-
he first glossary of common andnot-so-common terms and
buzz-words for reference to high perfor-
mance liquid chromatography (HPLC)columns and column technology
was pub-lished in 1988 (1). It is time for an updatebecause many
new terms have arisen or, in some
cases, their original meanings haveexpanded or changed;
the various techniques of capillary elec-trophoresis (CE) have
become well devel-oped and are used in many laboratoriesthroughout
the world; and
the International Union of Pure andApplied Chemistry (IUPAC)
publishedits massive undertaking titled Nomen-clature for
Chromatography, which pro-vides guidance and changes in some ofthe
more commonly accepted terms (2).This months Column Watch will
update the earlier glossary and will expandcoverage into
techniques beyond HPLC.This glossary is not intended to be an
in-depth or highly theoretical treatment. Forexample, we have
elected not to cover themyriad terms used in
instrumentation,detection, data handling, quantitativeanalysis, and
validation associated with liquid-phase analysis but instead have
cho-sen to use terms that analysts mayencounter in everyday
laboratory work withcolumns, phases, and method development.The
listing should be helpful to those juststarting in HPLC, CE, and
related tech-niques. It also may serve as a refresher forlong-time
users.
The entire glossary also can be found on the LCGC web site at
http://www.chromatographyonline.com.
AAa: See separation factor.A solvent: Usually the weaker solvent
in
a binary eluent or gradient elution separa-tion. In
reversed-phase liquid chromatogra-phy (LC), the A solvent typically
is water ora water-rich mixture.
A term: The first term in the vanDeemter equation. See eddy
dispersionterm and van Deemter equation.
Absorption: The process of retention inwhich the solute
partitions into a liquid-like coating.
Activity: The relative strength of the sur-face of the packing
in adsorption chro-matography. For silica gel, the more avail-able
the silanol groups, the more active thesurface. Activity can be
controlled byadding water or other polar modifier thathydrogen
bonds to the active sites, therebyreducing the surface
activity.
Additive: A substance added to themobile phase to improve the
separation ordetection characteristics; for example, acompeting
base to negate the effects ofsilanols, a chelating agent to block
metalsites, or a UV-absorbing compound to per-form indirect
photometric detection.
Adjusted retention time (tR9): A measureof the retention time
adjusted for theholdup time; tR9 5 tR 2 tM, where tR is
theretention time and tM is the holdup time(the time it takes for a
small, unretainedcompound that completely permeates thepores to be
eluted from the chromato-graphic column).
Adjusted retention volume (VR9):Adjusts the retention volume for
theholdup volume; VR9 5 VR 2 VM, whereVR is the retention volume of
the peak ofinterest and VM is the holdup volume (thevolume
corresponding to the total volumeof mobile phase in the column).
See alsodead volume and holdup volume.
Adsorbent: Packing used in adsorptionchromatography. Silica gel
and alumina arethe most frequently used adsorbents in
Ronald E. Majors andPeter W. Carr
This months Column
Watch column is an
extensive glossary of
definitions and terms
used in the liquid-phase
separation techniques of
high performance liquid
chromatography,
capillary electrophoresis,
and capillary
electrochromatography.
The glossary should be
useful to those just
starting to use these
separation techniques
and can serve as a
refresher for long-time
users. It provides some of
the newer nomenclature
recommended by the
International Union of
Pure and Applied
Chemistry.
Glossary of Liquid-PhaseSeparation Terms
T
Ronald E. MajorsColumn Watch Editor
Column WatchColumn
-
enzyme, antigen, or hormone for themacromolecule of interest to
a solid sup-port (or carrier). This immobilized ligandwill interact
only with molecules that canselectively bind to it. Molecules that
willnot bind will be eluted unretained. Theretained compound later
can be released ina purified state. Affinity chromatography
isnormally practiced as an onoff separationtechnique.
Agarose: High molecular weight polysac-charide used as a
separation medium inbiochromatography. It is used in bead
form,often in gel-filtration chromatography, withaqueous mobile
phases.
Alkoxysilane: A reactant used for thepreparation of chemically
bonded phases. Itwill react with silica gel as follows: R3SiOR1
[SiOH fi [SiOSiR3 1 ROH, whereR is an alkyl group.
Alumina: A normal-phase adsorbent usedin adsorption
chromatography. Aluminumoxide is a porous adsorbent that is
availablewith a slightly basic surface; neutral andacidic
modifications also can be made.Basic alumina can have advantages
over sil-ica, which is considered to have an acidicsurface.
Amino phase: A propylamino phase usedin normal bonded-phase
chromatography.It is somewhat reactive for solute moleculessuch as
aldehydes or mobile-phase additivesthat can react with amines. The
aminophase has found some applications as aweak anion exchanger,
and it also is usedfor the separation of carbohydrates with
awateracetonitrile mobile phase. It is a rela-tively unstable
phase.
Amphoteric ion-exchange resin: Ion-exchange resins that have
both positive andnegative ionic groups. These resins are mostuseful
for ion retardation in which all ionicmaterials can be removed from
solutionbecause the anionic and cationic function-alities coexist
on the same material.
Analyte: The compound of interest to beanalyzed by injection
into and elution froman HPLC column.
Anion exchange: The ion-exchange pro-cedure used for the
separation of anions.Synthetic resins, bonded-phase silicas,
andother metal oxides can be analyzed in thismode. A typical
anion-exchange functionalgroup is the tetraalkylammonium,
whichmakes a strong anion exchanger. An aminogroup on a bonded
stationary phase is anexample of a weak anion exchanger.
Asymmetry: Factor describing the shapeof a chromatographic peak.
Chromato-graphic theory assumes a Gaussian shapeand that peaks are
symmetrical. A quantita-
high performance liquid chromatography(HPLC).
Adsorption: The process of retention inwhich the interactions
between the soluteand the surface of an adsorbent dominate.The
forces can be strong forces (hydrogenbonds) or weak (van der Waals
forces). Forsilica gel, the silanol group is the drivingforce for
adsorption, and any solute func-tional group that can interact with
thisgroup can be retained on silica. The termadsorption places
emphasis on the surfaceversus penetration or embedding in the
sta-tionary phase coated or bonded to a sur-face.
Adsorption chromatography: One of thebasic LC modes that relies
upon adsorptionto the surface of an active solid to effect
theseparation. Silica gel and alumina are themost frequently used
normal-phase adsor-bents, and molecules are retained by
theinteraction of their polar function groupswith the surface
functional groups; forexample, silanols of silica. Carbon also
isused as an adsorbent in a reversed-phasemode.
Adsorption isotherm: A plot of the equi-librium concentration of
sample in themobile phase per unit volume versus theconcentration
in the stationary phase perunit weight in adsorption
chromatography.The shape of the adsorption isotherm candetermine
the chromatographic behavior ofthe solute; for example, peak
tailing, peakfronting, and column overload.
Aerogel: A packing prepared when thedispersing agent is removed
from a gel sys-tem without collapsing the gel structure.Silica gels
and glass beads used for size-exclusion chromatography (SEC) are
exam-ples of aerogels that can retain their struc-tures even at the
high pressures used inHPLC. See also xerogels.
Affinity chromatography: A techniquein which a biospecific
adsorbent is preparedby coupling a specific ligand such as an
tive measure is the peak asymmetry factor,which is the ratio of
the distance from thepeak apex to the back side of the
chro-matography curve over the distance fromthe peak apex to the
front side of the chro-matography curve at 10% of the peakheight.
Other measures of asymmetry arecommonly used, especially the U.S.
Phar-macopeia (USP) method. See Figure 1. Seealso FoleyDorsey
equation.
Asymmetry factor: A factor that denotesband shape. The asymmetry
factor is calcu-lated from the chromatographic peak bydropping a
perpendicular at the peak apexand a horizontal line at 10% of the
peakheight; at the intersection, the distance tothe tail of the
peak along the horizontal line(distance B) divided by the distance
alongthe horizontal line to the front of the peak(distance A)
produces a ratio called thepeak asymmetry factor (see Figure 1).
Theratio is 1 for a symmetrical peak, less than1 for a fronting
peak, and greater than 1 fora tailing peak. The higher the value,
the lesssymmetrical the peak; values greater than 2are
unacceptable.
Atmosphere (atm): A measure of thepressure drop across an HPLC
column; 1atm 5 14.7 lb/in.2 (psi). See also bar andpascals.
BBb: See phase ratio.Bo: See permeability.B solvent: Usually the
stronger solvent in
a binary eluent or gradient separation; typi-cally the organic
modifier or modifier-richbinary mixture with water in
reversed-phaseLC.
B term: The second term of the vanDeemter equation. See also
longitudinaldiffusion and molecular diffusion term.
Backflushing: A column-switching tech-nique in which a four-way
valve placedbetween the injector and the column allowsmobile-phase
flow in either direction. Back-flushing is used to elute strongly
held com-pounds at the head of a column. It can beused for
analyzing these compounds ormerely removing them from the
column.
Band: Refers to the chromatographicpeak as it moves down and is
eluted fromthe column.
Band broadening: The process ofincreasing width and concomitant
dilutingof the chromatographic band as it movesdown the column. The
peak is injected as anarrow slug and, ideally, each
separatedcomponent would be eluted as a narrowslug of pure compound
if not for theprocess of band broadening. The measure
Figure 1: Example of a tailing peak. (Modi-fied with permission
from reference 3.)
1.0
0.5
0.10.0
Nor
mal
ized
pea
k he
ight
32 36 40 44 48t1 tp t2
Time (s)
B A
wa 5 A 1 B hp
-
of band broadening is bandwidth (tw) or,more correctly, the
number of theoreticalplates (N ) in the column. Sometimescalled
band dispersion or band spreading.See Figure 2.
Bandwidth (tw): The width of the chro-matographic band during
elution from thecolumn. It usually is measured at the base-line by
drawing tangents to the inflectionpoints on the sides of the
Gaussian curvethat represents the peak. Small bandwidthsusually
represent efficient separations; alsocalled peak width. See Figure
2.
Bar: A unit of pressure measurement inHPLC equal to 1 atm, ;15
lb/in.2, or 0.1MPa.
