the terminology guide for chromatographers

7/23/2019 The Terminology Guide for Chromatographers

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Volume 31 Number S10 October 2013

www.chromatographyonline.com

THE CHROMATOGRAPHYAND SAMPLE PREPARATION

TERMINOLOGY GUIDE


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TERMINOLOGY GUIDE OCTOBER 2013 5www.chromatographyonline.com

In 2002, LCGC provided readers

with a gas chromatography (GC)glossary to organize the myriad

terms used in gas chromatography(1). Likewise, in 2008, the third glos-sary of common and not-so-common

terms and buzz words for referenceto high performance liquid chromatog-raphy (HPLC) columns and columntechnology was published (2). It istime for an update because new termshave arisen or, in some cases, theiroriginal meanings have expanded orchanged. Because there are a num-

ber of terms common to both GCand LC, we decided to combine theglossaries into one large listing. Inaddition, with new sample prepara-tion technologies also making theirappearance, expanding the glossary toinclude terminology specific to samplepreparation was another goal. Finally,ion chromatography (IC), which somefeel is a subset of LC, does have someof its own nomenclature and so thoseterms are included here.

We stick to the conventions of theInternational Union of Pure and Ap-plied Chemistry (IUPAC) in their No-

menclature for Chromatography thatprovides guidance and changes in some

of the more commonly accepted terms(3). Still, there are many terms in com-mon usage that are not in alignment

with the IUPAC def initions and thatnomenclature will be covered here as

well.

This terminology guide is not in-tended to be an in-depth listing orhighly theoretical coverage. For ex-ample, we have elected not to covermany of the myriad terms used ininstrumentation, detection, data han-dling, and validation associated withchromatographic analysis but have

chosen to use terms that may be en-countered in everyday laboratory workaround columns, injection techniques,phases, method development, samplepreparation tasks, and general usage.The listing should be helpful to thosejust starting in chromatography but itcan also serve as a refresher for long-time users in the field.

References(1) J.V. Hinshaw, LCGC20(11), 10341040

(2002).(2) R.E. Majors and P.W. Carr, LCGC

26(2),118168 (2008).(3) L.S. Ettre, Nomenclature for Chroma-

tography in Pure and Appl. Chem.65(4),

819872 (1993).

The Chromatographyand Sample PreparationTerminology GuideRonald E. Majors and John Hinshaw


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96-well collection plate:A fixed-sizepolyethylene rectangular plate (127.8mm85.5 mm): consisting of an array

of 812 (96) small test tubes calledwells; volumes of wells range from 0.5to 2 mL.96-well filtration plate:A fixed-sizepolyethylene rectangular plate (127.8mm85.5 mm) consisting of an arrayof 812 (96) of small filter tubes (vol-umes range from 0.5 to 2 mL); a mem-

brane filter placed at the bottom of thewell is used to filter liquid samples;sometimes a prefilter is placed abovethe membrane filter to prevent clogging

with particulate samples.96-well plate:A small rectangularplastic plate consisting of 96 individual

wells that are basically small-volume test

tubes arranged in an 8 12 well pat-tern; used for liquid handling and othersuch requirements.96-well solid-phase extraction plate:

A small rectangular plastic plate con-sisting of 96 individual f low-throughSPE wells arranged in an 8 12 arraythat have top and bottom frits to con-

tain solid particles of sorbent or resin toperform SPE on a miniaturized scale;generally 1 mg to 0.2 g of packing isplaced into the well, which can havea volume of up to 2 mL; used for au-tomated SPE withxyzliquid handlingsystems or customized workstations.

A

A solvent:Usually the weaker solvent ina binary eluent or gradient elution sepa-ration. In reversed-phase liquid chroma-tography (LC), the A solvent typically is

water or a water-rich mixture.Aterm:The first term in the van Deem-

ter equation. See eddy dispersion termand van Deemter equation.

Absorption:The process of retention inwhich the solute partitions into a liquid-like coating.Accelerated solvent extraction (ASE):

Trade name for a pressurized fluid ex-traction system introduced by Dionexand now sold by Thermo Fisher Scien-tific; seepressurized fluid extractionfor details of technique.Active flow technology:A conceptthat incorporates two types of columndesigns: curtain flow technologymeans

segmenting the flow at the injection endof the column to ensure the analyte seesthe middle of the packed bed where it isnot disturbed by wall effects;parallel seg-mented flowat the column outlet selectsjust the middle portion of the flow profileresulting in improved efficiency withoutthe presence of wall effects, giving the

best overall column efficiency; a specialendfitting design is used to sample thecenter of the parabolic flow profile.Active sampling:In active gas sam-pling, a pump is used to push the samplethrough a mass flow controller and intothe canister. Additional sample can becollected, relative to the amount that

can be collected by passive sampling,by pressurizing the canister with sample.Commonly the sample is pressurized to103 kPa (15 psig), effectively doublingthe sample volume.Active site:A reactive or strongly at-tracting site on the surface of a chro-matographic packing that may bindanalytes or cause peak tailing; some-times mobile phase additives (such as acompeting base) can negate the effectsof active sites.Activity:In adsorption chromatography,the relative strength of the surface of thepacking. For silica gel, the more avail-

able the silanol groups, the more activethe surface. Activity can be controlled


7/84

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by the addition of water or other polarmodifier that hydrogen-bonds to theactive sites thereby reducing the surfaceactivity; can also refer to biological ac-

tivity of a biomolecule.Additive:A substance added to themobile phase to improve the separationor detection characteristics; examples

would be a competing base to negatethe effects of silanols, a chelating agentto block metal sites, or addition of aUV-absorbing compound to perform

indirect photometric detection.Adsorbent:Packing used in adsorptionchromatography. Silica gel and aluminaare the most frequently used adsorbents inchromatography and sample preparation.Adsorption:A process of retention inwhich the interactions between thesolute and the surface of an adsorbent

dominate. The forces can be strongforces (for example, hydrogen bonds) or

weak (van der Waals forces). For silicagel, the silanol group is the driving forcefor adsorption and any solute functionalgroup which can interact with thisgroup can be retained on silica. Theterm adsorptionplaces emphasis on the

surface versus penetration or embeddingin the stationary phase coated or bondedto a surface.Adsorption chromatography:One ofthe basic separation and SPE modes thatrelies on the adsorption process to ef-fect a separation. Silica gel and aluminaare the most frequently used normal-phase adsorbents in LC. Moleculesare retained by the interaction of theirpolar function groups with the surfacefunctional groups (for example, silanolsof silica). Carbon is also used as an ad-sorbent in a reversed-phase LC mode.Porous polymer, carbonaceous, and

molecular sieve packings in GC exhibitadsorptive properties as well.

Adsorption isotherm:In adsorption, aplot of the equilibrium concentration ofsample in the mobile phase per unit vol-ume versus the concentration in the sta-

tionary phase per unit weight. The shapeof the adsorption isotherm can determinethe chromatographic behavior of the sol-ute such as tailing, fronting, or overload.Aerogel:A packing prepared when thedispersing agent is removed from a gelsystem without collapsing the gel struc-ture. Silica gels and glass beads used for

SEC are examples of aerogels that canretain their structures even at the highpressure used in HPLC. See xerogels.Affinity chromatography: A tech-nique in which a biospecific adsorbentis prepared by coupling a specific li-gand (such as an enzyme, antigen, orhormone) for the macromolecule of

interest to a solid support (or carrier).This immobilized ligand will interactonly with molecules that can selectivelybind to it. Molecules that will not bindare eluted unretained. The retainedcompound can later be released in apurified state. Affinity chromatogra-phy is normally practiced as an onoff

separation technique.Agarose:High-molecular-weight poly-saccharide used as a separation mediumin biochromatography. It is used in beadform and often used in gel filtration chro-matography using aqueous mobile phases.Alkoxysilane:A reactant used for thepreparation of chemically bondedphases. It will react with silica gel as fol-lows : R3SiOR +SiOHSiOSiR3+ ROH where R is an alkyl group.Alumina:A normal-phase adsorbentused in adsorption chromatography.

