![Terminology Guide October 2013 33
www.chromatographyonline.com
Fluoro phase: One of a family of
aliphatic and aromatic reversed-phase
materials in which a substantial frac-
tion of the bonded phase is fluorinated.
Sometimes called fluorous phases or
perfluoro phases. Typically have dif-
ferent selectivities than hydrocarbon
phases.
Fluted filter paper: Filter paper that
is folded in a systematic manner to
allow more air space in the filter fun-
nel; this allows liquid to flow faster
through the filter paper.
Foley-Dorsey equation: A correc-
tion of the plate count and retention
time to correct for peak tailing from
extracolumn sources of broadening.
(See J.P. Foley and J.G. Dorsey, Anal.
Chem. 55, 730–737 [1983].)
Forced-flow leaching: Solid mate-
rial is packed into stainless steel col-
umn and toluene (or other extraction
solvent) is pumped into the column
under pressure and with heating; hot
solvent leaches (extracts) out extract-
able compounds which are collected at
the exit of the column.
Fraction of analyte extraction (E):
The fraction of analyte extracted: E
= (CoVo)/(CoVo + CaqVaq) = (KDV)/(1 +
KDV ) where Vo is the volume of or-
ganic phase, Vaq the volume of aque-
ous phase, and V is the phase ratio
Vo/Vaq.
Fractionation range: In SEC, refers
to the operating range of a gel or pack-
ing. This is the range in which the
packing can separate molecules based
on their size. At one end of the range,
molecules that are too large to diffuse
into the pores are excluded. At the
other end of the range, molecules that
can diffuse into all of the pores totally
permeate the packing are eluted (un-
separated) at the permeation volume.
Freeze drying: The process of removing
water, mainly from biological samples,
by using vacuum sublimation.
Frictional heating: Viscous heating of
solvent molecules passing through very
small-diameter (micrometer) particles;
causes a rise in temperature over the
length of the column; reduced by using
smaller-diameter columns (smaller heat
dissipation) and superficially porous
particles, which also have improved heat
dissipation.
Frit: The porous element at either end
of a column that serves to contain the
column or SPE packing. It is placed at
the very ends of the column tube or in
the end fitting. Frits can be stainless
steel or another inert metal or plastic
such as porous PTFE or polypropylene.
The frit porosity must be less than the
smallest particle in the column; other-
wise particles will pass through the frit
and packing will be lost.
Frontal analysis: A chromatographic
technique that involves continuous ad-
dition of sample to the column with
the result that only the least sorbed
compound, which moves at the fast-
est rate, is obtained in a pure state.
The second-least-sorbed compound
is eluted with first eluted compound,
the third-least-sorbed compound with
the first and second compound, and so
forth until the original sample passes
through the column exit. Frontal
analysis is seldom used and is mainly
a preparative technique.
Frontal chromatography: The same as
frontal analysis.
Fronting: Peak shape in which the front
part of the peak (before the apex) in a
chromatogram tapers in advance of the
remainder of the peak (that is, the front
is less steep than the rear). There is an
asymmetric distribution with a leading](https://image.slidesharecdn.com/chromatographysamplepraperationguide-230128114400-d47dd8dc/85/Chromatography-sample-praperation-guide-pdf-33-320.jpg)
![38 Terminology Guide October 2013 www.chromatographyonline.com
chromatography. However, unlike
normal-phase chromatography, which
uses nonpolar solvents such as hexane
and methylene chloride and tries to
exclude water from the mobile phase,
HILIC requires some water in the
mobile phase to maintain a stagnant
enriched water layer on the surface
into which analytes may selectively
partition. In addition, water-miscible
organic solvents are used instead of
the water-immiscible solvents used in
normal-phase chromatography. With
HILIC, sorbents such as bare silica,
bonded diol, and polyhydroxyethylas-
partamide are used. Polar analytes are
well retained and are eluted in order
of increasing hydrophilicity, just the
inverse of reversed-phase LC.
Hydrophobic: “Water hating”; re-
fers both to stationary phases that
are not compatible with water or to
molecules in general that have little
affinity for water. Hydrophobic
molecules have few polar functional
groups; most have a high content of
hydrocarbon (aliphatic and aromatic)
functionality.
Hydrophobic interaction chroma-
tography (HIC): A technique in which
weakly polar (nonhydrocarboneous)
packings are used to separate molecules
by virtue of the interactions of their hy-
drophobic moieties and the hydropho-
bic sites on the packing surface. High
concentrations of salt solutions are used
in the mobile phases and separations
are affected by changing the salt con-
centration. The technique is analogous
to “salting out” molecules from solu-
tion. Gradients are run by decreasing
the salt concentration; often used for
the separation of proteins that are sen-
sitive to denaturization by the organic
solvents used in regular reversed-phase
chromatography; usually little or no
organic solvent is used in the mobile
phase in HIC.
Hydrophobic subtraction model:
Developed by Lloyd Snyder and John
Dolan, this model is used to charac-
terize reversed-phase columns; using
five types of probes, based on their
equations, they can predict if a cer-
tain column will be close in selectiv-
ity characteristics to another column
or totally different (orthogonal); over
300 columns have been characterized
using this model. (See L. Snyder and
J. Dolan, LCGC 20[11], 1016–1026
[2002].)
Hydroxyapatite: A porous calcium
hydroxyphosphate solid that chemi-
cally resembles bone and tooth used
as a packing material used in bio-
chromatography for nucleic acid con-
stituents, monoclonal antibodies, and
proteins.
Hypercrosslinking: Mainly refers to
a new way to synthesize a polymeric
monolith; hypercrosslinked monolithic
capillary columns contain an array of
small pores and have very high surface
areas.
Hyphenated techniques: Refers to
the family of techniques best known
by their abbreviations, including LC–
MS, LC–FTIR, and MS-MS.
I
Immobilized liquid extraction: Simi-
lar to SPE but a polymeric stationary
phase is bonded to the inside of a glass
vial; analytes partition into polymeric
phase and loading, washing, and elu-
tion steps are performed by addition of
various solvents.
Immobilized metal affinity chroma-
tography (IMAC): See metal affinity
chromatography.](https://image.slidesharecdn.com/chromatographysamplepraperationguide-230128114400-d47dd8dc/85/Chromatography-sample-praperation-guide-pdf-38-320.jpg)
![56 Terminology Guide October 2013 www.chromatographyonline.com
dissociates by splitting into A−, known
as the conjugate base of the acid, and
the hydrogen ion or proton, H+, which,
in the case of aqueous solutions, ex-
ists as the hydronium ion — in other
words, a solvated proton. The disso-
ciation constant is usually written as a
quotient of the equilibrium concentra-
tions (in mol/L), denoted by [HA], [A−],
and [H+]: Ka = ([H+] [A−])/[HA]; due to
the many orders of magnitude spanned
by Ka values, a logarithmic measure of
the acid dissociation constant is more
commonly used in practice. The loga-
rithmic constant, pKa, which is equal to
−log10 Ka, is sometimes also (but incor-
rectly) referred to as an acid dissociation
constant.
Planar chromatography: A separation
technique in which the stationary phase
is present as or on a plane (IUPAC).
Typical forms are paper and thin layer
chromatography.
Plate height (H): See theoretical plate
height.
Plate height, effective (Heff): The
column length divided by the number
of effective theoretical plates: Heff = L/
Neff
Plate number (N): See theoretical
plate number.
Polar: A polar molecule may be polar
as a result of polar bonds or as a result
of an asymmetric arrangement of non-
polar bonds and nonbonding pairs of
electrons; polar molecules are generally
able to dissolve in water (H2O) because
of the polar nature of water; polar mol-
ecules do not prefer nonpolar organic
solvents such as hexane. Polar molecules
have slightly positive and slightly nega-
tively charged ends; we often refer to a
compound’s polarity.
Polarity index (P’): The polarity index
is a measure of the relative polarity of a
solvent and is useful for identifying suit-
able mobile phase solvents or extraction
solvents. The polarity index increases
with polarity; examples: hexane, P′ =
0.0; isopropanol, P′ = 3.9; tetrahydrofu-
ran, P′ = 4.0; methanol, P′ = 5.1; aceto-
nitrile, P′ = 5.8; water, P′ = 9.0
Polyacrylamide gel: Neutral hydro-
philic polymeric packings used in
aqueous SEC. They are prepared by the
copolymerization of acrylamide with
N,N′-methylene-bis-acrylamide.
Polyaromatic hydrocarbon (PAH):
Members of a class of hydrocarbon
molecules characterized by one or more
fused aromatic rings.
Polychlorinated biphenyl (PCB): Bi-
phenyl molecule with two or more
chlorine substitutions.
Polyethylene glycol (PEG): Polymeric
hydrocarbon used as a GC stationary
phase; possesses moderately polar reten-
tion characteristics.
Polyethyleneimine (PEI): Polyethyl-
eneimine, an anionic polymeric phase
used to coat or bond onto silica or a
polymeric packing. Most often used for
the separation of proteins and peptides.
Polymeric packings: Packings based
on polymeric materials, usually in the
form of spherical beads. Typical poly-
mers used in LC as well as GC are
polystyrene–divinylbenzene (PS-DVB),
polydivinybenzene, polyacrylamide,
polymethylacrylate, polyethyleneoxide,
polydextran, or polysaccharide.
Polymeric phase: Refers to chemically
bonded phase where a polymer species
is bonded to silica-based particles or to
the wall of an open-tubular column.
Polymeric SPE: The use of a polymeric
base material (for example, PS-DVB or
methacrylate) rather than an inorganic
material (for example, silica or alu-
mina); polymers generally have a wider](https://image.slidesharecdn.com/chromatographysamplepraperationguide-230128114400-d47dd8dc/85/Chromatography-sample-praperation-guide-pdf-56-320.jpg)
![Terminology Guide October 2013 57
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pH range and higher sample capacity
than some of the inorganic materials.
Polystyrene–divinylbenzene (PS-
DVB) resin: The most common base
polymer for ion-exchange chromatog-
raphy. Ionic groups are incorporated
by various chemical reactions. Neutral
PS-DVB beads are used in reversed-
phase LC. Porosity and mechanical
stability can be altered by variation of
the crosslinking through the variation
of the DVB content. In GC, a number
of porous polymer stationary phases are
available for gas and light-compound
separations.
PONA: Refers to the analysis of paraf-
fins, olefins, naphthenes, and aromatics.
Poppe plot: A kinetic plot named after
Professor Hans Poppe (J. Chromatogr.
A 778, 3 [1997]), University of Amster-
dam, the Netherlands, where the plate
time log (t0/N) is depicted as a func-
tion of the number of theoretical plates
to assess the limits of column perfor-
mances as a function of particle size,
column pressure drop, and so forth.
Pore diameter: Same as mean pore
diameter.
Pore size: The average size of a pore in
a porous packing. Its value is expressed
in angstroms or nanometers. The pore
size determines whether a molecule can
diffuse into and out of the packing. See
mean pore diameter.
Pore volume (Vi): The total volume of
the pores in a porous packing, usually
expressed in mL/g. Better termed the
specific pore volume. It is measured by
the BET method of nitrogen adsorp-
tion or by mercury intrusion where Hg
is pumped into the pores under high
pressure.
Poroshell: Similar to nonporous par-
ticles and porous-layer beads; particles
are generally in the 2–5 µm range with
a submicrometer-thick shell; wide pore
versions (>300 Å) allow rapid diffusion
of macromolecules and smaller pore
versions (90–120 Å) are for small mol-
ecules; give similar efficiency to sub-
2-µm particles but at much lower pres-
sure because of their larger particle size.
Porosity: For a porous substrate, the
ratio of the volume of the pores in a
particle to volume occupied by the par-
ticle. The pore volume is a measure of
the porosity and is expressed in mL/g.
Porous layer bead: A small glass bead
coated with a thin layer of stationary
phase. The thin layer can be an ad-
sorbent, resin, or a phase chemically
bonded onto the adsorbent. These pack-
ings were among the first to be used in
HPLC. They were of larger particle size
(20–40 µm) than the microparticulate
packings of today but were easy to pack
and gave adequate efficiency. Also re-
ferred to as controlled surface porosity
supports and pellicular materials.
Porous particle: Refers to column
packing particles possessing intercon-
necting pores of specified diameter and
pore volume; generally in HPLC porous
particles with diameters below 10 µm
are the most popular, and in prepara-
tive chromatography larger particles
are used because of their lower cost and
higher column permeability.
Porous polymer: A packing material,
generally spherical, based on organic
polymers or copolymers; popular ex-
amples would be polystyrene–divinyl-
benzene, polyacrylates, polydextrans,
polyacrylamides, and polybutadienes.
Retains solutes by selective adsorption
or molecular size interaction.
Porous-layer open-tubular (PLOT) col-
umn: An open-tubular column used in
GC or HPLC that has particles coated
or uncoated with stationary phase](https://image.slidesharecdn.com/chromatographysamplepraperationguide-230128114400-d47dd8dc/85/Chromatography-sample-praperation-guide-pdf-57-320.jpg)
![Terminology Guide October 2013 63
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Removable well plates: See array 96-
well plate.
Representative sample: A sample re-
sulting from a statistically worked out
sampling plan; it can be expected to ad-
equately reflect the properties of interest
of the parent population.
Residual silanols: The silanol (-Si–OH)
groups that remain on the surface of a
packing after chemically bonding a
phase onto its surface. These silanol
groups, that may be present in very
small pores, may not be accessible to
the reacting bulky organosilane (such as
octadecyldimethylchlorosilane) but may
be accessible to small polar compounds.
Often they are removed by endcapping
with a small organosilane such as tri-
methylchlorosilane. See endcapping.
Resin: A solid polymeric packing used
in ion exchange separations. The most
popular resins are polystyrene–divinyl-
benzene copolymers of small particle size
(<10 µm). Ionic functionality is incorpo-
rated into the resin.
Resolution (Rs): Peak resolution; incor-
porates both efficiency and separation. A
resolution of 1.5 is said to be “baseline”
resolution, and a minimum resolution of
1.7–2.0 is considered essential for robust-
ness. For two closely eluted peaks: R =
(tR,2– tR,1)/wb where the subscripts 1 and
2 refer to the first and second peaks.
From N, k2 and α: Rs= (√N
—
/4)((α – 1)/α)
(k2/(k2 + 1)) (k2 is the retention factor of
the second peak).
Resolution equation: See resolution.
Response factor (RF): Defines the rela-
tionship between the measured peak area
or height and the quantity of substance
represented by a peak.
Restricted access media (RAM): RAM
are sorbents are used for the direct injec-
tion of biological fluids such as plasma
or serum into an HPLC flow stream.
They contain an outer hydrophilic sur-
face that provides minimal interaction
with proteins and when combined with
small pores on the sorbent exclude the
proteins. The inner surface is hydropho-
bic, and when small molecules diffuse
into the pores they interact by reversed-
phase mechanisms and are retained. The
small molecules such as drugs and their
metabolites can be removed by rinsing
with an organic solvent. RAMs are most
successfully used in a column switching
setup where the secondary column is
used to resolve the small molecules and
the proteins are directed to waste so as
not to foul the secondary column.
Retention factor (k): The measure
of time the sample component resides
in the stationary phase relative to the
time it resides in the mobile phase: k
= (tR – tM)/tM. Formerly, kʹ was used
and it was called the capacity factor or
capacity ratio.
Retention gap: A short piece of deac-
tivated but uncoated column placed
between the inlet and the analytical
column. A retention gap often helps
relieve solvent flooding. It also entrains
nonvolatile sample contaminants from
on-column injection.
Retention index (I): A uniform system
of retention classification according to a
solute’s relative location between a pair
of homologous reference compounds on
a specific column under specific condi-
tions. A series of normal straight-chain
hydrocarbons, fatty acid esters, or mul-
tiring polycyclic aromatic hydrocarbons
have been used for the reference com-
pounds. For a solute i that is eluted at tʹRi
between two hydrocarbons with chain
length z and z + 1: I = 100[z + (logt′
Ri –
logt′
Rz)/(logt′
Rz1 – logt′
Rz)].
Retention time (tR): The time between
injection and the appearance of the peak](https://image.slidesharecdn.com/chromatographysamplepraperationguide-230128114400-d47dd8dc/85/Chromatography-sample-praperation-guide-pdf-63-320.jpg)
![Terminology Guide October 2013 67
www.chromatographyonline.com
Selectivity coefficient (kA/B): In ion-ex-
change chromatography, the equilibrium
coefficient obtained by application of the
law of mass action to an ion exchanger
and characterizing the ability of an ion
exchanger to select one of two ions pres-
ent in the same solution. For example,
the exchange of Na+ for H+ in: KNa/H =
[Na]S
*[H]S/[Na]M[H]M
Selectivity triangle: An approach to
classify the properties of stationary
phases in reversed-phase LC. Results
can be represented in a “selectivity
triangle” in which the apices of the
triangle represent the relative con-
tributions of steric hindrance (χS),
hydrogen bonding basicity (χB) and
cation exchange capacity (χC) to se-
lectivity. A graphical visualization of
the column selectivity allows three-
dimensional data to be presented in
a two-dimensional space. Provides an
informative yet universal approach for
phase classification compared to other
models. With this model, selection of
columns of either equivalent or dif-
ferent selectivity is readily achievable,
which should further facilitate the ap-
plication of reversed-phase LC.
Selectivity tuning: Several techniques
for adjusting the selectivity of separa-
tions involving more than one column or
stationary-phase type. Serially coupled
columns and mixed-phase columns can
be selectivity-tuned.
Semipreparative chromatography: Re-
fers to preparative LC carried out on an
analytical size (4–5 mm i.d.) or a slightly
larger (6–10 mm i.d.) column. Normal
injection size would be in milligram to
low gram amounts.
Sensitivity (S): Degree of detector
response to a specified solute amount
per unit time or per unit volume, often
defined by lower limit of detection
(LOD). For a concentration-sensitive
detector such as a thermal conductivity
detector or UV–vis detector: S = mmax/c
where mmax is the peak height and c is
the solute concentration in the detec-
tor; units of sensitivity for a concentra-
tion-sensitive detector that responds in
millivolts are mV·mL/g. For mass-flow
sensitive detectors such as the flame-
ionization detector: S = mmax/Wt where
Wt is the mass of solute passing through
the detector per unit time; the units of
S are then expressed as A•s/g or C/g.
Separation: The degree of separation of
two peaks in time. See separation fac-
tor (α), relative retention, resolution.
Separation factor (α): The separa-
tion factor α expresses the relative
retention of two adjacent peaks: α =
t′
R2/t′
R1 = k2/k1 where t′
R2 is the reten-
tion time of the second peak and t′
R1
is the retention time of the first peak;
k2 and k1 are the corresponding reten-
tion factors.
Separation impedance (E): A figure
of merit developed by John Knox to
compare the efficiency of two chro-
matographic systems by normalizing for
both analysis time and pressure drop: E
= tRΔp/N2 ν(1 + k) where tR is the re-
tention time, Δp is the pressure drop, N
is the theoretical plate number, ν is the
reduced velocity, and k the retention fac-
tor. The lower the value of E, the better
the system.
Separation number (SN): Separation
number, or Trennzahl (TZ). A mea-
sure of the number of peaks that could
be placed with baseline resolution be-
tween two sequential peaks, z and z+1,
in a homologous series, such as two
hydrocarbons: SN = (tR(z+1) – tR(z))/
(wh(z+1) + wh(z))
Septum: Silicone or other elastiomeric
material that isolates inlet carrier flow](https://image.slidesharecdn.com/chromatographysamplepraperationguide-230128114400-d47dd8dc/85/Chromatography-sample-praperation-guide-pdf-67-320.jpg)
Chromatography sample praperation guide .pdf
- 1. Volume 31 Number S10 October 2013 www.chromatographyonline.com The Chromatography and Sample Preparation Terminology Guide
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- 5. Terminology Guide October 2013 5 www.chromatographyonline.com In 2002, LCGC provided readers with a gas chromatography (GC) glossary to organize the myriad terms used in gas chromatography (1). Likewise, in 2008, the third glos- sary of common and not-so-common terms and “buzz words” for reference to high performance liquid chromatog- raphy (HPLC) columns and column technology was published (2). It is time for an update because new terms have arisen or, in some cases, their original meanings have expanded or changed. Because there are a num- ber of terms common to both GC and LC, we decided to combine the glossaries into one large listing. In addition, with new sample prepara- tion technologies also making their appearance, expanding the glossary to include terminology specific to sample preparation was another goal. Finally, ion chromatography (IC), which some feel is a subset of LC, does have some of its own nomenclature and so those terms are included here. We stick to the conventions of the International Union of Pure and Ap- plied Chemistry (IUPAC) in their “No- menclature for Chromatography” that provides guidance and changes in some of the more commonly accepted terms (3). Still, there are many terms in com- mon usage that are not in alignment with the IUPAC definitions and that nomenclature will be covered here as well. This terminology guide is not in- tended to be an in-depth listing or highly theoretical coverage. For ex- ample, we have elected not to cover many of the myriad terms used in instrumentation, detection, data han- dling, and validation associated with chromatographic analysis but have chosen to use terms that may be en- countered in everyday laboratory work around columns, injection techniques, phases, method development, sample preparation tasks, and general usage. The listing should be helpful to those just starting in chromatography but it can also serve as a refresher for long- time users in the field. References (1) J.V. Hinshaw, LCGC 20(11), 1034–1040 (2002). (2) R.E. Majors and P.W. Carr, LCGC 26(2),118–168 (2008). (3) L.S. Ettre, “Nomenclature for Chroma- tography” in Pure and Appl. Chem. 65(4), 819–872 (1993). The Chromatography and Sample Preparation Terminology Guide Ronald E. Majors and John Hinshaw
- 6. 6 Terminology Guide October 2013 www.chromatographyonline.com 96-well collection plate: A fixed-size polyethylene rectangular plate (127.8 mm × 85.5 mm): consisting of an array of 8 × 12 (96) small “test tubes” called wells; volumes of wells range from 0.5 to 2 mL. 96-well filtration plate: A fixed-size polyethylene rectangular plate (127.8 mm × 85.5 mm) consisting of an array of 8 × 12 (96) of small filter tubes (vol- umes range from 0.5 to 2 mL); a mem- brane filter placed at the bottom of the well is used to filter liquid samples; sometimes a prefilter is placed above the membrane filter to prevent clogging with particulate samples. 96-well plate: A small rectangular plastic plate consisting of 96 individual wells that are basically small-volume test tubes arranged in an 8 × 12 well pat- tern; used for liquid handling and other such requirements. 96-well solid-phase extraction plate: A small rectangular plastic plate con- sisting of 96 individual flow-through SPE wells arranged in an 8 × 12 array that have top and bottom frits to con- tain solid particles of sorbent or resin to perform SPE on a miniaturized scale; generally 1 mg to 0.2 g of packing is placed into the well, which can have a volume of up to 2 mL; used for au- tomated SPE with xyz liquid handling systems or customized workstations. A A solvent: Usually the weaker solvent in a binary eluent or gradient elution sepa- ration. In reversed-phase liquid chroma- tography (LC), the A solvent typically is water or a water-rich mixture. A term: The first term in the van Deem- ter equation. See eddy dispersion term and van Deemter equation. Absorption: The process of retention in which the solute partitions into a liquid- like coating. Accelerated solvent extraction (ASE): Trade name for a pressurized fluid ex- traction system introduced by Dionex and now sold by Thermo Fisher Scien- tific; see pressurized fluid extraction for details of technique. Active flow technology: A concept that incorporates two types of column designs: curtain flow technology means segmenting the flow at the injection end of the column to ensure the analyte sees the middle of the packed bed where it is not disturbed by wall effects; parallel seg- mented flow at the column outlet selects just the middle portion of the flow profile resulting in improved efficiency without the presence of wall effects, giving the best overall column efficiency; a special endfitting design is used to sample the center of the parabolic flow profile. Active sampling: In active gas sam- pling, a pump is used to push the sample through a mass flow controller and into the canister. Additional sample can be collected, relative to the amount that can be collected by passive sampling, by pressurizing the canister with sample. Commonly the sample is pressurized to 103 kPa (15 psig), effectively doubling the sample volume. Active site: A reactive or strongly at- tracting site on the surface of a chro- matographic packing that may bind analytes or cause peak tailing; some- times mobile phase additives (such as a competing base) can negate the effects of active sites. Activity: In adsorption chromatography, the relative strength of the surface of the packing. For silica gel, the more avail- able the silanol groups, the more active the surface. Activity can be controlled
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- 8. 8 Terminology Guide October 2013 www.chromatographyonline.com by the addition of water or other polar modifier that hydrogen-bonds to the active sites thereby reducing the surface activity; can also refer to biological ac- tivity of a biomolecule. Additive: A substance added to the mobile phase to improve the separation or detection characteristics; examples would be a competing base to negate the effects of silanols, a chelating agent to block metal sites, or addition of a UV-absorbing compound to perform indirect photometric detection. Adsorbent: Packing used in adsorption chromatography. Silica gel and alumina are the most frequently used adsorbents in chromatography and sample preparation. Adsorption: A process of retention in which the interactions between the solute and the surface of an adsorbent dominate. The forces can be strong forces (for example, hydrogen bonds) or weak (van der Waals forces). For silica gel, the silanol group is the driving force for adsorption and any solute functional group which can interact with this group can be retained on silica. The term adsorption places emphasis on the surface versus penetration or embedding in the stationary phase coated or bonded to a surface. Adsorption chromatography: One of the basic separation and SPE modes that relies on the adsorption process to ef- fect a separation. Silica gel and alumina are the most frequently used normal- phase adsorbents in LC. Molecules are retained by the interaction of their polar function groups with the surface functional groups (for example, silanols of silica). Carbon is also used as an ad- sorbent in a reversed-phase LC mode. Porous polymer, carbonaceous, and molecular sieve packings in GC exhibit adsorptive properties as well. Adsorption isotherm: In adsorption, a plot of the equilibrium concentration of sample in the mobile phase per unit vol- ume versus the concentration in the sta- tionary phase per unit weight. The shape of the adsorption isotherm can determine the chromatographic behavior of the sol- ute such as tailing, fronting, or overload. Aerogel: A packing prepared when the dispersing agent is removed from a gel system without collapsing the gel struc- ture. Silica gels and glass beads used for SEC are examples of aerogels that can retain their structures even at the high pressure used in HPLC. See xerogels. Affinity chromatography: A tech- nique in which a biospecific adsorbent is prepared by coupling a specific li- gand (such as an enzyme, antigen, or hormone) for the macromolecule of interest to a solid support (or carrier). This immobilized ligand will interact only with molecules that can selectively bind to it. Molecules that will not bind are eluted unretained. The retained compound can later be released in a purified state. Affinity chromatogra- phy is normally practiced as an “on–off” separation technique. Agarose: High-molecular-weight poly- saccharide used as a separation medium in biochromatography. It is used in bead form and often used in gel filtration chro- matography using aqueous mobile phases. Alkoxysilane: A reactant used for the preparation of chemically bonded phases. It will react with silica gel as fol- lows : R3SiOR + ≡SiOH → ≡Si–OSiR3 + ROH where R is an alkyl group. Alumina: A normal-phase adsorbent used in adsorption chromatography. Aluminium oxide (Al2O3) is a porous adsorbent which is available with a slightly basic surface; neutral and acidic modifications can also be made. Basic
- 9. Terminology Guide October 2013 9 www.chromatographyonline.com alumina can have advantages over silica, which is considered to have an acidic surface; alumina is seldom used as an HPLC column packing in practice. In GC applications, alumina will separate low molecular-weight gases. Amino phase: A propylamino phase used in normal-phase chromatography. It is a somewhat reactive phase for any solute molecule (for example, aldehydes) or mobile phase additive that can react with amines. The amino phase has found some applications as a weak anion ex- changer and for the separation of carbo- hydrates using a water–acetonitrile mo- bile phase. It is a relatively unstable phase. Amperes full-scale (AFS): Extent of the maximum detector output, for detectors utilizing an electrometer. Amperometric detection: Electro- chemical detection applying a constant potential to the working electrode. Mea- sured current from oxidation or reduc- tion is proportional to the sample con- centration. Very selective and sensitive method. Works with electrode reactions not changing the electrode surface (for example, cyanide, nitrite, thiosulfate, phenols). Only approximately 10% of the analyte is oxidized or reduced. May be used standalone as well as in series or parallel to other detectors. Ampholyte: A substance that carries both positive and negative charges (they are amphoteric). Examples: amino acids, proteins. Amphoteric ion-exchange resin: Ion- exchange resins that have both positive andnegativeionicgroups.Theseresinsare most useful for ion retardation where all ionic materials can be removed from solu- tion since the anionic and cationic func- tionalities coexist on the same material. Analyte protectorant: In GC, a chemi- cal compound or compounds that are added to a sample before injection to cut down on interactions between analytes that are unstable or behave poorly in the GC flow path on active sites; the protectorants are chosen so that they do not interfere with the analysis of the compounds of interest yet prevent these compounds from interacting with the active sites in the flow path; these pro- tectorants are not generally required for LC and LC–MS. Analytical column: A chromatogra- phy column used for qualitative and quantitative analysis; a typical analyti- cal column for LC will be 50–250 cm × 4.6 mm, but columns with smaller diameters (down to 0.05 mm i.d.) can also be considered as analytical col- umns. GC analytical columns range in length from 1 m to as much as 60 m, with inner diameters ranging from less than 100 µm up to 2 mm. Station- ary phases can be coated or bonded onto the interior of the tubing; packed GC columns are generally wider and shorter and are less frequently used nowadays. Chromatography columns can be constructed of stainless steel, glass, glass-lined stainless steel, PEEK, fused silica, and other metallic and nonmetallic materials. Anion exchange: The ion-exchange procedure used for the separation of an- ions. Synthetic resins, bonded phase sili- cas, and other metal oxides are available for this mode. A typical anion-exchange functional group is tetraalkylammo- nium, making a strong anion exchanger. An amino group on a bonded stationary phase would be an example of a weak anion exchanger. Argentation SPE: The incorporation of a silver salt into the SPE stationary phase will help in retaining compounds with olefinic bonds. Normally used in
- 10. 10 Terminology Guide October 2013 www.chromatographyonline.com organic solvents to maximize charge- transfer interactions. Array 96-well plate: A 96-well SPE plate where the 96 individual wells are removable from the base plate; such a setup allows users to place different types and amounts of SPE sorbents into various configurations in each of the 96-wells. This type of 96-well plate has also been referred to as a flexible 96- well plate configuration. Asymmetry: Factor describing the shape of a chromatographic peak. The- ory assumes a Gaussian shape and that peaks are symmetrical. A quantitative measure is the peak asymmetry factor, which is the ratio of the distance from the peak apex to the back side of the chromatography curve over the distance from the peak apex to the front side of the chromatography curve at 10% of the peak height. Various other measures of asymmetry are in common use, espe- cially the USP method. See also Foley– Dorsey equation. Asymmetry factor: A factor that de- notes band shape; calculated from the chromatographic peak by dropping a perpendicular at the peak apex and a horizontal line at 10% of the peak height; at the intersection the distance to the tail of the peak along the hori- zontal line (distance B) divided by the distance along the horizontal line to the front of the peak (distance A); this ratio is the peak asymmetry fac- tor; for a symmetrical peak the value is one; for a fronting peak the value is less than one; for a tailing peak, the value is greater than one; the higher the value the less symmetrical the peak is; values greater than 2 are generally unacceptable. Atmosphere (atm): A unit of pressure. 1 atm = 101.325 kPa or 14.6959 psi. Average particle size (dp): The aver- age particle size of the packing in the column. A 5-µm LC column would be packed with particles with a definite particle size distribution because pack- ings are never monodisperse. Particle sizes in GC usually are expressed in terms of mesh size distribution; 80–100 mesh is a commonly used particle range. See particle size distribution. B B solvent: Usually the stronger solvent in a binary eluent or gradient separation. In reversed-phase LC, typically the organic modifier or modifier-rich binary mixture with water. B term: The second term of the van Deemter equation; the first term of the Golay equation. See longitudinal dif- fusion, molecular diffusion term, van Deemter equation, Golay equation. Back extraction: Used in liquid–liquid extraction to perform an additional ex- traction to further purify a sample; ini- tially the extraction may take place with an aqueous solvent buffered at a high pH and an immiscible organic solvent; after the initial extraction takes place and in- terferences are removed, then by having another aqueous solution at a low pH, one can back-extract the analyte into the organic layer based on the analyte now 1.0 0.5 0.1 0.0 Normalized peak height 32 36 40 44 48 t1 tp t2 Time (s) B A wa = A + B hp Figure 1: Example of a tailing peak. (Mod- ified with permission from reference 3.)
