower joint of vertical distg tube of distn app. Place tip of adapter into 0.75 mL 6% NH20H.HCI soln and 2-3 mL H20 to seal adapter. Cool receiver during distn. If sample is expected to have high diacetyl concn, e.g., if diacetyl can be smelled or tasted readily, use 1.50 mL NH20H.HCI soln and divide distillate into 2 equal portions. Use only 1 portion for distn, and multiply result by 2. Connect CO2line to feed into boiling flask well above liq. level, and adjust CO2gas flow to maintain CO2 atm. in entire system. Heat, and distil at ca 1 drop/sec. Collect 30 mL distillate. Place receiver in ca 80° H2 0 bath 15 min. Raise temp. of H20 bath to 100° or transfer receiver to boiling H20 bath. Let distillate evap. to 15 mL. Cool to room temp., and transfer quant. to 20 mL vol. flask. Add 1.0 mL 0.827M K2HPO. and let stand 5 min. Successively add 0.6 mL NH4 0H, 2.5 mL satd K Na tartrate soln, and 0.2 mL FeS04 soln. Dil. to 20 mL with H20 and mix. Within 20 min, read A of colored soln at 520 nm against blank.
- 188. 166 10. BEVERAGES: MALT BEVERAGES AND BREWING MATERIALS AOAC METHODS (1980) From calibration curve, det. mg diacetyl in 250 mL sample. Multiply by 4 to obtain mg diacetyl/L beer. Report result to 2 decimal places. 10.044 Iodine Reaction for Unconverted Starch--Procedure (a) For light beer.--Fill 15 mm diam. test tube to within 2.5 cm from rim with beer, 10.001. Carefully add 0.02N I from dropper to form distinct layer on top of beer. Observe at once, by transmitted light, color developed at interface. Report blue as indicating presence of starch; purple, amylodextrin; and reddish tinge, erythrodextrin. Qualify results by using terms faint trace, trace, and plain trace according to whether the color developed is faint, distinct, or strong. (b) For dark beer, but applicable also to a light beer.--To 5 mL beer in test tube add 25 mL alcohol, shake thoroly, and let stand. Decant, pouring off last trace of beer-alcohol mixt. Dissolve ppt (dextrin) in 5 mL H2 0 and to this soln add 0.02N I soln dropwise. Interpret as in (a). 10.045 Protein--Official Final Action To 25 mL prepd sample, 10.001, at 20° in Kjeldahl flask, add 2-3 mL H2S04 and conc. to sirupy consistency. Det N as in 2.057. %N x 6.25 = % protein. % protein = [(mL 0.1N acid - mL O.lN base) x 1.4 x 6.25 x 1001/(sp gr x mL sample x 1000). 10.046 Free Amino Nitrogen in Beer (26) Official Final Action Prep. sample as in 10.001. Dil. 1.0 mL prepd sample to 50 mL with H20. Transfer 2.0 mL to each of three 16 x 150 mm test tubes and proceed as in 10.180. 10.047 Ash--Official Final Action Evap. to dryness 50 mL prepd sample, 10.001, measured at 20°. Proceed as in 31.012 or 31.013. 10.048 Phosphorus--Official Final Action To 50 mL prepd sample, 10.001, measured at 20°, add 20 mL 2% Ca(OAc)2 soln, evap. to dryness, and ignite at low redness to white ash. Add 10-15 mL boiling HN03 (1 +9) and det. P20 S as in 7.119. (Washing phosphomolybdate ppt with 1% KN03 soln instead of H20 prevents creeping.) 10.049 Carbon Dioxide--Official Final Action Manometric Method (7) Apparatus (a) Piercing apparatus--(T) For bottles.-Consists of gas-tight packing box and fastening for adjustment over container, and hollow spike connected to accurate pressure gage and outlet valve. Check gages frequently. (2) For cans.-Consists of metal frame in which can is placed. Top of app., which is pressed or screwed down and locked over can top, contains hollow spike surrounded by compressible rubber sealing plug; hollow spike leads to accurate pressure gage and outlet valve. (One app., adjustable for use with both bottles and cans, may be employed.) Notes: Piercing devices can be obtained from Zahm and Nagel Co., Inc., 74 Jewett Ave, Buffalo, NY 14214; and Micromat, 185 Rt 17, North Mahwah, NJ 07430. For suitable manometer for calibrating gages, see Gray and Stone, Ind. Eng. Chem., Anal. Ed., 10, 15 (1938). Dead wt testing unit suitable for calibration can be obtained from AMETEK/- Mansfield & Green Division, 6185 Cochran Rd, Solon, OH 44139; Amthor Testing Instrument Co., Inc., 45-53 Van Sinderen Ave., Brooklyn, NY 11207; Dresser Industries, Inc., 250 E Main St, Stratford, CT 06497, and other companies. (b) Absorption buret.