BET method: Developed by Bruner,Emmett, and Teller (BET), a
method formeasuring surface area that uses
nitrogenadsorptioncondensation in pores at liquidnitrogen
temperature. Pore volume andpore size distribution also can be
obtainedfrom BET method calculations.
Bidentate silane: A specific type ofbonded phase in which a
short hydrocar-bon bridge connects two silicon atoms in asilane
that is bound to the surface throughtwo siloxane groups.
Binary mobile phase: Mobile phasecomprising two solvents or
buffers.
Biocompatible: A term to indicate thatthe column or instrument
component willnot irreversibly or strongly adsorb or deac-tivate
biomolecules such as proteins. Fre-quently means metal-free or
ceramic sur-faces and components.
Bonded-phase chromatography: Themost popular mode in LC in which
aphase chemically bonded to a support isused for separation. The
most popular sup-port for bonded-phase chromatography
ismicroparticulate silica gel, and the mostpopular type of bonded
phase is organo-silane such as octadecyl for
reversed-phasechromatography. Approximately 70% of allHPLC
applications are performed usingchemically bonded phases.
Bonded-phase concentration: See coverage.
Boxcar chromatography: See columnswitching.
Breakthrough volume: The volume atwhich a particular solute
pumped continu-ously through a column will begin to beeluted. It is
related to the column volumeand the retention factor of the solute.
It isuseful to determine the total sample capac-ity of the column
for a particular solute.
Buffer: A solution that maintains con-stant pH by resisting
changes in pH from
dilution or addition of small amounts ofacids and bases.
Buffer capacity: A quantitative measureof the potential of a
buffer solution(defined as the number of equivalents ofstrong acid
or base to cause a one pH unitchange in 1 L of a buffer solution)
or sim-ply the ability of a buffer to withstandinjections of a
buffered sample solutionwithout changing mobile-phase pH; capac-ity
determined by pH, buffer pKa, andbuffer concentration.
CCC term: The interphase mass transfer
term of the van Deemter equation. See alsomass transfer and van
Deemter equation.
C8: See octylsilane.C18: See octadecylsilane.C4, C8, C18, etc.:
Refer to the alkyl-chain
length of a reversed bonded phase.CS: See Langmuir
isotherm.Capacity: See sample capacity.Capacity factor (k9): Old
term for a
chromatographic parameter that measuresthe degree of retention.
Now defined as theretention factor (k) by the InternationalUnion of
Pure and Applied Chemistry(IUPAC). See also retention factor
formethod of calculation.
Capillary column: Refers to columnswith inner diameters less
than 0.5 mm.
Capillary electrochromatography (CEC):A hybrid technique in
which capillarycolumns are packed with chromatographicsorbents and
electroosmotic flow ratherthan pressure moves mobile phase
throughthe column; technique has the surface-mediated selectivity
potential of HPLCand the high efficiency of capillary
elec-trophoresis (CE).
Capillary gel electrophoresis (CGE): A technique in which a
capillary is filledwith, or the walls coated or covalentlybonded
with, cross-linked polyacrylamideto simulate slab gel
electrophoresis; thispolymer network uses a sieving mecha-nism;
used for protein, carbohydrate, andDNA separations such as
fingerprintingand sequencing.
Capillary isoelectric focusing : Separa-tion is based on
isoelectric points of pro-teins; the capillary is filled with
solution;the sample is introduced into the capillaryin the presence
of ampholytes; under theapplication of an electric field, the
proteinmigrates until it reaches a pH at which it isneutralized and
maintains that position inthe capillary.
Capillary LC: Generally refers to HPLCperformed in a
fused-silica or other type of
Figure 2: Widths of a Gaussian peak at various heights as a
function of the standard deviation(s) of the peak. (Modified with
permission from reference 2.)
1.000
0.882
0.607
0.500
0.324
0.134
0.044
Inflection points
Tangents drawn tothe inflection points
Nor
mal
ized
pea
k he
ight
wi 5 2s
wb5 4s
3s
4s
s
5s
wh 5 2.355s
wi
wh
-
Column performance (N): Refers to theefficiency of a column; the
number of the-oretical plates for a given test compound.
Column plate number (N): Denotes thecolumn efficiency; the
larger the platenumber, the more theoretical plates thecolumn
possesses; a typical well-packedcolumn with a 5-mm dp porous
packing ina 15 cm 3 4.6 mm column should provide10,00012,000
plates.
Column switching: Using multiplecolumns connected by switching
valves forbetter chromatographic separations or sam-ple cleanup.
Fractions from a primary col-umn can be switched to two or more
sec-ondary columns, which in turn can befurther diverted to
additional columns orto detectors; sometimes called
multidi-mensional chromatography.
Column volume (Vc): The volume of theunpacked column; Vc 5 AcL,
where Acand L are the cross-sectional area of thetube and the tube
length, respectively.
Competing base: Adding a small basiccompound such as
triethylamine ordimethyloctylamine at 1050 mM concen-tration to the
mobile phase in reversed-phase chromatography to inhibit basic
ana-lytes from interacting with residualsilanols; works by the law
of mass actionbecause concentration of competing base ismuch
greater than analyte. See also addi-tive.
Comprehensive two-dimensional chro-matography: Two-dimensional
chromatog-raphy applied to every fraction. See alsotwo-dimensional
chromatography.
Controlled surface porosity support:Same as porous-layer bead
and pellicularpacking.
Counterion: The ion in solution used todisplace the ion of
interest from the ionicsite in an ion-exchange process. In
ionpairing, it is the ion of opposite chargeadded to the mobile
phase to form a neu-tral ion pair in solution.
Coupled columns: A form of columnswitching that uses a primary
column con-nected to two secondary columns by aselector valve.
Fractions from the first col-umn can be selectively transferred to
thesecond and third columns for additionalseparations. This term
also is used todescribe two or more columns connectedin series to
provide an increased number ofplates.
Coverage: Refers to the amount ofbonded phase on a silica
support inbonded-phase chromatography. Coverageusually is described
in micromoles per
capillary column; the inner diameters typi-cally are less than
0.5 mm; has also beencalled micro-LC.
Capillary micellar electrochromatogra-phy: The CEC version of
micellar electro-kinetic capillary chromatography (MEKC).
Capillary tubing: Tubing to connect var-ious parts of a
chromatograph and directflow to the proper places. Most
capillarytubing used in HPLC is less than 0.020 in.in inner
diameter. The smallest usefulinner diameter is approximately 0.004
in.
Capillary zone electrophoresis (CZE):CE performed in an open
fused-silica cap-illary tube with and without various addi-tives
and capillary coatings; also calledopen-tube capillary zone
electrophoresis.
Capping: Same as endcapping.Carrier: A term most often used in
affin-
ity chromatography; refers to the supportthat binds the active
ligand, usually by acovalent bond; can also refer to the sup-port
in other chromatography modes suchas liquidliquid
chromatography.
Carrier gas: The mobile phase in gaschromatography (GC).
Cartridge column: A column type thathas no endfittings and is
held in a cartridgeholder. The column comprises a tube andpacking
contained by frits in each end ofthe tube. Cartridges are easy to
change andare less expensive and more convenientthan conventional
columns with endfit-tings.
Cation-exchange chromatography: Theform of ion-exchange
chromatography thatuses resins or packings with functionalgroups
that can separate cations. An exam-ple of a strong cation
functional groupwould be a sulfonic acid; a weak cation-exchange
functional group would be a car-boxylic acid.
CE: Capillary electrophoresis.CEC: See capillary
electrochromatogra-
phy.CGE: See capillary gel electrophoresis.CZE: See capillary
zone electrophoresis.Chain length: The length of carbon
chain in the hydrocarbon portion of areversed-phase packing. It
is expressed asthe number of carbon atoms (C8, C18,etc.). It
specifically excludes the shortchains typically methyl, isopropyl,
andsec-butyl groups that also are attachedto the silane.
Channeling: Occurs when voids createdin the packing material
cause mobile phaseand accompanying solutes to move morerapidly than
the average flow velocity,which in turn allows band broadening
to
occur. The voids are created by poor pack-ing or erosion of the
packed bed.
Chemisorption: Sorption caused by achemical reaction with the
packing. Mostof these interactions are irreversible andusually
occur on packings with reactivefunctional groups such as silanol
orbonded amino phases. Chemisorption iscommon with metal oxide
phases that havestrong Lewis acid sites.
Chiral recognition: The ability of a chi-ral stationary phase to
interact differentlywith two enantiomers leading to theirHPLC
separation.
Chiral stationary phases: Stationaryphases that are designed to
separate enan-tiomeric mixtures. The phases can becoated or bonded
to solid supports, createdin situ on the surface of the solid
support,or exist as surface cavities that allow spe-cific
interactions with one enantiomericform.
Chlorosilane: A chemical reagent used to prepare siloxane bonded
phases; reactiv-ity changes from a monochlorosilane ,dichlorosilane
, trichlorosilane; the alkylportion (octadecyl, octyl, etc.) will
dictatethe hydrophobicity of the resulting bondedphase;
alkoxysilanes can be used but areless reactive.
Chromatogram: A plot of detector signaloutput or sample
concentration versustime or elution volume during the
chro-matographic process.
Chromatograph: As a noun: a deviceused to implement a
chromatographic sep-aration. As a verb (IUPAC): the act of
sep-arating by elution through a chromato-graphic bed.
Classification: The process of sizing col-umn packing particles;
generally in HPLC,small particle-size distribution providesbetter
efficiency and a greater permeabilitybecause of the absence of
fines. Classifica-tion can be performed by
sedimentation,elutriation, and centrifugal air techniques.
Column back pressure: See head pres-sure.
Column chromatography: Any form ofchromatography that uses a
column ortube to hold the stationary phase. Open-column
chromatography, HPLC, andopen-tubular capillary chromatography
allare forms of column chromatography.Most often refers to
open-column chro-matography used for preparative-scalework.
Column length (L): The length of chro-matography column in HPLC
or capillaryin CE used to perform the liquid-phaseseparation.
-
square meter or in terms of percentage car-bon (w/w).
Critical micelle concentration: The con-centration of an ionic
surfactant abovewhich a micelle is formed by aggregation;micelles
added to a mobile phase improvethe separation of nonionic
substances inHPLC and CE (MEKC) by a partitioningmechanism.