Aluminium oxide (Al2O3) is a porousadsorbent which is available with a

slightly basic surface; neutral and acidicmodifications can also be made. Basic


9/84


alumina can have advantages over silica,which is considered to have an acidicsurface; alumina is seldom used as anHPLC column packing in practice. In

GC applications, alumina will separatelow molecular-weight gases.Amino phase:A propylamino phaseused in normal-phase chromatography.It is a somewhat reactive phase for anysolute molecule (for example, aldehydes)or mobile phase additive that can react

with amines. The amino phase has found

some applications as a weak anion ex-changer and for the separation of carbo-hydrates using a wateracetonitrile mo-bile phase. It is a relatively unstable phase.Amperes full-scale (AFS):Extent of themaximum detector output, for detectorsutilizing an electrometer.Amperometric detection: Electro-

chemical detection applying a constantpotential to the working electrode. Mea-sured current from oxidation or reduc-tion is proportional to the sample con-centration. Very selective and sensitivemethod. Works with electrode reactionsnot changing the electrode surface (forexample, cyanide, nitrite, thiosulfate,

phenols). Only approximately 10% ofthe analyte is oxidized or reduced. Maybe used standalone as well as in series orparallel to other detectors.Ampholyte:A substance that carriesboth positive and negative charges (theyare amphoteric). Examples: amino acids,proteins.Amphoteric ion-exchange resin:Ion-exchange resins that have both positiveand negative ionic groups. These resins aremost useful for ion retardation where allionic materials can be removed from solu-tion since the anionic and cationic func-tionalities coexist on the same material.Analyte protectorant:

In GC, a chemi-cal compound or compounds that are

added to a sample before injection to cutdown on interactions between analytesthat are unstable or behave poorly inthe GC flow path on active sites; the

protectorants are chosen so that theydo not interfere with the analysis of thecompounds of interest yet prevent thesecompounds from interacting with theactive sites in the flow path; these pro-tectorants are not generally required forLC and LCMS.Analytical column:A chromatogra-

phy column used for qualitative andquantitative analysis; a typical analyti-cal column for LC will be 50250 cm4.6 mm, but columns with smallerdiameters (down to 0.05 mm i.d.) canalso be considered as analytical col-umns. GC analytical columns rangein length from 1 m to as much as 60

m, with inner diameters ranging fromless than 100 m up to 2 mm. Station-ary phases can be coated or bondedonto the interior of the tubing; packedGC columns are generally wider andshorter and are less frequently usednowadays. Chromatography columnscan be constructed of stainless steel,

glass, glass-lined stainless steel, PEEK,fused silica, and other metallic andnonmetallic materials.Anion exchange:The ion-exchangeprocedure used for the separation of an-ions. Synthetic resins, bonded phase sili-cas, and other metal oxides are availablefor this mode. A typical anion-exchangefunctional group is tetraalkylammo-nium, making a strong anion exchanger.

An amino group on a bonded stationaryphase would be an example of a weakanion exchanger.Argentation SPE:The incorporationof a silver salt into the SPE stationary

phase will help in retaining compoundswith olefinic bonds. Normally used in


10/84


organic solvents to maximize charge-transfer interactions.Array 96-well plate:A 96-well SPEplate where the 96 individual wells are

removable from the base plate; such asetup allows users to place differenttypes and amounts of SPE sorbentsinto various configurations in each ofthe 96-wells. This type of 96-well platehas also been referred to as aflexible 96-well plate configuration.Asymmetry:Factor describing the

shape of a chromatographic peak. The-ory assumes a Gaussian shape and thatpeaks are symmetrical. A quantitativemeasure is the peak asymmetry factor,

which is the ratio of the distance fromthe peak apex to the back side of thechromatography curve over the distancefrom the peak apex to the front side of

the chromatography curve at 10% of thepeak height. Various other measures ofasymmetry are in common use, espe-cially the USP method. See also FoleyDorsey equation.Asymmetry factor:A factor that de-notes band shape; calculated from thechromatographic peak by dropping a

perpendicular at the peak apex anda horizontal line at 10% of the peakheight; at the intersection the distanceto the tail of the peak along the hori-zontal line (distance B) divided by thedistance along the horizontal line tothe front of the peak (distance A);this ratio is the peak asymmetry fac-tor; for a symmetrical peak the valueis one; for a fronting peak the value isless than one; for a tailing peak, thevalue is greater than one; the higherthe value the less symmetrical the peakis; values greater than 2 are generallyunacceptable.Atmosphere (atm):

A unit of pressure. 1atm = 101.325 kPa or 14.6959 psi.

Average particle size (dp):The aver-age particle size of the packing in thecolumn. A 5-m LC column would bepacked with particles with a definiteparticle size distribution because pack-ings are never monodisperse. Particlesizes in GC usually are expressed interms of mesh size distribution; 80100

mesh is a commonly used particle range.Seeparticle size distribution.

B

B solvent:Usually the stronger solvent ina binary eluent or gradient separation. Inreversed-phase LC, typically the organicmodifier or modifier-rich binary mixture

with water.Bterm:The second term of the vanDeemter equation; the first term of theGolay equation. See longitudinal dif-

fusion,molecular diffusion term, vanDeemter equation, Golay equation.Back extraction:Used in liquidliquidextraction to perform an additional ex-traction to further purify a sample; ini-tially the extraction may take place withan aqueous solvent buffered at a high pHand an immiscible organic solvent; afterthe initial extraction takes place and in-terferences are removed, then by havinganother aqueous solution at a low pH,

one can back-extract the analyte into theorganic layer based on the analyte now

1.0

0.5

0.10.0

Normalizedpe

akheight

32 36 40 44 48t1 tp t2

Time (s)

B A

wa=A

+ B

hp

Figure 1: Example of a tailing peak. (Mod-ified with permission from reference 3.)


11/84


being in a neutral form. An examplewould be for the cleanup of an acidicsubstance containing COOH groups;at high pH the carboxyl would be ionized

and prefer the aqueous layer and impu-rities may migrate to the organic phaseand discarded; then the pH of the aque-ous layer can be adjusted to a low value.Now the carboxyl group is in an union-ized form and readily extracted into theorganic layer as a purified substance.Backflushing:Useful in chromatogra-

phy to remove compounds that are heldstrongly at the head of a column. By re-versing the flow at the conclusion of arun, analytes trapped at the head of thecolumn can be flushed from the columnentrance because they have a shorter dis-tance to travel; sometimes a strong solventin LC or elevated temperatures in GC

will be needed to move them along. Avalve or fluidic device is used to effect thechange of mobile-phase flow direction.Backflushing can be used for analysis ofthese compounds or merely to removethem from the column.Back-pressure regulator:In LC, a de-vice placed on-line after the detector to

maintain a positive pressure on the flowcell minimizing solvent outgassing prob-lems in the detector. In GC, the termusually refers to a carrier-gas regulator inthe split vent line that maintains a con-stant pressure at the inlet as split flowschange.Bakeout:The process of removing con-taminants from a column by operationat elevated temperatures, which shouldnot exceed the maximum column tem-perature.Band:Refers to the chromatographicpeak as it moves along and is eluted fromthe column.Band broadening:

The process of in-crease in width and concomitant dilu-

tion of the chromatographic band as itmoves down the column. The peak isinjected as a narrow slug and ideallyeach separated component would elute

as a narrow slug of pure compound ifnot for the process of band broadening.The measure of band broadening is thepeak dispersion, , or more correctly N,the number of theoretical plates in thecolumn. Sometimes called band disper-sionor band spreading.Band width:Seepeak width at baseand

peak width at half-height.Baseline:The baseline is the line drawnby the recording device representing thesignal from the detector when only mo-bile phase is passing through, in the ab-sence of any solutes. It also represents thepoint from which calculations are oftenmade on peaks to determine peak area

or peak height.Baseline drift:Term for any regularchange occurring in baseline signalfrom an LC or GC detector; it mayarise from changes in flow rate of themobile phase or from stationary phasebleed and may trend in a positive ornegative direction. Baseline drift oc-

curs over a longer period of time thanbaseline noise.Baseline noise:Irregular variations(short term) in the chromatographicbaseline as a result of electrical noise ortemperature fluctuations, outgassing inthe flow cell, or poorly mixed mobile-phase solvents.Bed volume:See column volume.BEH:Bridged ethyl hybrid; an inorganicorganic HPLC particle; has higher pHlimits than silica gel.BET method:A method for measuringsurface area developed by Bruner, Em-mett, and Teller (BET) that uses nitro-

gen adsorptioncondensation in pores atliquid nitrogen temperature. Pore volume


12/84


and pore size distribution can also be ob-tained from BET calculations.Bidentate silane:A specific type ofbonded phase in which a short hydrocar-bon bridge connects two silicon atoms ina silane that is bounded to the surfacethrough two siloxane groups.Bimodal: In SEC, can be a porouspacking material that has two distinctpore sizes or pore size distributions; inion-exchange or HILIC chromatog-raphy or sample preparation can be apacking material that has two typesof functionalities (for example, cationexchange and reversed phase; cationand anion) on one packing; in somecases, mixed beds consisting of two

different packings in one column canbe bimodal.

