Dry Ashing

International Journal of Innovation and Applied Studies
ISSN 2028-9324 Vol. 5 No. 4 Apr. 2014, pp. 327-332
© 2014 Innovative Space of Scientific Research Journals
http://www.ijias.issr-journals.org/
Corresponding Author: Kassa Belay (kassa.belay1@gmail.com) 327
Validation of a Method for Determining Heavy Metals in Some Ethiopian Spices By Dry
Ashing Using Atomic Absorption Spectroscopy
Kassa Belay
1
, Abi Tadesse
2
, and Tesfahun Kebede
2
1
Adirat University Department of Chemistry, P.O. Box 50, Adigrat, Ethiopia
2
Haramaya University Department of Chemistry, P.O. Box 138, Dire dawa, Ethiopia
Copyright © 2014 ISSR Journals. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT: The optimal conditions for determination of Pb, Cd and Cr in spices sample by FAAS after dry mineralization 1g
spice samples for 5hr in a temperature of 500 °C followed dissolution in 4 mL HNO3 in Fenugreek, Black cumin, garlic and
ginger. Working linear ranges are given: for Cd, 0 -6mg/l, for Cr, 0.2-1mg/l and for Pb, 2-20μg/l. Found detection limits are
0.2mg/l, 0.13mg/l and 0.02μg/l for Cd, Cr and Pb, respectively. The result obtained Lead in Fenugreek, Black cumin, garlic and
ginger ranged from 0.0126 to 0.0155, 0.0205 to 0.0254, 0.0046 to 0.0066 and 0.0161 to 0.0178 mg/kg respectively. Cadmium
is detected only in Fenugreek in the ranged of ND to 0.0175mg/kg where as Chromium in Fenugreek, Black cumin, Garlic and
Ginger ranges from 0.0187 to 0.0219, 0.0134 to 0.0152, 0.0014 to 0.0016 and 0.0258 to 0.0346 mg/kg respectively which is
under the values recommended for concentration levels present in the analyzed samples.
KEYWORDS: dry ashing, fenugreek, black cumin, ginger, garlic, atomic absorption spectrometry.
1 INTRODUCTION
Spices are dried parts of plants, which have been used as dietary components of food often to improve its color, aroma,
palatability and acceptability. They consist of rhizomes, barks, leaves, fruits, seeds, and other parts of the plant. There have
been many definitions for ‘spices’ which are often used interchangeably with ‘herbs’. Spices are defined as the aromatic parts
of any plants that are used to add flavor to food. But herbs are the aromatic leaves of any plant that can add flavor to food.
The origins of herbs are leaves of plants that have soft stems or are shrubs but not trees. In general, spices are defined as un-
leafy dried substances whereas herbs are leafy un-dried substances [1].
In Ethiopia, there are between 6,000 to 7,000 higher plant species, out of about 500 (about 8%) are edible [1]. According
to [2]. on 50 species of aromatic and medicinal plants collected from eastern Ethiopia, it was indicated that a sizable number
of local merchants were involved in the trading of spices along with other aromatic and medicinal plants. Of these plant
species studied as such, a significant number of the plants (over 25) were spices.
Heavy metals are those metals which are toxic for our body even if at their lower concentration include cadmium (Cd),
copper (Cu), lead (Pb), zinc (Zn), mercury (Hg), arsenic (As), silver (Ag), and chromium (Cr). Most of these metal ions (Cd, Cu,
Zn, Hg, As, Ag, Cr, and Fe) can be release from the industries are in simple cationic forms [3], [4].
Many analytical methods including Atomic Absorption Spectrometry for trace element determination in plant materials
require the digestion of the sample [5].Because of its sensitivity, specificity, simplicity and precision, Atomic Absorption
Spectrometry (AAS) is the most widely recommended instrument utilized in analytical procedures for trace heavy metal
analysis. In order to separate the analyte from the matrix and to avoid organic matter which may react with the metal ions or
chemical reagents and interfere with the analyte in acid digestion methods are very important step. The most commonly
used methods for the sample treatment of spices are dry ashing, wet ashing and microwave assisted treatment.

Validation of a Method for Determining Heavy Metals in Some Ethiopian Spices By Dry Ashing Using Atomic Absorption
Spectroscopy
ISSN : 2028-9324 Vol. 5 No. 4, Apr. 2014 328
Dry ashing procedures use a high temperature muffle furnace. Most minerals are converted to oxides, sulfates,
phosphates, chlorides or silicates. The advantage of Dry ashing is safe, few reagents are required, many samples can be
analyzed simultaneously and not labor intensive. The disadvantage of this method is, it requires relatively expensive
apparatus (platinum crucibles and muffle furnace),losses due to volatilization, resistance to ash by some materials, difficult
dissolution of ashed material and high risk of contamination[6].