Cross-linking: During the process ofcopolymerization of resins
to form a three-dimensional matrix, a difunctionalmonomer is added
to form cross-linkagesbetween adjacent polymer chains. Thedegree of
cross-linking is determined bythe amount of the monomer added to
thereaction. For example, divinylbenzene is atypical cross-linking
agent for the produc-tion of polystyrene ion-exchange resins.The
swelling and diffusion characteristicsof a resin are governed by
its degree ofcross-linking.
Cyclodextrins: Cyclic oligomers of sev-eral D-(1)-glucopyranose
units used in chi-ral HPLC and CE separations; popularones are
named a-, b-, and g-cyclodex-trins; they have a truncated cone
shape, arelatively hydrophobic cavity, and primaryand secondary
hydroxyl groups at theirends; they separate on the basis of
differen-tial inclusion of enantiomers; modifiedcyclodextrins with
derivatized hydroxylgroups also are used for selectivity
modifi-cation.
DDDead volume (VM): The column dead
volume comprises the entire space accessi-ble to a small
molecule that can fully per-meate all pores of a packing material.
Itincludes the interstitial volume and theunoccupied pore volume.
It is denoted asVM. The system dead volume includes theadditional
volume in the tubing that con-nects the injector and detector to
the col-umn. The system dead volume usually isapproximated by
injecting a small, essen-tially unretained species. Uracil,
acetoneand thiourea are most commonly usedspecies in reversed-phase
chromatography.See also adjusted retention volume,holdup volume,
and void volume.
DEAE: See diethylaminoethyl.Degassing: The process of removing
dis-
solved gas from the mobile phase before orduring use. Dissolved
gas may come out ofsolution in the detector cell and causebaseline
spikes and noise. Dissolved air canaffect detectors such as
electrochemical (byreaction) or fluorescence (by
quenching)detectors. Dissolved gases also can cause
pumps to lose their prime. Degassing isperformed by heating the
solvent, heliumsparging, or using vacuum (in a vacuumflask) or
on-line evacuation from a tubemade of a gas-permeable substance
such aspolytetrafluoroethylene (PTFE).
Denaturing HPLC: Using reversed-phaseHPLC to investigate genetic
mutations bythe investigation of DNA base pairs.
Desalting: Technique in which low mol-ecular weight salts and
other compoundscan be removed from nonionic and highmolecular
weight compounds. An exampleis using a reversed-phase packing to
retainsample compounds by hydrophobic effectsyet allowing salts to
pass through unre-tained. Using an SEC column to excludelarge
molecules and retain lower molecularweight salts is another
example.
Dextran: Polydextran-based packingmaterial primarily used for
low-pressurebiochromatography; an example would beSephadex
(Amersham Pharmacia Biotech,Piscataway, New Jersey).
Diethylaminoethyl (DEAE): A popularweak anion-exchange
functionality (typi-cally attached to cellulose or
Sepharose[Amersham Pharmacia Biotech]) used forseparating
biomolecules.
Diffusion coefficient (DM or DS): A fun-damental parameter of a
molecule in gas,solution (DM), or the stationary phase(DS).
Expressed in square centimeters persecond. DM is dependent on the
molecularweight of the solute, temperature, solventviscosity, and
molar volume of the solute.A typical value for a 100-Da molecule
inreversed-phase chromatography at roomtemperature is 1025
cm2/s.
Diol phase: A hydrophilic phase that isuseful in normal and
reversed phase. It is adiol structure (two OH groups on adja-cent
carbon atoms in an aliphatic chain).In normal-phase work, it is
less polar thansilica. It has been used to separate proteinsand
polypeptides in reversed-phase chro-matography.
Displacement chromatography: A chro-matographic process in which
the sample isplaced onto the column head and then isdisplaced by a
compound that is morestrongly sorbed than the compounds of
theoriginal mixture. Sample molecules thenare displaced by each
other and by themore strongly sorbed compound. Theresult is that
the eluted sample solute zonesmay be sharpened; displacement
tech-niques have been used mainly in prepara-tive-scale HPLC
applications.
Distribution constant (coefficient) (Kc):The total equilibrium
concentration of a
component in all forms or on the station-ary phase divided by
the total equilibriumconcentration of the component in themobile
phase; also called the distributioncoefficient or the partition
coefficient inpartition chromatography. In partitionchromatography,
Kc is used when the con-centration in the stationary phase
isexpressed per unit volume of the phase (VR 5 VM 1 KcVS). In a
solid stationaryphase, Kg is used and is expressed per mass
(weight) of the dry solid phase. Inadsorption chromatography with a
well-characterized adsorbent of known surfacearea, the
concentration in the stationaryphase is expressed per unit surface
area.
DM: See diffusion coefficient.dp: See particle size.DS: See
diffusion coefficient.Dwell time: The time equivalent to
dwell volume; determined by the productof flow rate and the
dwell volume.
Dwell volume: The volume between thepoint of mixing of solvents
(usually in themixing chamber or at the proportioningvalves in the
liquid chromatograph) andthe head of an LC column. Important
ingradient elution or in isocratic elution situ-ations when changes
in solvent composi-tion are made so that the column experi-ences
the composition change in theshortest possible time. Low-pressure
mix-ing systems generally have larger dwell vol-umes than
high-pressure mixing systems.
Dynamic coating: The formation of in-situ coatings on the
packing in HPLC oron capillary walls in CE by adding a sub-stance
to the mobile phase that adsorbsonto (or absorbs into) the packing
or atthe wall surface. The purpose of a dynamiccoating is to
generate a new stationaryphase or to deactivate the packing
materialor capillary wall to prevent unwantedinteractions. One
simple example is theadjustment of the mobile phase or
runningbuffer to less than pH 3 to protonatesilanols and negate
their effect. Anotherexample is coating the phase with ahydrophilic
polymeric material to preventadsorption of proteins.
EEE: See separation impedance.: See interparticle porosity.Eddy
dispersion (diffusion) term (l):
The A term in the van Deemter equation.It is the contribution to
plate height fromthe heterogeneity in axial velocities as aresult
of the particle size and geometry ofthe packing, as well as wall
effects; A 5
-
2ldp, where l is an empirical column con-stant. Typical values
of l for well-packedcolumns are 0.81.0. Some theories
ofchromatography indicate a velocity-depen-dent contribution to the
height equivalentto a theoretical plate (HETP) from thisprocess.
Also known as eddy diffusion,flow-heterogeneity induced
broadening,and the multipath term. See also vanDeemter
equation.
e: See interstitial porosity.Effective capillary length: The
dis-
tance between the point of sample additionand the point of
detection in CE. For on-capillary detection in which the column
isused as the flow cell in UV detection, thislength is shorter than
the capillary length.
Effective plate height (Heff): The col-umn length divided by the
effective platenumber.
Effective theoretical plates (Neff): Alsocalled the effective
plate number byIUPAC. The true number of plates in acolumn, because
it corrects theoreticalplates for dead volume. Neff 516[(tR9/wb)2],
where tR9 is the adjustedretention time and wb is the bandwidth
ofthe peak (see Figure 2). It is a better figureof merit than
simple plate number forcomparing devices of very different
geome-tries and phase ratios.
Efficiency (N or H ): A measure typicallydetermined by the
number of theoreticalplates (N ) calculated from the equation N 5
16(VR/wb)2 5 16 (tR/wb)2, where wbis the peak width measured at the
base (seeFigure 2). If the peak width is measured athalf height,
the following equation is used:N 5 5.545 (VR/wh)2. The plate
height(H ) or HETP is determined by H 5 L/N.The efficiency of
asymmetric peaks is bet-ter determined from the peak centroid
andvariance by mathematical analysis of thepeak shape. See also
FoleyDorsey equa-tion.
Effluent: The mobile phase leaving thecolumn; same as
eluate.
i: See intraparticle porosity.Electroendosmotic flow: See
electro-
osmotic flow.Electromigration injection: Inlet end of
CE capillary is placed in sample solutionand voltage is applied
for a set time; ana-lytes move from sample vial into
capillary;discrimination effects may occur becausecompounds of
differing charges willmigrate at different rates.
Electroosmotic flow (veo): Bulk flow ofsolvent within capillary
caused by presenceof zeta potential (electric charge) at
thecapillary walls and absence of flow resis-
symmetrical peak; VR 5 FtR, where F isthe flow rate and tR is
the retention time ofthe peak of interest.
Elutriation: A technique used to frac-tionate packing particles
by size based onthe difference in their Stokes terminalvelocities.
It most often is used for the sep-aration of ion-exchange resins
that requirea particularly narrow size range, such asamino acid
resins. The technique involvesthe upward flow of water into a large
tube.The unsized beads are added to the mov-ing water, and the
particles seek their ownlevel, depending upon their density
andparticle size. They are removed at certainlevels in the tube.
High-purity spherical sil-ica gels sometimes are sized by
elutriation.
Endcapping: A technique used toremove silica gel silanol groups
that mayremain after reaction with a large silylatingagent such as
octadecyltrichlorosilane. Thecolumn is said to be endcapped when
asmall silylating reagent (such as trimethyl-chlorosilane or
dichlorodimethylsilane) isused to bond residual silanol groups on
asilica-gelbased packing surface. Mostoften used with
reversed-phase packings tominimize undesirable adsorption of
basic,ionizable, and ionic compounds. Endcap-ping reactions also
are used to remove ter-minal silanol groups from
polymericphases.
Endfitting: The fitting at the end of thecolumn that permits
connection to theinjector or detector. Most HPLC endfit-tings have
frits to contain the packing andlow dead volumes for minimum
bandspreading. They usually are constructed ofstainless steel, but
polyetherether ketone(PEEK) and other polymeric materials alsoare
used.
Enzymophoresis: A tandem format inwhich a short fused-silica
capillary contain-ing immobilized enzyme on the inner wallis
coupled with a CZE capillary; an enzy-matic reaction occurs with
the injectedsample, and the products and unreactedsubstances enter
the separation capillary;used to improve separations, detection,
oranalyte preconcentration.
T: See total porosity.Exchange capacity: See ion-exchange
capacity.Excluded volume: See interstitial vol-
ume.Exclusion chromatography: See ion-
exclusion chromatography and stericexclusion chromatography.