-(Fig. 10:01). Consists of graduated tube (one type has 0-5 mL graduated in 0.05 mL divisions, 5-15 mL in 0.1 mL, and 15-25 mL in 0.5 mL) with bulb marked at 40 mL, and closed at each end by stopcocks. Connect buret to valve of piercing app. and to leveling bulb by transparent alkali- resistant plastic or rubber tubing. (Burets are available from Zahm and Nagel Co. and Micromat Co., (a). and from New York Laboratory Supply Co.) (c) Leveling bulb.-Approx. 300 mL, with support. 10.050 Determination Bring samples to 25° by immersion in H20 bath at 25°. If sample is bottle, make scratch on bottle at beer level. If sample is can, weigh unopened can. Fill leveling bulb and then absorption buret with 15% NaOH soln. Completely displace air in tubing connecting it to piercing app. with H2 0 or NaOH soln and attach piercing device to bottle or can. Take care that no air is trapped in system that will be carried into buret during detn. With valve of piercing device closed, pierce bottle crown or can by depressing hollow steel spike. Shake bottle or can until pressure reaches const max. value; then stop, and record pressure reading. Open valve on piercing app. cautiously and let gas-foam mixt. flow into absorption buret until pressure gage reads zero. Close valve and shake or tip buret (depending on its construction) until CO2 is absorbed and gas vol. in buret reaches min. value. Adjust level ing bottle to equalize hydrostatic pressure and read vol. of "headspace air" contained in buret. TRANSPARENT PLASTIC TUBING A PIERCING DEVICE OPTIOKAL OTHER FORMS AVAILABLE . - GLASS BEND ...-- RUBBER TUBING FIG. 10:01-Absorption buret (other forms available)
- 189. AOAC METHODS (1980) If detn of "total air" is also desired, continue evolution of gas from bottle or can by shaking it. Absorb evolved CO, by swirling and shaking buret. Continue shaking and CO, absorption until there is no further increase in vol. of unabsorbed gas in buret. Final vol. of unabsorbed gas may be considered the "air content" or "total air" of container. Disconnect piercing device from package and insert thermom- eter to be sure that temp. is 25°. Det. headspace vol. as follows: (a) Bottles.-Fill bottle to top with H20 and pour from it into 100 mL graduate until liq. level in bottle corresponds to scratch mark placed on it. Vol. in mL of liq. poured off is headspace vol. (b) Cans.-Empty beer from weighed can and let it drain completely. Weigh empty can. Fill empty can with H2 0 and weigh. Subtract wt empty can.from wt unopened can of beer to obtain wt of beer before opening can. Divide beer wt by sp gr of beer to obtain vol. beer in can in mL. Subtract wt empty can from wt can filled with H20. Difference is wt H20, equiv. to vol. in mL required to fill can completely. Subtract vol. beer from vol. can to obtain headspace in can before opening. (This detn of headspace in cans is only approx. correct, due to unknown degree of bulging of cans under pressure, distortion of end on opening or puncturing can, and difficulty of accurately defining when can is completely filled with H20.) Calc. % CO2 by wt and vol. as follows: % CO2 by wt = [P - (mL "head-space air" x 14.7/mL headspacel) x 0.00965, where P = absolute pressure in psi = gage pressure + 14.7. Vol CO2 = % CO2 by wt x sp gr of beer/0.1976 = % CO2 by wt x 5.0607 x sp gr of beer. Report % by wt to second decimal, and vol CO2 to first decimal. Foam Collapse Rate (B)-Official Final Action Sigma Value Method 10.051 Apparatus (a) Special foam funnel.-Marked at 800 mL. Kontes Glass Co. drawing No. 9357B or CGW drawing No. XA-7396 (Science Products Div., Corning Glass Works). (b) Stopwatch.-Or clock that indicates sec. 10.052 Determination Perform detn at room temp. (22-2)0). Attemperate beer in container to 25±0.5° in H2 0 bath or const temp. room. Clean foam funnel thoroly with warm detergent soln; rinse well, first with warm H20 and then with H20 at ca 25°. Clamp funnel at suitable ht to ringstand and let drain 1 min. Make foam detns immediately after draining. Open beer container, rest side of container on funnel edge, and direct stream of beer into center offunnel, pouring smoothly to avoid entrapping air in beer. Pour until foam reaches 800 mL mark, start stopwatch, and cover funnel with ;;.100 mm watch glass. After 30 sec, open stopcock to let all beer flow out in 25-30 sec at as uniform rate as possible; open stopcock wide for last 1-2 sec until small amt of foam drains out. Immediately close stopcock, reset stopwatch to 0, and start it again. Discard drained beer. After exactly 200 sec, let beer formed from collapsed foam flow out into 100 mL graduate at such rate that all beer drains off in 25-30 sec (total time 225-230 sec). Just as last drop of beer drains off, close stopcock and stop stopwatch. Record time in sec as "t" and mL drained beer as "b." BEER 167 To collapse remaining foam, wash down inside of funnel with 2 mL isopropanol or n-BuOH delivered from fine-point pipet. Open stopcock wide and let liq. drain into 25 mL graduate 1 min. Record mL drained beer as "c" (mL liq. drained - 2 mL defoaming agent). 