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26
Common HPLC Detectors
UV-VIS65%RILS
10%
Electro10%
FLD8%
MS
RadioactivityConductivityLC/IR
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34
UV-VIS Detectors
b
c
Detector Flow Cell
I0 I
Log I 0 = A = abcI
Principles : The fraction of light transmitted through the detector cell isrelated to the solute concentration according to Beer’s Law.
Characteristics : Specific, Concentration Sensitive, good stability,gradient capability.
Special : UV-VIS Spectral capability (Diode Array Technology ).
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UV-VIS Detector with Spectra Capability
• Diode Array UV-VIS Detector allows online measurement of spectra.Wavelength range 190 - 900 nm.
• Wavelength Resolution: Up to 1 nm.• Wavelength calibration with Holmium oxide filter.
Diode Array
GratingOptical
Slit
Detector Flow Cell
HomiumFilter
AchromaticLens
UVLamp
VisLamp
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Online Spectra - UV-VIS Detector
Wavelength
Time
AbsorbanceSpectra
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Peak Purity UV Spectra Based
Spectral differences at different points of peak elution Homogeneity of spectra in the peak
Peak with time marker for spectra selection
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Isoabsorbance Plot
The acquisition wavelengthcan be optimized based onstored spectra.
Wave
length
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Necessity for More Than One Detector -Sensitivity
PAH's extracted from soil;Sup.LC-PAH 150x4.6mm;Solv.: H2O/CH3OH= 10:90
Fluorescence
UV-signal
W L
2 4 1 / 3 9 4
W L
2 7 0 / 3 8 8
W L
2 4 8 / 4 1 1
W L
3 0 2 / 4 2 0
W L
2 4 7 / 5 0 4
P y r e n e
C h r y s e
n e
B e n z o
( e ) p y r e
n e
P e r y l e
n e
B e n z o
( k ) f l u o
r a n t h e n e
B e n z o
( a ) p y r e
n e
B e n z o
( g h i ) p e
r y l e n e
I n d e n o
( 1 2 3 - c
d ) p y r e n e
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Necessity for More Than One Detector -Selectivity
Flecainide inSerum
Therapeutic concentration: 1.8mg/l, 20ul injected
UV and fluorescence signal
FL signal
UV signal
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Necessity for More Than One Detector -Qualitative Information
Qualitative Information
Take peak spectrum(UV)
Chlortoluron
?
44
68
58
96 132 138158
172
215
200
Take peak spectrum(MS)
104
Mass/Charge
Atrazine
?
Wavelength (nm)60 80 100 120 140 160 180 200 220
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Principles of Fluorescence Detection
S1
S0
S0…. Basic energy level of electrons
S1…Excited state of electrons by external energy e.g. UV Light (low wavelength / excitation WL )
Emission Energy
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Excitation - Emission
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Fluorescence Detection
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LC-MS
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HPLC-MSD API- Electrospray
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10
Select Gradient Steepness
0 10 20 30 40min.
100% B
100% B
0% B
0% B
tG = 40
tG = 20
100% B
100% B% B
% B
G = 10
tG = 5
10
Steep
Shallow
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Gradient Shape
%B
time
Linear Step
Concave Convex
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he rst 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 and Applied Chemistry (IUPAC) publishedits massive undertaking titled “Nomen-clature for Chromatography,” which pro-vides guidance and changes in some of the more commonly accepted terms (2).This month’s “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 withliquid-phase analysis but instead have cho-sen to use terms that analysts may encounter 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 foundon theLCGC web site at http://www.chromatographyonline.com.
A A : Seeseparation factor.
A solvent: Usually the weaker solvent ina binary eluent or gradient elution separa-tion. In reversed-phase liquid chromatography (LC), the A solvent typically is water oa water-rich mixture.
A term: The rst term in the van
Deemter equation. Seeeddy dispersionterm andvan Deemter equation.Absorption: The process of retention in
which 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 by adding water or other polar modier 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, a competing base to negate the effects of silanols, a chelating agent to block metalsites, or a UV-absorbing compound to per-form indirect photometric detection.
Adjusted retention time ( t R ): A measureof the retention time adjusted for theholdup time;t R t R t M, wheret R is theretention time andt M is the holdup time(the time it takes for a small, unretained
compound that completely permeates thepores to be eluted from the chromato-graphic column).