Exclusion limit: The upper limit of mol-ecular weight (or size)
beyond which mole-cules will be eluted at the same retention
tance. Most likely source of zeta potentialis presence of
ionized silanols at the fused-silica surface or intentional coating
of thecapillary wall with an ionic phase. Depend-ing upon zeta
potential, electroosmoticflow may be toward anode or cathode
andcontributes to overall retention in CE tech-niques.
Electrophoresis: The movement of sam-ple ions under the
influence of an appliedvoltage.
Electrophoretic mobility (m): Character-istic of a given ion in
a given medium andat a given temperature in CE analyses;
pro-portional to the charge of ion and inverselyproportional to
solution viscosity and theions radius.
Eluate: Combination of mobile phaseand solute exiting the
column; also calledeffluent.
Eluent: The mobile phase used to per-form a separation.
Eluite: The species being eluted, theanalyte, or the sample.
Eluotropic series: A series of solvents(eluents) with an
increasing degree of sol-vent strength generally used in
liquidsolidor adsorption chromatography. In normal-phase
chromatography, a nonpolar solventsuch as pentane would be at the
low end ofthe scale, an intermediate solvent such asmethylene
chloride would be in the middleof the scale, and a strongly polar
solventsuch as methanol would be near the upperend of the scale. In
reversed-phase chro-matography, the reverse order of strengthwould
be observed; water would be weakand acetonitrile strong. Thus, when
devel-oping a method or running a gradient, aneluotropic series is
useful for selecting sol-vents. See also Snyder o.
Elute: To chromatograph by elutionchromatography. The term elute
is pre-ferred over develop, which was used inolder
nomenclature.
Elution: The process of passing mobilephase through the column
to transportsolutes down a column.
Elution chromatography: The mostcommonly used chromatographic
methodin which a sample is applied to the head ofthe column as a
narrow zone and individ-ual analytes are separated and eluted
fromthe end of the column. Compare with dis-placement
chromatography and frontalanalysis.
Elution volume (VR): Refers to the vol-ume of mobile phase
necessary to elute asolute from a column. It is the volumefrom the
point of injection to the volumeat maximum concentration (apex) for
a
-
volume, called the exclusion volume. ManySEC packings are known
by their exclusionlimit. For example, a 105 column ofporous silica
gel will exclude any com-pounds with a molecular weight greaterthan
100,000, based on a polystyrene cali-bration standard.
Exclusion volume (V0, Vei): The mini-mum retention volume of a
molecule onan SEC packing in which all moleculeslarger than the
size of the largest pore aretotally excluded. These molecules are
inca-pable of penetrating the pores and areeluted at the
interstitial (interparticle) vol-ume of the column.
Exponentially modified Gaussian peak:An asymmetric peak
resulting from passinga Gaussian peak through a detector that
isexcessively slow or has an excessive volume.Frequently used to
model peak tailing aris-ing from the column per se. The basis
forthe FoleyDorsey equations. See alsoFoleyDorsey equation.
Extracolumn effects: The total bandbroadening effects of all
parts of the chro-matographic system outside of the columnitself.
Extracolumn effects must be mini-mized to maintain the efficiency
of a col-umn. Sources of band broadening caninclude the injector
design, injection vol-ume, connecting tubing, endfittings,
frits,detector cell volume, and internal detectortubing. The
variances of all of these contri-butions are additive.
Extracolumn volume: The volumebetween the effective injection
point andthe effective detection point, excluding thepart of the
column containing the station-ary phase. It comprises the volumes
of theinjector, connecting lines and frits, and thedetector. It
determines the extracolumneffects.
FFF: See flow rate.F: See flow resistance parameter.Fast LC: Use
of HPLC of short columns
(1.57 cm) with conventional inner diam-eters (26 mm) packed with
small particles(3- or 5-mm dp). Separation times in therange of
minutes, or even seconds, arecommon.
Fast protein LC (FPLC): A termed coinedto cover the specific use
of HPLC for sepa-rating proteins. Generally, glass columns,moderate
pressure, and sphericalmicrobeads are used for FPLC.
Flash chromatography: A very fast formof classic LC used by
synthetic organicchemists for rapid purification. Performed
primarily in the normal-phase mode,sometimes with reversed-phase
chromatog-raphy.
Flow rate (F ): The volumetric rate offlow of a mobile phase
through an LC col-umn. Typical flow rates are 12 mL/minfor a
conventional 4.6-mm i.d. HPLC column.
Flow resistance parameter (F): F 5dp2/Bo, where Bo is
permeability. See alsopermeability.
Fluoro phase: One of a family ofaliphatic and aromatic
reversed-phasematerials in which a substantial fraction ofthe
bonded phase is fluorinated. Some-times called fluorous phases or
perfluorophases. Typically these phases have differ-ent
selectivities than hydrocarbon phases.
FoleyDorsey equation: A correction ofthe plate count and
retention time for peaktailing from extracolumn sources of
broad-ening. See reference 3.
FPLC: See fast protein LC.Fractionation range: Refers to the
oper-
ating range of a gel or packing in SEC.This range is where a
packing can separatemolecules based on their size. At one endof the
range, molecules that are too largeto diffuse into the pores are
excluded. Atthe other end of the range, molecules thatcan diffuse
into all of the pores totally per-meate the packing and are eluted
(unsepa-rated) at the permeation volume.
Free solution CE: See capillary zoneelectrophoresis.
Frit: The porous element at either end ofa column that contains
the column pack-ing. It is placed at the very ends of the col-umn
tube or, more commonly, in the end-fitting. Frits can be stainless
steel or otherinert metal or plastic such as porous PTFEor
polypropylene. The frit porosity mustbe less than the smallest
particle in theHPLC column; otherwise particles willpass through
the frit, and the packing willbe lost.
Frontal analysis: A chromatographictechnique that involves
continuous addi-tion of sample to the column with theresult that
only the least sorbed com-pound, which moves at the fastest rate,
isobtained in a pure state. The second-least-sorbed compound is
eluted with the first-eluted compound, the
third-least-sorbedcompound with the first and second com-pound and
so on until the original sampleis eluted at the column exit.
Frontal analy-sis is seldom used and is mainly a prepara-tive
technique.
Frontal chromatography: Same asfrontal analysis.
Fronting: Peak shape in which the frontpart of the peak (before
the apex) in achromatogram tapers in advance of theremainder of the
peak; that is, the front isless steep than the rear. The peak has
anasymmetric distribution with a leadingedge. The asymmetry factor
for a frontingpeak has a value of less than one. Tailing isthe
opposite effect. Fronting can result athigh sample loads because of
positive cur-vature in the isotherm and from usingpoorly packed
columns.
GGg: The obstruction or tortuosity factor.
Molecular diffusing term. See also tortuos-ity.
Gaussian curve: A standard error curve,based on a mathematical
function, that is asymmetrical, bell-shaped band or peak.Most
chromatographic theory assumes aGaussian peak. Using the peak
maximumposition as a measure of retention and theefficiency
equations mentioned aboveassume Gaussian peak shape. See Figure
2.
Gaussian peak: A peak whose shapeconforms closely to the
equation: C 5Cmax exp[2(t 2 tR)2/2s2].
Gel: The solid packing used in gel chro-matography or
gel-permeation chromatog-raphy (GPC). An actual gel consists of
twoparts: the dispersed medium (solid por-tion) and the dispersing
medium (the sol-vent). Also defined as a colloidal dispersionof a
solid and liquid in which the solid isthe continuous phase.
Gel-filtration chromatography (GFC):Also called aqueous
size-exclusion chro-matography. Performed with aqueousmobile
phases. Generally refers to molecu-lar size separation performed on
soft gelssuch as polydextrans, but analysts also canuse highly
cross-linked polymers, silicagels, and other porous media. Most
gel-filtration separations involve biopolymersand water-soluble
polymers such as poly-acrylic acid.
Gel-permeation chromatography (GPC):SEC performed with organic
mobilephases used for the separation and charac-terization of
polymers. SEC with aqueousmobile phases is called aqueous GPC,GFC,
or aqueous SEC.
GFC: See gel-filtration chromatography.Gigapores: See perfusion
chromatog-
raphy.GPC: See gel-permeation chromatog-
raphy.Gradient: A process to change solvent
-
strength as a function of time (normallysolvent strength
increases) thereby elutingprogressively more highly retained
analytes.Typically gradients can be binary, ternary,and quaternary
solvent mixtures in whichsolvents are blended to achieve the
properstrength.
Gradient elution: Technique for decreas-ing separation time by
increasing themobile-phase strength over time during
thechromatographic separation. Also knownas solvent programming.
Gradients can becontinuous or stepwise. Binary, ternary,and
quaternary solvent gradients have beenused routinely in HPLC.
Graphitized carbon packing: A reversed-phase packing material
consisting of puregraphitic carbon. Possesses interesting sor-bent
properties such as preferential separa-tion of geometric isomers
such as o-, m-and p-aromatics and cistrans isomers.
Guard column: A small column placedbetween the injector and the
analytical col-umn. It protects the analytical columnfrom
contamination by sample particulatesand strongly retained species.
The guardcolumn usually is packed with the samematerial as that in
the analytical columnand is often of the same inner diameter. Itis
much shorter, costs less, and usually isdiscarded when it becomes
contaminated.Integrated guardanalytical column sys-tems often are
preferred to minimize extra-column effects caused by connecting
tub-ing with separate guard and analyticalcolumns.
HHh: Reduced plate height. Defined as
HETP/dp, where HETP is the heightequivalent to a theoretical
plate and dp isthe particle diameter. See also reducedplate
height.
H: Same as HETP. See also efficiency.h: See viscosity.Head
pressure (Dp): The difference in
pressure between the inlet and outlet of acolumn measured in
pounds per squareinch. Governed by the following approxi-mate
equation for a column packed withspherical particles of typical
internal poros-ity (0.5): Dp 5 3000Lh/tMdp2, where L isthe column
length in centimeters, h is themobile-phase viscosity in
centipoise, tM thecolumn holdup time in minutes, and dp isthe
particle diameter in micrometers. Pres-sure can be expressed in
pounds per squareinch, bars, atmospheres, or pascals.
