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;