Binary mobile phase:Mobile phase con-sisting of two solvents or buffers (or one

of each).Bindelute :In SPE, the normal mode ofoperation where upon loading the sampleonto a conditioned sorbent or resin, theanalytes of interest are retained (bound)

while interferences and perhaps some ofthe matrix is not retained by the pack-ing; after a wash step to remove some ofthe undesired sample components, theelution step uses a strong solvent to elutethe analytes of interest in a small volume.Biocompatible:A term to indicate thatthe column or instrument component

will not irreversibly or strongly adsorbor deactivate biomolecules, such as pro-

teins. Frequently means metal-free orceramic surfaces and components.

1.000

0.882

0.607

0.500

0.324

0.134

0.044

Inflection points

Tangents drawn tothe inflection points

Normalizedpea

kheight

wi= 2

wb= 4

3

4

5

wh= 2.355

wi

wh

Figure 2: Widths of a Gaussian peak at various heights as a function of the standarddeviation s) of the peak. (Modified with permission from reference 2.)


13/84

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Blank:More correctly named methodblank. A blank prepared to representthe matrix as closely as possible. Themethod blank is prepared and analyzed

exactly like the field samples. Purpose:Assess contamination introduced dur-ing sample preparation activities.Bleed:Loss of material from a columnor septum due to high-temperatureoperation. May result in ghost peaksplus increased detector baseline offsetand noise; in extreme cases, bleeding

chemicals from the stationary phasemay build up on detector surfaces.Blending:Refers to the process of mak-ing a heterogeneous sample into a moreconsistent and uniform sample by sometype of blending operation; the mostpopular type of blender is the mechani-cal blender that chops a semisoft mate-

rial into smaller parts.Bonded phase:A stationary phase thathas been chemically bonded to theinner wall of an open-tubular (capil-lary) column or to the support particles.In LC, the substrate is usually a silicagel particle or other base material.Bonded-phase chromatography:The

most popular mode in LC, in which aphase chemically bonded to a supportis used for the separation. The mostpopular support for bonded-phasechromatography is microparticulatesilica gel and the most popular type ofbonded phase is the organosilane, suchas octadecyl (for reversed-phase chro-matography). Approximately 70% ofall HPLC is carried out on chemicallybonded phases.Bonded-phase concentration:Seecoverage.Boxcar chromatography:See columnswitching; alternate name.Breakthrough capacity:

See break-through volume.

Breakthrough volume:The volume atwhich a particular solute pumped con-tinuously through a column will beginto be eluted. It is related to the column

volume plus the retention factor of thesolute. It is useful to determine thetotal sample capacity of the columnfor a particular solute.BTEX:Refers to benzene, toluene, eth-ylbenzene, and xylenes analysis.Buffer:A solution that maintains con-stant pH by resisting changes in pH as

a result of dilution or addition of smallamounts of acids and bases.Buffer capacity:A quantitative mea-sure of the potential of a buffer solu-tion (defined as the number of equiva-lents of strong acid or base to cause aone unit change in the pH of 1 L ofa buffer solution) or simply the abil-

ity of a buffer to withstand injectionsof a buffered sample solution withouta change in mobile-phase pH; capac-ity determined by pH, buffer pKa, andbuffer concentration.Buffer strength:See ionic strength.

C

C4, C8, C18:Refers to the alkyl chainlength of a reversed bonded phase.Cterm:The interphase mass transferterm of the van Deemter and Golayequations.Canister collection:A stainless steelvessel designed to hold vacuum toless than 1.3 Pa (10 mTorr) or pres-sure to 275 kPa (40 psig). Canistersare available in a range of volumes:400 mL, 1.0 L, 3.0 L, 6.0 L, and 15L. The size of canister used usuallydepends on the concentration of theanalytes in the sample, the samplingtime, the flow rate, and the sample

volume required for the sampling pe-riod. Typically, smaller canisters are


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used for more concentrated samples,such as soil gas collection, 3-L and6-L canisters are used to obtain in-tegrated (TWA) ambient air samples

at sampling times of up to 24 h, andlarge 15-L canisters are used for refer-ence standards. Sampling time will belimited by the combination of canis-ter size and the f low rate at which thesample is to be collected.Capacity:See sample capacity.Capacity factor (k):Deprecated name

for retention factor.Capillary column: Refers to chro-matography columns of small innerdiameter, ostensibly small enough todisplay a capillary effect with liquids.The diameter below which a column isconsidered capillary is poorly defined.See open-tubular column, capillary

LC.Capillary column, packed:A capillarycolumn that is packed with stationary-phase particles. In GC, 1/16-in. o.d. by1-mm i.d. columns are common.Capillary electrochromatography

(CEC): A hybrid technique wherecapillary columns are packed with

chromatographic sorbents and elec-troosmotic flow moves mobile phasethrough the column rather than pres-sure; the technique has the surface-mediated selectivity potential of HPLCand the high efficiency of CE.Capillary GC:See open-tubular col-umn.Capillary LC: Generally refers toHPLC carried out in a fused-silica orother type of capillary column; mostof the time the dimensions are in thesub-0.5-mm i.d. range. Has also beencalled micro LC.Capillary micellar electrochromatog-

raphy (CMEC):The CEC version ofMEKC.

Capillary tubing:Tubing to connectvarious parts of the chromatograph inorder to direct flow to the proper place.Most capillary tubing used in HPLC is

less than 0.020 in. in internal diameter.The smallest useful internal diameter isabout 0.004 in.Capping:Same as endcapping.Carbon load: For a bonded-phasesilica, term usually used to describethe surface coverage or the degree to

which the available si lanols on the

column packings surface have reactedand been replaced with the bondedphase; the higher the carbon load, thelower number of residual silanols. Thecarbon load is normally expressed as% carbon (for example, 12% carbon).In reversed-phase LC, the higher thecarbon load, the greater the analyte

retention.Carrier:A term most often used in af-finity chromatography; refers to thesupport that is used to attach the ac-tive ligand, usually by a covalent bond.Can also refer to the support in otherchromatography modes, such as LLC.Carrier gas:Term for the gaseous mo-

bile phase in GC.Cartridge:Generally refers to the con-tainer used in SPE or filtration; a car-tridge may be as simple as a medical-grade syringe barrel that is filled withpacking contained at both ends by frits;it can also be a molded device or evena stainless steel device that containssimilar sorts of packing material. InSPE, the device is also referred to asan SPE tube.Cartridge column:A column typethat has no endfittings and is held in acartridge holder. The column consistsof a tube and the packing is contained

by frits in each end of the tube. Car-tridges are easy to change and are less


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expensive and more convenient thanconventional columns with endfittings.Cation-exchange chromatography:The form of ion-exchange chroma-

tography that uses resins or packingswith functional groups that can sepa-rate cations. An example of a strongcation functional group would be asulfonic acid; a weak cation-exchangefunctional group would be a carbox-ylic acid.Centrifugation: Centrifugation is

a process that involves the use of thecentrifugal force for the sedimenta-tion of mixtures with a centrifuge (seecentrifuge). This process is used toseparate two immiscible liquids. More-dense components of the mixture mi-grate away from the axis of the centri-fuge, while less-dense components of

the mixture migrate toward the axis.Chemists and biologists may increasethe effective gravitational force on a testtube so as to more rapidly and com-pletely cause the precipitate (pellet)to gather on the bottom of the tube.The remaining solution is properlycalled the supernate or supernatant

liquid. The supernatant liquid is theneither quickly decanted from the tube

without disturbing the precipitate, orwithdrawn with a Pasteur pipette.Centrifuge:A centrifuge is a pieceof equipment, generally driven by anelectric motor (some older models werespun by hand), that puts an object inrotation around a fixed axis, applyinga force perpendicular to the axis (seecentrifugation).Certify:Specific SPE products fordrugs of abuse isolation and analysis.Chain length:The length of carbonchain in the hydrocarbon portion of a

reversed-phase packing. It is expressedas the number of carbon atoms (for ex-

ample, C8, C18). Specifically excludesthe short chains typical methyl, isopro-pyl, and sec-butyl groups also attachedto the silane.