- 11. Terminology Guide October 2013 11 www.chromatographyonline.com being in a neutral form. An example would be for the cleanup of an acidic substance containing –COOH groups; at high pH the carboxyl would be ionized and prefer the aqueous layer and impu- rities may migrate to the organic phase and discarded; then the pH of the aque- ous layer can be adjusted to a low value. Now the carboxyl group is in an union- ized form and readily extracted into the organic layer as a purified substance. Backflushing: Useful in chromatogra- phy to remove compounds that are held strongly at the head of a column. By re- versing the flow at the conclusion of a run, analytes trapped at the head of the column can be flushed from the column entrance because they have a shorter dis- tance to travel; sometimes a strong solvent in LC or elevated temperatures in GC will be needed to move them along. A valve or fluidic device is used to effect the change of mobile-phase flow direction. Backflushing can be used for analysis of these compounds or merely to remove them from the column. Back-pressure regulator: In LC, a de- vice placed on-line after the detector to maintain a positive pressure on the flow cell minimizing solvent outgassing prob- lems in the detector. In GC, the term usually refers to a carrier-gas regulator in the split vent line that maintains a con- stant pressure at the inlet as split flows change. Bakeout: The process of removing con- taminants from a column by operation at elevated temperatures, which should not exceed the maximum column tem- perature. Band: Refers to the chromatographic peak as it moves along and is eluted from the column. Band broadening: The process of in- crease in width and concomitant dilu- tion of the chromatographic band as it moves down the column. The peak is injected as a narrow slug and ideally each separated component would elute as a narrow slug of pure compound if not for the process of band broadening. The measure of band broadening is the peak dispersion, σ, or more correctly N, the number of theoretical plates in the column. Sometimes called band disper- sion or band spreading. Band width: See peak width at base and peak width at half-height. Baseline: The baseline is the line drawn by the recording device representing the signal from the detector when only mo- bile phase is passing through, in the ab- sence of any solutes. It also represents the point from which calculations are often made on peaks to determine peak area or peak height. Baseline drift: Term for any regular change occurring in baseline signal from an LC or GC detector; it may arise from changes in flow rate of the mobile phase or from stationary phase bleed and may trend in a positive or negative direction. Baseline drift oc- curs over a longer period of time than baseline noise. Baseline noise: Irregular variations (short term) in the chromatographic baseline as a result of electrical noise or temperature fluctuations, outgassing in the flow cell, or poorly mixed mobile- phase solvents. Bed volume: See column volume. BEH: Bridged ethyl hybrid; an inorganic– organic HPLC particle; has higher pH limits than silica gel. BET method: A method for measuring surface area developed by Bruner, Em- mett, and Teller (BET) that uses nitro- gen adsorption–condensation in pores at liquid nitrogen temperature. Pore volume
- 12. 12 Terminology Guide October 2013 www.chromatographyonline.com and pore size distribution can also be ob- tained from BET calculations. Bidentate silane: A specific type of bonded phase in which a short hydrocar- bon bridge connects two silicon atoms in a silane that is bounded to the surface through two siloxane groups. Bimodal: In SEC, can be a porous packing material that has two distinct pore sizes or pore size distributions; in ion-exchange or HILIC chromatog- raphy or sample preparation can be a packing material that has two types of functionalities (for example, cation exchange and reversed phase; cation and anion) on one packing; in some cases, mixed beds consisting of two different packings in one column can be bimodal. Binary mobile phase: Mobile phase con- sisting of two solvents or buffers (or one of each). Bind–elute : In SPE, the normal mode of operation where upon loading the sample onto a conditioned sorbent or resin, the analytes of interest are retained (bound) while interferences and perhaps some of the matrix is not retained by the pack- ing; after a wash step to remove some of the undesired sample components, the elution step uses a strong solvent to elute the analytes of interest in a small volume. Biocompatible: A term to indicate that the column or instrument component will not irreversibly or strongly adsorb or deactivate biomolecules, such as pro- teins. Frequently means metal-free or ceramic surfaces and components. 1.000 0.882 0.607 0.500 0.324 0.134 0.044 Inflection points Tangents drawn to the inflection points Normalized peak height wi = 2σ wb = 4σ 3σ 4σ σ 5σ wh = 2.355σ wi wh Figure 2: Widths of a Gaussian peak at various heights as a function of the standard deviation σs) of the peak. (Modified with permission from reference 2.)
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- 14. 14 Terminology Guide October 2013 www.chromatographyonline.com Blank: More correctly named method blank. A blank prepared to represent the matrix as closely as possible. The method blank is prepared and analyzed exactly like the field samples. Purpose: Assess contamination introduced dur- ing sample preparation activities. Bleed: Loss of material from a column or septum due to high-temperature operation. May result in ghost peaks plus increased detector baseline offset and noise; in extreme cases, bleeding chemicals from the stationary phase may build up on detector surfaces. Blending: Refers to the process of mak- ing a heterogeneous sample into a more consistent and uniform sample by some type of blending operation; the most popular type of blender is the mechani- cal blender that chops a semisoft mate- rial into smaller parts. Bonded phase: A stationary phase that has been chemically bonded to the inner wall of an open-tubular (capil- lary) column or to the support particles. In LC, the substrate is usually a silica gel particle or other base material. Bonded-phase chromatography: The most popular mode in LC, in which a phase chemically bonded to a support is used for the separation. The most popular support for bonded-phase chromatography is microparticulate silica gel and the most popular type of bonded phase is the organosilane, such as octadecyl (for reversed-phase chro- matography). Approximately 70% of all HPLC is carried out on chemically bonded phases. Bonded-phase concentration: See coverage. Boxcar chromatography: See column switching; alternate name. Breakthrough capacity: See break- through volume. Breakthrough volume: The volume at which a particular solute pumped con- tinuously through a column will begin to be eluted. It is related to the column volume plus the retention factor of the solute. It is useful to determine the total sample capacity of the column for a particular solute. BTEX: Refers to benzene, toluene, eth- ylbenzene, and xylenes analysis. Buffer: A solution that maintains con- stant pH by resisting changes in pH as a result of dilution or addition of small amounts of acids and bases. Buffer capacity: A quantitative mea- sure of the potential of a buffer solu- tion (defined as the number of equiva- lents of strong acid or base to cause a one unit change in the pH of 1 L of a buffer solution) or simply the abil- ity of a buffer to withstand injections of a buffered sample solution without a change in mobile-phase pH; capac- ity determined by pH, buffer pKa, and buffer concentration. Buffer strength: See ionic strength. C C4, C8, C18: Refers to the alkyl chain length of a reversed bonded phase. C term: The interphase mass transfer term of the van Deemter and Golay equations. Canister collection: A stainless steel vessel designed to hold vacuum to less than 1.3 Pa (10 mTorr) or pres- sure to 275 kPa (40 psig). Canisters are available in a range of volumes: 400 mL, 1.0 L, 3.0 L, 6.0 L, and 15 L. The size of canister used usually depends on the concentration of the analytes in the sample, the sampling time, the flow rate, and the sample volume required for the sampling pe- riod. Typically, smaller canisters are
- 15. Terminology Guide October 2013 15 www.chromatographyonline.com used for more concentrated samples, such as soil gas collection, 3-L and 6-L canisters are used to obtain in- tegrated (TWA) ambient air samples at sampling times of up to 24 h, and large 15-L canisters are used for refer- ence standards. Sampling time will be limited by the combination of canis- ter size and the flow rate at which the sample is to be collected. Capacity: See sample capacity. Capacity factor (k’): Deprecated name for retention factor. Capillary column: Refers to chro- matography columns of small inner diameter, ostensibly small enough to display a capillary effect with liquids. The diameter below which a column is considered “capillary” is poorly defined. See open-tubular column, capillary LC. Capillary column, packed: A capillary column that is packed with stationary- phase particles. In GC, 1/16-in. o.d. by 1-mm i.d. columns are common. Capillary electrochromatography (CEC): A hybrid technique where capillary columns are packed with chromatographic sorbents and elec- troosmotic flow moves mobile phase through the column rather than pres- sure; the technique has the surface- mediated selectivity potential of HPLC and the high efficiency of CE. Capillary GC: See open-tubular col- umn. Capillary LC: Generally refers to HPLC carried out in a fused-silica or other type of capillary column; most of the time the dimensions are in the sub-0.5-mm i.d. range. Has also been called micro LC. Capillary micellar electrochromatog- raphy (CMEC): The CEC version of MEKC. Capillary tubing: Tubing to connect various parts of the chromatograph in order to direct flow to the proper place. Most capillary tubing used in HPLC is less than 0.020 in. in internal diameter. The smallest useful internal diameter is about 0.004 in. Capping: Same as endcapping. Carbon load: For a bonded-phase silica, term usually used to describe the surface coverage or the degree to which the available silanols on the column packing’s surface have reacted and been replaced with the bonded phase; the higher the carbon load, the lower number of residual silanols. The carbon load is normally expressed as % carbon (for example, 12% carbon). In reversed-phase LC, the higher the carbon load, the greater the analyte retention. Carrier: A term most often used in af- finity chromatography; refers to the support that is used to attach the ac- tive ligand, usually by a covalent bond. Can also refer to the support in other chromatography modes, such as LLC. Carrier gas: Term for the gaseous mo- bile phase in GC. Cartridge: Generally refers to the con- tainer used in SPE or filtration; a car- tridge may be as simple as a medical- grade syringe barrel that is filled with packing contained at both ends by frits; it can also be a molded device or even a stainless steel device that contains similar sorts of packing material. In SPE, the device is also referred to as an SPE tube. Cartridge column: A column type that has no endfittings and is held in a cartridge holder. The column consists of a tube and the packing is contained by frits in each end of the tube. Car- tridges are easy to change and are less
- 16. 16 Terminology Guide October 2013 www.chromatographyonline.com expensive and more convenient than conventional columns with endfittings. Cation-exchange chromatography: The form of ion-exchange chroma- tography that uses resins or packings with functional groups that can sepa- rate cations. An example of a strong cation functional group would be a sulfonic acid; a weak cation-exchange functional group would be a carbox- ylic acid. Centrifugation: Centrifugation is a process that involves the use of the centrifugal force for the sedimenta- tion of mixtures with a centrifuge (see centrifuge). This process is used to separate two immiscible liquids. More- dense components of the mixture mi- grate away from the axis of the centri- fuge, while less-dense components of the mixture migrate toward the axis. Chemists and biologists may increase the effective gravitational force on a test tube so as to more rapidly and com- pletely cause the precipitate (“pellet”) to gather on the bottom of the tube. The remaining solution is properly called the “supernate” or “supernatant liquid.” The supernatant liquid is then either quickly decanted from the tube without disturbing the precipitate, or withdrawn with a Pasteur pipette. Centrifuge: A centrifuge is a piece of equipment, generally driven by an electric motor (some older models were spun by hand), that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis (see centrifugation). Certify: Specific SPE products for drugs of abuse isolation and analysis. Chain length: The length of carbon chain in the hydrocarbon portion of a reversed-phase packing. It is expressed as the number of carbon atoms (for ex- ample, C8, C18). Specifically excludes the short chains typical methyl, isopro- pyl, and sec-butyl groups also attached to the silane. Channeling: Occurs when voids cre- ated in the packing material cause mo- bile phase and accompanying solutes to move more rapidly than the average flow velocity allowing band broadening to occur. The voids are created by poor packing or erosion of the packed bed. Charged aerosol detection (CAD): The effluent from the LC column is nebulized and then vaporized in a heated drift tube, which results in a cloud of analyte particles; these par- ticles are charged and then the current from the charged particle flux is mea- sured. The ELSD technique measures the light scattering properties of the aerosol particles. CAD is more sensi- tive and gives a more linear response than ELSD; it is also a universal detec- tion method. Check valve: A device inserted into a moving fluid stream that allows flow of the stream in only one direction; most often used on the inlet and outlet sides of an HPLC pump. Chelating resin: Chelating resin con- tains functional groups that will inter- act with cationic species (for example, metals such as copper, iron, heavy metal ions); useful for concentrating trace quantities or for separation. Chemical filtration: A liquid sample is passed through a packing material (for example, adsorbent, ion exchange, and so forth) that selectively interacts with one or more compounds within the sample and acts as a chemical way to remove and purify the liquid sample. Regular filtration does not involve any chemical interaction but merely re- moves particulates.
- 17. Terminology Guide October 2013 17 www.chromatographyonline.com Chemical suppression: Remove back- ground conductivity by ion exchange. Converts the eluent into a low- or non- conducting component (for example, carbonate into carbonic acid, hydrox- ide into water, or nitric acid into water). Anion analysis: the counter cation (for example, sodium) is replaced by the proton (H+). Cation analysis: the counter anion (for example, nitrate) is replaced by hydroxide (OH-). The mea- sured signal is the corresponding acid or base of the anion or cation respec- tively. The dissociation of these acids and bases influences the signal. All sup- pressor devices work on this principle. The typical background conductivity after suppression is <20 µS/cm. Chemisorption: Sorption due to a chemical reaction with the packing. Most such interactions are irreversible. Usually occurs on packings with reac- tive functional groups such as silanol or bonded amino phases. Chemisorption is common with metal oxide phases that have strong Lewis acid sites. Chip format: A miniaturization tech- nique where small channels on a glass, polymer or other type of matrix are used instead of large bore columns, capillaries, and so forth. The result- ing format is greatly reduced in size compared to conventional chromato- graphic instruments; advantages are the reduction in sample, solvent, and so forth; can be more easily coupled to detection techniques (for example, MS and MS-MS) where smaller amounts of mobile phase can lead to sensitivity enhancements and reduction in ion suppression. Chiral recognition: The ability of a chiral stationary phase to interact dif- ferently with two enantiomers leading to their chromatographic separation. Chiral stationary phase (CSP): A sta- tionary phase that is designed to sepa- rate enantiomeric compounds. The phase can be coated or bonded to solid supports, created in situ on the surface of the solid support, or can include sur- face cavities that allow specific interac- tions with one enantiomeric form. Chlorosilane: A chemical reagent used to prepare siloxane bonded phases; re- activity changes from a monochlorosi- lane < dichlorosilane < trichlorosilane; the alkyl portion (for example, octa- decyl, octyl, and so forth) will dictate the hydrophobicity of the resultant bonded phase; alkoxysilanes can be used but are less reactive. Chopping: The process of mechanically cutting a sample into smaller parts. Chromatogram: A plot of detector signal output versus time or elution volume during the chromatographic process. Chromatograph: (n) A device used to implement a chromatographic separa- tion; (v) the act of separation by chro- matography. Chromatographic conditions: Those chromatographic method experimen- tal parameters that describe how an analysis was performed. Sufficient information must be presented so that the analysis can be duplicated for veri- fication purposes. Classification: The process of sizing column packing particles; generally, in HPLC a small particle size distribution provides better efficiency and a greater permeability because of the absence of fines. Classification can be performed by sedimentation, elutriation, and using centrifugal air classifiers. Co-ion: An ion of the same sign of charge as the ionic groups making up the stationary phase.
- 18. 18 Terminology Guide October 2013 www.chromatographyonline.com Coating efficiency (CE, UTE, UTE%): A metric for evaluating column quality. The minimum theoretical plate height divided by the observed plate height: CE = Hmin/H Cold injection: An injection that takes place at temperatures below the final oven temperature, usually at or below the solvent boiling point. Column: The tube and stationary phase through which mobile phase flows re- sulting in a chromatographic separa- tion. Column chromatography: Any form of chromatography that uses a column, tube, or plate to hold the stationary phase. Open-column chro- matography, HPLC, and open-tubu- lar capillary gas chromatography are all forms of column chromatography. Most often refers to open-column chromatography used for prepara- tive work. Column dead time: See hold-up time. Column equilibration: To provide re- producible results, a column should be equilibrated with the surrounding envi- ronment be it a temperature condition, mobile phase equilibrium, pressure condition, and so forth; in GC, it is important that the temperature of the column be stabilized after a tempera- ture programmed run and in LC, the column must be returned to its original conditions before another gradient is run. Column inner diameter (dc): The inner diameter of an uncoated chro- matography column. Column length (L): The length of the analytical chromatography column used to perform the chromatographic separation. Distinct from the length of a precolumn (LC) or retention gap (GC) connected in series. Column outlet flow rate, corrected (Fa): In GC, the column outlet flow rate corrected from column tempera- ture and outlet pressure to room tem- perature and pressure, for example, the flow rate as measured by a flow meter. Difficult to measure directly for narrow-bore open-tubular col- umns, the flow rate can be calcu- lated from the average carrier-gas linear velocity, pressure drop, temper- atures, and the column dimensions: Fa = (u –πdc 2T0)/(4jTc). Such calcula- tions are the basis of electronic pres- sure control. Column overload: If one exceeds the sample capacity (or loading capacity) of a column, peaks will become distorted and may be difficult to measure and to achieve reproducible chromatogra- phy from run to run. One can measure column capacity by running a break- through study (see breakthrough vol- ume). Column packing: The solid material, usually a porous solid with or without a chemically interactive surface, placed inside or on the walls of the column used to differentially retain analytes; also referred to as the stationary phase; common packings include un- bonded and bonded silica, resins, in- organic–organic hybrids, graphitized carbon, porous polymers, and molecu- lar sieves. Column performance: Denotes the column efficiency. See theoretical plate. Column plate number: Denotes the column efficiency. See theoretical plate. Column suppressor: Initial setup. Packed ion-exchanger columns were used for chemical suppression. Draw- backs: require regeneration, changes
- 19. Terminology Guide October 2013 19 www.chromatographyonline.com of the selectivity throughout the usage. Column switching: The use of mul- tiple columns connected by switching valves to effect better chromatographic separations or for sample cleanup. Frac- tions from a primary column can be switched to two or more secondary columns which in turn can be further diverted to additional columns or to the detector (or detectors); sometimes referred to as multidimensional chro- matography. Column temperature (Tc): Tempera- ture of the column. A uniform tem- perature across the column usually is desirable; however, GC separations also may be performed with a moving temperature gradient along the column length. Column volume (Vc): The volume of the unpacked, uncoated column: Vc = AcL = πrc 2L, where Ac and L are the cross-sectional area of the tube and the tube length, respectively. Cool-down time: Length of time re- quired to cool a GC oven from the final oven temperature to the initial oven temperature. Shorter cool-down times allow a greater number of analyses to be performed in a given time period. Competing base: In reversed-phase LC, addition of a small basic com- pound such as triethylamine or di- methyloctylamine at 25–50 mM concentration to the mobile phase to inhibit basic analytes from interact- ing with residual silanols; works by law of mass action because the con- centration of competing base is much greater than that of the analyte. See also additive. Comprehensive GC (GC×GC): Two- dimensional technique in which all compounds experience the selectivity of two columns connected in series by a retention modulation device, thereby generating much higher resolution than with any single column. Comprehensive two-dimensional chromatography: Two-dimensional chromatography applied to every frac- tion. See two-dimensional chroma- tography. Compressibility correction factor (j): Due to gas compressibility, the carrier gas expands and its velocity increases as it proceeds along a GC column from the inlet pressure pi to the outlet pres- sure po. The carrier gas compressibility correction factor corrects the carrier gas velocity at the outlet of a GC col- umn to the average carrier gas velocity: j = 3(P2 – 1)/2(P3 – 1), where P is the column pressure drop: P = pi/po Concentration: The process of increas- ing the strength or density of a diluted sample; a more concentrated sample will be easier to measure; concentration can be accomplished by a wide variety of sample preparation techniques such as evaporation, adsorption, diffusion, and so forth. Conditioning (SPE): This is gener- ally considered to be the first step in SPE; the stationary phase must first be put into a chemical or physical state that it can accept the sample solution loaded in the second SPE step; a conditioning solvent is passed through the SPE stationary phase where it will solvate the phase so that it will more easily sorb the sample of interest; for a reversed-phase SPE car- tridge, methanol or acetonitrile serves as a conditioning solvent; sometimes the excess conditioning solvent must be removed but the packing shouldn’t be allowed to dry out because that may affect the “conditioned” phase.
- 20. 20 Terminology Guide October 2013 www.chromatographyonline.com Conductivity: Conductivity is the in- herent parameter of all ions. Therefore it is the signal for measuring the chro- matogram. It may be measured directly or after chemical and sequential sup- pression respectively. As conductivity is strongly dependent on the temperature (around 2 %/°C), a thorough isolation and thermal stabilization of the detec- tor block is recommended. The mea- sured conductivity of a solution is given by the sum of the “single ion conduc- tivities.” The single ion conductivity is a linear function of the concentration of the ion and its equivalent conduc- tivity. At very low concentrations or at constant ion strength the equivalent conductivity is constant. The mea- sured signal in nonsuppressed IC is proportional to the concentration and the difference of eluent and sample ion: As the ionic strength of the solution is constant (stoichiometric ion exchange) also the equivalent conductivities are constant. This leads to very linear cali- bration curves. The measured signal in suppressed IC is proportional to the sum of the equivalent conductivities of the analyte ion and the counterion (which has been introduced by suppres- sion). As only the sample ion and its counterion is adding to the measured conductivity after suppression, the total concentration is changing during the peak. Therefore the equivalent conduc- tivities are no longer constant. This is one reason for the inherent nonlinear- ity of the calibration curves in sup- pressed IC. Coning and quartering: A sample size reduction technique where a portion of free-flowing solid material (powder) is systematically divided into quadrants to achieve a statistically representative sample. Coning and quartering is a method used by analytical chemists to reduce the sample size of a powder without creating a systematic bias. The technique involves pouring the sample so that it takes on a conical shape, and then flattening it out into a cake. The cake is then divided into quarters; the two quarters that sit opposite one another are discarded, and the other two are combined and constitute the reduced sample. The same process is continued until an appropriate sample size remains. Analyses are made with respect to the sample left behind. Continuous liquid–liquid extraction: Useful when the KD value is very low or the required sample volume is very large when multiple extractions are im- practical; also if the extraction is slow, a long time may be required for equi- librium to be established; in continu- ous LLE, fresh solvent is continually recycled through the aqueous sample; continuous extractors are available for heavier-than-water and lighter-than- water solvents. Controlled surface porosity support: Same as porous layer bead and pel- licular stationary phase. Cool on-column injection: Cool on- column injection is a technique of in- troducing a sample as a liquid directly into a GC column; this lack of prior vaporization offers the following advan- tages: It eliminates sample discrimina- tion; it eliminates sample alteration; and it provides high analytical preci- sion. However, there are some special requirements: It requires relatively clean samples; real samples are often too concentrated for on-column injec- tion and must be diluted; and peak splitting or peak distortion can occur due to differing polarities of solvent, stationary phase, and solutes.