10.053 Calculations Sigma value = t/(2.303 log[(b + c)/clt where t = time of foam collapse (225-230 sec), b = mL beer collapsed from foam in time t, c = mL beer from residual foam at time t. Report to nearest whole number. Foam Flashing Method 10.054 Apparatus (a) Compressed carbon dioxide gas.-Contained in steel cyl- inder with reducing valve. (b) Pressure surge tank.-Approx. 7.6 L (2 gaL), fitted with inlet and outlet· gas connections, bleeder valve, and pressure gage. (Or use '/.." Type 10 Pressure Regulator, 2-60 psi, Lexington Control, Inc., PO Box 132, Burlington, MA 01803.) (e) Orifice foam flashing apparatus.-Foam flashing orifice tube connected to stainless steel Master Volume Gage Bottle attachment seated pressure-tight on open bottle. Permits ap- plication of gas pressure to expel beer from bottle as foam. Furnished with inlet valve (No.1) and outlet valve (No.2), and adjustable ht sample tube (Micromat Co., 185 Rt 17, North Mahwah, NJ 07430). (d) Graduated cylinder.-200 mL, 4.6 cm id x 12 cm deep, graduated in 5 mL intervals to brim (Labtician Products Co., 190-99th Ave, Hollis, NY 11423) (500 mL graduate truncated smoothly at 200 mL mark may be used). (e) Stopwatch.-Or clock that indicates sec. 10.055 Determination Perform detns at room temp. (22-2)0). Clean grease from equipment, and connect CO2 cylinder, pressure surge tank, and orifice foam flashing app. in that order, with Tygon tubing. Be sure all connections are gas-tight. Set CO2 cylinder reducing valve to 31 Ib gage pressure (1600 mm Hg, 214 KPa). Replace air in pressure surge tank with CO2 • Close inlet valve (No.1) and adjust pressure on surge tank to 29 Ib (1500 mm Hg, 200 KPa), using bleeder valve to reduce excess pressure. Attemperate bottles of beer to 25±0.5° in H2 0 bath or const temp. room. Clean 200 mL graduate with detergent soln, rinse well with H20, and fill with H20. Let graduate drain free of H20 1 min; then secure graduate beneath foam flashing orifice tube in upright position with clamp and ringstand so placed that they do not interfere with reading graduations. Open attemperated beer bottle; fit its neck to bottle attachment of orifice foam flashing app. to secure bottle in place. Raise sample tube so that it is above liq. level in headspace. Open inlet and outlet valves (No.1 and No.2) to flush headspace and connecting Tygon tubing with CO2• Close valves. Bring sample tube to ca 1 cm from bottom of bottle and secure tube in this position by tightening gasket sealing nut at top of bottle attach- ment portion of app. Open inlet valve (No.1). If pressure on surge tank drops, readjust to 29 Ib (1500 mm Hg, 200 KPa). With orifice tube diverted from foam receiving cylinder, open outlet valve (No.2) and let outflowing foam go to waste for 10 sec. Then direct stream of foam into measuring cylinder by placing orifice tube at oblique angle below (tip ca 6 mm away
- 190. 168 10. BEVERAGES: MALT BEVERAGES AND BREWING MATERIALS AOAC METHODS (1980) from) rim of inside wall of cylinder. Fill cylinder just to over- flowing. Just as overflow begins, divert stream of foam out of cylinder, start stopwatch, and close outlet valve (No.2) to stop flow of foam. After exactly 90 sec, read mL liq. beer formed from collapsed foam. Wash down cylinder walls with 2 mL isopropanol from pipet and carefully swirl liq. in cylinder so all foam collapses. Read mL liq. at rest; this reading - 2 mL (added isopropanol) = total mL beer formed by collapse of 200 mL foam. 10.056 Calculations Foam Value Units (FVU) = 200 x (B2 - B,)/B2, where 200 = arbitrary factor chosen to give FVU generally near 100, B2 = total mL beer from collapse of 200 mL foam, B, = mL beer formed from foam collapsed in 90 sec. Report mL to nearest whole number. 10.057 Calcium (9)-Official Final Action ASBC Method I Reagents (a) Ammonium oxalate soln.