Channeling:Occurs when voids cre-ated in the packing material cause mo-bile phase and accompanying solutesto move more rapidly than the averageflow velocity allowing band broadeningto occur. The voids are created by poorpacking or erosion of the packed bed.Charged aerosol detection (CAD):

The effluent from the LC column isnebulized and then vaporized in aheated drift tube, which results in acloud of analyte particles; these par-ticles are charged and then the currentfrom the charged particle flux is mea-sured. The ELSD technique measuresthe light scattering properties of the

aerosol particles. CAD is more sensi-tive and gives a more linear responsethan ELSD; it is also a universal detec-tion method.Check valve:A device inserted into amoving fluid stream that allows flow ofthe stream in only one direction; mostoften used on the inlet and outlet sides

of an HPLC pump.Chelating resin:Chelating resin con-tains functional groups that will inter-act with cationic species (for example,metals such as copper, iron, heavymetal ions); useful for concentratingtrace quantities or for separation.Chemical filtration:A liquid sampleis passed through a packing material(for example, adsorbent, ion exchange,and so forth) that selectively interacts

with one or more compounds withinthe sample and acts as a chemical wayto remove and purify the liquid sample.Regular filtration does not involve any

chemical interaction but merely re-moves particulates.


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Chemical suppression:Remove back-ground conductivity by ion exchange.Converts the eluent into a low- or non-conducting component (for example,

carbonate into carbonic acid, hydrox-ide into water, or nitric acid into water).

Anion analysis: the counter cat ion(for example, sodium) is replaced bythe proton (H+). Cation analysis: thecounter anion (for example, nitrate) isreplaced by hydroxide (OH-). The mea-sured signal is the corresponding acid

or base of the anion or cation respec-tively. The dissociation of these acidsand bases influences the signal. All sup-pressor devices work on this principle.The typical background conductivityafter suppression is


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Coating efficiency (CE, UTE, UTE%):A metric for evaluating column quality.The minimum theoretical plate heightdivided by the observed plate height:

CE = Hmin/HCold injection:An injection that takesplace at temperatures below the finaloven temperature, usually at or belowthe solvent boiling point.Column:The tube and stationary phasethrough which mobile phase f lows re-sulting in a chromatographic separa-

tion.Column chromatography : Anyform of chromatography that usesa column, tube, or plate to hold thestationary phase. Open-column chro-matography, HPLC, and open-tubu-lar capillary gas chromatography areall forms of column chromatography.

Most often refers to open-columnchromatography used for prepara-tive work.Column dead time:See hold-up time.Column equilibration:To provide re-producible results, a column should beequilibrated with the surrounding envi-ronment be it a temperature condition,

mobile phase equilibrium, pressurecondition, and so forth; in GC, it isimportant that the temperature of thecolumn be stabilized after a tempera-ture programmed run and in LC, thecolumn must be returned to its originalconditions before another gradient isrun.Column inner diameter (dc): Theinner diameter of an uncoated chro-matography column.Column length (L):The length of theanalytical chromatography columnused to perform the chromatographicseparation. Distinct from the length

of a precolumn (LC) or retention gap(GC) connected in series.

Column outlet flow rate, corrected

(Fa):In GC, the column outlet flowrate corrected from column tempera-ture and outlet pressure to room tem-

perature and pressure, for example,the flow rate as measured by a flowmeter. Difficult to measure directlyfor narrow-bore open-tubular col-umns, the flow rate can be calcu-lated from the average carrier-gaslinear velocity, pressure drop, temper-atures, and the column dimensions:

Fa = (udc2T0)/(4jTc). Such calcula-tions are the basis of electronic pres-sure control.Column overload:If one exceeds thesample capacity (or loading capacity) ofa column, peaks will become distortedand may be difficult to measure andto achieve reproducible chromatogra-

phy from run to run. One can measurecolumn capacity by running a break-through study (see breakthrough vol-ume).Column packing:The solid material,usually a porous solid with or withouta chemically interactive surface, placedinside or on the walls of the column

used to differentially retain analytes;also referred to as the stationary

phase;common packings include un-bonded and bonded silica, resins, in-organicorganic hybrids, graphitizedcarbon, porous polymers, and molecu-lar sieves.Column performance:Denotes thecolumn efficiency. See theoretical

plate.Column plate number:Denotes thecolumn efficiency. See theoretical

plate.Column suppressor: Initial setup.Packed ion-exchanger columns were

used for chemical suppression. Draw-backs: require regeneration, changes


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of the selectivity throughout theusage.Column switching:The use of mul-tiple columns connected by switching

valves to effect better chromatographicseparations or for sample cleanup. Frac-tions from a primary column can beswitched to two or more secondarycolumns which in turn can be furtherdiverted to additional columns or tothe detector (or detectors); sometimesreferred to as multidimensional chro-

matography.Column temperature (Tc):Tempera-ture of the column. A uniform tem-perature across the column usually isdesirable; however, GC separationsalso may be performed with a movingtemperature gradient along the columnlength.

Column volume (Vc):The volume ofthe unpacked, uncoated column: Vc=AcL = rc

2L,whereAcand Lare thecross-sectional area of the tube and thetube length, respectively.Cool-down time:Length of time re-quired to cool a GC oven from the finaloven temperature to the initial oven

temperature. Shorter cool-down timesallow a greater number of analyses tobe performed in a given time period.Competing base:In reversed-phaseLC, addition of a small basic com-pound such as triethylamine or di-methyloctylamine at 2550 mMconcentration to the mobile phase toinhibit basic analytes from interact-ing with residual silanols; works bylaw of mass action because the con-centration of competing base is muchgreater than that of the analyte. Seealso additive.Comprehensive GC (GCGC):Two-

dimensional technique in which allcompounds experience the selectivity

of two columns connected in series bya retention modulation device, therebygenerating much higher resolution than

with any single column.

Comprehensive two-dimensionalchromatography:Two-dimensionalchromatography applied to every frac-tion. See two-dimensional chroma-tography.Compressibility correction factor (j):Due to gas compressibility, the carriergas expands and its velocity increases

as it proceeds along a GC column fromthe inlet pressurepito the outlet pres-surepo. The carrier gas compressibilitycorrection factor corrects the carriergas velocity at the outlet of a GC col-umn to the average carrier gas velocity:j = 3(P2 1)/2(P3 1), where Pis thecolumn pressure drop: P =pi/poConcentration:The process of increas-ing the strength or density of a dilutedsample; a more concentrated sample

will be easier to measure; concentrationcan be accomplished by a wide varietyof sample preparation techniques suchas evaporation, adsorption, diffusion,and so forth.Conditioning (SPE):This is gener-ally considered to be the f irst step inSPE; the stationary phase must f irstbe put into a chemical or physicalstate that it can accept the samplesolution loaded in the second SPEstep; a conditioning solvent is passedthrough the SPE stationary phase

where it will solvate the phase so thatit will more easily sorb the sample ofinterest; for a reversed-phase SPE car-tridge, methanol or acetonitrile servesas a conditioning solvent; sometimesthe excess conditioning solvent mustbe removed but the packing shouldnt

be allowed to dry out because thatmay affect the conditioned phase.