Heart cutting: Refers to collection of thecenter of the peak at
which purity should
used in SEC to define molecular shape andto explain why
molecules with the samemolecular weight often have different
elu-tion volumes. Measured by determiningthe Stokes radius.
Hydrophilic: Greek word for water lov-ing. Refers to stationary
phases that arefully compatible with water and to water-soluble
molecules in general. Many col-umns used to separate proteins such
asion-exchange, SEC, and affinity columns are hydrophilic in nature
and shouldnot irreversibly sorb or denature protein inan aqueous
environment.
Hydrophilic interaction chromatogra-phy: Using ion-exchange
columns to sepa-rate compounds on the basis of
nonionicinteractions. Columns are used to separatehydrophilic
peptides with a gradient fromorganic to aqueous solvents. Solutes
areseparated based on their hydrophilicityrather than
hydrophobicity.
Hydrophobic: Greek word for water fear-ing. Refers to stationary
phases that areincompatible with water or to moleculesthat in
general have little affinity for water.Hydrophobic molecules have
few polarfunctional groups. Most have a high con-tent of
hydrocarbon (aliphatic and aro-matic) functionality.
Hydrophobic interaction chromatogra-phy: A technique in which
weakly polar(nonhydrocarboneous) packings are usedto separate
molecules by the interactions oftheir hydrophobic moieties and the
hydro-phobic sites on their packing surface. Highconcentrations of
salt solutions are used inthe mobile phases, and separations are
gen-erated by changing the salt concentration.The technique is
analogous to salting-outmolecules from solution. Gradients are
runby decreasing the salt concentration. Thetechnique often is used
to separate proteinsthat are sensitive to denaturization by
theorganic solvents used in regular reversed-phase chromatography.
Usually little or noorganic solvent is used in the mobile phasein
hydrophobic interaction chromatogra-phy.
Hydrostatic injection: Also called hydro-dynamic injection.
Using gravity (differen-tial pressure) to make an injection into
aCE capillary. Vial containing sample solu-tion is raised at a set
distance above theground end of the column and columninlet end is
placed into vial for set time.After determining flow rate, users
candetermine injected volume. Good forwide-bore capillaries.
Hydroxyapatite: A porous calcium
be maximum in preparative LC. The termalso is used in column
switching.
Heff: See effective plate height.Helium sparging: See degassing.
Helium
has a very low solubility in most commonliquids.
HETP: Height equivalent to a theoreticalplate. A carryover from
distillation theory;a measure of column efficiency; HETP 5L/N,
where L is column length and N isthe number of theoretical plates.
HETPshould be approximately 23 dp for 5-mmparticles with a typical
well-packed HPLCcolumn, HETP (or H ) values usually arein the range
of 0.010.03 mm. See alsoefficiency and h.
High performance CE: A technique inwhich small-diameter
capillaries, bufferedconducting solutions, and high voltages
(asmuch as 30,000 V) separate ionic mole-cules based on their
differential electropho-retic mobilities. Nonionic (neutral)
mole-cules can be separated by MEKC.
High performance liquid chromatogra-phy (HPLC): The modern,
fully instrumen-tal form of liquid-phase chromatographytechnique
that uses small particles andhigh pressures. Sometimes called
high-pressure LC.
Holdup volume (VM): The total volumeof mobile phase in the
column regardlessof where it exists; VM 5 Ve 1 Vi, where Veis the
interstitial volume and Vi is theintraparticle volume. Also called
the col-umn void volume. IUPAC indicates thatuse of the term dead
volume should beeliminated for this concept. The use ofdead volume
is limited to regions notswept by the flowing mobile phase
system.Holdup volume is measured by injectingan unretained species
that fits in all thepores. See also interstitial porosity
andintraparticle porosity.
HPLC: See high performance liquidchromatography.
Hybrid silica: Silica gel comprising bothorganic and inorganic
moieties with hybridproperties of polymeric packings and
silicapackings. Synthesized from silanes contain-ing organic
functionality. Different selec-tivity but better high-pH stability
thanbare or uncoated silica gel.
Hydrodynamic injection: Used in CE.See also hydrostatic
injection.
Hydrodynamic volume: The molecularvolume defined by the
effective diameter ofa molecule in free solution at which
thehydrodynamic sphere would be a spheredefined by the molecule as
it revolvesaround its central axis in solution. Term
-
hydroxy phosphate solid that chemicallyresembles bone and tooth.
Used as a pack-ing material in biochromatography fornucleic acid
constituents, monoclonal anti-bodies, and proteins.
Hyphenated techniques: Refers to thefamily of techniques best
known by theiracronyms, including LCmass spectrome-try (MS),
LCFourier transform IR spec-troscopy (FTIR), and LCMSMS. Seealso
multidimensional chromatography.
IIIC: See ion chromatography.Immobilized metal-affinity
chromatog-
raphy: See metal-affinity chromatogra-phy.
Imprinted phases: Polymer and silicaphases generated in the
presence of a tem-plate or printing molecule. These phaseshave
enhanced selectivity for the templat-ing molecule.
Included volume: Also known as totallyincluded volume. The
volume at which asmall molecule that explores the entirepore space
of a column is eluted. See alsosize-exclusion chromatography.
Indirect detection: Used for non-UVabsorbing or nonfluorescing
analytes. AUV-absorbing or fluorescent compoundadded to the mobile
phase maintains ahigh background signal; when a nonab-sorbing or
nonfluorescing analyte is eluted,the background is diluted and a
negativepeak is observed for that analyte. When ananalyte acts to
increase the concentrationof the indicating species, it produces a
pos-itive peak. When a negative signal isdetected, the detector
signals are reversedto the output device.
Infinite diameter column effect: At acertain column length, a
sample injectedinto center of a packed bed spreads byradial
diffusion but never reaches columnwall, where wall effects can
cause bandbroadening. Phenomenon observed byJohn Knox, who showed
that a samplepeak collected in the exact center of thecolumn exit
displayed a higher efficiencythan a sample peak collected near the
wall.The infinite diameter effect depends oncolumn length, internal
diameter, particlesize, and mobile-phase properties. Very sel-dom
applied in HPLC.
Inlet: The initial part of the columnwhere the solvent and
sample enter. Aninlet frit usually holds the packing in placeand,
in some cases, protects the packedbed.
In-line filter: A device that prevents par-ticulate matter from
damaging the column.
Modern low-volume, in-line filters can beplaced between the
injector and the col-umn without major contributions to
bandbroadening. A filter in this position pre-vents sample
particles from entering thepacked bed or column inlet frit.
Interparticle porosity (e): The inter-particle volume of a
packed column perunit column volume; e 5 Ve/Vc, where Veis the
interstitial volume and Vc is the totalcolumn volume. See also
interstitialporosity.
Interparticle volume (Vo): The volumeof mobile phase located
outside the parti-cles.
Interstitial porosity (e): The fraction ofthe volume in the
column located in theinterparticle (interstitial) space; e 5
Ve/Vc.
Interstitial velocity (ue): The actualvelocity of the eluent as
it moves throughthe column flowing around the particles;ue 5 F/Ace.
The interstitial velocity is thebasis for computing the reduced
velocity.
Interstitial volume (Ve): The volumebetween the particles. It
does not includethe volume in the pores of the particles.Also
called the excluded volume (see SEC)and interparticle volume.
Measured byinjecting a molecule that does not perme-ate any pores
and does not interact withthe surface of the particles. In SEC,
thisvolume is denoted Vo.
Intraparticle porosity (i): The fractionof the particle volume
that is the pore vol-ume; i 5 Vpore/Vparticle.
Intraparticle volume (Vi): The volumeinside the pores of the
particles. Also calledthe internal and included volume. Can
bemeasured by the BET method or mercury-intrusion porosimetry.
Ion chromatography (IC): An ion-exchange technique in which low
concen-trations of organic and inorganic anions orcations are
determined using ion exchang-ers of low ion-exchange capacity
withdilute buffers. Conductivity detectors oftenare used. IC is
practiced in two forms: Insuppressed IC, a second column or a
mem-brane separator is used to remove thebuffer counter ion from
the analyte andsimultaneously replace it with a hydrogenor
hydroxide ion that concomitantly con-verts the buffer to an
uncharged speciesthereby suppressing background andenhancing
sensitivity. In nonsuppressed IC,low-concentration, weakly
conductingbuffers are carefully selected, the entireeffluent is
passed through the detector, andions are detected above the
backgroundsignal.
Ion-exchange capacity: The number of
ionic sites on the packing that can partici-pate in the exchange
process. The exchangecapacity is expressed in milliequivalents
pergram. A typical styrenedivinylbenzenestrong anion-exchange resin
may have 35mequiv/g capacity. Exchangers for IC havevery low
capacity. Capacity of weak anionand cation exchangers varies
dramaticallywith pH.
Ion-exchange chromatography: A modeof chromatography in which
ionic sub-stances are separated on cationic or anionicsites of the
packing. The sample ion, usu-ally with a counterion, will exchange
withions already on the ionogenic group of thepacking. Retention is
based on the affinityof different ions for the site and other
solu-tion parameters such as pH, ionic strength,and counterion
type. Ion chromatographybasically is an ion-exchange technique.
Ion exclusion: The process in which ionized solutes can be
separated from un-ionized or partially ionized solutesusing
ion-exchange resins. Separationresults from Donnan potential in
whichionic solutes exist at a higher concentrationin solution than
in the stationary phase,whereas nonionic solutes are evenly
distrib-uted between the mobile phase and resin.Therefore, ionic
solutes will move fasterdown the column than nonionic solutes.Ion
exclusion occurs in reversed-phasechromatography when anions are
separatedat pH values at which the silanol groupsare ionized.
Ion-moderated partitioning chroma-tography: A technique used for
separatingcarbohydrates using strong cation-exchangepackings that
are in specific cationic form(for example, calcium, hydrogen,
silver).The separation mechanism is complexationrather than ion
exchange.
Ion-pair chromatography: Form ofchromatography in which ions in
solutioncan be paired or neutralized and separatedas an ion pair on
a reversed-phase column.Ion-pairing agents usually are ionic
com-pounds that contain a hydrocarbon chain,which imparts a certain
hydrophobicity sothat the ion pair can be retained on
areversed-phase column. Retention is pro-portional to the length of
the hydrophobicchain and the concentration of the ion-pairadditive.