- 21. Terminology Guide October 2013 21 www.chromatographyonline.com Core–shell: See superficially porous particles (SPPs). Coulometric detector: Same as am- perometric detection, but with a con- version rate of 100%. All the analyte is oxidized or reduced at the working electrode. The response is larger than with amperometric detection. But on the other hand also the baseline noise is larger. Therefore the detection limits are almost the same. Counterion: In an ion-exchange pro- cess, the ion in solution used to displace the ion of interest from the ionic site. In ion pairing, it is the ion of opposite charge added to the mobile phase to form a neutral ion pair in solution. Coupled columns: A form of column switching that uses a primary column connected to two secondary columns via a selector valve. Fractions from col- umn one can be selectively transferred to columns two and three for addi- tional separation to occur. The term is also used to describe two or more col- umns connected in series to provide an increased number of plates. Coverage: Refers to the amount of bonded phase on a silica support in bonded phase chromatography. Cov- erage is usually described in µmol/m2 or in terms of %C (w/w). Crash plate: Refers to the process of precipitating protein from plasma by the addition of a miscible organic sol- vent such as acetonitrile; when a 96- well flow-through or fixed-well plate is used for this process, it is referred to as crashing and the plate a crash plate. Critical micelle concentration (CMC): The concentration of an ionic surfac- tant above which a micelle is formed by aggregation; micelles added to the mobile phase are used to improve the separation of nonionic substances in HPLC and CE (MEKC) by a partition- ing mechanism. Cross-linked phase: A stationary phase that includes cross-linked poly- mer chains. Usually it is also bonded to the column inner wall. See bonded phase. Crosslinking: For resins, during the process of copolymerization to form a three-dimensional matrix a difunc- tional monomer is added to form cross- linkages between adjacent polymer chains. The degree of cross-linking is determined by the amount of this monomer added to the reaction. For example, divinylbenzene is a typical cross-linking agent for the production of polystyrene ion-exchange resins. The swelling and diffusion characteristics of a resin are governed by its degree of cross-linking. Crushing: Tungsten carbide variable jaw crushers for reducing the size of large, extremely hard, brittle samples. Curtain flow technology: Curtain flow technology refers to the process of injection of sample across a radial cross section of an HPLC column to ensure the analyte sees the middle por- tion of the packed bed and not the wall where flow effects may be different; the technique is coupled with a parallel seg- mented flow fitting at the column out- let to select just the middle portion of the flow profile resulting in improved efficiency without the presence of wall effects. Cutting: Cutting mills can reduce soft- to-medium hard materials (diameter < 100 mm). Cyano phase: A chemically bonded phase that terminates with the -CN functional group; it can be used in normal-phase chromatography as a moderate polarity sorbent and in
- 22. 22 Terminology Guide October 2013 www.chromatographyonline.com reversed-phase chromatography as a short chain bonded phase. Cyclodextrins: Cyclic oligomers of several D-(+)-glucopyranose units used in chiral HPLC and CE separations; popular ones are named α-, β-, and γ-CDs; they have a truncated cone shape, a relatively hydrophobic cavity, and primary and secondary hydroxyl groups at their end; separate on basis of differential inclusion of enantio- mers; modified CDs with derivatized hydroxyl groups are also used for se- lectivity modification. D Data acquisition rate: A term refer- ring to the rate of sampling of a de- tector output. To characterize a chro- matographic peak at least 20–30 data points must be collected. The data acquisition rate, usually measured in hertz, defines how many data points per second are collected while the peak is moving through the detector. For fast chromatography, the data acquisition rate must be sufficiently rapid to char- acterize a narrow peak. Modern detec- tors have data rates up to 200 Hz; also known as data rate and sampling rate. See detector time constant. Dead volume: Dead volume is extra volume experienced by solutes as they pass through a chromatographic sys- tem, in particular any unswept vol- ume exposed to the mobile phase flow. Excessive dead volume causes addi- tional peak broadening. Related to the hold-up volume, which is the volume of mobile phase necessary to elute an unretained compound. See hold-up volume. Deep-well plate: A 96-well plate ca- pable of handling up to 2 mL of liquid volume per well. Degassing: The process of removing dissolved gas from the mobile phase prior or during use. Dissolved gas that may come out of solution in the detec- tor cell can cause baseline spikes and noise. Dissolved air can affect certain detectors, such as electrochemical (by reaction) or fluorescence (by quench- ing). Dissolved gases can also cause pumps to lose prime. Degassing is car- ried out by heating the solvent or by vacuum (in a vacuum flask), or on-line using evacuation of a tube made from a gas-permeable substance such as PTFE, or by helium sparging. Denaturing HPLC: Use of reversed- phase HPLC to investigate genetic mutations by the investigation of DNA base pairs. Derivatization: A technique used in chemistry that transforms a chemical compound into a product (the reac- tion’s derivate) of similar chemical structure, called a derivative. Gener- ally, a specific functional group of the compound participates in the de- rivatization reaction and transforms the compound into one with a differ- ent reactivity, solubility, boiling point (volatility), melting point, aggregate state, or chemical composition. The resulting new chemical properties can be used for quantification (for example, UV or fluorescence detection) or better separation properties. Desalting: Technique where low- molecular-weight salts and other com- pounds can be removed from nonionic and high-molecular-weight com- pounds. The use of a reversed-phase packing to retain sample compounds by hydrophobic effects yet allow salts to pass through unretained would be an example of desalting. The use of an SEC column to exclude large molecules
- 23. Terminology Guide October 2013 23 www.chromatographyonline.com and retain lower-molecular-weight salts is another example. Desalting using dialysis is commonly used in protein purification. Desorption: The process in chroma- tography where a molecule residing on the surface of a packing material or on another solid surface (for example, col- umn wall or frit) or stationary phase moves from the surface into the mobile phase. Detector response time: Time for a detector to respond to ~90% of the in- coming solute amount. The response time is generally taken as 2–4 times the time constant. See also detector time constant. Detector time constant (τ): The time for a detector to respond to 1/e = 63.2% of an instantaneous change in solute amount. In general the detector time constant should be less than 10% of the peak width at half-height. Excessive detector dead volume, slowly respond- ing electronics, digital data acquisition speeds, and signal filtering strongly in- fluence detector response times. Too- slow detector response times cause peak tailing, loss of peak height and detect- ability, plus loss of peak resolution for closely adjacent peaks. Dextran: Polydextran-based packing material primarily used for low pres- sure biochromatography; an example would be Sephadex (Amersham Phar- macia Biotech). Dialysis: Dialysis works on the prin- ciples of the diffusion of solutes and ultrafiltration of fluid across a semi- permeable membrane. Diffusion is a property of substances in water; substances in water tend to move from an area of high concentration to an area of low concentration. A semipermeable membrane is a thin layer of material that contains holes of various sizes, or pores. Smaller solutes and fluid pass through the membrane, but the membrane blocks the passage of larger sub- stances (for example, red blood cells, large proteins). It is a technique used in biological sample prep to desalt biological fluids. Diatomaceous earth: Also known as diatomite or kieselguhr, it is a natu- rally occurring, soft, siliceous sedi- mentary rock that is easily crumbled into a fine white to off-white powder. Diatomaceous earth consists of fos- silized remains of diatoms, a type of hard-shelled algae. It has a particle size ranging from less than 3 µm to more than 1 mm, but typically 10– 200 µm. Depending on the granular- ity, this powder can have an abrasive feel, similar to pumice powder, and is very light as a result of its high poros- ity. The typical chemical composition of oven-dried diatomaceous earth is 80–90% silica, with 2–4% alumina (attributed mostly to clay minerals) and 0.5–2% iron oxide; highly puri- fied diatomaceous earth is used as a support for chromatography and for supported liquid–liquid extraction. Diethylaminoethyl (DEAE): A popu- lar weak anion-exchange functionality typically attached to cellulose or Sep- harose (GE Healthcare); used for the separation of biomolecules. Diethylene glycol succinate (DEGS): A GC stationary phase. Diffusion coefficient (DM or DG; DS or DL): A fundamental parameter of a molecule in the liquid or gaseous mobile phase (DM or DG) or in the liquid stationary phase (DS or DL) that expresses the degree of free mobility of the molecule in solution. Expressed
- 24. 24 Terminology Guide October 2013 www.chromatographyonline.com in cm2/s, the diffusion coefficient is dependent on molecular weight of the solute, temperature, solvent viscosity, and molar volume of the solute. A typical value of a small molecule (100 Da) in a liquid phase at room tem- perature is on the order of 10-5 cm2/s. Gaseous solutes in helium carrier gas at 120 °C have diffusion coefficients that are several orders of magnitude higher, around 0.4 cm2/s. Digestion: The process of treating an insoluble chemical compound with a reactive substance (for example, for inorganic compounds it might be a strong acid; for a biological compound it might be an enzyme) that will break it down or disintegrate the compound into a more soluble form that can be further treated or analyzed. Digital chromatography: The pro- cess of solid-phase extraction (SPE) is sometimes referred to as digital chro- matography. A substance is either on the SPE stationary phase or is off the stationary phase during its retention and elution; in chromatography we are often trying to resolve closely re- lated substances by exploiting subtle differences in retention in more of an analog separation mode; in terms of k values, ideally the solute in SPE has a value of infinity when on the sorbent and zero when eluted into solution. Dilute and shoot: A simple sample preparation procedure where one merely dilutes the sample with solvent, mobile phase, or a compatible liquid and then injects that diluted sample into a chromatograph without any fur- ther sample preparation. Dilution: Reducing the concentration of a chemical by adding an inert sub- stance; the substance can be a liquid, solid, or gas. Dimethylchlorosilane (DMCS): Some- times used for silanizing glass GC parts such as inlet liners and endcapping sil- ica-based HPLC bonded phases. Dis- posable presilanized inlet liners are a preferable alternative that avoid use and storage of this hazardous reagent. Diode-array detection (DAD): Each wavelength of the UV and visible range of the light is measured with an indi- vidual diode. The optical resolution of the detector is defined by the number of diodes used (for example, 844 di- odes: optical resolution = 1.4 nm). Diol phase: A hydrophilic phase use- ful in both normal and reversed phase. It consists of a diol structure (two -OH groups on adjacent carbon atoms in an aliphatic chain). In normal-phase work, it is less polar than silica and in reversed phase work has been used for the separation of proteins and poly- peptides. Direct injection: Sample enters the inlet and is swept into the column by carrier gas flow. No sample splitting or venting occurs during or after the injection. Direct sampling: A method of sample collection where a sample is taken di- rectly from the source. For example, a cannister may be used to collect a gas sample exactly where the scientist desires. A river water sample can be obtained by lowering a collection vessel directly into the water. A thermal desorption tube can be used to concentrate volatile and semivolatile analytes by passing a gas stream through the adsorbent con- tained within the tube. This would be an example of direct sampling. Direct thermal sampling: Refers to the process of using temperature as a variable in sample volatile and semi- volatile substances; static headspace at
- 25. Terminology Guide October 2013 25 www.chromatographyonline.com a given temperature is an example of thermal sampling; by selecting a cer- tain temperature certain sample com- ponents can be ruled out because they may have extremely low volatility at a selected temperature; thermal sampling can occur in stages all the way up to pyrolysis where chemical bonds in the sample are purposely broken to access the structure of the material. Disk: A number of sample prepara- tion and separation media take the form of a disk; the most popular disks are used in filtration and may consist of any number of porous polymeric materials; the most popular types of SPE disks would have embedded particles in a disk made of PTFE or other inert polymeric material or a fiberglass matrix with interdispersed sorbent particles; some biological purification media employ the disk format. The stationary phases may contain ion exchange groups or other functionality that attracts solutes of interest or impurity that one may want to get rid of. Disk cartridge (SPE): See disk. Dispersion: See system dispersion. Dispersive liquid–liquid microextrac- tion (DLLME): The technique is based on a three-component solvent system. The container is usually a centrifuge tube and the appropriate mixture of immiscible organic extraction solvent (usually a few microliters, such as 8 µL of tetrachloroethylene) and a dispersive solvent (for example, ~1 mL of acetone) is rapidly injected with a syringe into an aqueous solution (~5 mL) contain- ing the analyte of interest. When the three solvents are rapidly mixed, a cloudy solution is formed consisting of droplets of extraction solvent; the entire mixture is centrifuged and the droplet of solvent containing extracted ana- lytes (tetrachloroethylene) is removed by a microsyringe for direct injection. Extraction is almost instantaneous and enrichment values are quite high. Dispersive SPE (dSPE): In dSPE, loose SPE packing material is added directly to a solution rather than pass- ing it through the packed material in a cartridge or tube; dSPE is most often used as the second step in QuEChERS where matrix compounds are removed from the organic solvent salting out ex- traction of the first step. Displacement chromatography: Chromatographic process where the sample is placed onto the head of the column and then is displaced by a compound that is more strongly sorbed than the compounds of the original mixture. Sample molecules are then displaced by each other and by the more strongly sorbed compound. The result is that the eluted sample solute zones may be sharpened; displacement techniques have been used mainly in preparative HPLC applications. Disposable filter: See syringe filter. Dissolution: The process of having a sample dissolve in an appropriate solvent. Distillation: A method of separating mixtures based on differences in vola- tility of components in a boiling liquid mixture. Distillation is a unit operation, or a physical separation process, and not a chemical reaction; it can be used to purify organic compounds or to re- move solvent; fractional distillation is used to separate compounds with close boiling points; azeotropic distillation is using an azeotrope to remove a solvent that has a boiling point too close or equal to another compound that can- not be separated.
- 26. 26 Terminology Guide October 2013 www.chromatographyonline.com Distribution constant (coefficient) (Kc): The equilibrium concentration of a component in or on the station- ary phase divided by the equilibrium concentration of the component in the mobile phase; also called the distribution coefficient or in partition chromatogra- phy the partition coefficient; in partition chromatography Kc is used and refers to the case where the concentration in the stationary phase is expressed per unit volume of the phase: VR = VM + KcVS; in the case of a solid stationary phase, Kg is used and is expressed as per mass (weight) of the dry solid phase; in the case of adsorption chromatography with a well characterized adsorbent of known surface area, the concentration in the stationary phase is expressed as per unit surface area. Dividers: A mechanical device used in subdividing solid powder samples into smaller units; they can be manual or au- tomated; sample dividers will subdivide material samples into two smaller por- tions by a single pass or further subdivi- sions can be attained by multiple passes. The important feature of sample divid- ers is that each subdivision retains the characteristics of the original sample. Dried blood spot (DBS) analysis: A newer method for sampling and trans- porting blood samples; a small (~15 µL) sample of whole blood is placed on a cel- lulose or other paper-like material and is dried for 2 h; the dried blood spot can be extracted to remove analytes of interest for further workup; has poten- tial to replace drawing large quantities for blood analysis; used in conjunction with LC–MS-MS for high sensitivity and specificity. Dried media spot (DMS) analysis: In addition to DBS, other biological fluids (for example, plasma, serum, cerebrospi- nal fluid, saliva) as well as other non- biological media have been investigated. Drying: Drying of sample extracts can be accomplished by heating (evapora- tion), vacuum dessication, and other means; water can be removed (dried) from organic solvents by using anhy- drous sodium sulfate. Dwell time: The time equivalent to dwell volume; determined by the prod- uct of flow rate times the dwell volume. Dwell volume: In LC, refers to the volume between the point of mixing of solvents (usually in the mixing chamber or at the proportioning valves in the liq- uid chromatograph) and the head of the chromatographic column; important in gradient elution or when changes in sol- vent composition are made in isocratic elution so that the column experiences the composition change in the shortest possible time. Low-pressure mixing sys- tems generally have larger dwell volumes than high-pressure mixing systems. Dynamic coating: The formation of in- situ coatings on the packing in HPLC or on capillary walls in CE by addition of a substance to the mobile phase that adsorbs (or absorbs) onto the pack- ing or at the wall surface; the purpose of a dynamic coating is to generate a new stationary phase or to deactivate the packing material or capillary wall to prevent unwanted interactions; one simple example is the adjustment of the mobile phase or running buffer to a pH < 3 to protonate silanols and ne- gate their effect; another is coating the phase with a hydrophilic polymeric ma- terial to prevent adsorption of proteins. In GC, dynamic coating applies a con- trolled-thickness coating to the inside of open-tubular columns as a solution flows through the column and leaves a thin coating behind, which is then
- 27. Terminology Guide October 2013 27 www.chromatographyonline.com evaporated to yield a thin coating of any non-volatile material from the solution. Dynamic headspace: See purge-and- trap sampling. E Eddy dispersion (diffusion) term (A term): The A term in the van Deemter equation. It is the contribution to plate height that arises from the heterogeneity in axial velocities as a result of the par- ticle size and geometry of the packing as well as wall effects: a = 2λdp. Typi- cal values of λ for well-packed columns are 0.8–1.0. Some theories of chroma- tography indicate a velocity dependent contribution to the HETP from this process. Also known as eddy diffusion, flow-heterogeneity induced broadening, and the multipath term. The A term is zero in open-tubular GC columns. See van Deemter equation. Efficiency: The ability of a column to produce sharp, well-defined peaks. More efficient columns have more theo- retical plates (N) and smaller theoretical plate heights (H). For asymmetric peaks it is better determined from the peak centroid and variance by mathematical analysis of the peak shape. See Foley- Dorsey equation. Effluent: The mobile phase leaving the column; the same as eluate. Electrochemical detector: Global term for all detection modes recording elec- trical potential or current (conductivity, amperometric, pulsed amperometric, coulometric, and potentiometric detec- tion). More often used for amperometric, pulsed amperometric, and coulometric detection only. Electrochemical suppression: Con- tinuously working chemical suppressor where H+ or OH- are electrochemically produced by the electrolysis of water. The expression is misleading as the suppres- sion is chemical. Only the supply of the respective ion is done electrochemically. Electrodialysis: Used to transport salt ions from one solution through ion-ex- change membranes to another solution under the influence of an applied elec- tric potential difference. This is done in a configuration called an electrodialysis cell. The cell consists of a feed (diluate) compartment and a concentrate (brine) compartment formed by an anion- exchange membrane and a cation-ex- change membrane placed between two electrodes; can provide good enrichment factors. Electrolytic suppression: Synonym to electrochemical suppression. Electrolytic conductivity detection (ELCD): An electrolytic conductivity de- tector catalytically reacts halogen-con- taining solute with hydrogen (reductive mode) to produce strong acid by-prod- ucts that are dissolved in a working fluid. The acids dissociate and the increased electrolytic conductivity is measured. Other operating modes modify the chemistry for response to nitrogen- or sulfur-containing substances. Electron-capture detection (ECD): An electron-capture detector ionizes solutes by collision with metastable carrier gas molecules produced by β-emission from a radioactive source such as 63Ni. ECD is one of the most sensitive detection methods and responds strongly to halo- genated solutes and others with a high electron-capture cross-section. Electronic or programmed pressure control (EPC or PPC): Any of a number of pressure and flow control devices that incorporate electronic pressure or flow sensing and can be programmed from the GC microcontroller. Such devices enable method control of flow, velocity,
- 28. 28 Terminology Guide October 2013 www.chromatographyonline.com and pressure for GC columns, as well as providing a convenient means of in- corporating gas-related parameters into electronic methods. Eluate: Combination of mobile phase and solute exiting the column; also called effluent. Eluent: The mobile phase used to carry out a separation. Eluite: The species being eluted: the analyte, or the sample. Eluotropic series: A series of solvents (eluents) with an increasing degree of solvent strength generally used in liquid–solid or adsorption chroma- tography. In normal-phase chroma- tography, a nonpolar solvent such as pentane would be at one end of the scale, an intermediate solvent such as dichloromethane would be in the middle of the scale, and a strongly polar solvent such as methanol would be near the upper end of the scale. In normal-phase chromatography, the reverse order of strength would be observed; water would be weak and hexane strong. Thus, when developing a method or running a gradient, an eluotropic series is useful for selecting solvents. Elute: To chromatograph by elution chromatography. The term elute is pre- ferred to the term develop used in older nomenclature. Elution: The process of passing mobile phase through the column to transport solutes down the column. Elution chromatography: The most commonly used chromatographic method where the sample is applied to the head of the column as a narrow zone and individual molecules are separated and eluted at the end of the column under the influence of a directed flow of mobile phase. Compare to displace- ment chromatography and frontal analysis. Elution step: This is generally consid- ered to be the fourth step in SPE and occurs after the washing (rinsing) step; in the elution step, analytes are removed from the SPE stationary phase by elu- tion with a strong solvent so that the analytes are now in a concentrated state; often, the strong solvent is removed by evaporation and reconstituted in a sol- vent more compatible with the chro- matographic system. Elution volume (VR): Refers to the vol- ume of mobile phase required to elute a solute from the column. For a symmet- ric peak, it is the volume from the point of injection to the volume at maximum concentration (apex): VR = FtR where F is the flow rate and tR is the retention time of the peak of interest. Elutriation: A technique used to frac- tionate packing particles by size based on the difference in their Stokes terminal velocities. It is most often used for the separation of ion-exchange resins that need to have a particularly narrow size range, such as amino acid resins. The technique involves the upward flow of water into a large tube. The unsized beads are added to the moving water and the particles seek their own level, de- pending on their density and particle size. They are then removed at certain levels in the tube. High purity spherical silica gels are sometimes sized by elutriation. Emulsion: A mixture of two or more liquids that are normally immiscible (nonmixable or unblendable). Emul- sion is usually referred to when both the dispersed and the continuous phase are liquids. In an emulsion, one liquid (the dispersed phase) is dispersed in the other (the continuous phase). Emulsions are bothersome in LLE because they
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- 30. 30 Terminology Guide October 2013 www.chromatographyonline.com sometimes are hard to break so that the layers can be separated and further treated, if necessary. There are numer- ous ways to break emulsions. Enantiomeric compound: Chemical compounds that display chiral activity; such compounds will require a separa- tion mechanism that can differentiate between the R- or S-enantiomer. Spe- cialty columns are available for this purpose. Endcapping: A technique used to re- move silanol groups on silica gel that may remain after reaction with a large silylating agent such as octadecyltrichlo- rosilane. The column is said to be end- capped when a small silylating reagent (for example, trimethylchlorosilane and dichlorodimethylsilane) is used to bond residual silanol groups on a silica gel-based packing surface. Most often used with reversed-phase packings to minimize undesirable adsorption of basic, ionizable, and ionic compounds. For polymeric phases with terminal si- lanol groups, endcapping reactions are also used to remove them. Endfitting: The fitting at the end of the column that permits connection to the injector or detector. Most HPLC endfittings contain a frit to hold the packing and have a low dead volume for minimum band spreading. They are usually constructed of stainless steel but PEEK and other polymeric materi- als are also used. Equilibration: See column equilibra- tion. Equivalent conductivity: The equiva- lent conductivity is a function of the concentration and the limiting conduc- tivity (equivalent conductivity at infinite dilution). Evaporation: The process of removing a volatile compound for the purposes of isolating a compound of interest; sol- vent evaporation is the most often used sample preparation technique. Evaporative light scattering detec- tion (ELSD): The effluent from the LC column is nebulized and then vapor- ized in a heated drift tube. This process results in a cloud of analyte particles that passes through a beam of light; the particles scatter the light and generate a signal at a photodiode or photomulti- plier; it is a universal detection method where molecules are not required to have a chromophore, be fluorescent, or be electrochemically active. Exchange capacity: See ion-exchange capacity. Excluded volume: See interstitial vol- ume. Exclusion chromatography: See steric- exclusion chromatography and ion ex- clusion. Exclusion limit: In SEC, the upper limit of molecular weight (or size) beyond which molecules will be eluted at the same retention volume, called the exclu- sion volume. Many SEC packings are referred to by their exclusion limit. For example, a 105 column of porous silica gel will exclude any compounds with a molecular weight over 100,000, based on a polystryene calibration standard. Exclusion volume (Vo): The minimum retention volume of a molecule on an SEC packing where all molecules larger than the size of the largest pore are to- tally excluded. These molecules are inca- pable of penetrating the pores and elute at the interstitial (interparticle) volume of the column. Exponentially modified Gaussian peak: A theoretical model for peak asymmetry arising from the column, inlet, and detector. The basis for the Foley-Dorsey equations.
- 31. Terminology Guide October 2013 31 www.chromatographyonline.com External calibration: Calibration mode using one or more standard solutions to establish the calibration function (cali- bration curve). With this function the concentration is calculated out of the measured peak area (or height) of the sample chromatogram. Linear or qua- dratic regressions are mainly applied but other regression modes are possible as well (for example, polynomial, point- to-point). Extracolumn effects: The total band- broadening effects of all parts of the chromatographic system outside of the column itself. Extracolumn effects must be minimized to maintain the ef- ficiency of the column. Areas of band broadening can include the injector, injection volume, connecting tubing, endfittings, frits, detector cell volume, and internal detector tubing. The vari- ances of all of these contributions are additive. Extracolumn volume: The volume be- tween the effective injection point and the effective detection point, excluding the part of the column containing the stationary phase. It is composed of the volumes of the injector, connecting lines and frits, and the detector; it determines the extra column effects. See dead vol- ume. Extraction: The general term for remov- ing analytes of interest from a matrix. F FAME: Fatty acid methyl ester. Fast GC: Gas chromatography per- formed on short, narrow-bore open- tubular columns, or on conventional columns at elevated linear velocities. Fast LC: The use in HPLC of short columns (1.5–7 cm in length) with conventional internal diameters ( 2–6 mm) packed with small particles (dp = 1.5–5 µm). Separation times in the range of minutes, sometimes seconds, are common; sometimes referred to as ultrafast LC. Fast protein liquid chromatography (FPLC): A term coined to cover the spe- cific use of HPLC for the separation of proteins. Generally, glass columns, mod- erate pressure, and spherical microbeads are used for FPLC. FFAP: Free fatty-acid phase. Fiberglass disks: A format where SPE particles are embedded in a fiberglass matrix; the disk format is especially useful for processing large volumes of sample (for example, water) given that the larger cross-sectional area allows for a higher flow rate than can be used for a typical cartridge. Filter funnel: A filter funnel is a laboratory funnel used for separating solids from liquids via the laboratory process of filtering. To achieve this, a disk-shaped piece of filter paper is usually folded into a cone and placed within the funnel. The suspension of solid and liquid is then poured through the funnel. The solid par- ticles are too large to pass through the filter paper and are left on the paper, while the much smaller liquid molecules pass through the paper to a vessel positioned below the funnel, producing a filtrate. The filter paper is used only once. If only the liquid is of interest, the paper is discarded; if the suspension is of interest, both the solid residue on the paper and the filtrate are kept for further analysis. Filter holder: Membrane filters or membrane disks are sometimes fur- nished loose and can be placed in a holder (usually of stainless steel con- struction) for processing samples; after the filter is used the holder is opened
- 32. 32 Terminology Guide October 2013 www.chromatographyonline.com and the used filter is replaced with a fresh one. Filter porosity: Pore size relates to the filter’s ability to filter out particles of a certain size. For example, a 0.5-µm membrane will filter out particles with a diameter of 0.5 µm or larger from a filtration stream. Filter porosity is typi- cally not related to, nor controlled by, pore size. These two parameters are es- sentially independent. Porosity is also unrelated to thickness. Rather, it is a function of the polymer and casting process used in the manufacture of the filter. Filter vial: A filter vial is a membrane filter unit that consists of two pieces. One is the filtration plunger, which con- tains a membrane filter suitable for the solvent being filtered. The second part of the filter vial is the vial body itself; once the sample is loaded and filtered, the filter vial can be placed directly in the autosampler without transferring the filtered sample to another vial. Filtration: The process of passing a liq- uid through a paper, membrane, glass, or other type of filter for the purposes of removing particulates that could cause problems downstream during a chro- matographic analysis; a chemical filter also removes certain chemical species. See chemical filtration. Fixed-well plate: A 96-well plate with fixed (nonremovable) wells, not an array. See 96-well plate and array 96-well plate. Flame ionization detection (FID): A flame ionization detector ionizes hydro- carbon-containing solutes in a hydro- gen–air flame. FID is a nearly universal detection method that responds strongly to most classes of organic compounds. Flame-photometric detection (FPD): A flame-photometric detector burns heteroatomic solutes in a hydrogen-air flame. The visible-range atomic emis- sion spectrum is filtered through an interference filter and detected with a photomultiplier tube. Different inter- ference filters may be selected for sul- fur, tin, or phosphorus emission lines. FPD is a sensitive and selective detec- tion method. Flash chromatography: A very fast form of classical liquid chromatography used by synthetic organic chemists to ef- fect rapid purification. Done primarily in the normal-phase mode, sometimes with reversed-phase LC. See column chromatography. Flexible well plate: See array 96-well plate. Flow injection extraction: An on-line extraction technique where a sample is injected as a plug into an aqueous flow stream that is divided into small seg- ments by an organic phase; the aqueous and organic segments are mixed during their passage down a coil, and eventu- ally the phases are separated at the end by a special fitting and the amount of extract compound can be measured in the organic phase. Flow programming: Used to decrease the retention time of slow-moving com- pounds. Flow programming is occasion- ally used in concert with temperature programming in GC. Flow rate (F, Fc): The volumetric rate of flow of mobile phase through a column. For a conventional 4.6-mm i.d. HPLC column, typical flow rates are 1–2 mL/ min. GC packed column flow rates typically range from 10 to 40 mL/min, and open-tubular column flows range from less than 1 mL/min up to about 10 mL/min. Flow resistance parameter (Φ): Φ = dp 2/Bo See permeability.