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Conductivity:Conductivity is the in-herent parameter of all ions. Thereforeit is the signal for measuring the chro-matogram. It may be measured directly

or after chemical and sequential sup-pression respectively. As conductivity isstrongly dependent on the temperature(around 2 %/C), a thorough isolationand thermal stabilization of the detec-tor block is recommended. The mea-sured conductivity of a solution is givenby the sum of the single ion conduc-

tivities. The single ion conductivity isa linear function of the concentrationof the ion and its equivalent conduc-tivity. At very low concentrations or atconstant ion strength the equivalentconductivity is constant. The mea-sured signal in nonsuppressed IC isproportional to the concentration and

the difference of eluent and sample ion:As the ionic strength of the solution isconstant (stoichiometric ion exchange)also the equivalent conductivities areconstant. This leads to very linear cali-bration curves. The measured signalin suppressed IC is proportional to thesum of the equivalent conductivities

of the analyte ion and the counterion(which has been introduced by suppres-sion). As only the sample ion and itscounterion is adding to the measuredconductivity after suppression, the totalconcentration is changing during thepeak. Therefore the equivalent conduc-tivities are no longer constant. This isone reason for the inherent nonlinear-ity of the calibration curves in sup-pressed IC.Coning and quartering:A sample sizereduction technique where a portion offree-flowing solid material (powder) issystematically divided into quadrants

to achieve a statistically representativesample. Coning and quartering is a

method used by analytical chemiststo reduce the sample size of a powder

without creating a systematic bias. Thetechnique involves pouring the sample

so that it takes on a conical shape, andthen flattening it out into a cake. Thecake is then divided into quarters;the two quarters that sit opposite oneanother are discarded, and the othertwo are combined and constitute thereduced sample. The same process iscontinued until an appropriate sample

size remains. Analyses are made withrespect to the sample left behind.Continuous liquidliquid extraction:Useful when the KDvalue is very lowor the required sample volume is verylarge when multiple extractions are im-practical; also if the extraction is slow,a long time may be required for equi-

librium to be established; in continu-ous LLE, fresh solvent is continuallyrecycled through the aqueous sample;continuous extractors are available forheavier-than-water and lighter-than-

water solvents.Controlled surface porosity support:Same as porous layer beadand pel-

licular stationary phase.Cool on-column injection:Cool on-column injection is a technique of in-troducing a sample as a liquid directlyinto a GC column; this lack of priorvaporization offers the following advan-tages: It eliminates sample discrimina-tion; it eliminates sample alteration;and it provides high analytical preci-sion. However, there are some specialrequirements: It requires relativelyclean samples; real samples are oftentoo concentrated for on-column injec-tion and must be diluted; and peaksplitting or peak distortion can occur

due to differing polarities of solvent,stationary phase, and solutes.


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Coreshell:See superficially porousparticles (SPPs).Coulometric detector:Same as am-perometric detection, but with a con-

version rate of 100%. All the analyteis oxidized or reduced at the workingelectrode. The response is larger than

with amperometric detection. But onthe other hand also the baseline noiseis larger. Therefore the detection limitsare almost the same.Counterion:In an ion-exchange pro-

cess, the ion in solution used to displacethe ion of interest from the ionic site.In ion pairing, it is the ion of oppositecharge added to the mobile phase toform a neutral ion pair in solution.Coupled columns:A form of columnswitching that uses a primary columnconnected to two secondary columns

via a selector valve. Fractions from col-umn one can be selectively transferredto columns two and three for addi-tional separation to occur. The term isalso used to describe two or more col-umns connected in series to provide anincreased number of plates.Coverage:Refers to the amount of

bonded phase on a silica support inbonded phase chromatography. Cov-erage is usually described in mol/m2or in terms of %C (w/w).Crash plate:Refers to the process ofprecipitating protein from plasma bythe addition of a miscible organic sol-vent such as acetonitrile; when a 96-well flow-through or fixed-well plate isused for this process, it is referred to ascrashingand the plate a crash plate.Critical micelle concentration (CMC):The concentration of an ionic surfac-tant above which a micelle is formedby aggregation; micelles added to the

mobile phase are used to improve theseparation of nonionic substances in

HPLC and CE (MEKC) by a partition-ing mechanism.Cross-linked phase: A stationaryphase that includes cross-linked poly-

mer chains. Usually it is also bondedto the column inner wall. See bonded

phase.Crosslinking:For resins, during theprocess of copolymerization to forma three-dimensional matrix a difunc-tional monomer is added to form cross-linkages between adjacent polymer

chains. The degree of cross-linkingis determined by the amount of thismonomer added to the reaction. Forexample, divinylbenzene is a typicalcross-linking agent for the productionof polystyrene ion-exchange resins. Theswelling and diffusion characteristicsof a resin are governed by its degree of

cross-linking.Crushing:Tungsten carbide variable

jaw crushers for reducing the size oflarge, extremely hard, brittle samples.Curtain flow technology:Curtainflow technology refers to the processof injection of sample across a radialcross section of an HPLC column to

ensure the analyte sees the middle por-tion of the packed bed and not the wall

where flow effects may be different; thetechnique is coupled with a parallel seg-mented flow fitting at the column out-let to select just the middle portion ofthe flow profile resulting in improvedefficiency without the presence of walleffects.Cutting:Cutting mills can reduce soft-to-medium hard materials (diameter 300 are used.Megapascal (MPa):A unit of pressure; 1MPa = 10 bar, 10.133 atm, or 145.0 psi.Megapores:Seeperfusion chromatog-raphy.Membrane extraction with sorbent

interface (MESI): A version of dy-namic headspace where a silicone hol-low fiber membrane is placed in theheadspace about the sample; an inertgas is passed through the membraneand analytes that are permeable tothe membrane pass from the head-space and are swept to an adsorbenttrap; after a period of concentration,

the trapped analytes are thermally de-sorbed to the GC column.


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Membrane filtration:Membrane filter;membrane disk.Membrane suppressor:Continuouschemical suppression. Ion exchange

through ion-exchange membranes. H+or OH-are supplied by the respectiveregeneration solution (for example, sul-furic acid).Metal affinity chromatography:Aspecial form of ligand exchange chro-matography used for the separation ofbiopolymers with a particular affin-

ity for a specific metal cation typicallycopper(II), zinc(II), and iron(II).Metalophile:A compound that hashigh affinity for active (acidic) silanolgroups on silicas surface. Usually astrongly basic amine.Method detection limit (MDL):Theminimum amount of solute that can be

analyzed within specified statistical lim-its of precision and accuracy, includingsample preparation.Method development:A process ofoptimizing the separation includingthe sample pretreatment so as to obtaina reproducible and robust separation.Usually it emphasizes the search for the

stationary phase, eluent, and columntemperature combination that providesan adequate separation.Method translation:Several math-ematical techniques for adjusting GCmethod parameters for variations in thecarrier gas type or column dimensions,

with the objective of maintaining eitherthe same or ratiometric retention times.Useful when changing from helium tohydrogen carrier gas, or when increas-ing speed of analysis or resolution byadjusting column dimensions. Not use-ful if changing the stationary phase to achemically different type.Method validation:

A process of test-ing a method to show that it performs

to the desired limits of precision andaccuracy in retention, resolution, andquantitation of the sample componentsof interest.

Micellar chromatography:The addi-tion of micelles to the mobile phaseto effect separations. The micellesmay act as displacing or partitioningagents and provide another parameter

which may be used to change selec-tivity. Surfactants above their criticalmicelle concentration are used in mi-

cellar chromatography and in MEKCform of CE.Micro LC:Refers collectively to tech-niques where a column of smaller thanconventional internal diameter is usedfor separation. The term micro LC ismost often used for HPLC in


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currently in its infancy, and applicationsshould expand with time.Microdialysis:Microdialysis is a mini-mally invasive sampling technique that

is used for continuous measurement offree, unbound analyte concentrationsin the extracellular fluid of virtuallyany tissue. Analytes may include en-dogenous molecules (for example, neu-rotransmitters, hormones, and glucose)to assess their biochemical functions inthe body, or exogenous compounds (for

example, pharmaceuticals) to determinetheir distribution within the body. Themicrodialysis technique requires the in-sertion of a small microdialysis catheter(also referred to as microdialysis probe)into the tissue of interest. After theprobe is inserted into the tissue or (body)fluid of interest, small solutes can cross

the semipermeable membrane by passivediffusion. The microdialysis probe is de-signed to mimic a blood capillary andconsists of a shaft with a semipermeablehollow fiber membrane at its tip, whichis connected to inlet and outlet tubing.Microextraction:The general processof liquid extraction using small amounts

of organic solvent where the phase ratioVo/Vaq is quite low; other techniquesusing hollow microfibers as a barrierare also referred to as microextraction.Microparticulate:Refers to the smallparticles used in HPLC. Generallypackings with a particle diameter of lessthan 10 m and that are totally porousare considered microparticle packings.Micropipette tip:A form of SPE in

which the packing material is embed-ded or adsorbed on the inner wallsof a pipette tip; useful for the SPE ofvery small amounts of liquid sample;often used withxyzliquid handling

systems for automation purposes. Seepipette tip.