-Satd soln (ca 6%) of (NH4)2C204.H20 in H20. (b) Buffer soln.-pH 10.0. Dissolve 67.5 9 NH4CI in 200 mL H20. Measure pH, and add NH40H (ca 200 mL) to pH 10.0. Oil. to 1 L. (c) Eriochrome black T indicator soln.-Dissolve 0.1 9 indi- cator (Eastman Kodak Co., No. P6361, or equiv.) in 25 mL MeOH contg 1 g H2NOH.HCI. Store <2 months. (d) Sodium sulfide soln.-2%. Dissolve 2 9 Na2S in 100 mL H20. (e) Magnesium std soln.-l.00 mg/mL. Dissolve 1.00 9 Mg turnings in 100 mL O.lN HCI and dil. to 1 L with double distd H20. (f) Disodium dihydrogen EDTA std soln.-O.l%. Dissolve 1 9 Na2H2EDTA in 1 L H20. Stdze against Mg std soln. 10.058 Standardization Pipet 5 mL Mg std soln into 250 mL erlenmeyer and add 50 mL H20 and 1 mL Na2S soln. Prep. blank soln with 50 mL H20 and 1 mL Na2S soln. Add 5 mL buffer soln and 10 drops indicator soln to each erlenmeyer. Titr. flask contg Mg with EDTA std soln until permanent blue color exactly matches blank. Titr. 3 aliquots and use avo to calc. titer Mg soln, M = 5/mL EDTA std soln. Calc. Ca factor, C = (40.08/24.32) x M. 10.059 Determination (a) Total calcium and magnesium.-Pipet 5 mL prepd sample, 10.001, into 250 mL erlenmeyer and add 40 mL H20, 1 mL Na2S soln, 5 mL buffer soln, and 10 drops indicator soln. Titr. immediately with EDTA std soln. mL EDTA std soln = X. (b) Magnesium.-Pipet 25 mL prepd sample, 10.001, into 125 mL erlenmeyer and add 0.5 mL (NH4)2C204.H20 soln and 2 drops buffer soln. Refrigerate 2 hr at 0-2°. Filter thru Whatman No. 40, or equiv., paper. Add 1 drop HCI to filtrate. Pipet 5 mL filtrate into 250 mL erlenmeyer and add 40 mL H20, 1 mL Na2S soln, 5 mL buffer soln, and 10 drops indicator soln. Titr. immediately with EDTA std soln. mL EDTA std soln = Y. ppm Ca = [X - (25.5Y/25)) x C x 200 ASBC Method /I 10.060 Reagents (a) Calcein indicator soln.-Dissolve 0.2 9 indicator (G. Fred- erick Smith Chemical Co., Eastman Kodak Co., or equiv.) in 100 mL H20 contg 1 mL 5N NaOH. (b) EDTA std soln.-l mL = 1 mg CaC03• Available from Hach Chemical Co., PO Box 389, Loveland, CO 80537; Betz Labora- tories, Inc., 4636 Somerton Rd, Trevose, PA 19047; or equiv. 10.061 Determination Pipet 20 mL prepd sample, 10.001, into 250 mL erlenmeyer. Add 100 mL H20, 3 mL 5N NaOH, and 0.5 mL calcein indicator soln. Swirl to mix. Titr. with EDTA soln at 1 drop/sec, using overhead light and black background until yellow-green fluo- rescence is replaced by orange-brown color. mg Call = mL EDTA std soln x 20. Copper Direct, Nonashing Method (10) Official Final Action 10.062 Reagents (a) Zinc dibenzyldithiocarbamate (ZDBT) soln.- 0.5%. Dis- solve 5 9 ZDBT (available from Uniroyal Chemical under trade name "Arazate") in toluene and dil. to 1 L with toluene. Filter, if necessary, thru Whatman No. 42 paper, and store in brown bottle in cool, dark place. CCI. may be used instead of toluene. (b) Copper std solns.-(1) Stock soln.-l mg/mL. Dissolve 3.93 9 CuS04.5H20 (free of whitish deposit of lower hydrates) and d il. to 1 L with H20. Or dissolve 1.000 9 pure Cu wire or foil in 75 mL HN03 (1 +4) by warming. Boil to expel fumes, cool. and dil. to 1 L with H20. (2) Working soln.-l0 JLg/mL. Prep. im- mediately before use by dilg 5 mL stock soln with Cu-free distd H20 to 500 mL in vol. flask. (c) Copper-free distilled water.-Ext distd H20 with ZDBT soln in separator. 10.063 Apparatus (a) Photometer.-Any com. instrument with blue filter (430-460 nm) or spectrophtr set at 435 nm. (b) Copper-free centrifuge tubes.-Clean and rinse 50 mL centrf. tubes; add 15 mL H20, 3 mL H2S04(1 +3), and 5 mL ZDBT soln. Stopper with corks or glass stoppers and shake thoroly. Discard soln and let tube drain. 10.064 Preparation of Standard Curve Into series of cleaned, corked or g-s, 50 mL centrf. tubes add 0.0, 1.0, 2.0, 3.0, 4.0, and 5.0 mL Cu working std soln, contg 0.0, 0.4,0.8, 1.2, 1.6, and 2.0 ppm Cu, resp. Add 25 mL beer, degassed as in 10.065, and 1 drop n-hexyl alcohol; mix, and proceed as in 10.066. Color over this range follows Beer's law. Calc. factor, F, to convert A to ppm Cu after subtracting A of std contg 0.0 ppm Cu from those contg added Cu. If instrument response is not linear, use calibration curve. 10.065 Preparation of Sample Cool bottle or can and shake thoroly immediately before opening. Let gas bubbles leave liq. before removing cap or puncturing can. Discard ca '13 of sample and degas by swirling. Remove sample directly from container.