In this study, the authors compare the efficacy of differing digestion procedures dry ashing on commercial spice samples.
The contents of Lead, Chromium and Cadmium in spice samples produced in Ethiopia were determined by flame atomic
absorption spectrometry after dry ashing.
2 MATERIALS AND METHODS
2.1 APPARATUS
Buck scientific 210 VGP flame atomic absorption spectrometer was used in the experiments. A deuterium background
corrector was used for background corrections. The operating parameters for the elements were set as recommended by the
manufacturer (Table 1). For flame measurements, a 10-cm long slot-burner head, a lamp and an air-acetylene flame were
used.
Furnace (type 1500) for dry ashing was used to ash the sample for the analysis of Heavy metals concentration. Platinum
and porcelain Crucibles was used to dry the sample at Furnace. Sample volume, digestion times for the drying, ashing,
atomization and cleaning temperatures were optimized prior to analysis in order to obtain the maximum absorbance with
minimum background.
Table 1. Working Conditions of Atomic Absorption Spectroscopy
Element Wavelength nm Slit Width nm Lamp Current mA IDL mg/L MDL mg/L Flame type
Cd 228.9 0.7 2 0.005 0.0062 Rich/yellow
Cr 357.9 0.7 2 0.05 0.051 Lean/blue
Pb 217.0 1.0 5 0.01 0.016 Lean/blue
2.2 REAGENTS
All reagents were of analytical reagent grade. Double distilled deionized water (Milli-Q Millipore 18.2 MΩ-cm resistivity)
was used for all dilutions. HNO3 was of suprapure quality (E. Merck, Darmstadt). All plastic and glassware were cleaned by
soaking in diluted HNO3 (10% v/v) and rinsed with distilled water prior to use. The element standard solutions used for
calibration were prepared by diluting stock solutions of 1000 mg/L of each element.
2.3 SAMPLING
Total of four spice sample types were collected randomly from different traders and Samples were washed thoroughly
with tap water followed by de-ionized water and dried in the oven at a temperature of 105
o
C for 24 hr for Fenugreek, Ginger,
and Garlic. But black cumin was dried at a temperature of 105
o
C for 48 hr. The dried samples were ground in a stainless steel
mill till obtaining fine particles that pass through a 0.5 mm mesh and kept dry in a polyethylene bag in desiccators until
analysis.
2.4 DIGESTION PROCEDURE
Dry ashing was applied for digesting the spice samples produced in Ethiopia. Optimum digestion conditions are given
below.

Kassa Belay, Abi Tadesse, and Tesfahun Kebede
ISSN : 2028-9324 Vol. 5 No. 4, Apr. 2014 329
2.5 DRY ASHING
One gram of each spice sample was placed into porcelain crucible. The furnace temperature was slowly increased from
room temperature to 500˚C. The sample was ashed for about 5 hr until a white or grey ash residue was obtained. The residue
was dissolved in 5 mL of HNO3 (25%, v/v). The solution was transferred to a 10 mL volumetric flask and made up to the mark
[7].
2.6 DIGESTION CONDITIONS
Different procedures for spices sample digestion were assessed based on varying reagent volume, digestion time and
digestion temperature. For optimizing the procedure Fenugreek (Trigonella foenumgraecum L.) was selected and it was
digested with the three digestion methods. The selection for the respective optimum digestion methods was made based on
the particular procedure which resulted to the following conditions: clear digestion solution, minimal reflux time/digestion
time, minimal reagent volume consumption, absence of undigested spice samples [8]. The results are given in Table 2. The
comparison of three digestion methods showed statistically significant differences in results. The recovery values were nearly
(≥ 95%) for all digestion methods. ANOVA was used in this study (p < 0.05). The relative standard deviations were less than
10% for all elements. The approximate time required for dry ashing was 10 hr.
2.7 PREPARATION OF STANDARD SOLUTIONS
Determination of the metal concentration in the experimental solution was based on the calibration curve. In plotting the
calibration curves lead, cadmium and chromium stock solutions of 1000 ppm were prepared by dissolving 1.6 g of Pb(NO3)2 ,
2.74 g Cd(NO3)2.4H2O and 2.83 g K2Cr2O7 in de- ionized water respectively. Blank solutions were prepared for the methods
and, for the standard working solutions, to prepare 100 ppm, 10 mL of the standard Pb(NO3)2, Cd(NO3)2.4H2O and K2Cr2O7
stock solution were pipetted and added into 100 mL calibrated flasks finally diluted with de-ionized water and the solution
was mixed thoroughly. Next, to prepare 50 ppm standard solution of each metal, 50 mL of each of 100 ppm stock solution
was pipetted into 100 mL volumetric flasks and diluted with de-ionized water. Finally to prepare 0.0, 0.5, 1.0, 2.0, 4.0, 6.0
ppm aliquots of this standard working solution 0.0, 0.5, 1.0, 2.0, 4.0, 6.0 mL was pipetted from 50 ppm standard solution into
50 mL calibrated flasks and made up to volume with De-ionized water [9].