Microporous resin:Same as microre-ticular resin.Microreticular resin: Cross-linkedsynthetic ion-exchange resins that

have pores with openings correspond-ing to molecular sizes. Diffusion intothe narrow pores can be impaired andlow exchange rates can occur, as well aspoor performance, especially for largemolecules.Microwave-assisted extraction

(MAE):The use of microwave energy

to heat samples in the presence of asolvent allowing for rapid extraction;MAE can be performed in open ves-sels. A nonmicrowave-absorbing sol-vent is used and the sample contain-ing a substance with a high dielectricconstant (for example, water) is rapidlyheated, with the extracted analytes

passing into the extraction solvent. Avariation of this technique involves theaddition of an inert microwave-ab-sorbing solid substance that transfersthe heated energy to the surroundingsolvent. MAE can also take place inclosed vessels that are non-microwave-absorbing containers.Migration time (tM):The time it takesfor a charged molecule to move from thepoint of injection to the point of detec-tion in a CE capillary.Milling:Devices for reducing the par-ticle sizes of solid materials. Disk millspulverize


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agate, tungsten carbide, or PTFE-coated stainless steel balls.Mincing:The process of breaking downa meat or vegetable product into smaller

parts by tearing, chopping, cutting, dic-ing, and so forth.Minimum detectable quantity (MDQ):The amount of solute that produces asignal twice the noise level.Mixed-bed column:Combination oftwo or more stationary phases in thesame column, used most often for ex-

change separations (IEC mixed anionand cation resins) and SEC (mixture ofdifferent pore size packings). The ad-vantage in IEC is the total removal ofboth cationic and anionic compounds;the technique is useful in SEC becausea wider molecular weight range can beaccommodated by the same column.

Mixed-mode separation:A separationthat takes place in a single column asa result of retention and selectivity pro-vided by a dual retention mechanism.For example, at intermediate-to-highpH values, a reversed-phase column

with residual silanols can separate byhydrophobic interactions as well as

ionic interactions by virtue of the ion-ized silanols; sometimes mixed-modeseparations can be quite beneficial tothe selectivity (band spacing) but cancause peak asymmetry; the precise bal-ance of interactions may be difficult toreproduce with subsequent batches ofpacking.Mobile phase:The fluid that movessolutes through the column. In LC, themobile phase interacts with both the sol-ute and the stationary phase and there-fore can have a powerful influence onthe separation. In GC, the mobile phase,as an inert gas, has little interaction with

stationary phase and analytes and servesto move the sample through the column.

Mobile-phase modifier:Modifiers arematerials (usually organic or inorganiccompounds ) added to the mobile phaseto alter its elution properties.

Mobile-phase strength:See solventstrength.Modifier:An additive that changesthe character of the mobile phase. Inreversed-phase LC, for example, wateris the weak solvent and methanol, thestrong solvent, is sometimes called themodifier; sometimes other additives

such as competing bases like triethyl-amine or ion pair reagents are referred toas modifiers but they should more cor-rectly be called additives. See additive.Molecular diffusion term (Bterm):Refers to the Bterm (second term) ofthe van Deemter and Golay equations.

Also called longitudinalor axial diffu-

sion term. It dominates band broaden-ing only at very low flow rates below theminimum plate height where the diffu-sion of individual solutes can occur in alongitudinal (lengthwise) direction onthe column. See van Deemter equation,Golay equation.Molecular sieve:GC column packing

that retains solute by combined mo-lecular size and adsorptive interactions.Molecular sieves can separate light gasesand hydrocarbons.Molecular weight distribution:Thedistribution of the molecular weight ofmolecules in a polymer sample. Distri-bution can be defined as weight averageand number average.Molecularly imprinted phases (MIPs):See imprinted phases.Monolith:A monolithic HPLC col-umn is a special type of column usedin HPLC with porous channels ratherthan beads; monoliths, in chromato-

graphic terms, are porous rod struc-tures characterized by mesopores


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and macropores. These pores providemonoliths with high permeability, alarge number of channels, and a highsurface area available for reactivity.

The backbone of a monolithic columnis composed of either an organic (poly-meric) or inorganic (silica) substrate,and the column can be chemically al-tered for specific applications.Monomeric phase:Refers to a bondedphase where single molecules are bondedto a support. For silica gel, monomeric

phases are prepared by the reaction of analkyl- or arylmonochloro- or alkoxysi-lane. Polymeric phases generally are pre-pared from a di- or trichlorosilane or analkoxysilane reactant.Moving zone:The moving zone is thatfraction of the mobile phase in the col-umn that occupies the interstitial spaces.

Multidimensional chromatography:The use of two or more columns orchromatographic techniques to ef-fect a better separation. It is useful forsample cleanup, increased resolution,and increased throughput. Separa-tion is carried out with two or morecolumns in which peaks are selectively

directed onto or removed from at leastone of the columns by use of a timedvalve system. In GC, a Deans fluidicswitch is often used. It also can beused off-line by collecting fractionsand reinjecting onto a second column.

Also called coupled column chromatog-raphy, column switching, multicolumnchromatography, andboxcar chromatog-raphy. See backflushing, heart cut-ting, precut.Multidimensional protein iden-

tification technology (MudPIT):Multidimensional protein identifi-cation technology combines both a

cation-exchange prefractionation andreversed-phase HPLC separation of

tryptic peptides to analyze an entireproteome of a cell or tissue type pro-tein extract. The approach uses a dualenzymatic digestion (Lys-C followed

by trypsin) to increase the numberof peptides observed. The peptidesare separated using strong cation ex-change and are identified by MS-MSdetection.Multimodal SPE:The practice of SPE

where two different phases or modes areused to clean up a sample; the process

can consist of two separate cartridgesplaced in series with the analytes sepa-rated on the two different cartridges; asecond process is where two differentphases are present in the same cartridgeor even on the same packing; sometimesreferred to as mixed-mode SPE.

N

Nano LC:LC practiced with columnsthat have internal diameters less than100 m; usually requires specialized in-strumentation; often used in proteomicstudies where sample is limited and sen-sitivity is required.Narrow-bore column:Columns of less

than 2 mm i.d. used in HPLC, and lessthan 320 m i.d. in GC; also referred toas microbore.Nitrogenphosphorus detection

(NPD):The nitrogenphosphorus detec-tor catalytically ionizes N- or P-contain-ing solutes on a heated rubidium or ce-sium surface in a reductive atmosphere.NPD is highly selective with sensitivitysomewhat better than FID.Noise:See baseline noise.Non-aqueous reversed phase chroma-

tography (NARP):Refers to reversed-phase chromatography performed with-out water as a component of the eluent

on a reversed-phase packing. Used forcompounds that are very nonpolar that


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either cannot be eluted or are poorlyeluted from a reversed-phase column

with 100% methanol or acetonitrile; inthese cases, solvent A would be aceto-

nitrile and solvent B would be a stron-ger solvent such as tetrahydrofuran; forNARP, reversed-phase rules apply (thatis, the more nonpolar the analyte, thegreater the retention).Nonpolar:A nonpolar molecule is onein which the electrons are distributedmore symmetrically and thus does not

have an abundance of charges at theopposite sides. The charges all cancelout each other. Nonpolar compounds,solvents, or bonded phases readily dis-solve in organic solvents, such as hexane,or prefer such solvents in place of water.Nonpolar substances do not readily dis-solve in water.

Nonporous particle:Refers to a solidparticle used as a support for a porouscoated or bonded phase; pellicular par-ticles are nonporous particles with largeparticle diameters (approximately 40 m)and nonporous silicas and resins withsmall particle diameters of less than 3 musually consist of a microbead with a thin

porous outer coating of silica gel, bondedsilica gel, or polymeric phase.Nonsuppressed ion chromatography:Direct ion chromatography. After theseparation column the eluent is directlyfed into the conductivity detection with-out prior chemical suppression. Theconductivity measurement takes placeon the high background conductivity.This requires a very high quality detec-tor with perfectly stable temperature.The nonsuppressed approach allows de-tection of weak acids or bases that areundissociated after suppression forexample, silicate or borate. For cation

analysis the peaks for the components ofinterest are larger than with suppression.