- 191. AOAC METHODS (1980) 10.066 Determination To cleaned 50 mL centrf. tube, add 25 mL cold sample, measured in graduate, 3 mL H,S04 (1 +3), and 1 mL 30% H,O,. If foam interferes with sample measurement, add 1 drop hexyl alcohol. Mix, and place tube in boiling H20 bath 0.5 hr. If excessive foaming occurs, add 1 drop hexyl alcohol. Remove tube and cool to 25°. Add 5 or 10 mL, accurately measured, lDBT soln, depending upon size of photometer cell, and stopper tube. Ext at 25° by shaking vigorously 60 times. Re-ext again 4 times, giving 60 snapping shakes each time to obtain fine emulsion, allowing partial sepn between extns. Digested sample must be shaken vigorously with the lDBT soln; thoro and complete emulsification must be obtained during each series of extns or results may be low. Centrf. tube 2-3 min and draw off clear, colored layer to photometer cell of same size used in calibration, and det. A. If droplets of aq. layer are carried into pipet, remove by flowing solv. from pipet down wall of clean, dry test tube. H20 droplets will adhere to test tube and clear solv. can be poured off into cell. Prep. reagent blank by extg, in clean 50 mL centrf. tube, 25 mL Cu-free H20 at 25° and 3 mL H2S04 (1 +3) with 5 (or 10) mL lDBT soln and det. A,. To correct for A of color extd by solv., perform entire detn, omitting lDBT soln, but shaking with toluene (or CCI4) and det. A 2• Do not give tubes used for this solv.-extractable beer color blank preliminary cleaning with lDBT soln, since carryover of lDBT may give high readings. ppm Cu = [A -(A, + A 2)] x F, where F is factor for converting A to ppm Cu. 10.067 Cuprethol Method {H)-Official Final Action Apparatus (a) Photometer.-Any com. instrument with blue-green or green filter, or spectrophtr set at 445 nm and with 40-50 mm cells. (b) Copper-free glassware.-Clean all glassware with O.lN HN03 and rinse thoroly with Cu-free distd H20. 10.068 Reagents (a) Diethanolamine soln.-Dissolve 4.0 mL (HOCH2CH2)2NH (Eastman Kodak Co., No. 1598) in 200 mL MeOH. (b) Carbon disulfide soln.-Add 1.0 mL CS2 (free of pptd S) to 200 mL MeOH. (c) Cuprethol soln.-Mix 3 vols soln (a) and 1 vol. soln (b). Prep. fresh daily. Also mix equal vols soln (a) and MeOH for blank. (d) Copper std solns.-Prep. as in 10.062(b). (e) Buffer soln.-pH 4.4. Dissolve 63.3 g anhyd. NaOAc in ca 800 mL H20 contg 65 mL HOAc. Oil. to 1 L with H20. (f) Copper-free distilled water.-Use distd H20 redistd from all-glass app. thruout method. 10.069 Preparation of Standard Curve Into series of g-s 100 mL vol. flasks add 0.0, 1.0, 2.0, 4.0, 8.0, and 12.0 mL Cu working std soln contg 0.0, 0.4, 0.8, 1.6,3.2, and 4.8 ppm Cu, resp. Add H20 to 12 mL in each flask. Oil. to vol. with degassed low-Cu beer, 10.065, mix, and proceed as in 10.070. Use 0.0 sample to zero instrument, and obtain A or scale readings for 0.1,0.2,0.4,0.8, and 1.2 ppm added Cu. A over this range follow Beer's law. Calc. avo factor, F, converting A or scale readings to ppm Cu. If instrument response is not linear, draw and use smooth curve for calcg ppm Cu. BEER 169 10.070 Determination Slowly pour 50 mL cold beer into 50 mL graduate; avoid foaming. Transfer to 125 mL flask, add 25 mL buffer soln, (e), and mix. Measure two 30 mL aliquots in 50 mL graduate and transfer to sep. 50 mL flasks. Add 3 mL cuprethol soln, (c), to one flask and 3 mL blank soln to other. Mix each and let stand 10 min. lero instrument with blank. Det. A in same size cell and at same wavelength used in calibration. Calc. ppm Cu by mUltiplying A or scale reading by F, or use curve. Iron (12)-Official Final Action 10.071 Apparatus Photometer.-Spectrophtr set at ca 505 nm or photometer with filter in blue-green region, 500-550 nm, or preferably, 505-520 nm. 10.072 Reagents (a) Color reagent: (1) 2,2'-Bipyridine.--O.2%. Dissolve 1 g 2,2'-bipyridine in 20 mL HOAc (1 +2) and dil. to 500 mL with H2 0; or- (2) o-Phenanthroline.--O.3%. Dissolve 1.5 g o-phenanthroline in 500 mL H20 at 70°. (b) Iron std soln.-O.l mg/mL. (1) From iron wire.-Dissolve 0.500 g reagent grade Fe wire, wiped free of oxide, in 5 mL HCI (1 +4) and 1 mL HN03• Cover with watch glass, heat, and evap. to dryness; add H20 and evap. to dryness again. Dissolve residue in 3-5 mL HCI, cool, and transfer quant. to 500 mL vol. flask. Add 2 drops satd Br-H20, dil. to vol. with H20, and mix. Transfer 50 mL of this soln to 500 mL vol. flask, add 2 drops Br-H2 0, dil. to vol. with H20, and mix. (2) From ferrous ammonium su/fate.-Dissolve 3.512 g Fe(NH4b(S04)2.6H20 in H20, add 5 mL Hel, transfer quant. to 500 mL vol. flask, dil. to vol. with H20, and mix. Transfer 50 mL of this soln to 500 mL vol. flask, dil. to vol. with H20, and mix. (c) Ascorbic acid.-USP, ground to fine powder. 