Adjusted retention volume ( V R ): Adjusts the retention volume for theholdup volume;V R V R V M, whereV R is the retention volume of the peak of interest andV M is the holdup volume (thevolume corresponding to the total volumeof mobile phase in the column). See alsodead volume andholdup volume.
Adsorbent: Packing used in adsorption
chromatography. Silica gel and alumina arethe most frequently used adsorbents in
Ronald E. Majors and Peter W. Carr
This month’s “ColumnWatch” column is anextensive glossary ofdenitions and termsused in the liquid-phaseseparation techniques ofhigh performance liquid
chromatography,capillary electrophoresis,and capillaryelectrochromatography.The glossary should beuseful to those juststarting to use theseseparation techniquesand can serve as arefresher for long-timeusers. It provides some ofthe newer nomenclaturerecommended by theInternational Union ofPure and AppliedChemistry.
Glossary of Liquid-PhaseSeparation Terms
T
Ronald E. MajorsColumn Watch Editor
Column WatchColumn
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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 puried state. Affinity chromatography isnormally practiced as an on–off separationtechnique.
Agarose: High molecular weight polysac-charide used as a separation medium inbiochromatography. It is used in bead form,often in gel-ltration chromatography, withaqueous mobile phases.
Alkoxysilane: A reactant used for thepreparation of chemically bonded phases. Itwill react with silica gel as follows: R 3SiOR
SiOH → Si–OSiR 3 ROH, whereR is an alkyl group.
Alumina: A normal-phase adsorbent used
in adsorption chromatography. Aluminumoxide is a porous adsorbent that is availablewith a slightly basic surface; neutral andacidic modications 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 a weak anion exchanger, and it also is usedfor the separation of carbohydrates with a water–acetonitrile 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 be
analyzed 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 shape
of 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 driving force for adsorption, and any solute func-tional group that can interact with thisgroup can be retained on silica. The termadsorptionplaces 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 the
interaction 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 of the solute; for example, peak tailing, peak fronting, 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 biospecic adsorbent is preparedby coupling a specic 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 peak height. Other measures of asymmetry arecommonly used, especially theU.S. Phar-macopeia (USP ) method. See Figure 1. SeealsoFoley–Dorsey equation.
Asymmetry factor: A factor that denotesband shape. The asymmetry factor is calculated from the chromatographic peak by dropping a perpendicular at the peak apex and a horizontal line at 10% of the peak height; at the intersection, the distance tothe tail of the peak along the horizontal lin(distanceB ) divided by the distance along the horizontal line to the front of the peak (distance A) produces a ratio called thepeak asymmetry factor (see Figure 1). The
ratio is 1 for a symmetrical peak, less than1 for a fronting peak, and greater than 1 foa tailing peak. The higher the value, the lessymmetrical the peak; values greater than 2are unacceptable.
Atmosphere (atm): A measure of thepressure drop across an HPLC column; 1atm 14.7 lb/in.2 (psi). See alsobar and
pascals.
BB: See phase ratio.
B o: See permeability.B solvent: Usually the stronger solvent in
a binary eluent or gradient separation; typically the organic modier or modier-richbinary mixture with water in reversed-phasLC.
B term: The second term of the vanDeemter equation. See alsolongitudinal diffusion andmolecular diffusion term.
Backushing: A column-switching tech-nique in which a four-way valve placedbetween the injector and the column allowmobile-phase ow in either direction. Back
ushing 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 of increasing width and concomitant diluting of the chromatographic band as it movesdown the column. The peak is injected as anarrow slug and, ideally, each separated
component would be eluted as a narrow slug of pure compound if not for theprocess of band broadening. The measure
Figure 1: Example of a tailing peak. (Modi-ed with permission from reference 3.)
1.0
0.5
0.10.0
N o r m a
l i z e
d p e a
k h e
i g h t
32 36 40 44 48t 1 t p t 2
Time (s)
B A
w A Bh
p
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of band broadening is bandwidth (t w ) or,more correctly, the number of theoreticalplates (N ) in the column. Sometimescalled band dispersion or band spreading.See Figure 2.
Bandwidth ( t w): The width of the chro-matographic band during elution from thecolumn. It usually is measured at the base-line by drawing tangents to the inectionpoints 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 nitrogenadsorption–condensation in pores at liquidnitrogen temperature. Pore volume and
pore size distribution also can be obtainedfrom BET method calculations.Bidentate silane: A specic type of
bonded phase in which a short hydrocar-bon bridge connects two silicon atoms in a silane 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 a phase 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 using chemically bonded phases.