2.8 VALIDATION OF EXPERIMENTAL RESULTS
2.8.1 DETERMINATION OF DETECTION LIMITS
Detection limit is the lowest concentration level that can be determined at 95% confidence level [10] or the minimum
concentration that can be detected by the analytical method with a given certainty [11] a general accepted definition of
detection limit is the concentration that gives a signal three times the standard deviation of the blank or background signal
[12]. In this study the detection limit of each element was calculated as three times the standard deviation of the blank (3σ
blank, n = 5).
2.8.2 RECOVERY TESTS
The efficiency and accuracy of the optimized methods were evaluated by analyzing the digests of spiked samples. 0.02,
0.02 ppm of Pb, Cr and 0.2 ppm Cd, respectively, were taken from stock solution of each metal and spiked in a 250 mL
Erlenmeyer flask containing 1g spice sample. The recoveries of metals in the spiked spice samples were 92 to 103 %. The
results are given in Table 3. Generally, good recoveries were obtained for all metals, (particularly in Garlic for metals like Cd,
Pb and Cr). In Cd the percentage recovery for all samples except Fenugreek were not calculated due to results obtained was
not within the method detection limit. Each determination was carried out at least three times in order to ensure precision.
The relative standard deviations were less than 10% for all measurements.
% =
–
[2].

Spectroscopy
ISSN : 2028-9324 Vol. 5 No. 4, Apr. 2014 330
2.8.3 OPTIMIZATION OF WORKING PROCEDURE
The optimal procedure chosen on the basis of these criteria for dry ashing 5 hr digestion time at a temperature 500
0
C in 4
mL HNO3 was employed 4 mL HNO3 was added after white ash is obtained. The optimized operating conditions were
compared with literature report on similar study made by Mustafa et al., (2004). The work had improved especially dry
ashing and wet digestion methods employed there significantly as far as digestion time is concerned. as it listed in Table 2.
Table 2. Optimum working conditions for the digestion methods used
Digestion
Methods
Previous work optimum
parameters (Mustafa etal., 2004)
Current work optimum
Parameters
Difference
Dry
Ashing
T (
o
C) V(mL) Time(hr) T (
o
C) V(mL) Time(hr) T(
o
C) V(mL) Time (hr)
450 5 8 500 4 5 50 1 3
Note; T = temperature; V = volume DA= dry ashing
3 RESULTS AND DISCUSSION
The method detection limit (MDL) is defined as the concentration corresponding to three times the standard deviation of
blanks. Method detection limit values of the investigated elements for AAS were found to be 0.016 mg/L for Pb, 0.0062 mg/L
for Cd and 0.051 mg/L for Cr.
Trace metal levels in the analyzed samples are given in Table 4. The metal contents in the samples studied depended on
the specific species. Levels of the essential metals in the spice samples were found to be higher than those of the non-
essential metals. The lowest and highest contents of copper were found in 4.1 μg/g for Diantus sp. and 28.7 μg/g for Rhus
coriaria, respectively. The lowest and highest levels of zinc were found as 7.84 μg/g for Capsicum annuum and 47.6 μg/g for
Papaver somniferum, respectively. Copper and zinc values in spices of Nigeria have been reported in the range of 0.40-13.3
μg/g and 0.20-53.7 μg/g, respectively(7). The highest cadmium level was found as 0.93 μg/g for Piper nigrum, whereas the
lowest cadmium level was 0.10 μg/g in Nigella sp. The lowest and highest contents of nickel were found as 0.65 μg/g for
Cassia sp. and 8.69 μg/g for Menta sp, respectively. The concentrations of cadmium and nickel in spices of Nigeria have been
reported in the range of 0.12-0.36 μg/g and 1.03-3.47 μg/g, respectively(8). The concentration of lead varied from 0.47 μg/g
in Rhus coriaria to 1.89 μg/g in Nigella sp [13]
3.1 OPTIMIZATION OF WORKING PROCEDURE
The optimal procedure chosen on the basis of these criteria for dry ashing 5 hr digestion time at a temperature 500
0
C in 4
mL HNO3 was employed 4 mL HNO3 was added after white ash is obtained as given in Table 2.
3.2 RECOVERY TESTS
The efficiency and accuracy of the optimized methods were evaluated by analyzing the digests of spiked samples. 0.02,
0.02 ppm of Pb, Cr and 0.2 ppm Cd respectively, were taken from stock solution of each metal and spiked in a 250 mL
Erlenmeyer flask containing 1g spice sample. The recoveries of metals in the spiked spice samples were 92 to 103 %. The
results are given in Table 3. Generally, good recoveries were obtained for all metals, (particularly in Garlic for metals like Cd,
Pb and Cr). In Cd the percentage recovery for all samples except Fenugreek were not calculated since results obtained was
not within the method detection limit. Each determination was carried out at least three times in order to ensure precision.