Ion pairing also can occur in normal-phase chromatography when
onepart of the pair is dynamically loaded ontoa sorbent, but this
technique is not as pop-ular as reversed-phase chromatography.Also
known as ion-interaction chromatog-
-
raphy or dynamic ion-exchange chro-matography, which stresses
that userssometimes do not know the precise mecha-nistic details of
how the additive controlsretention.
Ion retardation: Refers to using ampho-teric ion-exchange
resins, which retardionic molecules and allow nonionic mole-cules
or nonelectrolytes to be eluted prefer-entially.
Ion suppression: Buffering in an aque-ous mobile phase at a
particular pH tosuppress solute ionization. For example,weak
carboxylic acids can have their ioniza-tion suppressed by the
adjustment of thepH below their pKa value. Useful forimproving peak
shape of weak acids andbases in reversed-phase chromatography.
Irregular packing: Refers to the shape ofa column packing.
Irregular packings areavailable in microparticulate sizes.
Thepackings are obtained from grinding solidmaterials into small
particles and sizingthem into narrow fractions using
classifica-tion machinery. Spherical packings areused more often
than irregular packings inanalytical HPLC, but the
less-expensive,irregular packings are still widely used
inpreparative-scale LC.
Irreversible adsorption: When a com-pound with a very strong
affinity for anadsorbent is injected onto a column, it canbe
adsorbed so strongly that it cannot beeluted from the column. A
chemical reac-tion between the sample and the surface ofthe
adsorbent is an example of irreversibleadsorption. See also
chemisorption.
Isocratic: Using a time invarianteluentcomposition in LC.
Isotachophoresis: Ionic species are sepa-rated in CE according
to differences inmobilities by applying an electric field.
Iso-tachophoresis is performed with a discon-tinuous buffer in
which the sample zone islocated between the background
electrolyteof higher (leading electrolyte) and lower(terminal
electrolyte) electrophoreticmobilities.
Isotachophoretic focusing: Using theprinciples of
isotachophoresis to focus orconcentrate analytes at the head of the
col-umn to provide concentration sensitivityenhancement.
Isotherm: See adsorption isotherm.Isothermal chromatography:
Using
conditions of constant temperature. Thevast preponderance of all
LC is performedunder isothermal conditions.
JJJoule heating: Electrical heating of sol-
sample component ions for strong electro-lytes.
Ligand: In ligand-exchange chromatog-raphy, it refers to the
analyte that under-goes ligand exchange with the stationaryphase.
In affinity chromatography, it refersto the biospecific material
enzyme, anti-gen, or hormone coupled with the sup-port (carrier) to
form the affinity column.In bonded-phase chromatography, it
refersto the moiety covalently bound to the sur-face.
Ligand-exchange chromatography: A technique in which chelating
ligands areadded to the mobile phase and undergosorption onto a
packing. These sorbedmolecules can act as chelating agents
withcertain solutes. For example, copper saltcan be added to the
mobile phase for thechelation and separation of amino
acids.Chelating resins function in a similar man-ner: chelating
groups are chemicallybonded to the polystyrene backbone.
Linear elution adsorption chromatog-raphy: Refers to adsorption
chromatogra-phy performed in the linear portion of anadsorption
isotherm. A term coined byLloyd Snyder.
Linear velocity (u): The velocity of themobile phase moving
through the column.Expressed in centimeters per second.Related to
flow rate by the cross-sectionalarea of the column. Determined by
divid-ing the column length (L) by the retentiontime of an
unretained compound. See alsovoid time.
Liquid chromatography (LC): A separa-tion technique in which the
mobile phaseis a liquid. Most often performed in a col-umn.
Liquidliquid chromatography: One ofthe earliest separation modes
of HPLC; itgave way to chemically bonded phases inthe early 1970s.
Same as partition chro-matography.
Liquidsolid chromatography: Same asadsorption
chromatography.
Loading (phase loading versus sampleloading): The amount of
stationary phasecoated or bonded onto a solid support.
Inliquidliquid chromatography, the amountof liquid phase in
milligrams of per gramof packing. In bonded-phase chromatogra-phy,
the loading may be expressed inmicromoles per square meter or
percentagecarbon (w/w). Also called coverage or sur-face coverage.
An alternate and unrelatedmeaning is the amount of sample
massinjected on an analytical- or preparative-scale column;
preparative-scale columnsoften are operated in an overloaded
condi-
vent within a capillary caused by voltageapplied to the column
and the resultingcurrent. The temperature is related to elec-tric
field strength, conductivity of the solu-tion and the capillary
radius. Minimizingheat generation and maximizing heat dissi-pation
is critical in CZE because thermalgradient and other heat-related
effects canadversely affect CEs high efficiency.
KKk: See retention factor.k9: An old term that has been
replaced
by the IUPAC-approved term, retentionfactor (k).
K: See partition coefficient.kA/B: See selectivity
coefficient.Kc: See distribution constant (coeffi-
cient).Kieselguhr: A diatomaceous earth used
in column chromatography and also as asample cleanup media. Only
weaklyadsorptive, it can be used as a support inliquidliquid
chromatography. Rarely usedin HPLC.
Knox equation: A modification of thevan Deemter equation
developed by JohnKnox in which the A term that representseddy
dispersion multiplied by u
13, where uis the interstitial eluent velocity. Usuallywritten
in terms of the dimensionless orreduced plate height (h) and
reduced veloc-ity (n) as h 5 An
13 1 B/n 1 Cn. See alsovan Deemter equation.
LLL: See column length.Laminar flow: The smooth time-invari-
ant flow that develops when a liquid ismoving under conditions
in which viscousforces dominate inertial forces. Laminarflow is
characterized by a low Reynoldsnumber (see Reynolds number). In
acylindrical tube, fluid streams in the centerflow faster than
those at the tube wall,which results in a radially parabolic
distrib-ution in axial fluid velocity. This nonuni-formity of axial
velocities in the intersticesin a packed bed also causes
substantialpeak broadening in packed columns.
Langmuir isotherm: A specific form ofan isotherm; CS 5 N0CM/(Kd
1 CM),where CS and CM are the equilibrium sta-tionary and
mobile-phase concentrationsof the solute, N0 the total number of
sur-face sites available for sorption, and Kd thesorption binding
constant.
LC: See liquid chromatography.Leading electrolyte : In
isotachophore-
sis, the electrolyte that contains the ionwith the highest
mobility above that of any
-
tion for throughput reasons.log kw: The extrapolated intercept
of a
plot of log k versus volume fraction oforganic modifier in
reversed-phase LC. Seealso S.
Longitudinal diffusion: Same as molecu-lar diffusion term. B
term in van Deemterequation. See also van Deemter equation.
MMm: See electrophoretic mobility.Macroporous resin
(macroreticular):
Cross-linked ion-exchange resins that havemolecular-scale
micropores and alsomacropores of several hundred angstroms.These
highly porous resins have large inter-nal surface areas that are
accessible to largemolecules.
Mass transfer (interphase): The processof solute movement
between the movingand stationary zones. The C term of thevan
Deemter equation is called the inter-phase mass transfer term. The
faster themass transfer process, the better the col-umn efficiency.
In HPLC, slow mass trans-fer is the most important factor
affectingcolumn efficiency. Its rate can be increasedby using
small-particle packings, thin stationary-phase layers,
low-viscositymobile phases, and high temperatures.
Mean pore diameter: The averagediameter of the pore of a porous
packing.It most commonly is determined by theBET method and is
reported as fourfoldthe specific pore volume divided by thespecific
surface area (4V/A) based on theassumption of uniform cylindrical
pores.The pore diameter is important in that itmust allow free
diffusion of solute mole-cules into and out of the pore so that
thesolute can interact with the stationaryphase. Additionally, the
pores must bewell-connected, with a minimum of deadends, so many
paths can allow a moleculeto access any part of the pore space.
InSEC, the packings have different porediameters; therefore,
molecules of differentsizes can be separated. For a typical
sub-strate such as silica gel, 60- and 100- porediameters are most
popular. Pore diametersgreater than 300 are used for the
separa-tion of biomolecules. Pores usually are clas-sified as micro
(,20 ), meso (20500 ),and macro (.500 ).
MECC: See micellar electrokinetic capil-lary chromatography.
Megapores: See perfusion chromatog-raphy.
MEKC: See micellar electrokinetic capil-lary chromatography.
HPLC. Columns of 2 mm and less areconsidered to be microbore
sizes. Innerdiameters of 0.5 mm and smaller are con-sidered
micro-LC columns.
Microchip devices: Microdevices basedon silicon, glass, and
other types of micro-fabricated chips in which experiments canbe
miniaturized into single- or multichan-nel microfluidic circuits.
These devices canbe used for CE and CEC. They should below cost and
disposable. Using micro-devices for separation currently is in
itsinfancy, and applications should expandwith time.
Microparticulate: Refers to the smallparticles used in HPLC.
Generally pack-ings with a particle diameter of less than10 mm that
are totally porous are consid-ered microparticles.
Microporous resin: Same as microretic-ular resin.
Microreticular resin: Cross-linked, syn-thetic ion-exchange
resins that have poreswith openings that correspond to molecu-lar
sizes. Diffusion into the narrow porescan be impaired, and low
exchange ratesand poor performance can occur, especiallyfor large
molecules.
Migration rate: See electrophoreticmobility.
Migration time (tm): The time it takesfor a charged molecule to
move from thepoint of injection to the point of detectionin a CE
capillary. Distinct from holduptime (tM).
Minimum plate height: The minimumof the van Deemter curve that
results froma plot of H versus n. This value representsthe most
theoretical plates that can beobtained for a certain column and
mobile-phase system. Usually occurs at excessivelylow flow rates.
Also known as the opti-mum plate height. It typically is two-
tothreefold the particle diameter of well-packed columns.
Mixed-bed column: Combination oftwo or more stationary phases in
the samecolumn, used most often in IEC (mixedanion and cation
resins) and SEC (mixtureof different pore size packings). Its
advan-tage in IEC is the total removal of bothcationic and anionic
compounds. Useful inSEC because a wider molecular weightrange can
be accommodated by the samecolumn.