Nonporous packing (NPR, NPS, NPZ):Particles similar to porous-layer beadbut with particle diameters in the sub-5-m range, often particles are in the

sub-2-m range; used for high-speedseparations in short columns; NPS refersto nonporous silica, NPR to nonporousresins, and NPZ to nonporous zirconia.Normal-phase chromatography:Amode of chromatography in which thestationary phase is more polar than themobile phase. Adsorption chromatogra-

phy on silica gel or alumina using mix-tures of less polar eluents (for example,hexanediethethyl ether) as a mobilephase would be a typical normal-phasesystem. Also refers to the use of polarbonded phases, such as -CN or NH2.Sometimes referred to as straight phasechromatography.

O

Octadecylsilane (ODS, C18):The mostpopular reversed phase in HPLC. Oc-tadecylsilane phases are bonded to silicaor polymeric packings. Both monomericand polymeric phases are available.Octylsilane (C8):A popular stationary

phase in reversed-phase chromatogra-phy; usually has slightly less retentionthan the more popular C18; both mo-nomeric and polymeric phases availableOff-line SPE:The normal practice ofSPE where SPE cartridges, disks, pipettetips, and so forth are handled usingmanual processes (for example, vacuummanifolds or pipette transfer); oppositeto on-line SPE.On-column detection:The column it-self serves as the flow cell in HPLC, CE,or CEC. Generally the term used whenfused-silica capillaries are employed; theouter polyimide layer is removed and op-

tical beam is directed through the capil-lary; a measuring device (for example, a


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photomultiplier tube) is located on theopposite side of the capillary.On-column injection (OCI):In GC, re-fers to the process of injecting the entire

liquid sample directly onto the head ofthe column using a fine needle that willfit inside the capillary. Usually carriedout at an initial column temperatureless than the solvent boiling point, alsotermed cold on-column injection.On-line column switching:See multi-dimensional chromatography, on-line

SPE.On-line preconcentration:A precol-umn is placed in front of the separationcolumn to concentrate analytes beforetheir separation; different mechanismsmay be used (for example, hydrophobicinteraction, adsorption, or enzymatic re-action) to retain analytes as a function of

time and then by a displacement process(such as solvent elution or pH change)concentrated analytes are transferred tothe separation column.On-line SPE:Refers to the use of smallstainless steel cartridges packed withSPE packings placed across two portsof a 6- or 10-port injection or column-

switching valve. The SPE trap is loadedwith sample by an external pump orsyringe transfer and then the valve isswitched so that the SPE trap becomespart of the HPLC flow stream and ana-lytes can be swept into the column basedon the solvent being used for displace-ment. On-line SPE columns are usuallyused multiple times whereas off-line SPEcartridges are generally used once.Open-tubular column:Also termedcapillary columns, open-tubular col-umns for GC have the stationary phasecoated or chemically bonded on theinner walls or have support particles

deposited on the inner walls. Internaldiameters range from ~100 m up to

750 m. In HPLC, SFC, and capillaryelectrophoresis, small internal diameter(less than 100 m) columns are used.The most frequently used column mate-

rial is fused-silica tubing. Used very littlein routine HPLC or SFC but routinelyin CE and GC. Also termed capillarycolumn.Open-tubular column, packed:A cap-illary-dimension column that is packed

with stationary phase particles. Alsocalled micropacked columns, particularly

in GC.Optically active resin:Incorporationof optically active groups into an ion-exchange resin to allow separation ofoptically active isomers. Not many arecommercially available in HPLC.Organic modifier: Water-miscibleorganic solvent added to an aqueous

mobile phase to effect separations in re-versed-phase HPLC. Common organicmodifiers are acetonitrile, methanol, iso-propanol, and tetrahydrofuran.Orthogonality:Refers to two separationdimensions for which the elution timesin the two dimensions can be treatedas statistically independent; ideally, the

two dimensions should have totally dif-ferent retention mechanisms (for exam-ple, reversed phase and normal phase;ion exchange and reversed phase; polarand nonpolar)Overload:In preparative chromatog-raphy, the overload is defined as thesample mass injected onto the column

where efficiency and resolution beginsto be affected if the sample size is furtherincreased. See sample capacity.

P

Packing:The adsorbent, gel, or solid sup-port used in the chromatography column.

Most modern analytical HPLC packingsare less than 10 m in average diameter


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with 5 m currently the favorite. GC col-umn packings range in size from 6080mesh down to 100120 mesh.Paired ion chromatography:The same

as ion-pair chromatography.Paper filtration:Using porous filterpaper (mainly cellulose) to removeparticulates from liquid samples; pa-pers with different porosities are avail-able. Low porosity filters will removevery fine particulates but may have alower flow rate while high porosity

filters filter out larger particulates ata higher f low rate; paper filtration isoften used in wet chemistry to filter,combust, and then weigh insolublematerials; ashless filter paper is usedfor this purpose.Particle diameter (dp):Average diam-eter of the column packing particles.

Particle size distribution:A measure ofthe distribution of the particles used topack the LC column. In HPLC, a nar-row particle size distribution is desirable.

A particle size distribution of dp 10%would mean that 90% of the particlesfall between 9 and 11 m for a 10-maverage dppacking.Particle size reduction:The generalprocess of reducing larger particles downto a size that can be more convenientlyextracted; the smaller the particle themore quickly it will dissolve or if in-soluble the more quickly analytes canbe extracted for further sample cleanup.Typical methods for reducing particlesize include pulverizing, milling, ho-mogenizing, chopping, blending, andso on.Particulates:Generally refers to asmall particles found in the mobilephase that can cause back pressureproblems by lodging in frits; it can

also refer to the small particles packedinto HPLC columns. Particulates that

escape from the column exit may causedetector noise.Partition chromatography:Separa-tion process where one of two phases is

held stationary on a solid support or thecolumn inner wall (stationary phase orliquid phase) while the other is allowedto flow freely down the column (mo-bile phase or carrier gas). Solutes parti-tion themselves between the two phasesbased on their individual partition co-efficients. LLC is an example; modern

bonded-phase LC can be considered tobe a form of partition chromatography

where one of the liquid phases is actu-ally bonded to the solid support. Mech-anistically, partition chromatographyimplies that the solute becomes at leastpartially embedded within the station-ary phase, which is impregnated, coated,

or bonded to the substrate, in contrastto an adsorption process in which thesolute does not penetrate into the reten-tive surface or interphase.Partition coefficient (K):The ratio ofthe equilibrium concentration of solutein the stationary phase relative to theequilibrium concentration of solute in

the mobile phase. In GC, the relativeconcentration of solute in the mobileand stationary phases is a function ofkand : K= k. Also called distribu-tion coefficient, KD, and distributionconstant, Kc.Passive sampling:In passive gas sam-pling, an air sample is pulled through aflow controller into an evacuated canis-ter over a chosen period of time, rang-ing from 5 min to 24 h. The samplingperiod and the flow rate determine thecanister volume required.Peak:The profile of an analyte com-pound as it is eluted from a column

through a detector; usually depicted ona visual output on a recorder or printer


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based on the detectors electrical re-sponse.Peak area (Ap):The area measuredunder a chromatographic peak; usu-

ally measured by an integrator or datasystem; the peak area is related to theamount of substance eluted in a peak.Peak capacity (n): The number ofequally well resolved peaks that can be fitin a chromatogram between the hold-upvolume and some upper limit in retentionk

n. For R= 1, ncan be expressed by the

approximation n= 1 + N

/4 ln(1 + kn)where Nis the plate number and k

nis the

retention factor of peak n.Peak dispersion:See band broadening.Peak doublet:A split peak generallycaused by column void, poor injectiontechnique, or solvent flooding in GC.Split peaks also could be closely eluted

compounds.Peak height (hp):The maximum heightof a chromatographic peak as measuredfrom the baseline to the peak apex; thepeak height is related to the amount ofsubstance eluted in a peak.Peak overload:When too much of anyone solute is injected its peak may bedistorted into a triangular shape.Peak shape:Describes the profile of achromatography peak. Theory assumesa Gaussian peak shape (perfectly sym-metrical); peak asymmetry factor de-scribes shape as a ratio. See asymmetry.Peak tracking:A method of matchingof peaks that contain the same com-pound between different experimentalruns during method development; reliesupon detection parameters of each pureanalyte; diode-array detectors and massspectrometers are among the best detec-tors for peak tracking because of theirspecificity. Also refers to data-system

tracking of gradual changes in retentiontimes caused by stationary-phase loss or

other column degradation or drift in thechromatographic conditions.Peak variance (2):The second centralmoment of the peak about the retention

time. For a Gaussian peak the varianceis the fundamental parameter control-ling peak width. See Gaussian peak.Peak volume (Vp):The volume occu-pied by a chromatographic peak fromstarting basepoint to ending basepointas it passes through the detector: Vp =Fcwb