- 33. Terminology Guide October 2013 33 www.chromatographyonline.com Fluoro phase: One of a family of aliphatic and aromatic reversed-phase materials in which a substantial frac- tion of the bonded phase is fluorinated. Sometimes called fluorous phases or perfluoro phases. Typically have dif- ferent selectivities than hydrocarbon phases. Fluted filter paper: Filter paper that is folded in a systematic manner to allow more air space in the filter fun- nel; this allows liquid to flow faster through the filter paper. Foley-Dorsey equation: A correc- tion of the plate count and retention time to correct for peak tailing from extracolumn sources of broadening. (See J.P. Foley and J.G. Dorsey, Anal. Chem. 55, 730–737 [1983].) Forced-flow leaching: Solid mate- rial is packed into stainless steel col- umn and toluene (or other extraction solvent) is pumped into the column under pressure and with heating; hot solvent leaches (extracts) out extract- able compounds which are collected at the exit of the column. Fraction of analyte extraction (E): The fraction of analyte extracted: E = (CoVo)/(CoVo + CaqVaq) = (KDV)/(1 + KDV ) where Vo is the volume of or- ganic phase, Vaq the volume of aque- ous phase, and V is the phase ratio Vo/Vaq. Fractionation range: In SEC, refers to the operating range of a gel or pack- ing. This is the range in which the packing can separate molecules based on their size. At one end of the range, molecules that are too large to diffuse into the pores are excluded. At the other end of the range, molecules that can diffuse into all of the pores totally permeate the packing are eluted (un- separated) at the permeation volume. Freeze drying: The process of removing water, mainly from biological samples, by using vacuum sublimation. Frictional heating: Viscous heating of solvent molecules passing through very small-diameter (micrometer) particles; causes a rise in temperature over the length of the column; reduced by using smaller-diameter columns (smaller heat dissipation) and superficially porous particles, which also have improved heat dissipation. Frit: The porous element at either end of a column that serves to contain the column or SPE packing. It is placed at the very ends of the column tube or in the end fitting. Frits can be stainless steel or another inert metal or plastic such as porous PTFE or polypropylene. The frit porosity must be less than the smallest particle in the column; other- wise particles will pass through the frit and packing will be lost. Frontal analysis: A chromatographic technique that involves continuous ad- dition of sample to the column with the result that only the least sorbed compound, which moves at the fast- est rate, is obtained in a pure state. The second-least-sorbed compound is eluted with first eluted compound, the third-least-sorbed compound with the first and second compound, and so forth until the original sample passes through the column exit. Frontal analysis is seldom used and is mainly a preparative technique. Frontal chromatography: The same as frontal analysis. Fronting: Peak shape in which the front part of the peak (before the apex) in a chromatogram tapers in advance of the remainder of the peak (that is, the front is less steep than the rear). There is an asymmetric distribution with a leading
- 34. 34 Terminology Guide October 2013 www.chromatographyonline.com edge. The asymmetry factor for a front- ing peak has a value less than 1. The opposite effect is tailing. Fronting can result at high sample loads because of positive curvature in the isotherm and from use of poorly packed columns. See asymmetry factor. Fused-core packing: See superficially porous particles (SPPs). Fused silica: Open-tubular GC col- umns and LC nanocolumns are com- monly manufactured from fused-silica tubing that is coated externally with a protective polymeric material such as polyimide. Fused-silica open-tubular column (FSOT): Open-tubular GC columns made of fused silica. See open-tubular column. G Gas–liquid (phase) chromatography (GLC, GLPC): Solutes partition between a gaseous mobile phase and a liquid stationary phase. Selective interactions between the solutes and the liquid phase give rise to different retention times in the column. Gas-phase extraction: See direct ther- mal sampling. Gas–solid chromatography (GSC): Sol- utes partition between a mobile gaseous phase and a solid stationary phase. Se- lective interactions between the solutes and the solid phase give rise to different retention times in the column. Gaussian curve: A standard error curve, based on a mathematical function, that is a symmetrical, bell-shaped band or peak. Most chromatographic theory assumes a Gaussian peak. Use of the peak maximum position as a measure of retention and equations mentioned above for efficiency assume a Gaussian peak shape. Gaussian peak: The equation of a Gaussian distribution can be written in terms of chromatography parameters as C = Cmaxexp(–(t – tR )2/2σ2) Gel: The solid packing used in gel chromatography or GPC. An actual gel consists of two parts: the dispersed me- dium (solid portion) and the dispersing medium (the solvent). Also defined as a colloidal dispersion of a solid and liq- uid in which the solid is the continuous phase. Gel filtration chromatography (GFC): Also called aqueous size-ex- clusion chromatography; carried out with aqueous mobile phases. Gener- ally refers to molecular size separa- tions performance on soft gels such as polydextrans but highly crosslinked polymers, silica gels, and other po- rous media may also be used. Most gel filtration separations involve bio- polymers and water-soluble polymers such as polyacrylic acid. Gel permeation chromatography (GPC): Size-exclusion chromatography (SEC) carried out with organic mobile phases used for the separation and characterization of polymers. SEC with aqueous mobile phases is referred to as gel filtration chromatography. Ghost peaks: Peaks not present in the original sample. Ghost peaks can be caused by septum bleed, solute decom- position, or carrier-gas contamination. Gigapores: See perfusion chromatog- raphy. Golay equation: M.J.E Golay formu- lated an equation for the theoretical plate height versus the average linear velocity of open-tubular (capillary) col- umns. The Golay equation is similar to the van Deemter equation, except that the A term is dropped because there is no column packing, and the B and C
- 35. Terminology Guide October 2013 35 www.chromatographyonline.com terms are modified accordingly as well: h = (B/u –) + u –(CM + CS) Grab sampling: In gas sampling, an evacuated sample canister can be opened and sample rapidly collected at an uncontrolled rate, usually over several seconds, until the container reaches equilibrium with atmospheric pressure. Generally this qualitative ap- proach is used when unknown analytes must be identified, when the air con- tains high concentrations of analytes at certain (short) times, or when an odor is noticed and a sample must be obtained quickly. Paired grab samples (before–after or smell–no smell) often are used to qualitatively diagnose a per- ceived problem. Gradient: A process to change solvent strength as a function of time (normally solvent strength increases) thereby elut- ing progressively more highly retained analytes; typically gradients can be bi- nary, ternary, and quaternary solvent mixtures where solvents are blended to achieve the proper strength. In GC, an older term that refers to column temperature programming to achieve a similar effect. Gradient delay volume: See dwell volume. Gradient elution: Technique for de- creasing separation time by increasing the mobile-phase strength over time during the chromatographic separa- tion. Also known as solvent program- ming. Gradients can be continuous or stepwise. Binary, ternary, and quater- nary solvent gradients have been used routinely in HPLC. Graphitized carbon: Graphitized car- bon is a graphitic carbon with more or less perfect three-dimensional hex- agonal crystalline order prepared from non-graphitic carbon by graphitization heat treatment; this packing material has a strong affinity for polar com- pounds in aqueous samples and water miscible organic extracts. Commonly used in pesticide analysis of food sam- ples; also known as graphitized carbon black (GCB). Also used as a GC station- ary phase. Graphitized carbon packing: A re- versed-phase packing material which consists of pure graphitic carbon; possesses interesting sorbent proper- ties such as preferential separation of geometric isomers such as o-, m- and p-aromatics and cis–trans isomers. Grinding: Both manual and automated mortar and pestles are available; grind- ing can be performed under wet or dry conditions; by this process particles of approximately 10 µm can be achieved. Guard column: A small column placed between the injector and the analyti- cal column. It protects the analytical column against contamination by sam- ple particulates and strongly retained species. The guard column is usually packed with the same material as is in the analytical column and is often of the same inner diameter. Its length is much shorter, it costs less, and is usu- ally discarded when it becomes con- taminated. Integrated guard–analytical column systems are often preferred to minimize extracolumn effects caused by the use of connecting tubing with separate guard and analytical columns. For GC, see also retention gap. H Headspace sampling: Gas-phase sam- pling technique in which solute is re- moved from an enclosed space above a solid or liquid sample. The headspace refers to the vapors that form above liquids and solids; if the sample is in
- 36. 36 Terminology Guide October 2013 www.chromatographyonline.com thermodynamic equilibrium with the gas phase in a closed thermostated vessel, the method is called static headspace sampling; if an inert gas passes over or through the sample and stripped sample volatiles accumulate in an adsorbent or cryogenic trap, the method is dynamic headspace or purge- and-trap sampling. Headspace single-drop microextrac- tion (HS-SDME): A single drop of sol- vent (1–2 µL) suspended in the head- space can partition volatile analytes into the solvent; the drop can be with- drawn into the syringe and injected into a GC system. Headspace solid-phase microextrac- tion (HS-SPME): Instead of a drop of solvent, a polymer-coated fiber can be placed in the headspace and once the analytes adsorb on the polymer coat, the fiber can be transferred to a GC inlet and the sorbed analytes volatilized by thermal desorption. Heart cutting: In preparative LC, re- fers to collection of the center of the peak where purity should be maximum. The term is also used for analytical col- umn switching for LC or GC in which two or more partially resolved peaks eluted from one column are directed onto another column of different po- larity, or at a different temperature, for improved resolution. Helium ionization detection (HID): An ionization detection method for GC. Helium is ionized by a radioac- tive or other energetic source; the re- sulting He2+ ions interact with solutes to produce ions that measured with a sensitive electrometer. Helium plasma or microwave-in- duced detection (HPD): A helium plasma is created in a microwave RF field. The plasma emits UV radiation that ionizes solute molecules, which are measured with an electrometer. Meta- stable He may also be harnessed in an electron-capture mode for halogen- specific response. See also pulsed dis- charge detection. Helium sparging: See degassing. He- lium has a very low solubility in most common liquids. High performance capillary electro- phoresis (HPCE): A technique where small diameter capillaries, buffered conducting solutions, and high voltage (up to 30,000 V) are used to separate ionic molecules based on their differ- ential electrophoretic mobilities; non- ionic (neutral) molecules can be sepa- rated by MEKC. High performance liquid chromatog- raphy (HPLC): Modern liquid-phase chromatography technique using small particles and high pressures. High-pressure mixing: A configura- tion of a gradient HPLC system where the solvents are mixed on the high- pressure side of multiple pumps (usu- ally two, binary); such a system offers a lower gradient delay volume than low pressure mixing systems where the solvents are mixed by proportioning valves before a single pump. High-abundance protein depletion: By using antibody columns specific for the highest abundance proteins, they can be selectively removed from plasma. This process enables investiga- tion of the lower abundance proteins, which may be biomarkers or other com- pounds of interest. Hold-up time (tM, t0): The time re- quired for an unretained compound to be eluted, or the time required for one column volume (VM) of mobile phase to pass through the column. In reversed-phase LC, uracil is often used
- 37. Terminology Guide October 2013 37 www.chromatographyonline.com to measure hold-up volume and hold- up times. In GC, methane or another unretained compound is used to mea- sured the column hold-up time. Also called the unretained peak time or dead time. Hold-up volume (VM or V0): The total volume of mobile phase in the column regardless of where it exists. In LC: The hold-up volume consists of the entire space accessible to a small molecule able to fully permeate all the pores of the packing material. It comprises the interstitial volume and the unoccupied pore volume. It is denoted as Vo or VM. The system hold-up volume includes the ad- ditional volume in the tubing used to connect the injector and detector to the column. The hold-up volume is usually approximated by inject- ing a small essentially unretained species. In reversed-phase LC, ura- cil, acetone, and thiourea are most commonly used: VM = tMFc. In GC: The gas-phase volume of the column that corresponds to the hold-up time. Measured by injecting an unretained species such as methane that fits in all the pores. See hold-up time. Hold-up volume, corrected (VM 0): The hold-up volume corrected for carrier- gas expansion along the column: VM 0 = V j Hollow-fiber liquid-phase microex- traction (HF-LPME): A hollow-fiber (HF) membrane technique where an HF membrane separates two extrac- tion phases; the membrane serves as a barrier and can be impregnated with solvent to permit liquid–liquid or liq- uid–liquid–liquid extractions to take place; the membrane can be selected to allow certain analytes to pass through but not others. Homogenization: The process of mak- ing a sample more uniform by size re- duction and blending; homogenizers with high speed blades are available to do the job. Hybrid silica: Silica gel consisting of both organic and inorganic moieties with hybrid properties of polymeric packings and silica packings; synthe- sized from silanes containing organic functionality; different selectivity but higher high pH stability than bare or uncoated silica gel. Hydrodynamic volume: The molecu- lar volume defined by the effective di- ameter of a molecule in free solution where the hydrodynamic sphere would be a sphere defined by the molecule as it revolves around its central axis in solution; termed used in size-exclusion chromatography to define molecular shape and to explain why molecules with the same molecular weight often have totally different elution volumes; measured by determining the Stokes radius. Hydrophilic: “Water loving”; refers both to stationary phases that are fully compatible with water or to water-sol- uble molecules in general. Many col- umns used to separate proteins (ion ex- change, SEC, affinity) are hydrophilic in nature and should not irreversibly sorb or denature protein in the aque- ous environment. Hydrophilic interaction liquid chro- matography (HILIC): An alternative technique to reversed-phase HPLC for the separation of highly polar analytes that may be only slightly re- tained or unretained by reversed-phase LC, HILIC requires a high percent- age of a nonpolar mobile phase and a polar stationary phase, similar to the requirements in normal-phase
- 38. 38 Terminology Guide October 2013 www.chromatographyonline.com chromatography. However, unlike normal-phase chromatography, which uses nonpolar solvents such as hexane and methylene chloride and tries to exclude water from the mobile phase, HILIC requires some water in the mobile phase to maintain a stagnant enriched water layer on the surface into which analytes may selectively partition. In addition, water-miscible organic solvents are used instead of the water-immiscible solvents used in normal-phase chromatography. With HILIC, sorbents such as bare silica, bonded diol, and polyhydroxyethylas- partamide are used. Polar analytes are well retained and are eluted in order of increasing hydrophilicity, just the inverse of reversed-phase LC. Hydrophobic: “Water hating”; re- fers both to stationary phases that are not compatible with water or to molecules in general that have little affinity for water. Hydrophobic molecules have few polar functional groups; most have a high content of hydrocarbon (aliphatic and aromatic) functionality. Hydrophobic interaction chroma- tography (HIC): A technique in which weakly polar (nonhydrocarboneous) packings are used to separate molecules by virtue of the interactions of their hy- drophobic moieties and the hydropho- bic sites on the packing surface. High concentrations of salt solutions are used in the mobile phases and separations are affected by changing the salt con- centration. The technique is analogous to “salting out” molecules from solu- tion. Gradients are run by decreasing the salt concentration; often used for the separation of proteins that are sen- sitive to denaturization by the organic solvents used in regular reversed-phase chromatography; usually little or no organic solvent is used in the mobile phase in HIC. Hydrophobic subtraction model: Developed by Lloyd Snyder and John Dolan, this model is used to charac- terize reversed-phase columns; using five types of probes, based on their equations, they can predict if a cer- tain column will be close in selectiv- ity characteristics to another column or totally different (orthogonal); over 300 columns have been characterized using this model. (See L. Snyder and J. Dolan, LCGC 20[11], 1016–1026 [2002].) Hydroxyapatite: A porous calcium hydroxyphosphate solid that chemi- cally resembles bone and tooth used as a packing material used in bio- chromatography for nucleic acid con- stituents, monoclonal antibodies, and proteins. Hypercrosslinking: Mainly refers to a new way to synthesize a polymeric monolith; hypercrosslinked monolithic capillary columns contain an array of small pores and have very high surface areas. Hyphenated techniques: Refers to the family of techniques best known by their abbreviations, including LC– MS, LC–FTIR, and MS-MS. I Immobilized liquid extraction: Simi- lar to SPE but a polymeric stationary phase is bonded to the inside of a glass vial; analytes partition into polymeric phase and loading, washing, and elu- tion steps are performed by addition of various solvents. Immobilized metal affinity chroma- tography (IMAC): See metal affinity chromatography.
- 39. Terminology Guide October 2013 39 www.chromatographyonline.com Impinger: Impingers are glass bubble tubes designed for the collection of airborne hazards into a liquid medium. When using a personal air sampler, a known volume of air bubbles is pumped through the glass tube that contains a liquid specified in the method. The liq- uid is then analyzed to determine air- borne concentrations. An impinger may be mounted on the side of an air sample pump or put into a holster and placed near a worker’s breathing zone. Imprinted phases: Polymer and silica phases generated in the presence of a “template” or “printing” molecule. Such phases have enhanced selectivity for the templating molecule; also called molec- ularly imprinted phases (MIPs). In-line filter: A device that prevents particulate matter from damaging the column. Modern low-volume, in-line filters can be placed between the injec- tor and the column without major con- tributions to band broadening. A filter in this position is used to prevent sample particles from entering the packed bed or column inlet frit. In situ derivatization: The act of de- rivatizing a compound of interest in its native environment; for example, for a tenaciously held analyte on a soil sample changing its chemical nature by performing an in situ derivatization, the compound may be more easily re- leased and isolated for further workup or analysis; derivatization may also be formed in solution and the derivatized compound extracted by LLE. Included volume: Also known as totally included volume. The volume at which a small molecule that explores the en- tire pore space of a column is eluted. See steric-exclusion chromatography. Indirect detection: Used for non-ul- traviolet-absorbing or nonfluorescing analytes; a UV-absorbing or fluores- cent compound is added to the mo- bile phase that maintains a high back- ground signal; when a nonabsorbing or nonfluorescing analyte is eluted, the background is diluted and a nega- tive peak is observed for that analyte; when an analyte acts to increase the concentration of the indicating species, a positive peak is observed. When a negative signal is detected, the detec- tor signals are reversed to the output device. Infinite diameter column effect (IDE): Name given by John Knox to the following phenomenon: At a cer- tain column length, a sample injected into center of a packed bed spreads by radial diffusion but never reaches col- umn wall, where wall effects can cause band broadening. Knox showed that a sample peak collected in the exact center of the column exit displayed a higher efficiency than a sample peak collected near the wall. The infinite diameter effect depends on column length, internal diameter, particle size, and mobile-phase properties. Very sel- dom applied in HPLC. Injection solvent: Solvent used to inject sample into an HPLC column; solvent should be of equal or lower strength than the mobile phase to prevent pre- mature movement down the column due to the presence of a stronger solvent. Inlet: In LC, the initial part of the col- umn where the solvent and sample enter. There is usually an inlet frit that holds the packing in place and, in some cases, protects the packed bed. In GC, a de- vice between the carrier gas source and the column inlet that transfers sample from outside the chromatograph into the column, often by vaporizing the sample. See split injection, splitless
- 40. 40 Terminology Guide October 2013 www.chromatographyonline.com injection, on-column injection, pro- grammed temperature vaporizer. Inlet filter: Filtration devices attached to the inlet lines of the pump that re- moves particulate matter from the mo- bile phase before the solvent reaches the pump; reservoir filters are an inlet filter that resides in the solvent bottle. Inlet liner: Deactivated glass tube in an inlet system into which liquid sample is injected. An inlet liner may be open or packed with deactivated glass wool, and it may have various internal structures. The purpose is to vaporize and disperse evaporating sample into the carrier gas stream as uniformly as possible while not causing significant sample break- down, adsorption, or discrimination. Inlet/outlet check valves: The check valve (or valves) on an LC pump that allows mobile phase to flow in one di- rection but not in the reverse direction. The inlet check valve allows flow from the reservoir into the pump, and the outlet check valve allows mobile phase to flow to the column from the pump. Instrumental bandwidth: The contri- bution of the analytical instrument to peak broadening; see extracolumn ef- fects for explanation. Instrumental dispersion: See extracol- umn effects. Internal standard (IS): In quantitative analysis, precision and accuracy are greatly improved by use of internal stan- dards (IS). The procedure involves the addition of a fixed amount of internal standard to a series of increasing con- centrations of reference sample and to the unknown concentration. The ratio of the areas of the reference concentra- tions and the areas of the IS is plotted against the known concentration of the reference samples. The internal standard should be chemically similar to the sub- stance being quantitatively determined and should have a retention time fairly close to it. Interparticle porosity (ε): The inter- particle volume of a packed column per unit column volume: ε = Vo/Vc. See also interstitial porosity. Interstitial porosity (εe): The fraction of the volume in the column located in the interparticle (interstitial) space: εe = Ve/Vc Interstitial volume (Ve): The volume between the particles. Does not include the volume in the pores of the particles. Also called the excluded volume (see steric-exclusion chromatography) and interparticle volume. Measured by injecting a molecule which does not per- meate any pores and does not interact with the surface of the particles. In SEC this volume is denoted Vo. Intraparticle porosity (εi): The fraction of the particle volume which lies in the pores: εi = Vpore/Vparticle Intraparticle volume (V): The volume inside the pores of the particles. Also called the internal volume and included volume. Can be measured by BET or mercury intrusion porosimetry. Ion chromatography (IC): An ion- exchange technique in which low con- centrations of organic and inorganic anions or cations are determined using ion exchangers of low ion-exchange ca- pacity with dilute buffers. Conductivity detectors are often used. Ion chroma- tography is practiced in two forms. In suppressed IC, a second column or a membrane separator is used to simulta- neously remove the buffer counterion to the analyte and replace it with hydrogen or hydroxide ion, which concomitantly converts the buffer to an uncharged spe- cies, thereby supressing background and enhancing sensitivity. In nonsuppressed IC, weakly conducting buffers at low
- 41. Terminology Guide October 2013 41 www.chromatographyonline.com concentration are carefully selected and the entire effluent is passed through the detector — ions are detected above the background signal. Ion exclusion: The process in which ionized solutes can be separated from unionized or partially ionized solutes using ion-exchange resins. Separation results from Donnan potential where ionic solutes exist at a higher concen- tration in solution than in the station- ary phase whereas nonionic solutes are evenly distributed between the mobile phase and resin. Therefore, ionic sol- utes will move faster down the column than non-ionic solutes. Ion exclusion is known to take place in reversed-phase LC when anions are separated at pH val- ues where the silanol groups are ionized. Ion moderated partitioning chroma- tography: A technique used for the separation of carbohydrates using strong cation-exchange packings that are in specific cationic form (for example, cal- cium, hydrogen, silver); the separation mechanism is complexation rather than ion exchange. Ion retardation: Refers to the use of amphoteric ion-exchange resins, which are used to retard ionic molecules and allow nonionic molecules or nonelectro- lytes to be eluted preferentially. Ion suppression: Buffering in an aque- ous mobile phase at a particular pH to suppress solute ionization. For example, weak carboxylic acids can have their ion- ization suppressed by the adjustment of the pH below their pKa. Useful for im- proving peak shape of weak acids and bases in reversed-phase LC. Ion-trap detector: Mass spectrometric detector that uses an ion-trap device to generate mass spectra. Ion-exchange capacity: The number of ionic sites on the packing that can take place in the exchange process. The exchange capacity is expressed in mil- lieqivalents per gram. A typical styrene- divinylbenzene strong anion exchange resin may have 3–5 meq/gm capacity. Exchangers for IC have very low capac- ity. Capacity of weak anion and cation exchangers varies dramatically with pH. Ion-exchange chromatography: A mode of chromatography in which ionic substances are separated on cationic or anionic sites of the packing. The sample ion (and usually a counterion) will ex- change with ions already on the iono- genic group of the packing. Retention is based on the affinity of different ions for the site and a number of other solution parameters (pH, ionic strength, coun- terion type, and so forth). Ion chroma- tography is basically an ion-exchange technique. Ion-pair chromatography: Form of chromatography in which ions in solu- tion can be “paired” or neutralized and separated as an ion pair on a reversed- phase column. Ion-pairing agents are usually ionic compounds that contain a hydrocarbon chain that imparts a cer- tain hydrophobicity so that the ion pair can be retained on a reversed-phase col- umn. Retention is proportional to the length of the hydrophobic chain and the concentration of the ion-pair additive. Ion pairing can also occur in normal- phase chromatography when one part of the pair is dynamically loaded onto a sorbent, but this technique is not as pop- ular as reversed-phase LC. Also known as ion-interaction chromatography or dynamic ion-exchange chromatogra- phy, stressing the fact that the precise mechanistic details of how the additive controls retention are not always known. Ionic strength: Ionic strength is a characteristic of an electrolyte solution.
- 42. 42 Terminology Guide October 2013 www.chromatographyonline.com It is typically expressed as the average electrostatic interactions among an electrolyte’s ions. It is related to elec- trolyte concentration, but the main difference between ionic strength and electrolyte concentration is that the ionic strength is higher if some of the ions are more highly charged. The higher the ionic strength of a mo- bile phase the more the mobile phase competes with the analyte for ionic or adsorptive sites. Irregular packing: Refers to the shape of a column packing. These irregular packings are available in micropartic- ulate sizes. The packings are obtained from grinding solid materials into small particles and then sizing them into narrow fractions using classifica- tion machinery. Spherical packings are now used more than irregular pack- ings in analytical HPLC but the less- expensive irregular packings are still widely used in preparative LC. Irreversible adsorption: When a compound that has a very strong af- finity for an adsorbent is injected onto a column, it can be adsorbed so strongly that it cannot be eluted from the column. A chemical reaction be- tween the sample and the surface of the adsorbent is an example of irre- versible adsorption. Isocratic: Use of a time-invariant eluent composition in LC. Isolate: Analyte to be isolated from ma- trix background and then analyzed. Isotherm: See adsorption isotherm. Isothermal chromatography: Use of conditions of constant column tem- perature. The vast preponderance of all LC is done under isothermal condi- tions, while most GC separations are performed with column temperature programming. Isotope-coded affinity tags (ICAT): Isotope-coded affinity tags (ICATs) are a gel-free method for quantitative proteomics that relies on chemical la- beling reagents. These chemical probes consist of three general elements: a reac- tive group capable of labeling a defined amino acid side chain (for example, iodoacetamide to modify cysteine resi- dues), an isotopically coded linker, and a tag (for example, biotin) for the af- finity isolation of labeled proteins and peptides. For the quantitative compari- son of two proteomes, one sample is labeled with the isotopically light (d0) probe and the other with the isotopi- cally heavy (d8) version. To minimize error, both samples are then combined, digested with a protease (such as tryp- sin), and subjected to avidin affinity chromatography to isolate peptides labeled with isotope-coded tagging reagents. These peptides are then ana- lyzed by liquid chromatography-mass spectrometry (LC–MS). The ratios of signal intensities of differentially mass- tagged peptide pairs are quantified to determine the relative levels of proteins in the two samples. The original tags were developed using deuterium, but later the same group redesigned the tags using 13C instead to circumvent issues of peak separation during LC because of the deuterium interacting with the stationary phase of the column. K Kieselguhr: A diatomaceous earth used both in column chromatography and as a sample cleanup media. Only weakly adsorptive, it is also used as a support in liquid–liquid chromatography and in supported liquid extraction, see sup- ported liquid extraction (SLE). Rarely used in HPLC.