10.073 Preparation of Standard Curve Prep. series of beer stds contg 0.0, 0.25, 0.50, 1.00, 2.00, and 3.00 ppm Fe as follows: Pipet 0.0, 0.25, 0.50,1.00,2.00, and 3.00 mL Fe std soln to series of 100 mL vol. flasks, add, from pipet, enough H20 to total 3.00 mL, and dil. to vol. with decarbonated beer, 10.074. Depending upon size cell to be used, develop color in 25 or 50 mL aliquots of each of the beer stds as in 10.075(a) or (b). If T values are obtained, convert to A = -log T, and plot A against ppm Fe. If straight line results, calc. factor, m, for converting A to ppm Fe, y, by use of equation y = mA + b (b = 0 if line passes thru origin). If instrument response is such that curve is obtained, use this curve to calc. results. 10.074 Preparation of Sample Adjust temp. of beer to 20-25°. Decarbonate by transferring sample to large erlenmeyer and shaking, first gently and then vigorously, until all gas is released. Do not filter unless necessary. If filtration is required, make sure filter paper is Fe-free by spotting sample of paper with drop of reagent prepd by dis- solving 25 mg ascorbic acid in 2 mL color reagent. 10.072(a). If beer sample is suspected of high Fe content, degas by shaking only, and permit foam to subside before sampling. 10.075 Determination Pipet 2 aliquots of degassed beer (25 or 50 mL as used in prepn of std curve) irito 50 mL or 125 mL erlenmeyers; add 25
- 192. 170 10. BEVERAGES: MALT BEVERAGES AND BREWING MATERIALS AOAC METHODS (1980) mg ascorbic acid to each aliquot, and add 2 mL color reagent, (a), to one and 2 mL H,O to other. (a) Stopper and heat both aliquots 15 min at 60°, or (b) let stand 30 min at room temp. Cool, and read both solns in photometer against H,O as ref., or read colored aliquot against beer blank as ref. Use same size cell and wavelength as used in prepn of std curve. 10.076 Calculations If H,O is used as ref. and factor is used, ppm Fe = (Asample - ADlank) X factor. If values are taken from std curve, ppm Fe = ppm Fe in sample - ppm Fe in blank. If beer blank is used as ref. and factor is used, ppm Fe = AsamPle x factor. If values are taken from std curve, ppm Fe = ppm Fe directly. 10.077 Other Metals See Chapter 25. 10.078 Chlorides-Official Final Action Place 50 mL sample in Pt dish, add 20 mL 5% Na,C03 soln, and proceed as in 3.071. Det CI as in 3.072 or 3.074. Sulfur Dioxide (13)-Official Final Action 10.079 Reagents (a) Color reagent.-Weigh 100 mg p-rosaniline.HCI into 250 mL vol. flask and dissolve in ca 200 mL H,O. Add 40 mL HCI (1+1), mix, and dil. to vol. with H,O. Let stand ca 15 min before use. Store in brown, g-s bottle in refrigerator. (b) Formaldehyde soln.-Dil. 5 mL 40% HCHO soln to 1 L with H,O and store in brown, g-s bottle in refrigerator. (c) Mercury stabilizing soln.-Dissolve 27.2 g HgCl, and 11.7 g NaCI in H,O, and dil. to 1 L with H,O. (Caution: See 51.079.) 10.080 Calibration Accurately weigh ca 250 mg NaHS03 into exactly 50 mL O.lN I soln in g-s flask. Let stand at room temp. 5 min. Add 1 mL HCI, and titr. excess I with O.lN Na,S,03' using 1% aq. starch soln as indicator (1 mL O.lN I consumed = 3.203 mg SO, or 5.20 mg NaHS03). From results of NaHS03 assay, prep. soln contg 10 mg SO,/mL (ca 8.6-9.0 g NaHS03/500 mL) (Soln I). Transfer 100 mL Hg stabilizing soln to 500 mL g-s vol. flask. Add 1.00 mL Soln I, and dil. to vol. with H,O (1 mL = 20 JLg SO,) (Soln 11). Using 10 mL graduate contg 1 drop n-hexyl alcohol as antifoam, transfer 10 mL portions of cold, undegassed beer (preferably of low SO, content) into series of eight 100 mL vol. flasks. To series add 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, and 8.0 mL Soln /I (0--160 JLg SO,). Oil. to vol. with H,O, and mix. Transfer 25 mL aliquots of each soln to sep. 50 mL vol. flasks. To each flask, add 5 mL color reagent. Mix, and add 5 mL HCHO soln. Mix, dil. to vo!. with H,O, mix, and hold in 25° H,O bath 30 min. Read color in spectrophtr at 550 nm or in photometer with green filter. Plot A as ordinates against JLg SO, added to beer as abscissas (color follows Beer's law over range). Calc. calibration factor F, converting readings to JLg SO, in 25 mL aliquot used, or convert directly to ppm SO,. 10.081 Preparation of Sample Using pipets, add 2 mL Hg stabilizing soln and 5 mL O.lN H,S04 to 100 mL vol. flask. Measure 10 mL cold, undegassed beer into 10 mL graduate contg 1 drop n-hexyl alcohol, and add to vol. flask. Swirl gently, and add 15 mL O.lN NaOH. Swirl, and hold 15 sec. Add 10 mL O.lN H,S04, then H,O to vol., and mix thoroly. Transfer 25 mL aliquot to 50 mL vol. flask. 