The relative standard deviations were less than 10% for all measurements.

Kassa Belay, Abi Tadesse, and Tesfahun Kebede
ISSN : 2028-9324 Vol. 5 No. 4, Apr. 2014 331
Table 3. Recovery test of the methods at P≤0.0
Metal
Dry Ashing
Fenugreek Black Cumin Garlic Ginger
Pb
Amount Recovered 0.032 0.040 0.025 0.035
percentage recovery 95 96 97 96
concentration in spice 0.0132 0.0209 0.0055 0.0154
Cr
concentration in spice 0.0187 0.0134 0.0016 0.0346
percentage recovery 94 93 95 96
Cd
concentration in spice 0.0125 - - -
percentage recovery 92 - - -
Since Cd is not detected in all except fenugreek the %R is also not calculated
Table 4. Metal Concentration (mg/kg) in Spices from Dire Dawa Market (Mean± S.D)
Digestion Methods Spices Pb Cr Cd
DA Fenugreek 0.0132±0.0006 0.0187±0.0008 0.0125±0.0016
Black cumin 0.0209±0.0003 0.0134±0.0005 ND
Garlic 0.0055±0.0006 0.0016±0.0002 ND
Ginger 0.0154±0.0002 0.0346±0.0018 ND
Note; DA= dry ashing and ND = not detected at P≤0.
4 SUMMARY AND CONCLUSION
4.1 SUMMARY
The general objective of the study was to compare the efficiency of digestion methods for determination of Cr (VI) Pb (II)
and Cd (II) in some selected Ethiopian spices using AAS. All experiments were done at Haramaya University soil science and
central laboratory. The Pb concentrations were found to be in the range of 0.0132 to 0.0140, 0.0209 to 0.0224, 0.0055 to
0.0058, and 0.0154 to 0.0172 mg/kg for Fenugreek, Black Cumin, Garlic and Ginger respectively. Whereas Cr concentrations
ranged from 0.0187 to 0.0269, 0.0134 to 0.0152, 0.0014 to 0.0016, 0.0258 to 0.0346 mg/kg in Fenugreek, Black Cumin, Garlic
and Ginger respectively, Cd was not detected in any of the samples except in Fenugreek which is ranging from 0.0125 to
0.0175 mg/kg. The results obtained showed that the spices in this study are not likely to pose any health risk to the public
through consumption of the spices for both the toxic elements determined. The recoveries of the trace metals were in the
range of 92 to 103% and the standard deviations were less than 10%.
4.2 CONCLUSIONS
This study is focused on check the extraction efficiency of dry ashing methods in different spices including Garlic, Ginger,
Black Cumin and Fenugreek for the determination of heavy metals like Cd, Pb and Cr by using Atomic Absorption
spectrometry (AAS).
Spice samples were ashed by using furnace and digested by using HNO3 as digestion reagents. The concentrations of
these reagents were optimized to minimize possible matrix interferences. Different temperature programs, reagent volume
and digestion time were investigated to obtain maximum digestion efficiency with minimum digestion reagent consumption
for all digestion methods. All methods gave almost similar results for the elements studied.
But dry ashing methods are more time consuming and complicated than the other digestion methods in terms of
digestion efficiency. Example the use of microwave digestion system in sample digestion provides very fast, safer, simple and

Spectroscopy
ISSN : 2028-9324 Vol. 5 No. 4, Apr. 2014 332
cleaner method of sample preparation, increases analyte recoveries and useful volatile elements. Effects of HNO3 and H2O2
concentrations on Pb (II), Cd (II) and Cr (VI) signals were investigated to see the effect of matrix matching on AAS and it was
found that increasing concentrations of HNO3 has suppression effect on Pb and Cr concentration whereas the effect on Cd
concentration were not detected by using neither direct calibration method or standard addition method by AAS.
Based on this knowledge direct calibration method was used for Pb and Cr determination whereas for Cd determination
standard addition method was applied but similar result was obtained. In the last part of the study the results of previous
analyses for Cd, Cr and Pb determination in spice samples were evaluated together with the results of this study for statistical
analysis. For each spice sample the results of all elements obtained by different digestion methods were compared by using
ANOVA at 95% confidence interval.
ACKNOWLEDGMENTS
The financial support provided by the Ministry of Education, and the guidance given by Dr. Abi Tadesse College of
natural and computational science (Haramaya University) to conduct the metal analysis are greatfully acknowledged. This
work is a part of the research carried for postgraduate programme (MSc) at faculty of Graduate Studies, Haramaya
University.
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