- 43. Terminology Guide October 2013 43 www.chromatographyonline.com Kilopascal (kPa): A unit of pressure. 100.0 kPa = 1.0 bar = 0.9869 atm. 101.325 kPa = 1.0 atm. Kinetic plot: Kinetic plots are methods to characterize the practical limits of column performance, where theoretical plates (H) and separation impedance (E) are plotted as a function of the pressure- drop limited plate number (N). The ki- netic plot retains the information shown in van Deemter plots but completes it with the information on the bed perme- ability. See Poppe plot. Knox equation: A modification of the van Deemter equation developed by John Knox that uses reduced terms, in which the A term that represents eddy dispersion is replaced with Aν1⁄3 where ν is the reduced interstitial eluent velocity. L Laminar flow: The smooth time-in- variant flow that develops when a liq- uid is moving under conditions where viscous forces dominant over inertial forces. Laminar flow is characterized by a low Reynolds number. For flow in a cylindrical tube fluid stream-lines near the center move faster than those at the tube wall which are static result- ing in a radially parabolic distribution in axial fluid velocity. This nonuniformity of axial velocities in the interstices in a packed bed also leads to substantial peak broadening in packed columns. Langmuir adsorption isotherm: A spe- cific form of an isotherm CS = (N0CM)/ (Kd + CM) where CS and CM are the equilibrium stationary and mobile phase concentrations of the solute, N0 the total number of surface sites avail- able for sorption and Kd the sorption binding constant. Large-volume injection (LVI): A tech- nique for introduction of larger than normal volumes of sample in a solvent into a capillary GC column; in this approach, the bulk of the solvent is evaporated before the sample transfers to the analytical column; there are two popular LVI techniques: programmed temperature vaporization and cool on- column injection with solvent vapor exit; both are approaches to lowering detec- tion limits. Ligand: In ligand-exchange chromatog- raphy, refers to the analyte that under- goes ligand exchange with the stationary phase. In affinity chromatography, re- fers to the biospecific material (enzyme, antigen, or hormone) coupled to the support (carrier) to form the affinity col- umn. In bonded-phase chromatography this term denotes the moiety covalently bound to the surface. Ligand-exchange chromatography: A technique in which chelating li- gands are added to the mobile phase and undergo sorption onto a pack- ing. These sorbed molecules can act as chelating agents with certain sol- utes. An example would be the use of copper salt added to the mobile phase for the chelatation and separation of amino acids. Chelating resins function in a similar manner, where chelating groups are chemically bonded to the polystyrene backbone. Limit of detection (LOD): The concen- tration of the analyte at which the re- sulting peak can be distinguished from baseline noise. Literature and norms describe different ways of determining the LOD. Limit of quantitation (LOQ): The minimum concentration of the analyte at which the resulting peak can be quan- tified with a defined level of certainty. Typically 3–5 times higher than the LOD.
- 44. 44 Terminology Guide October 2013 www.chromatographyonline.com Linear elution adsorption chromatog- raphy (LEAC): A term coined by Lloyd Snyder; refers to adsorption chromatog- raphy carried out in the linear portion of an adsorption isotherm; sometimes referred to as linear chromatography. Linear range (LR): Also, linear dynamic range. The range of solute concentration or amount over which detector response per solute amount is constant within a specified percentage. Linearity: In quantitative analysis, it is essential for the detector to yield a linear response with respect to solute concen- tration; some detectors may show non- linear performance in certain concentra- tion ranges, especially on the high end but also on the low end. Liquid chromatography: A separation technique in which the mobile phase is a liquid; most often carried out in a column. Liquid phase: In GC, a stationary liq- uid layer coated on the inner column wall (WCOT) or on a support (packed, PLOT, SCOT) that selectively interacts with different solutes to produce differ- ent retention times. Also refers to the stationary phase in LLC. Liquid–liquid chromatography (LLC): Same as partition chromatography. One of the earliest separation modes of HPLC; it gave way to chemically bonded phases in the early 1970s. Liquid–liquid diffusion coefficient (DL): See diffusion coefficient. Liquid–liquid extraction (LLE): LLE is an extraction technique for separat- ing interferences from the analytes by partitioning the analytes between two immiscible liquids (or phases); one phase is usually aqueous and the second phase an organic solvent; more hydrophilic compounds prefer the aqueous phase while more hydrophobic compounds will be found in the organic solvent; by the use of additives (for example, buf- fers or ion-pair reagents), equilibria can be shifted to “force” analytes or matrix compounds into one or other of the two layers. Liquid-phase microextraction (LPME): A liquid extraction technique where there is a great reduction in the accep- tor-to-donor phase ratio; a hollow fiber is impregnated with an organic solvent used to accommodate or protect micro- volumes of acceptor solution. This novel methodology proved to be an extremely simple, low-cost, and virtually solvent- free sample-preparation technique that provided a high degree of selectivity and enrichment by additionally eliminating the possibility of carryover between runs. Liquid–solid chromatography (LSC): Same as adsorption chromatography. Liquid–solid extraction: The general expression for extraction techniques that uses an organic solvent to extract analytes from a solid material. In its simplest form, the “shake flask” extrac- tion takes place at room temperature and works well for the case where the matrix is porous and the analytes are easily extractable. Loadability: The maximum amount of analyte that can be injected onto a col- umn above which it no longer permits the isolation of product at the desired level of purity or recovery level; impor- tant in preparative chromatography. Loading (phase loading versus sam- ple loading): The amount of stationary phase coated or bonded onto a solid sup- port. In liquid–liquid chromatography, the mass of liquid phase per gram of packing. In bonded-phase chromatog- raphy, the loading may be expressed in µmol/m2 or %C (w/w); also called cov- erage or surface coverage. An alternate
- 45. Terminology Guide October 2013 45 www.chromatographyonline.com (unrelated) meaning is the amount of sample mass that is injected on an ana- lytical or preparative column; prepara- tive columns are often operated in an overloaded condition for throughput reasons. Loading step (SPE): The second step in SPE (after conditioning) where the sample is loaded onto the SPE phase (cartridge). Log KW: In reversed-phase LC, the ex- trapolated intercept of a plot of log k ver- sus volume fraction of organic modifier in reversed-phase LC. See also solvent strength (S). Longitudinal diffusion: Same as mo- lecular diffusion term; B term in van Deemter equation; see van Deemter equation. Low pressure mixing: See high pres- sure mixing. Lyophilization: The process of dehy- drating a sample, often biological, con- taining water by using vacuum sublima- tion; also referred to as freeze drying. M Maceration: The process of breaking down a soft material into smaller parts by tearing, chopping, cutting, and so forth. Macroporous resin (macroreticular): Cross-linked ion-exchange resins that have both micropores of molecular di- mensions but also macropores of sev- eral hundred angstroms wide. These are highly porous resins with large internal surface area accessible to large molecules. Magnetic-bead technology: Micro magnetic beads are uniform polymer particles, typically 0.5–500 µm in di- ameter, that have iron oxide particles (or other particles that may be at- tracted to magnets) contained within the polymer matrix. Bioreactive mol- ecules can be adsorbed or coupled to their surface, and used to separate biological materials such as cells, pro- teins, or nucleic acids; by the use of magnets or magnetic fields, the beads can be easily manipulated in test tubes or 96-well plates. These microbeads are used for isolation and handling of specific material or molecules, as well as for analyzing sensitive molecules or those that are in low abundance (for example, in miniaturized and auto- mated settings). Make-up gas: Extra carrier gas or other gas added to the carrier gas as it flows into or through a detector. Make-up gas serves to improve peak shapes for open-tubular columns with detectors not necessarily designed for them exclusively by reducing the ef- fects of detector dead volume. Also, make-up gas may play an active role in detector operation, as for example when hydrogen serves as both make- up and combustion gas in a flame ion- ization detector. Mass spectrometric (MS) detector: Chromatography detector that records mass spectra of solutes as they are eluted from the column. Mass transfer (inter-phase) (C term): The process of solute movement be- tween the moving and stationary zones. The C term of the van Deemter equa- tion is referred to as the interphase mass transfer term. The faster the process of mass transfer the better the efficiency of column. In HPLC, slow mass transfer is the most important fac- tor affecting column efficiency. Its rate can be increased by the use of small- particle packings, thin layers of station- ary phase, low viscosity mobile phases, and high temperatures.
- 46. 46 Terminology Guide October 2013 www.chromatographyonline.com Matrix: In sample preparation, the ma- trix normally refers to the substance in which the analyst is attempting to mea- sure a solute or series of solutes; often the matrix is of no interest, and its concen- tration must be reduced or eliminated for a separation and measurement to take place; the matrix can be organic, inorganic, or biological. Matrix adsorption mode (SPE): A lesser used mode of SPE where the sorbent is chosen to maximize retention of the matrix and other interferences while the analyte of interest is unretained; the op- posite of the normal process (bind–elute) of SPE; there is no concentration of the analytes. Matrix solid phase dispersion (MSPD): Technique uses bonded phase solid supports as abrasives to produce disruption of sample architecture and as a bound solvent to aid complete sample disruption during the sample blending process; the finely divided sample is gently blended with a mor- tar and pestle, transferred to a column, and the analytes eluted with appropri- ate solvents. Matrix-solid phase extraction (MASE): See matrix solid phase dispersion. Maximum allowable operating tem- perature (MAOT): Highest continuous column operating temperature that will not damage a column, if the carrier gas is free of oxygen and other contami- nants. Slightly higher temperatures may be permissible for short periods of time during column bake-out. McReynolds constants: System for stationary-phase characterization. McReyonolds expanded on the ear- lier Rohrschneider polarity probes. The retention indices of a series of test probes such as benzene, 1-buta- nol, methyl-n-propylketone, nitropro- pane, and pyridine are taken together to express the overall phase polarity, or separately to express the station- ary-phase behavior toward individual compound classes. Mean pore diameter: The average pore diameter of the pore in a porous packing. It is most commonly deter- mined by BET analysis and is reported as four times the specific pore volume divided by the specific surface area based on the assumption of uniform cylindrical pores. The pore diameter is important in that it must allow free dif- fusion of solute molecules into and out of the pore so that the solute can inter- act with the stationary phase. Addition- ally, the pores must be well-connected, with a minimum of dead ends, so that there are many paths that can allow a molecule to access any part of the pore space. In SEC, the packings have dif- ferent pore diameters and therefore molecules of different sizes can be separated. For a typical substrate such as silica gel, 60- and 100-Å pore diam- eters are most popular. For packings used for the separation of biomolecules, pore diameters > 300 Å are used. Megapascal (MPa): A unit of pressure; 1 MPa = 10 bar, 10.133 atm, or 145.0 psi. Megapores: See perfusion chromatog- raphy. Membrane extraction with sorbent interface (MESI): A version of dy- namic headspace where a silicone hol- low fiber membrane is placed in the headspace about the sample; an inert gas is passed through the membrane and analytes that are permeable to the membrane pass from the head- space and are swept to an adsorbent trap; after a period of concentration, the trapped analytes are thermally de- sorbed to the GC column.
- 47. Terminology Guide October 2013 47 www.chromatographyonline.com Membrane filtration: Membrane filter; membrane disk. Membrane suppressor: Continuous chemical suppression. Ion exchange through ion-exchange membranes. H+ or OH- are supplied by the respective regeneration solution (for example, sul- furic acid). Metal affinity chromatography: A special form of ligand exchange chro- matography used for the separation of biopolymers with a particular affin- ity for a specific metal cation typically copper(II), zinc(II), and iron(II). Metalophile: A compound that has high affinity for active (acidic) silanol groups on silicas surface. Usually a strongly basic amine. Method detection limit (MDL): The minimum amount of solute that can be analyzed within specified statistical lim- its of precision and accuracy, including sample preparation. Method development: A process of optimizing the separation including the sample pretreatment so as to obtain a reproducible and robust separation. Usually it emphasizes the search for the stationary phase, eluent, and column temperature combination that provides an adequate separation. Method translation: Several math- ematical techniques for adjusting GC method parameters for variations in the carrier gas type or column dimensions, with the objective of maintaining either the same or ratiometric retention times. Useful when changing from helium to hydrogen carrier gas, or when increas- ing speed of analysis or resolution by adjusting column dimensions. Not use- ful if changing the stationary phase to a chemically different type. Method validation: A process of test- ing a method to show that it performs to the desired limits of precision and accuracy in retention, resolution, and quantitation of the sample components of interest. Micellar chromatography: The addi- tion of micelles to the mobile phase to effect separations. The micelles may act as displacing or partitioning agents and provide another parameter which may be used to change selec- tivity. Surfactants above their critical micelle concentration are used in mi- cellar chromatography and in MEKC form of CE. Micro LC: Refers collectively to tech- niques where a column of smaller than conventional internal diameter is used for separation. The term micro LC is most often used for HPLC in <0.5-mm i.d. columns; micro LC is used in high sensitivity analysis when the sample amount is limited and with certain ion- ization techniques in LC–MS where the volume of solvent flowing into the ion- ization source must be minimized. Microbore: Refers to the use of col- umns with smaller-than-usual internal diameters in HPLC. Columns with internal diameters of 2 mm and below are considered to be microbore columns; columns with internal diameters below 0.5 mm are referred to as micro LC col- umns. In GC, microbore may signify columns with inner diameters less than 200 µm. Microchip devices: Microdevices based on silicon, glass, and other types of mi- crofabricated chips where experiments can be miniaturized into single- or mul- tichannel microfluidic circuits; these devices can be used for CE and CEC, and should be low cost and disposable. Microchip-based GC devices have been available since approximately 1995. The use of these devices for separations is
- 48. 48 Terminology Guide October 2013 www.chromatographyonline.com currently in its infancy, and applications should expand with time. Microdialysis: Microdialysis is a mini- mally invasive sampling technique that is used for continuous measurement of free, unbound analyte concentrations in the extracellular fluid of virtually any tissue. Analytes may include en- dogenous molecules (for example, neu- rotransmitters, hormones, and glucose) to assess their biochemical functions in the body, or exogenous compounds (for example, pharmaceuticals) to determine their distribution within the body. The microdialysis technique requires the in- sertion of a small microdialysis catheter (also referred to as microdialysis probe) into the tissue of interest. After the probe is inserted into the tissue or (body) fluid of interest, small solutes can cross the semipermeable membrane by passive diffusion. The microdialysis probe is de- signed to mimic a blood capillary and consists of a shaft with a semipermeable hollow fiber membrane at its tip, which is connected to inlet and outlet tubing. Microextraction: The general process of liquid extraction using small amounts of organic solvent where the phase ratio Vo/Vaq is quite low; other techniques using hollow microfibers as a barrier are also referred to as microextraction. Microparticulate: Refers to the small particles used in HPLC. Generally packings with a particle diameter of less than 10 µm and that are totally porous are considered microparticle packings. Micropipette tip: A form of SPE in which the packing material is embed- ded or adsorbed on the inner walls of a pipette tip; useful for the SPE of very small amounts of liquid sample; often used with xyz liquid handling systems for automation purposes. See pipette tip. Microporous resin: Same as microre- ticular resin. Microreticular resin: Cross-linked synthetic ion-exchange resins that have pores with openings correspond- ing to molecular sizes. Diffusion into the narrow pores can be impaired and low exchange rates can occur, as well as poor performance, especially for large molecules. Microwave-assisted extraction (MAE): The use of microwave energy to heat samples in the presence of a solvent allowing for rapid extraction; MAE can be performed in open ves- sels. A nonmicrowave-absorbing sol- vent is used and the sample contain- ing a substance with a high dielectric constant (for example, water) is rapidly heated, with the extracted analytes passing into the extraction solvent. A variation of this technique involves the addition of an inert microwave-ab- sorbing solid substance that transfers the heated energy to the surrounding solvent. MAE can also take place in closed vessels that are non-microwave- absorbing containers. Migration time (tM): The time it takes for a charged molecule to move from the point of injection to the point of detec- tion in a CE capillary. Milling: Devices for reducing the par- ticle sizes of solid materials. Disk mills pulverize <20-mm diameter hard sam- ples by feeding between stationary and rotating disks with adjustable gap set- tings; samples are generally reduced to a 100-µm diameter; rotor speed mills combine impact and shearing processes to grind soft-to-medium hard and fi- brous materials to 80 µm; ball mills grind material to submicrometer size by developing high-grinding energy via centrifugal or planetary actions using
- 49. Terminology Guide October 2013 49 www.chromatographyonline.com agate, tungsten carbide, or PTFE- coated stainless steel balls. Mincing: The process of breaking down a meat or vegetable product into smaller parts by tearing, chopping, cutting, dic- ing, and so forth. Minimum detectable quantity (MDQ): The amount of solute that produces a signal twice the noise level. Mixed-bed column: Combination of two or more stationary phases in the same column, used most often for ex- change separations (IEC mixed anion and cation resins) and SEC (mixture of different pore size packings). The ad- vantage in IEC is the total removal of both cationic and anionic compounds; the technique is useful in SEC because a wider molecular weight range can be accommodated by the same column. Mixed-mode separation: A separation that takes place in a single column as a result of retention and selectivity pro- vided by a dual retention mechanism. For example, at intermediate-to-high pH values, a reversed-phase column with residual silanols can separate by hydrophobic interactions as well as ionic interactions by virtue of the ion- ized silanols; sometimes mixed-mode separations can be quite beneficial to the selectivity (band spacing) but can cause peak asymmetry; the precise bal- ance of interactions may be difficult to reproduce with subsequent batches of packing. Mobile phase: The fluid that moves solutes through the column. In LC, the mobile phase interacts with both the sol- ute and the stationary phase and there- fore can have a powerful influence on the separation. In GC, the mobile phase, as an inert gas, has little interaction with stationary phase and analytes and serves to move the sample through the column. Mobile-phase modifier: Modifiers are materials (usually organic or inorganic compounds ) added to the mobile phase to alter its elution properties. Mobile-phase strength: See solvent strength. Modifier: An additive that changes the character of the mobile phase. In reversed-phase LC, for example, water is the weak solvent and methanol, the strong solvent, is sometimes called the modifier; sometimes other additives such as competing bases like triethyl- amine or ion pair reagents are referred to as modifiers but they should more cor- rectly be called additives. See additive. Molecular diffusion term (B term): Refers to the B term (second term) of the van Deemter and Golay equations. Also called longitudinal or axial diffu- sion term. It dominates band broaden- ing only at very low flow rates below the minimum plate height where the diffu- sion of individual solutes can occur in a longitudinal (lengthwise) direction on the column. See van Deemter equation, Golay equation. Molecular sieve: GC column packing that retains solute by combined mo- lecular size and adsorptive interactions. Molecular sieves can separate light gases and hydrocarbons. Molecular weight distribution: The distribution of the molecular weight of molecules in a polymer sample. Distri- bution can be defined as weight average and number average. Molecularly imprinted phases (MIPs): See imprinted phases. Monolith: A monolithic HPLC col- umn is a special type of column used in HPLC with porous channels rather than beads; monoliths, in chromato- graphic terms, are porous rod struc- tures characterized by mesopores
- 50. 50 Terminology Guide October 2013 www.chromatographyonline.com and macropores. These pores provide monoliths with high permeability, a large number of channels, and a high surface area available for reactivity. The backbone of a monolithic column is composed of either an organic (poly- meric) or inorganic (silica) substrate, and the column can be chemically al- tered for specific applications. Monomeric phase: Refers to a bonded phase where single molecules are bonded to a support. For silica gel, monomeric phases are prepared by the reaction of an alkyl- or arylmonochloro- or alkoxysi- lane. Polymeric phases generally are pre- pared from a di- or trichlorosilane or an alkoxysilane reactant. Moving zone: The moving zone is that fraction of the mobile phase in the col- umn that occupies the interstitial spaces. Multidimensional chromatography: The use of two or more columns or chromatographic techniques to ef- fect a better separation. It is useful for sample cleanup, increased resolution, and increased throughput. Separa- tion is carried out with two or more columns in which peaks are selectively directed onto or removed from at least one of the columns by use of a timed valve system. In GC, a Deans fluidic switch is often used. It also can be used off-line by collecting fractions and reinjecting onto a second column. Also called coupled column chromatog- raphy, column switching, multicolumn chromatography, and boxcar chromatog- raphy. See backflushing, heart cut- ting, precut. Multidimensional protein iden- tification technology (MudPIT): Multidimensional protein identifi- cation technology combines both a cation-exchange prefractionation and reversed-phase HPLC separation of tryptic peptides to analyze an entire proteome of a cell or tissue type pro- tein extract. The approach uses a dual enzymatic digestion (Lys-C followed by trypsin) to increase the number of peptides observed. The peptides are separated using strong cation ex- change and are identified by MS-MS detection. Multimodal SPE: The practice of SPE where two different phases or modes are used to clean up a sample; the process can consist of two separate cartridges placed in series with the analytes sepa- rated on the two different cartridges; a second process is where two different phases are present in the same cartridge or even on the same packing; sometimes referred to as mixed-mode SPE. N Nano LC: LC practiced with columns that have internal diameters less than 100 µm; usually requires specialized in- strumentation; often used in proteomic studies where sample is limited and sen- sitivity is required. Narrow-bore column: Columns of less than 2 mm i.d. used in HPLC, and less than 320 µm i.d. in GC; also referred to as microbore. Nitrogen–phosphorus detection (NPD): The nitrogen–phosphorus detec- tor catalytically ionizes N- or P-contain- ing solutes on a heated rubidium or ce- sium surface in a reductive atmosphere. NPD is highly selective with sensitivity somewhat better than FID. Noise: See baseline noise. Non-aqueous reversed phase chroma- tography (NARP): Refers to reversed- phase chromatography performed with- out water as a component of the eluent on a reversed-phase packing. Used for compounds that are very nonpolar that
- 51. Terminology Guide October 2013 51 www.chromatographyonline.com either cannot be eluted or are poorly eluted from a reversed-phase column with 100% methanol or acetonitrile; in these cases, solvent A would be aceto- nitrile and solvent B would be a stron- ger solvent such as tetrahydrofuran; for NARP, reversed-phase rules apply (that is, the more nonpolar the analyte, the greater the retention). Nonpolar: A nonpolar molecule is one in which the electrons are distributed more symmetrically and thus does not have an abundance of charges at the opposite sides. The charges all cancel out each other. Nonpolar compounds, solvents, or bonded phases readily dis- solve in organic solvents, such as hexane, or prefer such solvents in place of water. Nonpolar substances do not readily dis- solve in water. Nonporous particle: Refers to a solid particle used as a support for a porous coated or bonded phase; pellicular par- ticles are nonporous particles with large particle diameters (approximately 40 µm) and nonporous silicas and resins with small particle diameters of less than 3 µm usually consist of a microbead with a thin porous outer coating of silica gel, bonded silica gel, or polymeric phase. Nonsuppressed ion chromatography: Direct ion chromatography. After the separation column the eluent is directly fed into the conductivity detection with- out prior chemical suppression. The conductivity measurement takes place on the high background conductivity. This requires a very high quality detec- tor with perfectly stable temperature. The nonsuppressed approach allows de- tection of weak acids or bases that are undissociated after suppression — for example, silicate or borate. For cation analysis the peaks for the components of interest are larger than with suppression. Nonporous packing (NPR, NPS, NPZ): Particles similar to porous-layer bead but with particle diameters in the sub- 5-µm range, often particles are in the sub-2-µm range; used for high-speed separations in short columns; NPS refers to nonporous silica, NPR to nonporous resins, and NPZ to nonporous zirconia. Normal-phase chromatography: A mode of chromatography in which the stationary phase is more polar than the mobile phase. Adsorption chromatogra- phy on silica gel or alumina using mix- tures of less polar eluents (for example, hexane–diethethyl ether) as a mobile phase would be a typical normal-phase system. Also refers to the use of polar bonded phases, such as -CN or NH2. Sometimes referred to as straight phase chromatography. O Octadecylsilane (ODS, C18): The most popular reversed phase in HPLC. Oc- tadecylsilane phases are bonded to silica or polymeric packings. Both monomeric and polymeric phases are available. Octylsilane (C8): A popular stationary phase in reversed-phase chromatogra- phy; usually has slightly less retention than the more popular C18; both mo- nomeric and polymeric phases available Off-line SPE: The normal practice of SPE where SPE cartridges, disks, pipette tips, and so forth are handled using manual processes (for example, vacuum manifolds or pipette transfer); opposite to on-line SPE. On-column detection: The column it- self serves as the flow cell in HPLC, CE, or CEC. Generally the term used when fused-silica capillaries are employed; the outer polyimide layer is removed and op- tical beam is directed through the capil- lary; a measuring device (for example, a
- 52. 52 Terminology Guide October 2013 www.chromatographyonline.com photomultiplier tube) is located on the opposite side of the capillary. On-column injection (OCI): In GC, re- fers to the process of injecting the entire liquid sample directly onto the head of the column using a fine needle that will fit inside the capillary. Usually carried out at an initial column temperature less than the solvent boiling point, also termed cold on-column injection. On-line column switching: See multi- dimensional chromatography, on-line SPE. On-line preconcentration: A precol- umn is placed in front of the separation column to concentrate analytes before their separation; different mechanisms may be used (for example, hydrophobic interaction, adsorption, or enzymatic re- action) to retain analytes as a function of time and then by a displacement process (such as solvent elution or pH change) concentrated analytes are transferred to the separation column. On-line SPE: Refers to the use of small stainless steel cartridges packed with SPE packings placed across two ports of a 6- or 10-port injection or column- switching valve. The SPE trap is loaded with sample by an external pump or syringe transfer and then the valve is switched so that the SPE trap becomes part of the HPLC flow stream and ana- lytes can be swept into the column based on the solvent being used for displace- ment. On-line SPE columns are usually used multiple times whereas off-line SPE cartridges are generally used once. Open-tubular column: Also termed capillary columns, open-tubular col- umns for GC have the stationary phase coated or chemically bonded on the inner walls or have support particles deposited on the inner walls. Internal diameters range from ~100 µm up to 750 µm. In HPLC, SFC, and capillary electrophoresis, small internal diameter (less than 100 µm) columns are used. The most frequently used column mate- rial is fused-silica tubing. Used very little in routine HPLC or SFC but routinely in CE and GC. Also termed capillary column. Open-tubular column, packed: A cap- illary-dimension column that is packed with stationary phase particles. Also called micropacked columns, particularly in GC. Optically active resin: Incorporation of optically active groups into an ion- exchange resin to allow separation of optically active isomers. Not many are commercially available in HPLC. Organic modifier: Water-miscible organic solvent added to an aqueous mobile phase to effect separations in re- versed-phase HPLC. Common organic modifiers are acetonitrile, methanol, iso- propanol, and tetrahydrofuran. Orthogonality: Refers to two separation dimensions for which the elution times in the two dimensions can be treated as statistically independent; ideally, the two dimensions should have totally dif- ferent retention mechanisms (for exam- ple, reversed phase and normal phase; ion exchange and reversed phase; polar and nonpolar) Overload: In preparative chromatog- raphy, the overload is defined as the sample mass injected onto the column where efficiency and resolution begins to be affected if the sample size is further increased. See sample capacity. P Packing: The adsorbent, gel, or solid sup- port used in the chromatography column. Most modern analytical HPLC packings are less than 10 µm in average diameter