10.082 Determination To soln in 50 mL vol. flask, add 5 mL color reagent, swirl, add 5 mL HCHO soln, swirl, and dil. to vol. with H,O. Mix, and hold in 25° bath 30 min. Read color as in 10.080, using cells of same size and same instrument settings. Correct for blank as follows: Measure 10 mL cold, undegassed beer into 100 mL vol. flask. Add 0.5 mL 1% aq. starch soln, then 0.05N I soln, dropwise until permanent bluish tinge persists. Add 1 drop more, dil. to vol., and mix thoroly. When blue fades, develop color in 25 mL aliquot as above. (Color readings for I blanks are usually low and uniform; when test is performed on series of similar beers, blank tests on all may be unnecessary.) ppm SO, = (As - A b) x F, where As = A of sample (or photometric reading with green filter equiv. to A), Ab = A of I blank, and F = factor derived from 10.080 for converting A to JLg SO, in aliquot, or directly to ppm SO,. 10.083 Caramel-Official Final Action See 11.055. Beer Bitterness (14)-Official Final Action (Certain preservatives, such as heptyl-p-hydroxybenzoate and sorbates and possibly some brewing adjuncts or coloring agents, may contribute toA at wavelengths specified. Interference from UV-absorbing material is greater for Bitterness Units method than for Iso-Alpha Acids.) Bitterness Units 10.084 Reagents (a) 2,2,4- Trimethylpentane (isooctane).-Spectral grade or equiv. (ASTM certified ref. fuel grade isooctane may be used after 1 distn or use practical grade isooctane, purified by passage thru silica gel column (12-28 mesh, Fisher Scientific Co. No. 5- 156, grade 408).) A at 275 nm in 1 cm cell should be equiv. to that of H,O (A .s 0.005). (b) Detyl aleohol.-Reagent grade or redistd equiv. One drop added to 20 mL isooctane increases A at 275 nm .s0.005 in 1 cm cell. 10.085 Apparatus (a) Mechanical shaker.-Platform or wrist-action type with extending arm adjusted vertically so that tube is held horizon- tally. (b) Spectrophotometer.-For use in UV range. (c) Centrifuge tubes.-50 mL, g-s or screw-cap with Teflon lining. 10.086 Determination Transfer 10.0 mL chilled (10°C, 50°F) carbonated beer to 50 mL centrf. tube, using pipet which has minute amt octyl alcohol in tip. Add 1 mL 3N HCI and 20 mL isooctane. Tightly stopper centrf. tube and shake vigorously 15 min on mech. shaker. If required, centrf. long enough to sep. phases. Immediately transfer portion clear upper (isooctane) layer to cell. Set instru- ment to read 0 A at 275 nm for isooctane-octyl alcohol blank (20 mL isooctane + 1 drop octyl alcohol). Record A in 1 cm cell at 275 nm. Calc. bitterness units (BU) = Am X 50. Report BU to nearest 0.5 unit.
- 193. AOAC METHODS (1980) Iso-Alpha Acids 10.087 Reagents (a) 2,2,4-Trimethylpentane (isooctane).-See 10.084(a). A at 255 nm in 1 cm cell should be equiv. to that of freshly redistd H20 from all-glass still. (b) Methanol.-Reagent grade, with A ~0.04 at 260 nm in 1 cm celi compared with freshly redistd H20 from all-glass still. (c) Acid methanol.-Mix 6.8 parts MeOH with 3.2 parts 4N HCI. (d) Alkaline methanol.-Just before use, mix 1.0 mL 1.5N NaOH with 500 mL MeOH. (e) Dctyl alcohol.-See 10.084(b). 10.088 Apparatus See 10.085. 10.089 Determination Transfer 15.0 mL chi lied carbonated beer to 50 mL centrf. tube, using pipet with minute amt octyl alcohol in tip. Add 2.0 mL 6N HCI and 15.0 mL isooctane. Close tube tightly and shake vigorously on mech. shaker ~30 min until completely extd. Place tube in centrf. set to run at highest permissible speed. Centrf. long enough to sep. phases. Break difficult emulsions by adding 1 drop detergent to centrf. tube. Verify that detergent does not contribute to A at 255 nm. (Union Carbide Corp. "Tergitol Anionic 7" (Na heptadecyl sulfate) is satisfactory.) Transfer 10.0 mL clear upper layer to 50 mL g-s graduate contg 10.0 mL acid MeOH. Stopper and invert 100 times at rate causing contents to pass from end to end. Let phases sep. Transfer 5.0 mL upper layer to 25 mL vol. flask and dil. to vol. with alk. MeOH. Read A in 1 cm celi at 255 nm in spectrophtr set to read 0 A for reagent blank consisting of 5 mL isooctane dild to 25 mL with alk. MeOH. ppm Iso-alpha acids of beer = 96.15 A 255 + 0.4. Report to nearest 0.5 ppm. 10.090 Proteolytic Chillproofing Enzymes (15) Official Final Action Casein Coagulation Method Reagent Substrate mixture.-Thoroly mix by grinding in large mortar and pestle 50 g com. skim milk powder (do not use special casein powder prepd for microbiological or other uses), 5.0 g L( +)-cysteine.HCI.H2 0, 4.4 g Na2 HPD4, 2.5 g NaCI, and 1.8 g citric acid.H20. Store at 0-40 and let warm to room temp. before use. 10.091 Test Place ca 100 mL degassed beer into 150 mL beaker and adjust pH to 6.4±0.1 with 1N NaOH. Transfer 50 mL aliquot to 25 x 200 mm test tube contg 250±30 mg substrate mixt. Suspend substrate mixt. with rubber-tipped glass stirring rod. Invert tube twice to mix uniformly, and place in 60° H20 bath. Progressive change in appearance of suspension, initially clouding, then formation of "pebbles," followed by coagulation and settling of casein indicates pas. test. Control beers, without chill-proofing enzymes, should remain unchanged. If semi- quant. data are desired, record time required to reach first stage of "pebbling." MALT 171 MALT 10.092 Sampling-Official Final Action For complete descriptions of trier, divider, sampler, and bushel weight tester, see "Grain Inspection Manual" GR Instruction 918-6 (latest edition available from Federal Grain Inspection Service, Standardization Div., US Dept. of Agriculture, 1400 Independence Ave SW, Washington, DC 20250). (a) Bulk malt in cars or bins.