Mixed-mode separation: A separationthat occurs in a single
column caused bythe retention and selectivity provided by
adual-retention mechanism. For example, areversed-phase column with
residual
Metal-affinity chromatography: A spe-cial form of
ligand-exchange chromatogra-phy used to separate biopolymers with
aparticular affinity for a specific metalcation, typically
copper(II), zinc(II), andiron(II).
Metalophile: A compound that has highaffinity for active acidic
silanol groups onsilicas surface. Usually a strongly basicamine or
multifunctional carboxylate orphenol.
Method development: A process foroptimizing the separation,
including thesample pretreatment, to obtain a repro-ducible and
robust separation. Usually, itemphasizes the search for the
stationaryphase, eluent, and column temperaturecombination that
provides an adequate, ifnot optimum, separation.
Method validation: A process of testinga method to show that it
performs to thedesired limits of precision and accuracy
inretention, resolution, and quantitation ofthe sample components
of interest.
Micellar chromatography: Addingmicelles to the mobile phase to
cause sepa-ration. The micelles may act as displacingor
partitioning agents and provide anotherparameter to change
selectivity. Surfactantsat concentrations greater than their
criticalmicelle concentration are used in micellarchromatography
and in MEKC.
Micellar electrokinetic capillary chro-matography (MEKC, MECC):
Similar tomicellar chromatography. Used for the CEseparation of
neutral compounds underelectroosmotic flow conditions. Detergentor
surfactant is added to the runningbuffer at a concentration to make
it greaterthan its critical micelle concentration. Ana-lytes
partition by hydrophobic interactionsinto and out of the micelles
while they aremoving through the capillary under theinfluence of
electroosmotic flow; the resultis that neutral compounds are
separated bytheir differential migration down the capil-lary.
Micro-LC: Refers collectively to tech-niques in which a column
of smaller thanconventional inner diameter is used forseparation.
The term micro-LC most oftenis used for HPLC in columns with
innerdiameters smaller than 0.5 mm; micro-LCis used in
high-sensitivity analysis when thesample amount is limited and with
certainionization techniques in LCMS in whichthe volume of solvent
flowing into the ion-ization source must be minimized.
Microbore: Refers to the use of smaller-than-usual inner
diameter columns in
-
silanols at intermediate-to-high pH valuescan separate by
hydrophobic interactionand ionic interaction by the
ionizedsilanols. Sometimes mixed-mode separa-tions can be quite
beneficial to the selectiv-ity (band spacing), but they can cause
peakasymmetry, and the precise balance ofinteractions may be
difficult to reproducewith subsequent packing batches.
Mobile phase: The solvent that movesthe solute through the
column. In LC, themobile phase interacts with both the soluteand
the stationary phase and, therefore,can have a powerful influence
on the sepa-ration.
Mobile-phase strength: See solventstrength.
Mobile-phase velocity (uM): The veloc-ity at which the mobile
phase percolatesthrough the bed of particles; uM 5 L/tM,where L is
column length and tM is holduptime. See also adjusted retention
volume,holdup volume, and dead volume.
Mobility: See electrophoretic mobility.Modifier: An additive
that changes the
character of the mobile phase. For exam-ple, methanol is the
strong solvent inreversed phase and sometimes is called themodifier
(water is the weak solvent); some-times other additives competing
basessuch as triethylamine or ion-pairingreagents are referred to
as modifiers, butthey more correctly should be called addi-tives.
See also additives.
Molecular diffusion term (B term):Refers to the B term (second
term) of thevan Deemter equation. Also called longitu-dinal or
axial diffusion term. It dominatesband broadening only at very low
flowrates below the minimum plate height atwhich the diffusion of
individual solutescan occur in a longitudinal (lengthwise)direction
on the column. The contributionto the B term arises from diffusion
in themobile phase and is 2gDM, where g is theobstruction factor
(typically 0.60.8) andDM is the diffusion coefficient. See alsovan
Deemter equation.
Molecular weight distribution: The dis-tribution of molecular
weight of moleculesin a polymer sample. Distribution can bedefined
as weight average and numberaverage.
Molecularly imprinted phases: Seeimprinted phases.
Monodisperse particles: Particles thatfall into a narrow range
of diameters. Seealso polydisperse particles.
Monomeric phase: Refers to a bondedphase in which single
molecules are
Common column abbreviations includeNPS, which refers to
nonporous silica;NPR, which refers to nonporous resins;and NPZ,
which refers to nonporous zirconia.
Nonporous particle: Refers to a solidparticle used as a support
for a porouscoated or bonded phase; pellicular particlesare
nonporous particles of large particlediameter (;40 mm). Nonporous
silicasand resins with small particle diameters ofless than 3 mm
usually are microbeads withthin porous outer coatings of silica
gel,bonded silica gel, or polymeric phase.
Normal-phase chromatography: Amode of chromatography performed
whenthe stationary phase is more polar than themobile phase. A
typical normal-phase sys-tem would be adsorption chromatographyon
silica gel or alumina using mixtures ofless polar eluents such as
hexanediethethylether as a mobile phase. Also refers to theuse of
polar bonded phases such as cyanoand alumina. Sometimes called
straight-phase chromatography.
OOOctadecylsilane: The most popular
reversed phase in HPLC. Octadecylsilanephases are bonded to
silica or polymericpackings. Both monomeric and polymericphases are
available. Abbreviated in columnnames as C18 and ODS.
Octylsilane: A popular stationary phasein reversed-phase
chromatography. Usuallyprovides slightly less retention than
themore popular C18. Both monomeric andpolymeric phases are
available. Abbreviatedin column names as C8.
ODS: See octadecylsilane.On-column detection: The column
itself
serves as the flow cell in HPLC orCECEC. Generally, the term
used withfused-silica capillary applications. Outerpolyimide layer
is removed, an opticalbeam is directed through the capillary, anda
measuring device such as a photomulti-plier tube is located on the
opposite side ofthe capillary.
On-line preconcentration: A precolumnis placed in front of the
separation columnto concentrate analytes before their separa-tion.
Different mechanisms hydropho-bic interaction, adsorption, or
enzymaticreaction may be used to retain analyteas a function of
time. Then concentratedanalytes are transferred to the
separationcolumn by a displacement process such assolvent elution
or pH change.
Open-tube capillary zone electrophore-sis: The application of CE
principles in an
bonded to a support. For silica gel,monomeric phases are
prepared by thereaction of an alkyl- or aryl- monochloro-or
alkoxysilane. Polymeric phases generallyare prepared from a di- or
trichlorosilaneor an alkoxysilane reactant in the presenceof
water.
Moving zone: To be distinguished fromthe mobile phase, this zone
is the fractionof the mobile phase in the column thatoccupies the
interstitial spaces. See also sta-tionary phase.
Multidimensional chromatography:The use of two or more columns
or chro-matographic techniques to generate a bet-ter separation. It
is useful for samplecleanup, increased resolution,
increasedthroughput, and increased peak capacity. Itcan be used
off-line by collecting fractionsand reinjecting them onto a second
col-umn or on-line by using a switching valve.Also called coupled
column chromatogra-phy, column switching, multicolumn
chro-matography, and boxcar chromatography.
NNn: See peak capacity.N: The number of theoretical plates;
N 5 16(tR/wb)2, where tR is retention timeand wb is the base
width of the peak. Ameasure of the efficiency of a column.Sometimes
measured as N 5 5.54(tR/wh)2,where wh (or w 12) is the peak width
at halfheight. See also efficiency and theoreticalplate.
n: See reduced velocity.Narrow-bore column: Columns of less
than 2-mm i.d. used in HPLC. Also calledmicrobore.
Neff: See effective theoretical plates.Nonaqueous reversed-phase
chro-
matography: Refers to reversed-phasechromatography performed
without wateras a component of the eluent on areversed-phase
packing. Used for very nonpolar compounds that cannot be elutedor
are difficult to elute from a reversed-phase column with 100%
methanol or acetonitrile. In these cases, solvent Ashould be
acetonitrile, and solvent Bshould be a stronger solvent such
astetrahydrofuran. Reversed-phase rulesapply to nonaqueous
reversed-phase chro-matography; that is, the more nonpolar
theanalyte, the greater the retention.
Nonporous packing: Particles similar toporous-layer bead but
with particle diame-ters in the sub-5-mm range; particles oftenare
in the sub-2-mm dp range. Used forhigh-speed separations in short
columns.
-
open capillary tube. Separations are basedon differential
electrophoretic mobility ofcharged compounds or ions. Typical
capil-lary dimensions are 150 cm 3 10200mm.
Open tubular columns: Small innerdiameter columns (less than 100
mm) cur-rently being investigated for use in HPLC,supercritical
fluid chromatography (SFC),and CE. Stationary phases can be
bondedon the internal walls of these smallcolumns. The most
frequently used col-umn material is fused-silica tubing. Usedvery
little in routine HPLC or SFC butfrequently in CE.
Optically active resin: Incorporation of optically active groups
into an ion-exchange resin to allow separation of opti-cally active
isomers. Few commerciallyavailable resins for HPLC
applications.
Organic modifier: Water-miscibleorganic solvent added to an
aqueousmobile phase to obtain separations inreversed-phase HPLC.
Common organicmodifiers are acetonitrile, methanol, iso-propanol,
and tetrahydrofuran.
Overload: In preparative chromatogra-phy the overload is defined
as the samplemass injected onto the column at whichefficiency and
resolution begins to beeffected if the sample size is increased
fur-ther. See also sample capacity.
PPDp: See head pressure.Pa: See pascal.Packing: The adsorbent,
gel, or solid
support used in an HPLC column. Mostmodern analytical HPLC
packings are lessthan 10 mm in average diameter, and 5mm is the
current favorite.
Paired-ion chromatography: Same asion-pair chromatography.
Particle size (dp): The average particlesize of the packing in
the LC column. A 5-mm dp column would be packed withparticles with
a definite particle-size distri-bution because packings are
nevermonodisperse. See also monodisperse par-ticles, particle size
distribution, and poly-disperse particles.