- 53. Terminology Guide October 2013 53 www.chromatographyonline.com with 5 µm currently the favorite. GC col- umn packings range in size from 60–80 mesh down to 100–120 mesh. Paired ion chromatography: The same as ion-pair chromatography. Paper filtration: Using porous filter paper (mainly cellulose) to remove particulates from liquid samples; pa- pers with different porosities are avail- able. Low porosity filters will remove very fine particulates but may have a lower flow rate while high porosity filters filter out larger particulates at a higher flow rate; paper filtration is often used in wet chemistry to filter, combust, and then weigh insoluble materials; ashless filter paper is used for this purpose. Particle diameter (dp): Average diam- eter of the column packing particles. Particle size distribution: A measure of the distribution of the particles used to pack the LC column. In HPLC, a nar- row particle size distribution is desirable. A particle size distribution of dp ± 10% would mean that 90% of the particles fall between 9 and 11 µm for a 10-µm average dp packing. Particle size reduction: The general process of reducing larger particles down to a size that can be more conveniently extracted; the smaller the particle the more quickly it will dissolve or if in- soluble the more quickly analytes can be extracted for further sample cleanup. Typical methods for reducing particle size include pulverizing, milling, ho- mogenizing, chopping, blending, and so on. Particulates: Generally refers to a small particles found in the mobile phase that can cause back pressure problems by lodging in frits; it can also refer to the small particles packed into HPLC columns. Particulates that escape from the column exit may cause detector noise. Partition chromatography: Separa- tion process where one of two phases is held stationary on a solid support or the column inner wall (stationary phase or liquid phase) while the other is allowed to flow freely down the column (mo- bile phase or carrier gas). Solutes parti- tion themselves between the two phases based on their individual partition co- efficients. LLC is an example; modern bonded-phase LC can be considered to be a form of partition chromatography where one of the liquid phases is actu- ally bonded to the solid support. Mech- anistically, partition chromatography implies that the solute becomes at least partially embedded within the station- ary phase, which is impregnated, coated, or bonded to the substrate, in contrast to an adsorption process in which the solute does not penetrate into the reten- tive surface or interphase. Partition coefficient (K): The ratio of the equilibrium concentration of solute in the stationary phase relative to the equilibrium concentration of solute in the mobile phase. In GC, the relative concentration of solute in the mobile and stationary phases is a function of k and β: K = βk. Also called distribu- tion coefficient, KD, and distribution constant, Kc. Passive sampling: In passive gas sam- pling, an air sample is pulled through a flow controller into an evacuated canis- ter over a chosen period of time, rang- ing from 5 min to 24 h. The sampling period and the flow rate determine the canister volume required. Peak: The profile of an analyte com- pound as it is eluted from a column through a detector; usually depicted on a visual output on a recorder or printer
- 54. 54 Terminology Guide October 2013 www.chromatographyonline.com based on the detector’s electrical re- sponse. Peak area (Ap): The area measured under a chromatographic peak; usu- ally measured by an integrator or data system; the peak area is related to the amount of substance eluted in a peak. Peak capacity (n): The number of equally well resolved peaks that can be fit in a chromatogram between the hold-up volume and some upper limit in retention kn. For R = 1, n can be expressed by the approximation n = 1 + √N — /4 · ln(1 + kn) where N is the plate number and kn is the retention factor of peak n. Peak dispersion: See band broadening. Peak doublet: A split peak generally caused by column void, poor injection technique, or solvent flooding in GC. Split peaks also could be closely eluted compounds. Peak height (hp): The maximum height of a chromatographic peak as measured from the baseline to the peak apex; the peak height is related to the amount of substance eluted in a peak. Peak overload: When too much of any one solute is injected its peak may be distorted into a triangular shape. Peak shape: Describes the profile of a chromatography peak. Theory assumes a Gaussian peak shape (perfectly sym- metrical); peak asymmetry factor de- scribes shape as a ratio. See asymmetry. Peak tracking: A method of matching of peaks that contain the same com- pound between different experimental runs during method development; relies upon detection parameters of each pure analyte; diode-array detectors and mass spectrometers are among the best detec- tors for peak tracking because of their specificity. Also refers to data-system tracking of gradual changes in retention times caused by stationary-phase loss or other column degradation or drift in the chromatographic conditions. Peak variance (σ2): The second central moment of the peak about the retention time. For a Gaussian peak the variance is the fundamental parameter control- ling peak width. See Gaussian peak. Peak volume (Vp): The volume occu- pied by a chromatographic peak from starting basepoint to ending basepoint as it passes through the detector: Vp = Fcwb Peak width at base (wb): The width of the chromatographic peak at the baseline as eluted from the column. It is measured at the baseline by drawing tangents to the inflection points on the sides of the Gaussian curve representing the peak. Smaller peak widths usually represent efficient separations; also re- ferred to as band width. It is sometimes convenient to estimate the peak width at base from the peak area and height: wb = 1.596 Ap/hp (see Figure 2). Peak width at half-height (wh): The width of the chromatographic peak at half of the peak height above the base- line. Smaller peak widths usually repre- sent efficient separations; also referred to as band width. It is sometimes conve- nient to estimate the peak width at half- height from the peak area and height: wb = 0.94 Ap/hp (see Figure 2). PEEK: Polyether ether ketone (PEEK) is a colorless organic polymer. It is used as a material for inert capillaries and fit- tings in HPLC and IC systems Pellicular: See porous layer bead. Percent B (%B): Refers to the stronger solvent in a binary solvent mixture; %A would be the weaker solvent analog. Perfusion chromatography: Re- fers to chromatography performed using particles with very large pores (for example, 4000–8000 Å)
- 55. Terminology Guide October 2013 55 www.chromatographyonline.com called throughpores (megapores or gigapores). Eluent flows through the particle as well as smaller in- terconnecting pores (for example, 300–1000 Å) called diffusive pores between the large pores. Best suited for the preparative separation of mac- romolecules. Permeability (Bo): Also referred to as column permeability and specific perme- ability; a term expressing the resistance of the packed column to the flow of mobile phase. For a packed column: Bo = (dp 2/180) · ε3/(1 - ε)2 ≈ (dp 2)/1012.For an open-tubular column: Bo = dc 2/32. A column with high permeability gives a low pressure drop. Permeation: In SEC, refers to the pro- cess where a solute can enter a mobile phase filled pore of the packing. Phase collapse: See phase dewetting. Phase dewetting: A term used in reversed-phase LC where very dense bonded-phase coverage and a high per- centage of aqueous content in mobile phase can lead to expulsion of water from the pores, which prevents the normal partitioning process from tak- ing place. Phase dewetting may occur with as high as 10% organic content in the mobile phase but can occur at lower %B values; results in earlier than normal elution of analytes, poor peak shape, and nonreproducible retention times. Phase ratio (β): The relative amount of stationary to mobile phase in the col- umn. In partition chromatography: β = VS/VM where VS and VM are the volume of stationary and mobile phase in the column respectively. For open-tubular columns: β ≈ rc/2df. Thicker station- ary phase films or higher phase loading gives longer retention and higher peak capacity. Phenol extraction: A sample prepara- tion technique used for the isolation of DNA from biological samples. Phenyl phase: A popular nonpolar bonded phase prepared by the reac- tion of dimethylphenylchlorosilane or alkoxysilane with silica gel for LC, or as the components of a cross-linked or bonded phase for GC. Claimed to have affinity for aromatic-containing compounds and does impart a different selectivity compared to alkyl bonded phases. Photoionization detection (PID): The photoionization detector ionizes solute molecules with photons in the UV en- ergy range. PID is a selective detection method that responds to aromatics and olefins when operated in the 10.2-eV photon range. It can respond to other materials with a more energetic light source. PIONA: Refers to the analysis of paraf- fins, isoparaffins, olefins, naphthenes, and aromatics. Pipette tip: Replaceable tips used in automation of liquid handling chores; used once and discarded to avoid con- tamination. Pirkle column: Chiral “brush type” sta- tionary phases based on 3,5-dinitroben- zoyl-phenylglycine silica that are used in the separation of a wide variety of en- antiomers. Named after the developer, Dr. William Pirkle, University of Illnois. pKa: An acid dissociation constant, Ka, (also known as acidity constant, or acid-ionization constant) is a quantita- tive measure of the strength of an acid in solution. It is the equilibrium con- stant for a chemical reaction known as dissociation in the context of acid- base reactions. The equilibrium can be written symbolically as: HA ←→ H+ + A- where HA is a generic acid that
- 56. 56 Terminology Guide October 2013 www.chromatographyonline.com dissociates by splitting into A−, known as the conjugate base of the acid, and the hydrogen ion or proton, H+, which, in the case of aqueous solutions, ex- ists as the hydronium ion — in other words, a solvated proton. The disso- ciation constant is usually written as a quotient of the equilibrium concentra- tions (in mol/L), denoted by [HA], [A−], and [H+]: Ka = ([H+] [A−])/[HA]; due to the many orders of magnitude spanned by Ka values, a logarithmic measure of the acid dissociation constant is more commonly used in practice. The loga- rithmic constant, pKa, which is equal to −log10 Ka, is sometimes also (but incor- rectly) referred to as an acid dissociation constant. Planar chromatography: A separation technique in which the stationary phase is present as or on a plane (IUPAC). Typical forms are paper and thin layer chromatography. Plate height (H): See theoretical plate height. Plate height, effective (Heff): The column length divided by the number of effective theoretical plates: Heff = L/ Neff Plate number (N): See theoretical plate number. Polar: A polar molecule may be polar as a result of polar bonds or as a result of an asymmetric arrangement of non- polar bonds and nonbonding pairs of electrons; polar molecules are generally able to dissolve in water (H2O) because of the polar nature of water; polar mol- ecules do not prefer nonpolar organic solvents such as hexane. Polar molecules have slightly positive and slightly nega- tively charged ends; we often refer to a compound’s polarity. Polarity index (P’): The polarity index is a measure of the relative polarity of a solvent and is useful for identifying suit- able mobile phase solvents or extraction solvents. The polarity index increases with polarity; examples: hexane, P′ = 0.0; isopropanol, P′ = 3.9; tetrahydrofu- ran, P′ = 4.0; methanol, P′ = 5.1; aceto- nitrile, P′ = 5.8; water, P′ = 9.0 Polyacrylamide gel: Neutral hydro- philic polymeric packings used in aqueous SEC. They are prepared by the copolymerization of acrylamide with N,N′-methylene-bis-acrylamide. Polyaromatic hydrocarbon (PAH): Members of a class of hydrocarbon molecules characterized by one or more fused aromatic rings. Polychlorinated biphenyl (PCB): Bi- phenyl molecule with two or more chlorine substitutions. Polyethylene glycol (PEG): Polymeric hydrocarbon used as a GC stationary phase; possesses moderately polar reten- tion characteristics. Polyethyleneimine (PEI): Polyethyl- eneimine, an anionic polymeric phase used to coat or bond onto silica or a polymeric packing. Most often used for the separation of proteins and peptides. Polymeric packings: Packings based on polymeric materials, usually in the form of spherical beads. Typical poly- mers used in LC as well as GC are polystyrene–divinylbenzene (PS-DVB), polydivinybenzene, polyacrylamide, polymethylacrylate, polyethyleneoxide, polydextran, or polysaccharide. Polymeric phase: Refers to chemically bonded phase where a polymer species is bonded to silica-based particles or to the wall of an open-tubular column. Polymeric SPE: The use of a polymeric base material (for example, PS-DVB or methacrylate) rather than an inorganic material (for example, silica or alu- mina); polymers generally have a wider
- 57. Terminology Guide October 2013 57 www.chromatographyonline.com pH range and higher sample capacity than some of the inorganic materials. Polystyrene–divinylbenzene (PS- DVB) resin: The most common base polymer for ion-exchange chromatog- raphy. Ionic groups are incorporated by various chemical reactions. Neutral PS-DVB beads are used in reversed- phase LC. Porosity and mechanical stability can be altered by variation of the crosslinking through the variation of the DVB content. In GC, a number of porous polymer stationary phases are available for gas and light-compound separations. PONA: Refers to the analysis of paraf- fins, olefins, naphthenes, and aromatics. Poppe plot: A kinetic plot named after Professor Hans Poppe (J. Chromatogr. A 778, 3 [1997]), University of Amster- dam, the Netherlands, where the plate time log (t0/N) is depicted as a func- tion of the number of theoretical plates to assess the limits of column perfor- mances as a function of particle size, column pressure drop, and so forth. Pore diameter: Same as mean pore diameter. Pore size: The average size of a pore in a porous packing. Its value is expressed in angstroms or nanometers. The pore size determines whether a molecule can diffuse into and out of the packing. See mean pore diameter. Pore volume (Vi): The total volume of the pores in a porous packing, usually expressed in mL/g. Better termed the specific pore volume. It is measured by the BET method of nitrogen adsorp- tion or by mercury intrusion where Hg is pumped into the pores under high pressure. Poroshell: Similar to nonporous par- ticles and porous-layer beads; particles are generally in the 2–5 µm range with a submicrometer-thick shell; wide pore versions (>300 Å) allow rapid diffusion of macromolecules and smaller pore versions (90–120 Å) are for small mol- ecules; give similar efficiency to sub- 2-µm particles but at much lower pres- sure because of their larger particle size. Porosity: For a porous substrate, the ratio of the volume of the pores in a particle to volume occupied by the par- ticle. The pore volume is a measure of the porosity and is expressed in mL/g. Porous layer bead: A small glass bead coated with a thin layer of stationary phase. The thin layer can be an ad- sorbent, resin, or a phase chemically bonded onto the adsorbent. These pack- ings were among the first to be used in HPLC. They were of larger particle size (20–40 µm) than the microparticulate packings of today but were easy to pack and gave adequate efficiency. Also re- ferred to as controlled surface porosity supports and pellicular materials. Porous particle: Refers to column packing particles possessing intercon- necting pores of specified diameter and pore volume; generally in HPLC porous particles with diameters below 10 µm are the most popular, and in prepara- tive chromatography larger particles are used because of their lower cost and higher column permeability. Porous polymer: A packing material, generally spherical, based on organic polymers or copolymers; popular ex- amples would be polystyrene–divinyl- benzene, polyacrylates, polydextrans, polyacrylamides, and polybutadienes. Retains solutes by selective adsorption or molecular size interaction. Porous-layer open-tubular (PLOT) col- umn: An open-tubular column used in GC or HPLC that has particles coated or uncoated with stationary phase
- 58. 58 Terminology Guide October 2013 www.chromatographyonline.com attached to the inner walls, which al- lows more rapid mass transfer. In GC, small porous particles such as polymer, alumina, silica, and so forth are attached to the walls or the wall may be modified by etching or other treatment to increase the inner surface area and provide gas– solid chromatographic retention behav- ior. In LC, porous polymers have been used occasionally. In GC, the technol- ogy is more developed. Postcolumn derivatization: See postcol- umn reaction. Postcolumn reaction: In LC and IC, after the analytical column a UV-trans- parent ion or molecule is converted into a component with better detectability (that is, UV–vis absorbance, fluores- cence) by adding a specific reagent. This product is then detected with UV–vis or fluorescence detection. The reaction of the analyte and the reagent is usually very selective and yields often in a col- ored product (visible detection), that is, chromate + diphenylcarbazide complex (540 nm); bromate + iodide → triiodide (352 nm). Parameters that will influence the sensitivity are the reaction time (flow rate/length of reaction coil), the reaction temperature, pH, concentration of cata- lysts. In GC, postcolumn methanization may be used to convert CO and CO2 to CH4 with hydrogen and a heated nickel catalyst to achieve flame ionization de- tection, more sensitive than thermal conductivity detection. Potentiometric detection: Ion selec- tive electrodes in a flow-through cell are used to detect the analyte ions. Not a very common type of detection. Pounds per square inch (psi): A unit of pressure: 14.6959 psi = 1 atm = 101.325 kPa = 1.013 bar. Precolumn derivatization: See precol- umn reaction. Precolumn reaction: Analytes are con- verted into components with better detectability (for example, UV–vis ab- sorbance) by a chemical reaction before injection. The analytes are separated and detected by UV–vis detection. Complex- ing agents such as EDTA, NTA, and so forth are used as their Fe(III) complexes. Precolumn: A short section of similar column placed before the analytical col- umn; used to physically retain undesired compounds or to saturate the mobile phase with stationary phase that may be packed into the precolumn (for example, a silica precolumn saturates the mobile phase with dissolved silica and prevents mobile phase from dissolving silica in the analytical column). Precolumn filter: A filter used between the injector and the column (or guard column) to keep unwanted sample components from reaching the column; sometimes called in-line filter, occasion- ally inlet filter. Preconcentration: See also trace enrich- ment. Precut: Peaks in the beginning of a chro- matogram are removed to vent or are di- rected onto another column of different polarity, or at a different temperature, for improved resolution. See heart cutting, multidimensional chromatography. Prefilter (SPE): In cases where samples contain a large amount of particulates, regular SPE cartridges and disks may become clogged and flow is reduced. Prefilters are filter devices that have higher porosity that will filter out large particles and allow the SPE bed to oper- ate more efficiently. Some SPE devices have prefilters built in; in others one can add a prefilter. In some cases, the use of an inert packing such as glass beads serves the same purpose as an actual filter.
- 59. Terminology Guide October 2013 59 www.chromatographyonline.com Preparative chromatography: Refers to the process of using chromatogra- phy as a technique for the isolation of a sufficient amount of material for other experimental or functional purposes. For pharmaceutical or biotechnological purifications, large columns of several feet in diameter can be used for puri- fying multiple grams of material. For isolating a few micrograms of valuable natural product, a 4.6-mm i.d. analytical column can be used. Both of these sepa- rations can be considered preparative chromatographic approaches. Prepara- tive LC is often employed; preparative GC is seldom used. Pressing: The general process of squeez- ing liquid from a semisolid material (such as plants, fruit, or meat). Pressure drop (Δp): The pressure drop across a column: Δp = pi – po, where pi and po are pressure at the column inlet and outlet, respectively. Pressure injection (CE): Pressure-in- duced injection; the use of pressure or vacuum to inject small (nanoliter) vol- umes of sample into a capillary column; best for narrow-bore capillaries (<10 µm i.d.); a version of hydrostatic injection. Pressure, absolute inlet (pi): The col- umn inlet pressure expressed relative to a vacuum. Pressure, absolute outlet (po): Pressure at the column outlet, relative to vacuum. Pressure, back: Same as head pressure, column pressure. Pressure, head (Δp): The pressure dif- ference between the inlet and outlet of the column. In LC, governed by the following approximate equation for a column packed with spherical particles: Δp ≈ (3000Lη)/(t0dp 2) where η is the mobile phase viscosity, t0 the column holdup time, and dp is the particle diameter. In packed- column GC the pressure drop can be ap- proximated as Δp ≈ (1012L η u –)/(dp 2 ), where u – is the average linear velocity. In open-tu- bular column GC, the pressure drop is Δp ≈ (8 L η u –)/(rc 2). The equations for GC will overestimate the required pressure drops by more than 10% at inlet pressures above 400 kPa (58 psig) because of gas compressibility effects. Pressure drop can be expressed in pressure units of psig, bar, atm, kPa, or MPa. The above equations will yield pressures in pascals if the dimensions are expressed in centimeters, times in seconds, and viscosi- ties in pascal-seconds. Pressure, relative (P): Relative pressure across the column: P = pi /po Pressurized-fluid extraction (PFE): Pressured fluid extraction is a liq- uid–solid extraction process where the sample and solvent are placed in a closed container and heated well above the solvent’s normal boiling point. The combination of increased temperature and resultant pressure extracts analytes and matrix compounds into the super- heated fluid, often in a few minutes. Because the technique extracts a wide variety of soluble compounds, addi- tional cleanup steps may be required after PFE is completed; method de- velopment involves selecting the best solvent for analytes and the poorest solvent for the matrix and other in- terferences that may be present. The technique has been approved for vari- ous environmental samples by the U.S. EPA under the generic name of PFE or pressurized-solvent extraction. See accelerated solvent extraction. Pressurized-solvent extraction (PSE): See accelerated solvent extraction, pressurized-fluid extraction. Primary sampling: The collection of one or more increments or units ini- tially taken from a population; the primary sample is that taken from the
- 60. 60 Terminology Guide October 2013 www.chromatographyonline.com primary source; proper statistical sam- pling protocols are recommended. Process-scale chromatography: Re- fers to the use of liquid chromatography at the industrial scale level outside the laboratory; generally requires specially designed columns (usually with di- ameters > 5 cm), recoverable solvents, lower-cost packings (with larger and ir- regular-shaped particles), and overloaded operating conditions compared to those of laboratory-scale HPLC. Programmed elution: A procedure in which the conditions of separation are changed in a programmed manner. Un- like LC, in GC and SFC both tempera- ture and pressure can be programmed, separately or simultaneously. Programmed temperature chroma- tography: Use of conditions in which the temperature is varied during the run in a controlled manner. Widely used in GC; seldom seen in LC. Programmed temperature injection (PTI): A cold injection technique in which the inlet temperature is specifi- cally programmed from the GC. Programmed temperature rate: The rate, in °C/min, at which the GC oven temperature is increased during a con- trolled temperature program ramp. The program may consist of multiple ramps with variable hold times before and after each. Typical GC programming rates range from <0.5 °C/min up to 40 °C/ min. Programming rates up to 200 °C/ min and higher have been applied to high-speed gas chromatography. Programmed temperature vaporiza- tion (PTV): In PTV, the sample is intro- duced into the inlet liner at a tempera- ture slightly below the boiling point of the solvent; the solvent is continually evaporated and vented through the inlet split line; after the solvent is gone, the temperature of the inlet is heated very rapidly to transfer the sample into the column; using PTV there is the potential for less sample discrimination and less thermal degradation of sensi- tive compounds compared to hot inlet injections. Programmed temperature vaporizer (PTV): An inlet system designed to per- form programmed-temperature injection. Protein crashing: The term used in removing or reducing the protein con- centration in a biological fluid such as plasma. After slight dilution, an organic solvent such as acetonitrile is added to the plasma and the proteins, which are insoluble, precipitate (crash). Centrifuga- tion or filtration is used to remove the protein, and the supernatant liquid is in- jected into an HPLC system or worked up further. Protein precipitation: See protein crashing. Pulsating flow: Flow originating from a reciprocating pump. Normally the pulses are dampened out by a pulse damper, an electronic pressure feedback circuit, or an active damper pump head. Some detectors (for example, electro- chemical, refractive index) are greatly affected by flow pulsations. Pulsed amperometric detection: Electrochemical detection applying dif- ferent potentials (pulses) to the working electrode. Components that can be an- alyzed include those that are oxidized or reduced at the electrode and those that react with the electrode surface or cover it. To remove reaction products that could foul the electrode, highly oxidative and reductive potentials are applied to the working electrode after the measuring potential. This removes the reaction products from the previ- ous measuring cycle and renews the
- 61. Terminology Guide October 2013 61 www.chromatographyonline.com electrode surface. Typical applications are carbohydrates and amino acids. Pulsed discharge detection (PDD): Several ionization detectors use a pulsed- discharge ion source to improve detec- tivity compared to constant-discharge detectors. Pulsed-splitless injection: A form of GC injection recommended for large volumes (up to 5 µL) of sample where a short-term high pressure pulse is im- posed on the inlet such that there is not a large volume of solvent vapor gener- ated and most or all of the sample is di- rected to the column; after the sample is transferred, then normal pressure is resumed. Using this technique, highly volatile compounds are less likely to be lost through the split vent line and ther- mally unstable compounds spend less time in the hot injection port so there is less degradation. Pulverizing: Electromechanically driven rod or vibrating base devised to reduce the particle size of solid samples. A freezer mill can be used with liquid nitrogen to treat malleable polymers or those with low glass transition tempera- tures. Purge-and-trap sampling: Dynamic headspace technique where the head- space vapors over a liquid or solid sample are continuously removed by a flow of gas over the sample (purging) or through the sample (sparging); volatil- ized analytes are usually concentrated by trapping on an adsorbent or by cryogenic means. The trap is then heated to desorb trapped components into a GC column. Most often used for volatile trace ana- lytes where concentration is needed. Pyrolysis gas chromatography: The process of heating a sample enough to break its chemical bonds, thereby form- ing smaller molecules that can be ana- lyzed by GC. Often applied to polymer characterization. Q Quaternary methyl amine (QMA): A strong anion-exchange functionality popular in resin-based packings; usually supplied in chloride form. Quaternary mobile phase: See quater- nary-solvent mobile phase. Quaternary-solvent mobile phase: A mobile phase consisting of four separate solvents that allow the mobile-phase composition to be fine-tuned; most often this mobile phase is delivered by a low-pressure quaternary pump. QuEChERS: A technique initially used for the extraction of pesticides from fruits and vegetables. It consists of two steps: salting out extraction using buffered or unbuffered solvent (usually acetonitrile), and dispersive SPE where a solid adsorbent is used to treat an aliquot from the first step to remove interferences and matrix compounds. QuEChERS (which stands for quick, easy, cheap, effective, rugged, and safe) is mostly used with GC–MS and LC– MS (or MS-MS) to more selectively an- alyze pesticide extracts. More recently, QuEChERS has expanded to matrices such as cooking oil, meat, fish, and biological fluids, and to other analytes, such as pharmaceuticals, antioxidants, and toxins. R Radial compression: The use of radial pressure applied to a flexible wall col- umn to cut down on wall effects. Radial diffusion or dispersion: Diffu- sion or dispersion across the column in a radial direction. If the sample is injected into the exact center of a column, it will spread not only in a longitudinal
- 62. 62 Terminology Guide October 2013 www.chromatographyonline.com direction as it moves down the column but radially as well, allowing the solute to reach the wall region where the eluent velocity is different than in the center of the column. Recovery: The amount of solute (sam- ple) that is eluted from a column rela- tive to the amount injected. Excellent recovery is important for good quanti- tation, for preparative separations, es- pecially for biomolecules, and for good peak shape and resolution. Reasons for inadequate recovery can be solute inter- action with active sites on the packing, with column frits, and with column tubing. Compound decomposition during the separation process can also effect recovery. Recycling chromatography: A tech- nique where the column effluent is re- circulated onto the head of the column in an attempt to gain the advantage of extended column length. Can be car- ried out on a single column by passing the effluent back through the pump. An alternative technique uses two columns connected by a switching valve where the effluent of one column is directed onto the head of the other column. Very seldom used in HPLC, and then only in exclusion chromatography. Reduced plate height (h): The plate height expressed in terms of the average particle diameter for packed columns: h = H/dp where dp is the particle diameter, or in terms of the column inner diam- eter for open-tubular columns: h = H/dc where dc is the column inner diameter. Refractive index detection (RI detec- tion): Based on differential refractive index between the mobile-phase sol- vent and the eluted analyte in mobile- phase background; not useful in gradi- ent work; often used in size-exclusion chromatography. Refractive index peak: A pseudo peak normally found near the hold-up volume resulting from the refractive index sensi- tivity of absorbance and other detectors. Regeneration: Regeneration of the packing in the column to its initial state after a gradient elution. Mobile phase is passed through the column stepwise or in a gradient. The stationary phase is restored (solvated) to its initial condition. In ion exchange, regeneration involves replacing ions taken up in the exchange process with the original ions which occupied the exchange sites. Regenera- tion can also refer to bringing back any column to its original state (for example, the removal of impurities with a strong solvent). Relative retention (r): Retention rela- tive to a standard peak: r = t′ R/t′ R(st) where t′ R is the retention time of the compo- nent of interest and t′ R(st) is the retention time of the standard peak. Also: r = ki/ kst where ki and kst are the correspond- ing retention factors. For two adjacent peaks, the separation factor, α, expresses the relative retention. See separation factor, resolution. Relative standard deviation (RSD, %RSD): In probability theory and statistics, the relative standard devia- tion (RSD or %RSD) is the absolute value of the coefficient of variation. It is often expressed as a percentage. A similar term that is sometimes used is the relative variance which is the square of the coefficient of variation. Also, the relative standard error is a measure of a statistical estimate’s reliability obtained by dividing the standard error by the estimate; then multiplied by 100 to be expressed as a percentage. The rela- tive standard deviation is widely used in analytical chemistry to express the precision and repeatability of an assay.