-Using 60" (1.5 m) trier, take ~6 probes from different parts of car, preferably 2 from center and 2 from each end. (b) Bulk malt during discharge thru spouts or openings.-At different times during filling or unloading of car, take, with trier or Pelican sampler, ~6 samples, each representing complete cross section of grain stream from spout. (c) Bagged malt.-Sample lengthwise thru center of open bags, ~2% of bags selected from different parts of car or storage room. Use 36" (0.9 m) trier. Indicate approx. proportion of inferior grain and take repre- sentative samples from each portion as outlined above. Im- mediately place each portion of sample in suitable large dry container and keep tightly closed. 10.093 Preparation of Sample-Official Final Action Divide samples, either by quartering or by using sample divider, until ca 1.4 kg (3 Ib) remains. Place reduced sample in air-tight container (preferably tin with screw or friction-type cover); do not use cartons, bags, wooden boxes, glass Mason jars, or wrapping paper. Remove foreign particles, such as stone, wood, and twine. Do not remove foreign seeds or dust particles. Bushel Weight (16)-Official Final Action 10.094 Method I Place sample in filling hopper of Winchester tester, open slide underneath, and let malt fill measuring cylinder to overflowing. Without jarring, level off with straight-edge longer than diam. of measuring cylinder, making one forward stroke consisting of 3 distinct zigzag motions. Weigh and report to nearest 100 g (~ Ib). 10.095 Method II Weigh 110 g sample to nearest 0.1 g and pour evenly into metal funnel provided with plunger discharge and placed on top of 250 mL cylinder graduated to meet NBS specifications. (Funnel must fit snugly into graduate and be large enough to hold the grain without danger of spilling when plunger is raised.) Then drop material into cylinder by pulling plunger up. Do not jar or tap cylinder during operation or before reading vol., and do not read uppermost grain level, as compensation must be made for ends of few kernels that protrude. If grain surface has slant, repeat test. Calc. bushel wt of malt (lb) as = 8545/vol. in mL of 110 g. Canst 8545 is derived from W, wt in Ib of US (Winchester) bushel of 2150.42 cu in. (35,239 mL). If V = vol. in mL of 110 g malt, 110/453.6W = V/35,239. W = 8545/V. 10.096 Length of Acrospire--Procedure For methods (a) and (bl. quarter sample until ca 200 kernels remain in 2 opposite quarters, and count out 100 kernels, rejecting those that are broken or those in which growth is not ascertainable.
- 194. 172 10. BEVERAGES: MALT BEVERAGES AND BREWING MATERIALS AOAC METHODS (1980) (a) Cutting.-Hold each kernel, furrow downward, on flat surface with pair of tweezers, cut thru kernel longitudinally with razor blade or other sharp instrument, and examine cut acrospire in both halves, comparing its length with that of kernel. Tally according to classifications below. (b) Peeling.-Remove husk covering acrospire with sharp instrument and examine acrospire length in comparison with kernel length. Tally according to classifications below. (c) Boiling.-Boil 10-15 g avo sample with 100-150 mL H20 20-30 min. After boiling, add cold H20 to cool contents of beaker. Decant, and pour grain on glass plate. Select 100 kernels at random, inspect acrospire, and tally according to classifica- tions below. Classify kernels as follows and report % in each group: 0-'/.0: those kernels without apparent growth, or having ac- rospire development up to, but not including, '/.0 length of grain. '/.o-Y:,: those kernels having acrospire development from '/.0 up to, but not including, 'I, length of grain. Y:,-%: those kernels having acrospire development from Y:, up to, but not including, % length of grain. %-1: those kernels having acrospire development of % but not greater than entire length of grain. Overgrown: those kernels having acrospire development in excess of length of grain. If it is apparent that overgrown acrospire has been broken off during processing, include kernel in overgrown classification regardless of length of remaining stub. 10.097 Mealiness-Procedure Count out 100 kernels remaining from preceding test if method 10.096(a) or (b) was used. Otherwise select 100 kernels as in 10.096 and cut kernels in longitudinal halves. Det. % mealy, half glassy, and glassy kernels. In case of uncertainty, pierce starch body with sharp point; if mealy, it will break away and crumble from point. Classify kernels as follows: Mealy kerne