Particle-size distribution: A measure of the distribution of the
particles used topack the LC column. In HPLC, a narrowparticle-size
distribution is desirable. A particle-size distribution of dp 6
10%would mean that 90% of the particles fallbetween 9 and 11 mm for
an average 10-mm dp packing.
Partition chromatography: Separation
Peak width (wb): Same as bandwidth.See Figure 2.
Pellicular packing: See porous-layerbead.
Percent B solvent (% B solvent): Refersto the stronger solvent
in a binary solventmixture. % A solvent would be the weakersolvent
analog.
Perfusion chromatography: Refers tochromatography performed
using particleswith very large pores (40008000 ) calledthroughpores
(megapores or gigapores).Eluent flows between the large pores
andthrough the particles 3001000 inter-connecting pores, called
diffusive pores.Best suited for the preparative separation
ofmacromolecules.
Permeability (Bo): Also called columnpermeability and specific
permeability. A term expressing the resistance of thepacked column
to the flow of mobilephase. For a packed column, Bo 'dp23/[180(1 2
)2] 5 dp2/1000. A col-umn with high permeability gives a
lowpressure drop.
Permeation: Refers to the SEC processin which a solute can enter
a mobile-phase-filled pore of the packing.
Phase ratio (b): The relative amount ofstationary to mobile
phase in the column.In partition chromatography, b 5 VS/VM,where VS
and VM are the volume, of sta-tionary and mobile phase in the
column,respectively. The retention factor is theproduct of the
phase ratio and the parti-tion coefficient.
Phenyl phase: A popular nonpolarbonded phase prepared by the
reaction ofdimethylphenylchloro- or alkoxysilanewith silica gel.
Reportedly has affinity foraromatic-containing compounds and
doesimpart a different selectivity comparedwith alkyl-bonded
phases.
Pirkle column: Chiral, brush-type stationary phases based on
3,5-dinitroben-zoylphenylglycine silica used in the separa-tion of
a wide variety of enantiomers.Named after its developer, William
Pirkleof the University of Illinois.
Planar chromatography: A separationtechnique in which the
stationary phase ispresent as or on a plane (IUPAC). Typicalforms
are paper and thin-layer chromatog-raphy.
Plate height (H): See HETP.Plate number: See column plate
num-
ber.Plate or plate number: Refers to theo-
retical plates in a packed column (IUPAC).See also theoretical
plate.
process in which one of two liquid phasesis held stationary on a
solid support (sta-tionary phase) while the other is allowed toflow
freely down the column (mobilephase). Solutes partition
themselvesbetween the two phases based on theirindividual partition
coefficients. Liquidliquid chromatography is an example;modern
bonded-phase chromatographycan be considered to be a form of
partitionchromatography in which one of the liquidphases is
actually bonded to the solid sup-port. Mechanistically partition
chromatog-raphy implies that the solute becomes atleast partially
embedded within the station-ary phase, which is impregnated,
coated, orbonded to the substrate. In contrast to anadsorption
process in which the solute doesnot penetrate into the retentive
surface orinterphase.
Partition coefficient (K ): The ratio ofthe equilibrium
concentration of solute inthe stationary phase relative to the
equilib-rium concentration of solute in the mobilephase. Also
called distribution coefficient,KD, and distribution constant (Kc
).
Pascal (Pa): A unit of pressure. 1 MPa isapproximately 10 bar
(atm) or 150 psi.
Peak capacity (n): The number ofequally well-resolved peaks (n)
that can befit in a chromatogram between the holdupvolume and some
upper limit in retention.For R 5 1, n is given by the
approximation1 1 0.25[(N)12 ln(1 1 kn)], where R is theresolution,
N is the number of theoreticalplates, and kn is the retention
factor forpeak n.
Peak dispersion: See band broadening.Peak doublet: A split peak
generally
caused by a column void. Could be closelyeluted compounds.
Peak shape: Describes the profile of achromatography peak.
Theory assumes aGaussian peak shape (perfectly symmetri-cal). Peak
asymmetry factor describes shapeas a ratio. See Figures 1 and 2.
See alsoasymmetry.
Peak tracking: A way of matching peaksthat contain the same
compound betweendifferent experimental runs during
methoddevelopment. Relies upon detection pa-rameters of each pure
analyte. Diode-arraydetectors and mass spectrometers areamong the
best detectors for peak trackingbecause of their specificity.
Peak variance (s2): The second centralmoment of the peak about
the retentiontime. For a Gaussian peak, the variance isthe
fundamental parameter controllingpeak width. See Figure 2. See also
Gauss-ian peak.
-
Polyacrylamide gel: Neutralhydrophilic polymeric packings usedin
aqueous SEC. Prepared by thecopolymerization of acryl-amide with
N,N9-methylenebisacry-lamide.
Polydisperse particles: Particlesthat have a substantial range
ofdiameters (.10%).
Polyethyleneimine: An anionicpolymeric phase used to coat orbond
onto silica or a polymeric pack-ing. Most often used for
separatingproteins and peptides.
Polymeric packings: Packingsbased on polymeric materials,
usuallyin the form of spherical beads. Typi-cal polymers used in LC
are poly-styrenedivinylbenzene (PSDVB),polydivinylbenzene,
polyacryl-amide, polymethylacrylate, polyeth-ylene-oxide,
polydextran, and polysaccha-ride.
Polymeric phase: Refers to achemically bonded phase in which
apolymer species is bonded to silica-based particles.
Polystyrenedivinylbenzene resin(PSDVB): The most common
basepolymer for ion-exchange chro-matography. Ionic groups are
incor-porated by various chemical reac-tions. Neutral PSDVB beads
areused in reversed-phase chromatogra-phy. Porosity and mechanical
stabil-ity can be altered by varying thecross-linking through the
DVB con-tent.
Pore diameter: Same as meanpore diameter.
Pore size: The average size of apore in a porous packing. Its
value isexpressed in angstroms or innanometers. The pore size
deter-mines whether a molecule can dif-fuse into and out of the
packing. Seealso mean pore diameter.
Pore volume: The total volume ofthe pores in a porous packing,
usu-ally expressed in milliliters per gram.More appropriately
called the specificpore volume. It is measured by theBET method of
nitrogen adsorption or by mer-cury-intrusion porosimetry in which
mer-cury is pumped into the pores under high pressure.
matographic approaches.Pressure (pressure drop) (Dp): See
head
pressure.Pressure injection: Pressure-induced
injection in CE. Using pressure or vacuumto inject
nanoliter-level volumes of sampleinto a capillary column. Best for
narrow-bore capillaries that have inner diametersless than 10 mm. A
version of hydrostaticinjection.
Process-scale chromatography: Refersto the use of LC at the
industrial-scale leveloutside of laboratories. Generally
requiresspecially designed columns (usually withdiameters . 5 cm),
recoverable solvents,low-cost packings (larger and irregular-shaped
particles), and overloaded operatingconditions compared with
laboratory-scaleHPLC.
Programmed-temperature chromatog-raphy: Varying temperature
during a chro-matographic run. Seldom used in LC.
PSDVB: See polystyrenedivinylben-zene resin.
Pulsating flow: Flow originating from areciprocating pump.
Normally, the pulsesare dampened by a pulse damper, an elec-tronic
pressure feedback circuit, or anactive damper pump head. Detectors
suchas electrochemical and refractive indexdetectors are greatly
affected by flow pulsa-tions.
QQQuaternary methyl amine: A strong
anion-exchange functionality popular inresin-based packings.
Usually supplied inchloride form.
Quaternary mobile phase: A mobilephase comprising four solvents
or buffers.
RRr: See relative retention.Radial compression: Using radial
pres-
sure applied to a flexible wall column toreduce wall
effects.
Radial diffusiondispersion: Diffusiondispersion across the LC
column in a radialdirection. If the sample is injected into
theexact center of a column, it will spread notonly in a
longitudinal direction as it movesdown the column but also
radially, whichallows the solute to reach the wall regionwhere the
eluent velocity is different thanin the center of the column.
Re: See Reynolds number.Recovery: The amount of solute or
sam-
ple that is eluted from a column relative tothe amount injected.
Excellent recovery isimportant for good quantitation, prepara-
Porosity: For a porous substrate, theratio of the volume of the
pores in a parti-cle to volume occupied by the particle.The pore
volume is a measure of theporosity and is expressed in milliliters
pergram.
Porous-layer bead: A small glass beadcoated with a thin layer of
stationaryphase. The thin layer can be an adsorbent,resin, or a
phase chemically bonded ontothe adsorbent. These packings were
amongthe first to be used in HPLC. They had2040 mm particle sizes,
which were largerthan the microparticulate packings oftoday, but
were easy to pack and providedadequate efficiency. Also called
controlledsurface-porosity supports and pellicularmaterials.
Porous particle: Refers to column pack-ing particles that
possess interconnectingpores of specified diameter and pore
vol-ume. For HPLC applications, analysts gen-erally use porous
particles with diametersless than 10 mm. Larger particles are
usedin preparative-scale chromatographybecause of lower cost and
higher columnpermeability.
Porous polymer: A packing material,generally spherical, that is
based on organicpolymers or copolymers. Popular examplesinclude
PSDVB, polyacrylates, polydex-trans, polyacrylamides, and
polybutadi-enes.
Precolumn: A small column placedbetween the pump and the
injector. Itremoves particulate matter that may bepresent in the
mobile phase, presaturatesthe mobile phase with stationary phase
orwith dissolved substrate to prevent a loss ofstationary phase or
dissolution of the ana-lytical column, and chemically
absorbssubstances that might interfere with theseparation. Its
volume has little effect onisocratic elution but contributes a
delay tothe gradient in gradient elution.
Preconcentration: See trace enrich-ment.
Preparative chromatography: Refers tothe process of using LC as
a technique forthe isolation of a sufficient amount ofmaterial for
other experimental or func-tional purposes. For pharmaceutical
orbiotechnological purifications, largecolumns of several feet in
diameter can beused for multiple grams of material. Forisolating a
few micrograms of valuable nat-ural product an analytical column
with a4.6-mm i.d. can be used. Based on theintended need of the
chromatographer,both size of columns are preparative chro-
-
The total retention volume (VR) is deter-mined by multiplying
the retention timeby the flow rate. The adjusted retentiontime
(tR9) adjusts for the column void vol-ume;