- 63. Terminology Guide October 2013 63 www.chromatographyonline.com Removable well plates: See array 96- well plate. Representative sample: A sample re- sulting from a statistically worked out sampling plan; it can be expected to ad- equately reflect the properties of interest of the parent population. Residual silanols: The silanol (-Si–OH) groups that remain on the surface of a packing after chemically bonding a phase onto its surface. These silanol groups, that may be present in very small pores, may not be accessible to the reacting bulky organosilane (such as octadecyldimethylchlorosilane) but may be accessible to small polar compounds. Often they are removed by endcapping with a small organosilane such as tri- methylchlorosilane. See endcapping. Resin: A solid polymeric packing used in ion exchange separations. The most popular resins are polystyrene–divinyl- benzene copolymers of small particle size (<10 µm). Ionic functionality is incorpo- rated into the resin. Resolution (Rs): Peak resolution; incor- porates both efficiency and separation. A resolution of 1.5 is said to be “baseline” resolution, and a minimum resolution of 1.7–2.0 is considered essential for robust- ness. For two closely eluted peaks: R = (tR,2– tR,1)/wb where the subscripts 1 and 2 refer to the first and second peaks. From N, k2 and α: Rs= (√N — /4)((α – 1)/α) (k2/(k2 + 1)) (k2 is the retention factor of the second peak). Resolution equation: See resolution. Response factor (RF): Defines the rela- tionship between the measured peak area or height and the quantity of substance represented by a peak. Restricted access media (RAM): RAM are sorbents are used for the direct injec- tion of biological fluids such as plasma or serum into an HPLC flow stream. They contain an outer hydrophilic sur- face that provides minimal interaction with proteins and when combined with small pores on the sorbent exclude the proteins. The inner surface is hydropho- bic, and when small molecules diffuse into the pores they interact by reversed- phase mechanisms and are retained. The small molecules such as drugs and their metabolites can be removed by rinsing with an organic solvent. RAMs are most successfully used in a column switching setup where the secondary column is used to resolve the small molecules and the proteins are directed to waste so as not to foul the secondary column. Retention factor (k): The measure of time the sample component resides in the stationary phase relative to the time it resides in the mobile phase: k = (tR – tM)/tM. Formerly, kʹ was used and it was called the capacity factor or capacity ratio. Retention gap: A short piece of deac- tivated but uncoated column placed between the inlet and the analytical column. A retention gap often helps relieve solvent flooding. It also entrains nonvolatile sample contaminants from on-column injection. Retention index (I): A uniform system of retention classification according to a solute’s relative location between a pair of homologous reference compounds on a specific column under specific condi- tions. A series of normal straight-chain hydrocarbons, fatty acid esters, or mul- tiring polycyclic aromatic hydrocarbons have been used for the reference com- pounds. For a solute i that is eluted at tʹRi between two hydrocarbons with chain length z and z + 1: I = 100[z + (logt′ Ri – logt′ Rz)/(logt′ Rz1 – logt′ Rz)]. Retention time (tR): The time between injection and the appearance of the peak
- 64. 64 Terminology Guide October 2013 www.chromatographyonline.com maximum. It is usually measured from the point of injection to the apex of the peak. For asymmetric peaks it should be measured to the center of the mass of the peak. Also called the total retention time (IUPAC). See retention volume; reten- tion time, adjusted. Retention time, adjusted (t’R): A mea- sure of the retention time adjusted for the void time or unretained peak time: t′ R = tR – tM where tR is the retention time and tM (or t0) is the hold-up time, void time, or unretained peak time (that is, the time it takes for a small, unretained compound that completely permeates the pores to be eluted from the chro- matographic column). Retention volume (VR): The volume of mobile phase required to elute a sub- stance from the column: VR = FctR or VR = VM + KDVS where VM is the void volume, KD the distribution coefficient, and VS the stationary phase volume. Also termed the total retention volume. See re- tention time. Retention volume, adjusted (V′R): Ad- justs the retention volume for the holdup volume (or V0) where VR is the retention volume of the peak of interest and VM is the hold-up or void volume, the volume corresponding to the holdup time: V′ R = VR – VM Retention volume, corrected (VR 0): Corrects the retention volume for the effect of carrier-gas expansion along the column: VR 0 = VR j Reversed-phase chromatography: The most frequently used mode in HPLC. It uses low polarity packings such as octa- decylsilane or octylsilane phases bonded to silica or neutral polymeric beads. The mobile phase is usually water or water- miscible organic solvents such as metha- nol or acetonitrile. Elution usually occurs based on the relative hydrophobicity (or lipophilicity) of the solutes; the more hydrophobic, the stronger the retention. The greater the water solubility of the analyte, the less it is retained. There are many variations of reversed-phase LC where various mobile phase additives are used to impart a different selectivity. For example, for the reversed-phase LC of anions, the addition of a buffer and a tetraalkylammonium salt would allow ion pairing to occur and effect separa- tions that rival ion-exchange chromatog- raphy. More than 90% of HPLC users employ reversed-phase LC. Reynolds number (Re): For flow in a smooth unpacked pipe where u – is the average velocity (cm/s), dc is the pipe diameter, η is the viscosity (Pa·s) and ρ is the density (g/cm3): Re = (u –dcρ)/η. The Reynolds number is the ratio of viscous to inertial energy of the mov- ing fluid. At low Re viscous friction dominates and controls fluid motion, making it slow and steady. In an un- packed tube flow becomes fully turbu- lent when Re exceeds 4200. In a packed bed u – is replaced with the average inter- stitial velocity and dc with the average particle diameter. Flow becomes turbu- lent in a packed bed at Re above about 10 but is not fully turbulent until Re exceeds 100–200. Riffler: A mechanical device used in subdividing solid powder samples into smaller units. Rifflers can be manual or automated. Rifflers will subdivide mate- rial samples into two smaller portions by a single pass or further subdivisions can be attained by multiple passes. Rinse step: In SPE, the rinse (wash) step is the third step in the process. After the sample is loaded, the rinse step is designed to eliminate interferences including various matrix compounds. A solvent (or solvents) or buffer is selected
- 65. Terminology Guide October 2013 65 www.chromatographyonline.com to remove interferences but not the ana- lytes of interest. Room temperature (To): The room or laboratory temperature can be used as a reference temperature for gas measure- ments, for example 20 °C or 25 °C. Round-well plates : 96-well plates that have round-shaped wells resembling 96 small test tubes. See 96-well plate. S Salting-out effect: The use of a high concentration of salt buffer in the mo- bile phase to cause a low polarity ana- lyte to have a decreased solubility in water and therefore precipitate or come out of solution; most often used for the hydrophobic interaction chromatog- raphy of proteins where proteins are first precipitated at high salt con- centrations then eluted by gradual dilution using reverse gradient elu- tion. Salting-out is also used in headspace sampling to increase the ionic strength of the sample solution and thereby decrease the solubility of dissolved analytes and in- crease their headspace concentrations. Can also be used in liquid–liquid extraction; see salting-out extraction. Salting-out extraction: In this extrac- tion approach, high concentrations of salt in the aqueous phase will cause certain compounds to migrate into an organic phase or perhaps vice-versa; high concentrations of salt also will force normally miscible solvents (such as water and acetonitrile) to become immiscible and be used for further partitioning more polar analytes than could be achieved by an extraction using a non-polar organic solvent. See QuEChERS or salting-out effect. Sample capacity: Refers to the amount of sample that can be injected onto a column without overload and loss of column efficiency. Often ex- pressed as grams of sample per gram of packing. Overload is defined as the sample mass injected that will cause the column efficiency to decrease by 10% from its normal value. Sometimes referred to as sample loading. Sample discrimination: The charac- teristic of systematic change in sample composition according to a specific sample property. For example, a GC inlet may exhibit mass discrimination and admit relatively higher amounts of low-boiling sample components than high-boiling components in the same sample or injection. Sample division: The process of sam- ple reduction to divide the sample into smaller portions while retaining repre- sentative characteristics of the primary sample. See sample size reduction. Sample loop: Part of an injection valve that delivers an accurate volume of liquid or gas to the column, giv- ing a “slug” injection; loops come in different volumes depending on the needs of the analysis and the size of the column. Sample pretreatment: Often syn- onymous with sample preparation; the process of manipulating the sample to make it easier to analyze. Sample size reduction: The process of sample reduction to divide the sample into smaller portions while retaining representative characteristics of the primary sample. See sample division. Sample tracking: The process of track- ing primary, secondary, laboratory, and further samples through the analyti- cal cycle; it is important for chain of custody reasons to be able to ensure that the sample analyzed in the instru- ment was the original sample collected
- 66. 66 Terminology Guide October 2013 www.chromatographyonline.com at the source; sample tracking can be as simple as writing a sample number on a container but can be more complex, such as using bar-coded vials or radio frequency identification (RFID) tags to automatically keep track of sample flow. Sampling: The process of collecting a representative sample at the source. Sampling can also refer to further sam- ple division as it more closely approaches the laboratory analysis; it is important to make sure that the final sample ana- lyzed represents a subsample of the origi- nal sample without any imposed bias or discrimination. Sampling error: In statistics, sampling error is incurred when the statistical characteristics of a population are esti- mated from a subset, or sample, of that population. Because the sample does not include all members of the population, statistics on the sample, such as means and quantiles, generally differ from parameters on the entire population. Because sampling typically is done to determine the characteristics of a whole population, the difference between the sample and population values is consid- ered a sampling error. Exact measure- ment of sampling error generally is not feasible because the true population values are unknown; however, sampling error can often be estimated by probabi- listic modeling of the sample. Sampling rate: See data acquisition rate. Sandwich technique: Injection tech- nique in which a sample plug is placed between two solvent plugs in the syringe so as to wash the syringe needle with sol- vent and obtain better sample transfer into the inlet. Saturator column: See precolumn. Scaleability: In going from analytical to preparative chromatography, refers to the reproducibility of results on columns of different internal diameters when using the same particle size and bonded phase; normally a larger diameter column is used to increase capacity; a linear scale- up process minimizes time required to optimize preparative separations. Scavenger: Special type of solid-phase particle that uses chemical reactions (un- like SPE, which uses molecular interac- tions) to remove undesired species such as undesired reaction products or excess starting material from an organic syn- thesis. Scavengers mostly operate on the basis of covalent bonding. Packing ma- terials contain reactive groups that can be used for organic or inorganic species such as catalysts. Secondary sampling: Refers to the pro- cess of taking a representative portion of the primary sample to further reduce its particle size or to prepare a laboratory sample for eventual analysis. Sedimentation: A technique used for the sizing of resins for ion-exchange chromatography; a broad distribution of beads is placed in a solvent, often water, in a container that is affixed to a station- ary surface. Based on particle size and particle density the beads will settle at different velocities into a gradient of sizes and the fraction of interest is removed. Very narrow cuts of particle size can be obtained by sedimentation. Selectivity: The fundamental ability of a stationary phase to selectively re- tain substances based on their chemical characteristics, including vapor pres- sure and polarity. In LC, selectivity is strongly influenced by the mobile- phase composition. In GC, carrier gas has less, if any, impact on chromato- graphic selectivity. Selectivity (α): Term replaced by the separation factor. See separation factor.
- 67. Terminology Guide October 2013 67 www.chromatographyonline.com Selectivity coefficient (kA/B): In ion-ex- change chromatography, the equilibrium coefficient obtained by application of the law of mass action to an ion exchanger and characterizing the ability of an ion exchanger to select one of two ions pres- ent in the same solution. For example, the exchange of Na+ for H+ in: KNa/H = [Na]S *[H]S/[Na]M[H]M Selectivity triangle: An approach to classify the properties of stationary phases in reversed-phase LC. Results can be represented in a “selectivity triangle” in which the apices of the triangle represent the relative con- tributions of steric hindrance (χS), hydrogen bonding basicity (χB) and cation exchange capacity (χC) to se- lectivity. A graphical visualization of the column selectivity allows three- dimensional data to be presented in a two-dimensional space. Provides an informative yet universal approach for phase classification compared to other models. With this model, selection of columns of either equivalent or dif- ferent selectivity is readily achievable, which should further facilitate the ap- plication of reversed-phase LC. Selectivity tuning: Several techniques for adjusting the selectivity of separa- tions involving more than one column or stationary-phase type. Serially coupled columns and mixed-phase columns can be selectivity-tuned. Semipreparative chromatography: Re- fers to preparative LC carried out on an analytical size (4–5 mm i.d.) or a slightly larger (6–10 mm i.d.) column. Normal injection size would be in milligram to low gram amounts. Sensitivity (S): Degree of detector response to a specified solute amount per unit time or per unit volume, often defined by lower limit of detection (LOD). For a concentration-sensitive detector such as a thermal conductivity detector or UV–vis detector: S = mmax/c where mmax is the peak height and c is the solute concentration in the detec- tor; units of sensitivity for a concentra- tion-sensitive detector that responds in millivolts are mV·mL/g. For mass-flow sensitive detectors such as the flame- ionization detector: S = mmax/Wt where Wt is the mass of solute passing through the detector per unit time; the units of S are then expressed as A•s/g or C/g. Separation: The degree of separation of two peaks in time. See separation fac- tor (α), relative retention, resolution. Separation factor (α): The separa- tion factor α expresses the relative retention of two adjacent peaks: α = t′ R2/t′ R1 = k2/k1 where t′ R2 is the reten- tion time of the second peak and t′ R1 is the retention time of the first peak; k2 and k1 are the corresponding reten- tion factors. Separation impedance (E): A figure of merit developed by John Knox to compare the efficiency of two chro- matographic systems by normalizing for both analysis time and pressure drop: E = tRΔp/N2 ν(1 + k) where tR is the re- tention time, Δp is the pressure drop, N is the theoretical plate number, ν is the reduced velocity, and k the retention fac- tor. The lower the value of E, the better the system. Separation number (SN): Separation number, or Trennzahl (TZ). A mea- sure of the number of peaks that could be placed with baseline resolution be- tween two sequential peaks, z and z+1, in a homologous series, such as two hydrocarbons: SN = (tR(z+1) – tR(z))/ (wh(z+1) + wh(z)) Septum: Silicone or other elastiomeric material that isolates inlet carrier flow
- 68. 68 Terminology Guide October 2013 www.chromatographyonline.com from the atmosphere and permits sy- ringe penetration for injection. Septum purge: Carrier gas is swept across the septum face and out to a separate vent so that material emitted from the septum does not enter the column. Sequential suppression: Combina- tion of chemical suppression (MSM) and CO2 suppression (MCS). The background conductivity of carbonate and hydrogencarbonate eluents after suppression is approximately 10–20 µS/cm. This is a result of the dissolved carbonic acid that partially dissociates. The MCS removes the CO2 from the suppressed eluent and therefore reduces the background even further (typically >1 µS/cm). Shell particle: See superficially porous particles. Sieving: Process of passing a sample of solid particles through a metal or plastic mesh of a uniform cross-sectional area (square opening from 3 µm to 123 mm) to separate particles into uniform sizes; can be performed under wet and dry conditions. Signal-to-noise ratio (S/N): The ratio of the peak height to the noise level. A detector gives a valid signal if there is some measurable response above the normal background noise; both detec- tor sensitivity and limit of detection are dependent on the level at which the sig- nal can be distinguished. A minimum S/N is equivalent to 2 but for quantita- tive methods sometimes a higher value is chosen (such as S/N = 6), meaning that the signal is 6 times that of the baseline noise. Silanol: The Si-OH group found on the surface of silica gel. There are dif- ferent strengths of silanols depending on their location and relationship to each other and based on the metal con- tent of the silica. The strongest silanols are acidic and often lead to undesirable interactions with basic compounds dur- ing chromatography. Silanophile: A compound that has high affinity for active (acidic) silanol groups on the silica surface. Usually a strongly basic amine. Silica gel: The most widely used HPLC packing. It has an amorphous structure, is porous, and consists of siloxane and silanol groups. It is used in all modes of LC as a bare packing for adsorption, as the support for LLC or for chemically bonded phases, and, with various pore sizes, as an SEC packing. Microparticu- late silicas of 3-, 5-, and 10-µm average particle diameter are used in HPLC. Compared to irregular silicas, in mod- ern analytical HPLC columns, spheri- cal silicas are preferred because of their packing reproducibility and because they have lower pressure drops; sometimes re- ferred to as silica. Also used as a gas-solid adsorbent in GC. Siloxane: The Si-O-Si bond. A principal bond found in silica gel or for a silylated compound or bonded phase. Stable ex- cept at high pH values; has little effect on the HPLC separation. Silylation: The process of reaction of an organochlorosilane or organoalk- oxysilane with a compound containing an reactive group. In LC it refers to the process of derivatizing the solute before chromatography to make the solute de- tectable or to prevent unwanted station- ary phase interactions. It can also refer to the process of adding a chemically bonded phase to a solid support or to deactivating the packing to cut down on surface activity. Simulated distillation (SIMDIS): Boiling-point separation technique
- 69. Terminology Guide October 2013 69 www.chromatographyonline.com that simulates physical distillation of petroleum products. Simulated moving bed : A chromato- graphic system involving a series of columns and valves set up to simulate the countercurrent movement of the mobile and stationary phases to allow for the continuous removal of product and reapplication of sample. A complex form of recycle chromatography used in preparative-scale chromatography. Single drop microextraction (SDME): A single drop of solvent (1–2 µL) sus- pended in the headspace can partition volatile analytes into the solvent; the drop can be withdrawn into the syringe and injected into a GC instrument. Single-ion conductivity (Ki): The single-ion conductivity is proportional to the concentration and the equivalent conductivity of the respective ion. Size-exclusion chromatography (SEC): Same as steric exclusion chromatogra- phy. Slurry packing: The technique most often used to pack HPLC columns with microparticles. The packing is suspended in a slurry (~10% w/v) and rapidly pumped into the empty column. Special high pressure pumps are used. Snyder solvent strength parameter (E0): Solvent strength parameter in ad- sorption chromatography; the energy of solvent adsorption per unit surface area occupied by the solvent. Soap chromatography: The earlier name for ion-pair chromatography. Long- chain soaps or detergents were used as the mobile phase additives. Sol gel: Silica gel formed by the aggrega- tion of silica sol; results in type B silica gel with lower surface acidity, lower trace metal, lower surface area and porosity, and higher high pH stability than older type A silica gels. Solid-phase extraction (SPE): A tech- nique for sample preparation using a solid phase packing (dp of 20–40 µm) contained in a small plastic car- tridge or disk or in a well of a 96-well flow-through plate. The solid station- ary phases used are no different than HPLC packings. However, although related to chromatography, the prin- ciple is different and is sometimes referred to as digital chromatography. The process as most often practiced requires four steps: conditioning the sorbent; adding the sample; washing away the impurities; and eluting the sample in as small a volume as possible with a strong solvent. SPE can be per- formed in a variety of formats, such as cartridges, disks, pipette tips, and 96- well plates, and in a variety of modes such as reversed phase, ion exchange, and normal phase. It is a widely used sample preparation technique. Solid support: The same as support. Solid-core packing: See superficially porous particles. Solid-phase microextraction (SPME): A technique in which a small polymer- coated solid fiber is placed into a solu- tion or above the headspace of a solid or liquid sample; analytes will diffuse into the coating until equilibrium is estab- lished; for GC, the fiber containing the sorbed sample is transferred to the GC and the trapped analytes are thermally desorbed into the column. In HPLC, solvent is used to rinse the sorbed ana- lytes for eventual injection into the LC column; less popularly used in LC than in GC. Solid-phase trapping: The use of an SPE cartridge or packed column to trap specific analytes that flow through the device; the packing material is chosen to selectively retain the analytes of interest
- 70. 70 Terminology Guide October 2013 www.chromatographyonline.com and let other compounds pass through unretained. Solute: The dissolved component of a mixture that is to be separated in the chromatographic column. May be re- ferred to as the analyte. Solvent: The liquid used to dissolve a sample for injection into a chromatogra- phy column or CE capillary; sometimes refers to the mobile phase used in LC. Solvent demixing: Occurs when two solvents with very different strengths (A = weak solvent and B = strong sol- vent) are used with unmodified silica or alumina; the strong solvent (B) will be preferentially adsorbed by the active surface of the stationary phase until it is saturated; until this occurs, the weak solvent (A) will be enriched (demixed) as it travels down the column; eventually when the entire column is saturated with B, this solvent will elute mixed with A at the initial strength and sample compo- nents are eluted with the sudden change in solvent strength. Solvent effect: A solute-profile sharp- ening technique used with splitless and on-column injection. Condensed solvent in the column during and shortly after injection traps volatile solutes into a nar- row band. See also retention gap. Solvent exchange: The process of ex- changing one solvent that may not be compatible with the analysis method for a solvent that is more compatible. In some cases, evaporation is used to re- move a volatile solvent and the sample is reconstituted in a different solvent. Solvent flooding: A source of peak- shape distortion caused by excessive solvent condensation inside the column during and after splitless or on-column injection. Solvent flushing: A column rinsing technique that may remove nonvola- tile sample residue and partially restore column performance. Some stationary phases may be damaged by solvent rins- ing or flushing. Solvent selectivity: Ability of a solvent to influence selectivity; there is solvent strength selectivity where a change in solvent strength (say from 5% B to 10% B) will change band spacing or solvent-type selectivity where change from methanol to acetonitrile as a reversed-phase organic modifier will change band spacing. Solvent selectivity triangle: A useful guide for choosing among different sol- vents for the purposes of changing band spacing; solvent selectivity is dependent on dipole moment, acidity, and basicity of the solvent molecule. For details, see L.R. Snyder, P.W. Carr, and S.C. Rutan, J. Chromatogr. A 656, 537–547 (1993). Solvent strength (S): Refers to the abil- ity of a solvent to elute a particular solute or compound from a column. Described for HPLC by Lloyd Snyder for linear elution adsorption chromatography on alumina, solvents were quantitatively rated in an eluotropic series; S varies with modifier type, stationary phase, and temperature. Less extensive data are available for silica and carbon adsorbents. Sonication: The use of ultrasound to create vigorous agitation at the surface of a finely divided solid material. The direct method uses a specially designed inert acoustical tool (a horn or probe, called a sonotrode) placed in sample–solvent mix- ture. In the indirect method, a sample container is immersed in an ultrasonic bath with solvent and subjected to ul- trasonic radiation. Dissolution is aided by the ultrasonic process. Heat can be added to increase the rate of extraction. The method is safe and rapid and is best for coarse, granular material. With the
- 71. Terminology Guide October 2013 71 www.chromatographyonline.com indirect method, multiple samples can be done simultaneously. Sorb: The process of being retained by a stationary phase when the retention mechanism — adsorption, absorption. partitioning — is not clear. Sorbent: Refers to a packing used in ad- sorptive chromatography LC. Common sorbents are polymers, silica gel, alumina, titania, and zirconia and chemically modified materials. Soxhlet extraction: A well accepted technique for the extraction of com- pounds from a solid sample; the sample is placed in a disposable porous container (thimble); constantly refluxing fresh condensed solvent flows through the thimble and dissolves analytes that are continuously collected in a boiling flask; special glassware called a Soxhlet extrac- tor is designed to perform the extraction unattended. Specific surface area: The surface area of an LC packing based on measure- ment by an accepted technique such as the BET method using nitrogen adsorption. Spherical packing: Refers to spherical solid packing materials. In analytical HPLC spherical packings generally are preferred over irregular particles but in preparative work irregular particles are often used because of their lower cost. Spin column: A small column that usually contains a packing material for sample cleanup or isolation; the sample is added to the column, which has a se- lective packing material; the column, in turn, fits into a small collector tube that is placed in a centrifuge, and the liquids in the tube are separated by centrifu- gation. Spin tubes are very popular in handling biological samples for isolating DNA, RNA, and other biocompounds of interest. Spin filter: Similar to a spin column but instead of the column packing a mem- brane filter is used; the purpose of the filter is to remove particulates. Spin tube: See spin column. Split injection: An injection technique for GC where only a portion of the sample is directed to the column. The ratio of the vented volume to the injected volume is called the split ratio, which has typical values of 100:1, 50:1, and so on. Split injection tries to avoid overloading the column; it ensures a representative sample reaches the column. The tech- nique is simple and rugged and protects the column. However, sample discrimi- nation is possible; splitless injections are usually performed automatically. Split ratio (s): The ratio of the sample amount vented to the sample amount entering the column in split injection. Higher split ratios place less sample on the column. Usually measured as the ratio of total inlet flow to column flow: s = (Fs + Fc)/Fc Split vent flow rate (Fs): Carrier gas flow rate from the split vent, measured at room temperature and pressure. Splitless injection: Derivative of split in- jection. During the first 0.5 to 4 min of sampling the sample is not split, and en- ters only the column. Splitting is restored afterwards to purge sample remaining in the inlet. Up to 99% of sample enters the column. Ensures higher sensitivity than split injections but flashback can occur and a higher possibility of sample deg- radation is possible as a result of longer residence time in the hot injection port. Square-well plates: 96-well plates that have square-shaped wells instead of the normal round-bottom wells. Stagnant mobile phase: The fraction of the mobile phase contained with the pores of the particle.