Peak width at base (wb):The widthof the chromatographic peak at thebaseline as eluted from the column. Itis measured at the baseline by drawingtangents to the inflection points on thesides of the Gaussian curve representingthe peak. Smaller peak widths usuallyrepresent efficient separations; also re-

ferred to as band width. It is sometimesconvenient to estimate the peak widthat base from the peak area and height:wb = 1.596Ap/hp(see Figure 2).Peak width at half-height (wh):Thewidth of the chromatographic peak athalf of the peak height above the base-line. Smaller peak widths usually repre-sent efficient separations; also referredto as band width. It is sometimes conve-nient to estimate the peak width at half-height from the peak area and height: wb= 0.94Ap/hp(see Figure 2).PEEK:Polyether ether ketone (PEEK) isa colorless organic polymer. It is used asa material for inert capillaries and fit-tings in HPLC and IC systemsPellicular:Seeporous layer bead.Percent B (%B):Refers to the strongersolvent in a binary solvent mixture; %Awould be the weaker solvent analog.Perfusion chromatography: Re-fers to chromatography performed

using part ic les with very largepores (for example, 40008000 )


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cal led throughpores (megapores orgigapores). Eluent flows throughthe particle as well as smaller in-terconnecting pores (for example,

3001000 ) called diffusive poresbetween the large pores. Best suitedfor the preparative separation of mac-romolecules.Permeability (Bo):Also referred to ascolumn permeabilityand specific perme-ability; a term expressing the resistanceof the packed column to the flow of

mobile phase. For a packed column: Bo= (dp

2/180) 3/(1 - )2 (dp2)/1012.For

an open-tubular column: Bo = dc2/32. A

column with high permeability gives alow pressure drop.Permeation:In SEC, refers to the pro-cess where a solute can enter a mobilephase filled pore of the packing.Phase collapse:Seephase dewetting.Phase dewetting: A term used inreversed-phase LC where very densebonded-phase coverage and a high per-centage of aqueous content in mobilephase can lead to expulsion of waterfrom the pores, which prevents thenormal partitioning process from tak-

ing place. Phase dewetting may occurwith as high as 10% organic contentin the mobile phase but can occur atlower %B values; results in earlier thannormal elution of analytes, poor peakshape, and nonreproducible retentiontimes.Phase ratio ():The relative amount ofstationary to mobile phase in the col-umn. In partition chromatography: =VS/VMwhere VSand VMare the volumeof stationary and mobile phase in thecolumn respectively. For open-tubularcolumns: rc/2df. Thicker station-ary phase films or higher phase loading

gives longer retention and higher peakcapacity.

Phenol extraction:A sample prepara-tion technique used for the isolation ofDNA from biological samples.Phenyl phase:A popular nonpolar

bonded phase prepared by the reac-tion of dimethylphenylchlorosilane oralkoxysilane with silica gel for LC, oras the components of a cross-linkedor bonded phase for GC. Claimed tohave affinity for aromatic-containingcompounds and does impart a differentselectivity compared to alkyl bonded

phases.Photoionization detection (PID):Thephotoionization detector ionizes solutemolecules with photons in the UV en-ergy range. PID is a selective detectionmethod that responds to aromatics andolefins when operated in the 10.2-eVphoton range. It can respond to other

materials with a more energetic lightsource.PIONA:Refers to the analysis of paraf-fins, isoparaffins, olefins, naphthenes,and aromatics.Pipette tip:Replaceable tips used inautomation of liquid handling chores;used once and discarded to avoid con-

tamination.Pirkle column:Chiral brush type sta-tionary phases based on 3,5-dinitroben-zoyl-phenylglycine silica that are used inthe separation of a wide variety of en-antiomers. Named after the developer,Dr. William Pirkle, University of Illnois.pK

a

:An acid dissociation constant,Ka, (also known as acidity constant, oracid-ionization constant) is a quantita-tive measure of the strength of an acidin solution. It is the equilibrium con-stant for a chemical reaction knownas dissociation in the context of acid-base reactions. The equilibrium can be

written symbolically as: HA

H

+

+A-where HA is a generic acid that


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dissociates by splitting into A, knownas the conjugate base of the acid, andthe hydrogen ion or proton, H+, which,in the case of aqueous solutions, ex-

ists as the hydronium ion in otherwords, a solvated proton. The disso-ciation constant is usually written as aquotient of the equilibrium concentra-tions (in mol/L), denoted by [HA], [A],and [H+]:Ka= ([H

+] [A])/[HA]; due tothe many orders of magnitude spannedby Kavalues, a logarithmic measure of

the acid dissociation constant is morecommonly used in practice. The loga-rithmic constant, pKa, which is equal to

log10Ka, is sometimes also (but incor-rectly) referred to as an acid dissociationconstant.Planar chromatography:A separationtechnique in which the stationary phase

is present as or on a plane (IUPAC).Typical forms are paper and thin layerchromatography.Plate height (H):See theoretical plateheight.Plate height, effective (Heff):Thecolumn length divided by the numberof effective theoretical plates: Heff = L/NeffPlate number (N): See theoretical

plate number.Polar:A polar molecule may be polaras a result of polar bonds or as a resultof an asymmetric arrangement of non-polar bonds and nonbonding pairs ofelectrons; polar molecules are generallyable to dissolve in water (H2O) becauseof the polar nature of water; polar mol-ecules do not prefer nonpolar organicsolvents such as hexane. Polar moleculeshave slightly positive and slightly nega-tively charged ends; we often refer to acompounds polarity.Polarity index (P):

The polarity indexis a measure of the relative polarity of a

solvent and is useful for identifying suit-able mobile phase solvents or extractionsolvents. The polarity index increases

with polarity; examples: hexane, P=

0.0; isopropanol, P= 3.9; tetrahydrofu-

ran, P= 4.0; methanol, P= 5.1; aceto-nitrile, P= 5.8; water, P= 9.0Polyacrylamide gel:Neutral hydro-philic polymeric packings used inaqueous SEC. They are prepared by thecopolymerization of acrylamide withN,N-methylene-bis-acrylamide.

Polyaromatic hydrocarbon (PAH):Members of a class of hydrocarbonmolecules characterized by one or morefused aromatic rings.Polychlorinated biphenyl (PCB):Bi-phenyl molecule with two or morechlorine substitutions.Polyethylene glycol (PEG):Polymeric

hydrocarbon used as a GC stationaryphase; possesses moderately polar reten-tion characteristics.Polyethyleneimine (PEI):Polyethyl-eneimine, an anionic polymeric phaseused to coat or bond onto silica or apolymeric packing. Most often used forthe separation of proteins and peptides.Polymeric packings:Packings basedon polymeric materials, usually in theform of spherical beads. Typical poly-mers used in LC as well as GC arepolystyrenedivinylbenzene (PS-DVB),polydivinybenzene, polyacrylamide,polymethylacrylate, polyethyleneoxide,polydextran, or polysaccharide.Polymeric phase:Refers to chemicallybonded phase where a polymer speciesis bonded to silica-based particles or tothe wall of an open-tubular column.Polymeric SPE:The use of a polymericbase material (for example, PS-DVB ormethacrylate) rather than an inorganic

material (for example, silica or alu-mina); polymers generally have a wider


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pH range and higher sample capacitythan some of the inorganic materials.Polystyrenedivinylbenzene (PS-

DVB) resin:The most common base

polymer for ion-exchange chromatog-raphy. Ionic groups are incorporatedby various chemical reactions. NeutralPS-DVB beads are used in reversed-phase LC. Porosity and mechanicalstability can be altered by variation ofthe crosslinking through the variationof the DVB con

the terminology guide for chromatographers

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