- 72. 72 Terminology Guide October 2013 www.chromatographyonline.com Standard addition: Process used to im- prove quantitation; necessary to have a pure standard of known concentration. An unknown concentration of sample is first injected to give a peak area; then to the unknown concentration a measured amount of pure compound is added. As a result of the new peak area, one can determine the original concentration. An alternative procedure is to add a constant amount of unknown concentration to a series of standards of pure substances and to plot the peak areas obtained against the known concentrations of the original standards. The slope of the line obtained gives the concentration of the unknown. Standards: A sample that contains known quantities of the compounds of interest. Standards are used to help identify sample peaks by comparing the time in which they are eluted to the retention times obtained through the injection of the sample under the same conditions. For quantitation, external standards are compounds that are used to construct calibration curves of detec- tor output (peak area or peak height) versus concentration; the concentration of unknowns is determined by fitting the detector output to the calibration curve. Internal standards are com- pounds of known concentration with different retention times that are added to the sample and relative detector re- sponses between the internal standard and the unknown are compared in order to quantitatively measure un- known compounds. Static coating: A technique for station- ary-phase deposition in open-tubular columns. The column is filled with a solution of stationary phase and one end sealed. A vacuum, heat, or both are applied to the open end. As the solvent evaporates, a thin uniform film of sta- tionary phase is left behind. Stationary phase: The immobile phase involved in the chromatographic process. The stationary phase in LC can be a solid, a bonded, immobilized, or coated phase on a solid support, or a wall-coated phase. The stationary phase used often characterizes the separation LC mode. For example, in LC, silica gel is used in adsorption chromatography, whereas an octadecylsilane bonded phase is used in reversed-phase chromatography. In GC, liquid or polymeric stationary phases are used for liquid–liquid partitioning separations, and porous-polymer, silica, alumina, or molecular sieve packings are used for adsorption and molecular size- based separations. Stationary phase film thickness (df): The average thickness of the stationary- phase film coated on the walls of an open-tubular (capillary) GC column. Most open-tubular GC columns have film thicknesses of 0.1–5 µm. Stationary zone: To be distinguished from the stationary phase. The station- ary zone includes the stagnant mobile phase and the chromatographically ac- tive stationary phase. Step gradient: See stepwise elution. Stepwise elution: Use of eluents of different compositions during the chromatographic run. These eluents are added in the stepwise manner with a pump or by a selector valve. Gradi- ent elution is the continuous version of changing of solvent composition. Steric exclusion chromatography (SEC): A major mode of LC in which samples are separated by virtue of their size in solution. Also known as size exclu- sion chromatography, gel permeation chro- matography, gel filtration chromatography, or gel chromatography. SEC is most often
- 73. Terminology Guide October 2013 73 www.chromatographyonline.com used for polymer separation and charac- terization, the separation of proteins, and the desalting of biological samples. Sterically protected bonded phase: Bonded phase that has sterically pro- tecting bulky functional groups such as isopropyl and isobutyl surrounding siloxane covalent surface bond; prevents attack on siloxane bond and prevents catalyzed hydrolysis and loss of bonded phase at low pH (< 3). Stir-bar sorbent extraction (SBSE): Principle similar to solid-phase mi- croextraction (SPME) but instead of a coated fiber a polymer-coated stir bar is used, greatly increasing the surface area, thus providing higher capacity and greater mass sensitivity. Similar to SPME, equilibration requires tens of minutes. For GC, a special thermal desorption unit is needed to handle the stir bar; in LC, the stir bar is usually rinsed off-line. Straight phase chromatography: Same as normal-phase chromatography. Strong anion exchanger: Anion- exchange packing with strongly basic ionogenic groups (for example, tetraal- kylammonium). Strong cation exchanger: Cation- exchange packing with strongly acidic ionogenic groups (for example, sulfonic). Strong solvent: In general, refers to a solvent which is a good solvent for a chemical compound; in chromatography, refers to the mobile phase constituent that provides a higher solvent strength that causes an analyte to elute more quickly from the column; in a water- acetonitrile binary solvent system for reversed-phase liquid chromatograhy, acetonitrile would be considered to be the strong solvent. Sub-2-µm packing: A term that refers to the use of porous packings below 2 µm average particle diameter; current prod- ucts vary from 1.5 to 2.0 µm. Sulfonyl cation exchanger: A strong cation-exchange functionality found in resin-based packings; usually propyl- SO3H; may come in other cationic forms such as sodium, ammonium, silver, and calcium. Sulfur chemiluminescence detection (SCD): Detection method that responds to sulfur-containing compounds by gen- erating and measuring light from chemi- luminescence. Supercritical fluid: The defined state of a compound, mixture, or element above its critical pressure and critical temperature. Supercritical fluid chromatography (SFC): A technique that uses a supercriti- cal fluid as the mobile phase. The tech- nique has been applied to the separation of substances which cannot be handled effectively by LC (because of detection problems) or GC (because of the lack of volatility). Examples are separations of triglycerides, hydrocarbons, and fatty acids. GC detectors and HPLC pumps have been used together in SFC. Supercritical fluid extraction (SFE): Uses supercritical fluid, most often carbon dioxide alone or containing a small percent of organic modifier for more polar analytes, to extract ana- lytes from solid materials; supercritical fluid has the diffusivity of a gas and the solvent power of a liquid; requires a special SFE unit where the pressure and temperature can be precisely controlled; analytes are collected in a cold trap, on an adsorbent or in a liquid; a “green” extraction technique. Superficially porous particles (SPPs): Same as porous-layer bead. Recently there has been a revival of superficially porous particles based on smaller
- 74. 74 Terminology Guide October 2013 www.chromatographyonline.com particles (1.3–5.0 µm) with thicker po- rous shells (0.3–0.6 µm); such particles give similar or better performance than sub-2-µm particles. Superheated water extraction: Water is heated well above its boiling point in a closed pressurized system; heating changes dielectric constant and increases the solvating power such that it becomes “organic-like.” It is a “green” method for extracting organic analytes from solid matrices. Support: Refers to solid particles. Sup- port can be naked, coated, or have a chemically bonded phase. The solid support doesn’t contribute to the liquid– liquid chromatographic process but is active for adsorptive processes. Support-coated open-tubular column (SCOT): A capillary column in which stationary phase is coated onto a sup- port material that is distributed over the column inner wall. A SCOT column generally has a higher peak capacity than a wall-coated open tubular col- umn (WCOT) with the same average film thickness. See wall-coated open tubular column WCOT. Supported liquid extraction (SLE): A technique based on the principles of liq- uid–liquid extraction where the aqueous phase is supported on a bed of highly purified, high surface area diatomaceous earth (in a tube, cartridge, or 96-well format); this aqueous phase may be buffered and may contain the sample to be partitioned; the organic phase is then percolated through the packed bed allowing for intimate contact with the dispersed aqueous phase. The effluent collected at the exit of the column con- tains the extracted analytes; compared to LLE, the SLE technique is miniatur- ized, easily automated, and provides ex- cellent extraction efficiency. Suppression: Method to reduce the background signal before detection (see electrochemical suppression, chemi- cal suppression, and sequential sup- pression). Typically used together with conductivity detection. Suppressor column: In ion chromatog- raphy, refers to the column placed after the ion-exchange column. Its purpose is to remove or suppress the ionization of buffer ions so that sample ions can be observed in a weakly conducting back- ground with a conductivity detector; sometimes rather than a column, mem- brane suppressors are used. Surface area: In an adsorbent, refers to the total area of the solid surface as de- termined by an accepted measurement technique such as the BET method, which uses nitrogen adsorption. The surface area of a typical porous adsor- bent such as silica gel can vary from 100 to 600 m2/g. Surface coverage: Usually refers to the mass of stationary phase per unit area bonded to an LC support. Often ex- pressed in micromoles per square meter of surface. Sometimes %C is given as an indicator of surface coverage. Surrogate samples: A pure analyte that is extremely unlikely to be found in any sample, and which is added to a sample aliquot in known amounts before ex- traction and is measured with the same procedures used to measure other sample components. A surrogate behaves simi- larly to the target analyte and is most often used with organic analytical pro- cedures. The purpose of a surrogate ana- lyte is to monitor method performance with each sample. Swelling or shrinking: Process where resins and gels increase (or decrease) their volume because of their solvent en- vironment. Swelling is dependent on the
- 75. Terminology Guide October 2013 75 www.chromatographyonline.com degree of crosslinking; low cross-linking resins will swell and shrink more than highly cross-linked resins. If swelling occurs in a packed column blockage or increased back pressure can occur. In ad- dition, column efficiency can be affected. Syringe filter: A small plastic holder containing a membrane filter that has Luer-lock fittings at both the top and the bottom so that it can be affixed to a sy- ringe (which also has a Luer-lock fitting) to pass a sample through the filtration media. Syringe filter diameters can range up to 90 mm. System dispersion: The contribu- tion to band broadening outside of the column itself; it generally refers to the instrumental contributions as well as other extra column contributions. With newer high efficiency columns, decreas- ing the system dispersion contributions will result in better chromatographic performance. System peak: The system peak is the peak of the eluent ion. There is no pos- sibility of quantifying that peak. It is an unwanted peak in the chromatogram. A possible explanation of the system peak: Because of the sample injection, the ion exchange equilibrium of the eluent ions get disturbed. The re-equilibration pro- cess yields in this additional peak. It ap- pears in suppressed and nonsuppressed IC, but it is pretty small with suppression. Using sequential suppression minimizes the system peak. T Tailing: The phenomenon where the normal Gaussian peak has an asymme- try factor greater than one. The peak will have an extended trailing edge. Tailing is caused by sites on the packing that have both a stronger-than-normal retention for the solute and slower desorption kinetics. A typical example of a tailing phenom- enon would be the strong adsorption of amines on the residual silanol groups of a low coverage reversed-phase packing at intermediate pH values. Tailing can also result from injecting an excessive mass or sample, from badly packed columns, from excessive extracolumn volume, poor fittings, and excessive detector volume, or slow detector response. Tailing peaks show an asymmetry factor greater than 1.0; see asymmetry factor. Tailing factor: U.S. Pharmacopeia mea- sure of peak asymmetry defined as the ratio of the peak width at 5% of the apex to two times the distance from the apex to the 5% height on the short time side of the peak. Greater than unity for tailed peaks. See Figure 1 and asymmetry fac- tor. TCDD: Tetrachlorodibenzo-p-dioxin. TCEP: Stationary phase for GC: tris-cya- noethoxypropane. Tedlar bags: Used for grab sampling of air or other gases; Tedlar (Dupont) sampling bags are a whole-air sampling device for high-level volatile organic com- pounds (VOCs) and permanent gases. Several EPA, NIOSH, and OSHA meth- ods exist for bag sampling for a variety of applications: stationary sources emissions, workplace atmospheres, ambient, indoor air quality, and breath analysis. The unique design of these sample bags in- corporates the sampling septum directly in the valve (polypropylene or stainless steel construction), providing easier use and lighter weight than other styles. Ternary mobile phase: Mobile phase consisting of a mixture of three individ- ual solvents or buffers or both. Theoretical plate: A hypothetical entity inside a column that exists by analogy to a multiplate distillation column. As sol- utes migrate through the column they
- 76. 76 Terminology Guide October 2013 www.chromatographyonline.com partition between the stationary phase and the carrier gas. Although this process is continuous, a stepwise model is often visualized. One step roughly corresponds to a theoretical plate. Theoretical plate height (H): The dis- tance along a chromatographic column that corresponds to a single theoretical plate. H = L ⁄N where L is column length and N is the number of theoretical plates. A carryover from distillation theory; a measure of efficiency of a column. For a typical well packed HPLC column, H should be about 2–3 dp for 5-µm par- ticles, usually in the range of 0.01–0.03 mm; modern superficially porous and sub-2-µm particles sometimes show plate heights of less than 2 dp. In open-tubular column GC, H should be between 0.5–2 times the column inner diameter. HETP is a deprecated term for the plate height. The combined van Deemter–Golay equa- tion gives the theoretical plate height for a chromatography column: H = A + B/u – +u – (CM + CS) where A is the contribution due to eddy diffusion and multipath flow and B is the contribution from longitudinal solute diffusion in the mobile phase. The C terms are related to the effects of diffu- sion on mass transfer; CM in the mobile phase and CS in the stationary phase. See A term, B term, C term, Golay equa- tion, and van Deemter equation. Theoretical plate height, minimum (Hmin): The minimum of the van Deem- ter curve that results from a plot of H versus u (LC) or H versus u – (GC). This value represents the most theoretical plates that can be obtained for a certain column and mobile phase system. Usu- ally occurs at excessively slow flow rates. Also known as the optimum plate height. For well-packed columns it is typically 2–3 times the particle diameter; for open-tubular columns 0.5–2 times the inner column diameter and, ignoring stationary-phase contributions to band broadening: Hmin = (dc/2)((1 + 6k + 11k2)/ (3(1 + k)2 ))1/2 Theoretical plate height, reduced (h): Used to compare efficiencies of different columns. A reduced plate-height value of 2 or less at the optimum velocity is considered to be a well-packed column. For packed columns: h = H/dp. For open- tubular columns: h = H/dc Theoretical plate number (N): The number of theoretical plates measured in a column. A concept described by Mar- tin and Synge. Relates chromatographic separation to the theory of distillation. The length of column that corresponds to a single theoretical plate relating to this concept is called the plate height or height equivalent to a theoretical plate. The larger the plate number, the more theoretical plates the column possesses. A typical well-packed HPLC column with a 5-µm porous packing in a 15-cm column of 4.6-mm i.d. should show 10,000–12,000 plates, which is the same number of plates for a 5-cm column of the same internal diameter packed with sub-2-µm particles or superficially po- rous particles. A typical 25-m, 0.25-mm i.d. open-tubular GC column with a thin stationary-phase film of 0.25 µm or less should exhibit 50,000 theoretical plates or more. The theoretical plate number is calculated from a chromatogram as fol- lows: N = 16(tR/wb)2 = 5.54(tR/wh)2 where wb is the width at the peak base and wh is the peak width at half-height. See theo- retical plate height. Theoretical plates, effective (Neff): The true number of theoretical plates in a col- umn. The number of effective theoretical plates corrects theoretical plates (N) for hold-up volume: Neff = 16(t′ R/wb)2 where t′ R is the adjusted retention time and wb
- 77. Terminology Guide October 2013 77 www.chromatographyonline.com is the peak width at base. It is a better figure of merit than simple plate number when comparing devices of very different geometries and phase ratios; sometimes referred to as effective plate number. Theoretical plates, required (Nreq): Number of theoretical plates required to yield a particular resolution (R) at a specific peak separation (α) and reten- tion factor (k): Nreq = 16R2 (α/(α – 1))2 ((k + 1)/k)2 Thermal desorption: The use of heat to desorb analytes from SPME fibers, an SBSE bar, or solid matrices placed in a thermal desorption tube. Thermal extraction: Uses high temper- atures (below pyrolysis temperatures) to extract stable analytes from porous solid matrices; samples are placed in thermal desorption tubes just as in thermal de- sorption. Thermal-conductivity detection (TCD): A thermal-conductivity detector mea- sures the differential thermal conductiv- ity of carrier gas and reference gas flows. Solutes emerging from a column change the carrier-gas thermal conductivity and produce a response. TCD is a universal detection method with moderate sensi- tivity. Thermally tuned tandem column chro- matography (T3C): A form of LC in which two columns with distinctly dif- ferent selectivities are placed in tandem and operated at two different tempera- tures so as to optimize the resolution and/ or speed of analysis. A common eluent is used in both columns and the entire sample passes through both columns and is detected with a single detector. It is not a two-dimensional technique in that each sample component gives only a single peak. Thermionic specific detection (TSD): See nitrogen–phosphorus detection. Time-integrated sampling: In gas sampling, to obtain a more representa- tive sample requires time-integrated sampling. A flow restrictor is used to spread the sample collection flow over a specific time period to ensure an “aver- age” composited or time-weighted aver- age (TWA) sample. A TWA sample will accurately reflect the mean conditions of the ambient air in the environment and is preferred when, for regulatory or health reasons, a typical exposure concentration is required for a situation that may have high variability, as in an occupational setting. Titania: TiO2, is an uncommon adsor- bent used in adsorption chromatography; also used as an SPE sorbent primarily for removal of phosphorous-containing com- pounds such as phospholipids. TMS: Trimethylsilyl (a chemical deriva- tive). In LC, the TMS group is frequently found on endcapped silica gel-based col- umns. Tortuosity (tortuousity factor) (ω): A property of a packed column that con- trols the inhibition of longitudinal dif- fusion of the solute as it diffuses along the column axis. The B term in the van Deemter equation is proportional to the tortuousity. Total mobile-phase volume (Vt): In SEC the total volume of mobile phase in the column. The same as VM. Also known as the totally included volume. Total permeation volume (Vp): The retention volume on an SEC packing where all molecules smaller than the smallest pore will elute. In other words, at Vp all molecules totally permeate all of the pores and are eluted as a single peak. Total porosity (εT ): The ratio of the total volume of mobile phase in the column to the total column volume: εT = VM/Vc = εe + εi(1 – εe)
- 78. 78 Terminology Guide October 2013 www.chromatographyonline.com Totally porous packing: A stationary phase that is a porous matrix. Solutes penetrate the porous to interact with the stationary phase. Trace enrichment: Technique where trace amounts of compounds are re- tained on an HPLC or precolumn packing out of a weak mobile phase or solution and then are eluted by the addition of a stronger mobile phase in a concentrated form. The technique has been most successfully applied in the concentration of trace amounts of hydrophobic compounds (for example, polynuclear aromatic hydrocarbons) out of water using a reversed-phase packing. A strong solvent such as acetonitrile serves to elute the enriched compounds. Trapping: Process of using a solid mate- rial (such as silica gel, polymer, or in- organic sorbent) or liquid solution to physically or chemically retain solutes of interest from a diluted stream of liquid or gas. Frequently used to concentrate analytes for more sensitive analysis. Trennzahl (TZ): See separation num- ber. Triethyl amine: A very common addi- tive used to block silanol groups in re- versed-phase LC when separating basic analytes. Trifluoroacetic acid: A very common mobile phase additive in reversed-phase LC for peptides and proteins. Also a derivatization reagent for amines and carboxylic acids. Tryptic digestion: A method of selec- tively and reproducibly dissecting pep- tide chains of proteins to yield a char- acteristic pattern of smaller units that allows analysis of the parent protein by gradient elution reversed-phase liquid chromatography. Turbulence: In fluid dynamics, turbu- lence or turbulent flow is a flow regime characterized by chaotic and stochastic property changes. Flow in which the ki- netic energy dies out as a result of the ac- tion of fluid molecular viscosity is called laminar flow. Although there is no theo- rem relating the nondimensional Reyn- olds number (Re) to turbulence, flows at Reynolds numbers larger than 4200 are typically (but not necessarily) turbulent, whereas those at low Reynolds numbers usually remain laminar. Turbulent flow: A form of fluid motion in which the flow ceases to be smooth and steady, and becomes chaotic and fluctuates with time. It is characterized by a pressure drop significantly higher than that which would be extrapolated from the laminar region to achieve the same volumetric flow rate. Turbulent flow chromatography: Chromatography performed at very high linear velocities with large particles, if present, under conditions using high Reynolds numbers. At these conditions the H versus u curves show a decrease in H with increase in u. Turbulent flow chromatography can be used for separa- tion or sample preparation. Two-dimensional chromatography: A procedure in which part or all of the sep- arated sample components are subjected to additional separation steps. This can be done by conducting a particular frac- tion eluted from the first column into a second column or system having a different separation characteristic. It includes techniques such as two-dimen- sional thin-layer chromatography using two eluent systems, where the second eluent is applied after rotating the plate through 90°. This also includes LC or GC followed by GC, or one LC mode followed by a different mode — for ex- ample, reversed-phase LC followed by SEC.
- 79. Terminology Guide October 2013 79 www.chromatographyonline.com Two-dimensional electrophoresis: Two-dimensional gel electrophoresis, abbreviated as 2DE or 2D electropho- resis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2D electrophoresis be- gins with 1D electrophoresis but then separates the molecules by a second property in a direction 90° from the first. In 1D electrophoresis, proteins (or other molecules) are separated in one dimension, so that all the proteins will lie along a lane; the molecules are spread out across a 2D gel. Because it is unlikely that two molecules will be similar in two distinct properties, molecules are more effectively sepa- rated in 2D electrophoresis than in 1D electrophoresis. Type A silica: Silica gel formed by gell- ing soluble silicates; generally higher acidity, higher surface area and poros- ity, more trace metals, poorer high-pH stability than Type B silicas. Type B silica: See sol gel. U Ultrahigh-pressure liquid chroma- tography (UHPLC): Ultrahigh-pressure liquid chromatography is often used loosely for any separation performed at pressures greater than provided by conventional pumps (400 bar); origi- nal meaning was for separations in the 20,000 psi+ range. Ultrafiltration: Variety of membrane filtration in which hydrostatic pres- sure forces a liquid against a semiper- meable membrane. Suspended solids and high-molecular-weight solutes are retained, and water and low-mo- lecular-weight solutes pass through the membrane. This separation pro- cess is used for purifying and concen- trating macromolecular (103–106 Da) solutions, especially protein solutions. Ultrafiltration is not fundamentally different from microfiltration, nano- filtration or gas separation, except in terms of the size of the molecules it retains. Ultrafiltration is applied in cross-flow or dead-end mode and separation in ultrafiltration undergoes concentration polarization. Ultrasonic sieving: Used for the accel- eration of sieving processes alternatively or complementary to the classical low frequency vibrators. Especially useful for very fine powders where ultrasound is often the only possibility to enable the sieving process at all. Ultrasonication: The irradiation of a liquid sample with ultrasonic (>20 kHz) waves resulting in agita- tion. Sound waves propagate into the liquid media result in alternating high-pressure (compression) and low- pressure (rarefaction) cycles. Dur- ing rarefaction, high-intensity sonic waves create small vacuum bubbles or voids in the liquid, which then collapse violently (cavitation) dur- ing compression, creating very high local temperatures; several regulatory methods for environmental samples (for example, soils or solid waste) specify ultrasonication. USP categories for chromatographic columns: United States Pharmacopeia characterizes columns for use in their HPLC methods by an “L” designation: L1 = octadecylsilane, L7 = octylsilane, L8 = aminopropyl, and so on. For GC columns, a “G” designation is used: G1 and G2 are dimethylpolysiloxane columns, G3 is a 50% phenylmethyl– polysiloxane column, G16 is a polyeth- ylene glycol column, and so on.
- 80. 80 Terminology Guide October 2013 www.chromatographyonline.com UV–vis detection: The absorbance of light is the signal for measuring the chromatogram. There are four differ- ent ways of applying UV–vis detection in IC: direct UV–vis, indirect UV–vis, UV–vis after postcolumn reaction, and UV–vis after precolumn reaction. V Vacancy chromatography: Technique where a mobile-phase additive causes a positive detector signal output. When a solute is eluted from the column, it dilutes the signal and yields a negative peak (“a vacancy”). The technique has been mostly been applied to single col- umn ion chromatography where mo- bile phases such as citrate and phthal- ate buffers absorb in the UV. When a nonabsorbing anion is eluted it dilutes the UV-absorbing background and causes a negative peak; the detector output leads are usually reversed so that the chromatogram looks normal. The technique has also been used in CE for detection. Vacuum compensation: Method of carrier-gas pressure control in GC with the column exit at mass-selective de- tector vacuum levels. Enabling vacuum compensation adjusts the column inlet pressure to maintain a set flow or ve- locity when the column exit is not at room pressure. Vacuum filtration: Using a vacuum to help pull liquids through a filter; espe- cially useful for viscous liquids or very fine, low porosity filters. Vacuum manifold: A manifold de- signed for SPE cartridges and SPE disks that uses a vacuum to pull liquids through the packed beds; pressurized manifolds are also available. Vacuum manifolds can process multiple samples from ranging from 8 to 24 at a time. van Deemter equation: An equation used to explain the band broadening in chromatography. The equation rep- resents the height of a theoretical plate (H) and has three terms. The A term is used to describe eddy dispersion (dif- fusion) that results from axial velocity heterogeneity. The B term is for the contribution from molecular diffusion or longitudinal diffusion for the solute while passing through the column. The C term is the contribution from inter- phase mass transfer and allows for the finite rate of transfer of the solute be- tween the stationary phase and mobile phase. In its simplest representation it is expressed as follows: H = A + B/u + C u. The van Deemter equation applies to packed columns both for LC and for GC. The related Golay equation ap- plies to open-tubular or capillary GC columns. See Golay equation. Velocity (u): The same as velocity, lin- ear. Velocity, average linear (u –): The aver- age speed at which a molecule of GC carrier gas or LC liquid mobile phase passes through a column: u – = L/tM where L is the column length and tM is the hold-up or unretained peak time. Velocity, column outlet (uo): In GC, the carrier gas velocity at the column outlet. Equal to the average carrier gas velocity divided by the compressibility correction factor: uo = u –/j. The carrier gas expands as it pass through the col- umn from the inlet to the outlet pres- sure, which causes the local carrier-gas velocity to increase along the column. The outlet velocity is always greater than the average velocity. See com- pressibility correction factor. Velocity, interstitial (ue): The ac- tual velocity of the eluent as it moves through the column flowing around
- 81. Terminology Guide October 2013 81 www.chromatographyonline.com the particles: ue = F/(Ac εc). The inter- stitial velocity is the basis for computa- tion of the reduced velocity. Velocity, linear (u): The velocity of the mobile phase moving through the column. Expressed in cm/s. In LC it is directly related to column flow rate by the cross-sectional area of the column and is determined by dividing the col- umn length (L) by the retention time of an unretained compound: u = L/tM. In GC, the speed at which carrier gas moves through the column usually is expressed as the average linear velocity to account for carrier-gas compressibil- ity. See hold-up time; velocity, aver- age linear; velocity, column outlet. Velocity, mobile phase (uM): The ve- locity at which the liquid mobile phase percolates through the bed of particles in an LC column: uM = L/tM. See veloc- ity, linear; velocity, average linear. Velocity, optimum linear (Uopt): The mobile-phase velocity corresponding to the minimum theoretical plate height, ignoring stationary-phase contributions to band broadening. In open-tubular GC: uopt = 8 (DG/dc) ((3(1 + k)2)/(1 + 6K + 11k2))1/2 Velocity, reduced (ν): Along with the reduced plate height, the reduced veloc- ity is used to compare different chro- matographic columns. It relates the solute diffusion coefficient (DM) in the mobile phase to the particle size of the column packing (dp): ν = u dp/Dm where u is the interstitial mobile-phase linear velocity in packed columns, or the average carrier-gas linear velocity in GC. For open-tubular columns, the column internal diameter is used in- stead of the particle diameter. Velocity, superficial (us): The hy- pothetical velocity the mobile phase would have if the same column were operated unpacked but with the same flow rate: us = F/Ac Velocity, zone (uz): The velocity of travel of the solute zone: uz = u/(1 + k) = L/tR Viscosity (η): Also referred to as mo- bile phase viscosity. The viscosity of the mobile phase varies with the tem- perature of the column. Column back pressure is directly proportional to solvent viscosity. Low-viscosity mobile phases generally give better efficiency than less viscous ones because diffu- sion coefficients are inversely related to solvent viscosity. For example, in reversed-phase LC, column efficiency is higher with acetonitrile as an organic modifier than with isopropanol which is more viscous. In GC, the viscosity of the gaseous mobile phase increases with temperature, which causes the carrier-gas flow rate and linear veloc- ity to decrease during temperature pro- gramming if the inlet pressure is held constant. Different GC carrier gases such as nitrogen, helium, or hydrogen have different viscosities. Void: The formation of a space or gap, usually at the head of the column, caused by a settling or dissolution of the column packing. A void in the col- umn leads to decreased efficiency and loss of resolution. Even a small void can be disastrous for small particle microparticulate columns. The void can be removed sometimes by filling it with glass beads or the same porous packing used in the remainder of the column. Void time: See hold-up time. Void volume: See hold-up volume. Volume, liquid phase: See volume, stationary phase. Volume, mobile phase (VG or VM): For wall-coated open-tubular columns
- 82. 82 Terminology Guide October 2013 www.chromatographyonline.com (WCOT), ignoring the stationary phase film thickness (df = 0): VG ≈ L(πdc 2)/4 Volume, stationary phase (VL or VS): Volume of the liquid stationary phase contained in the column. The ratio of the mobile-phase volume to the station- ary liquid-phase volume is the phase ratio of a GC column. See phase ratio. W Wall effect: The consequence of a looser packing density near the walls of a rigid HPLC column. The mobile phase has a tendency to flow slightly faster near the wall because of the increased local permeability. The solute molecules that happen to be near the wall are carried along faster than the average of the solute band and, consequently, band spreading results and there is a loss of column efficiency. Wall-coated open-tubular column (WCOT): An open-tubular (capillary) GC column in which a uniform stationary phase film is coated directly onto the col- umn wall. See also porous-layer open tu- bular (PLOT) column, support-coated open-tubular column (SCOT). Wash step: See rinse step. Water dip: Indicates the hold-up time in suppressed IC. Usually a negative peak that corresponds to the volume of sample water. The area depends on the difference in concentration of the eluent anions be- tween eluent and sample. The water dip is large if the sample is almost ultrapure water. If the sample is diluted with eluent there will be almost no water dip. Weak anion exchanger: Anion- exchange packing with weakly basic ionogenic groups (for example, amino or diethylaminoethyl). Weak cation exchanger: Cation-ex- change packing with weakly acidic iono- genic groups (for example, carboxyl). Weak solvent: In general, refers to a solvent that is a poor solvent for a par- ticular chemical compound; in chro- matography, refers to the mobile phase constituent that provides a low solvent strength and causes an analyte to be eluted more slowly from the column. In a water–acetonitrile binary solvent sys- tem for reversed-phase LC, water would be considered the weak solvent; in a bi- nary solvent eluent would normally be the “A” solvent. Wide-bore open-tubular column (WBOT): Open-tubular (capillary) GC column with a nominal inner diameter dc of ≥530 μm. Wilke-Chang equation: A semiempiri- cal equation used to estimate diffusion coefficients in liquids as a function of molecular size of solute and solvent viscosity. XYZ Xerogels: Gels used in SEC that will swell and shrink in different solvents; also refers to silica-based packings that are prepared from acidification of solu- ble silicates to give a amorphous, high surface, high porosity, rigid particle. Zero dead volume: Any fitting or com- ponent in which all of the volume is swept by the eluent. See dead volume. Zirconia: Porous zirconium oxide; used as a chromatographic sorbent usually coated or bonded with polymeric or- ganic phase. Zone: See band. Zwitterionic packing: A packing ma- terial for HPLC that carries both posi- tive and negative charges on its surface; zwitterionic packings are useful in the HILIC mode. Zwitterions: Compounds that carry both positive and negative charges in solution.
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