Bonded-phase concentration: Seecoverage.
Boxcar chromatography: Seecolumnswitching.
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 of acids and bases.
Buffer capacity: A quantitative measureof the potential of a buffer solution(dened as the number of equivalents of strong 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 pK a , andbuffer concentration.
CCC term: The interphase mass transfer
term of the van Deemter equation. See alsomass transfer andvan Deemter equation.
C8: See octylsilane.C18: Seeoctadecylsilane.C4, C8, C18, etc.: Refer to the alkyl-chain
length of a reversed bonded phase.
C S: SeeLangmuir isotherm.Capacity: Seesample capacity.Capacity factor ( k ): Old term for a
chromatographic parameter that measuresthe degree of retention. Now dened as theretention factor (k ) by the InternationalUnion of Pure and Applied Chemistry (IUPAC). See alsoretention factor formethod of calculation.
Capillary column: Refers to columnswith inner diameters less than 0.5 mm.
Capillary electrochromatography (CEC): A hybrid technique in which capillary columns are packed with chromatographicsorbents and electroosmotic ow 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 lledwith, or the walls coated or covalently bonded with, cross-linked polyacrylamideto simulate slab gel electrophoresis; this
polymer network uses a sieving mecha-nism; used for protein, carbohydrate, andDNA separations such as ngerprinting and sequencing.
Capillary isoelectric focusing : Separa-tion is based on isoelectric points of pro-teins; the capillary is lled with solution;the sample is introduced into the capillary in the presence of ampholytes; under theapplication of an electric eld, the proteinmigrates until it reaches a pH at which it isneutralized and maintains that position in
the capillary.Capillary LC: Generally refers to HPLCperformed in a fused-silica or other type o
Figure 2: Widths of a Gaussian peak at various heights as a function of the standard deviation( ) of the peak. (Modied 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
N o r m a
l i z e
d p e a
k h e
i g h t
w i 2
w b 4
3
4
5
w h 2.355
w i
w h
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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-m d p porous packing ina 15 cm 4.6 mm column should provide10,000–12,000 plates.
Column switching: Using multiplecolumns connected by switching valves fobetter chromatographic separations or sample 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 calledmultidi-mensional chromatography.
Column volume ( V c): The volume of theunpacked column;V c AcL, where Ac
andL are the cross-sectional area of thetube and the tube length, respectively.Competing base: Adding a small basic
compound such as triethylamine ordimethyloctylamine at 10–50 mM concen-tration to the mobile phase in reversed-phase chromatography to inhibit basic analytes from interacting with residualsilanols; works by the law of mass actionbecause concentration of competing base imuch greater than analyte. See alsoaddi-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 pellicular
packing.Counterion: The ion in solution used to
displace 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 a selector valve. Fractions from the rst 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 oplates.
Coverage: Refers to the amount of bonded phase on a silica support in
bonded-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 directow to the proper places. Most capillary tubing 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 asendcapping.Carrier: A term most often used in affin-
ity chromatography; refers to the supportthat binds the active ligand, usually by a
covalent bond; can also refer to the sup-port in other chromatography modes suchas liquid–liquid chromatography.
Carrier gas: The mobile phase in gaschromatography (GC).
Cartridge column: A column type thathas no endttings and is held in a cartridgeholder. The column comprises a tube andpacking contained by frits in each end of the tube. Cartridges are easy to change andare less expensive and more convenientthan conventional columns with endt-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:Seecapillary electrochromatogra-
phy.CGE:Seecapillary gel electrophoresis.CZE:Seecapillary zone electrophoresis.Chain length: The length of carbon
chain in the hydrocarbon portion of a reversed-phase packing. It is expressed asthe number of carbon atoms (C8, C18,etc.). It specically 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 more
rapidly than the average ow 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 a chemical 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 differently with two enantiomers leading to theirHPLC separation.
Chiral stationary phases: Stationary phases 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-cic interactions with one enantiomeric
form.Chlorosilane: A chemical reagent usedto prepare siloxane bonded phases; reactiv-ity changes from a monochlorosilanedichlorosilane 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.
Classication: The process of sizing col-umn packing particles; generally in HPLC,small particle-size distribution providesbetter efficiency and a greater permeability because of the absence of nes. Classica-tion can be performed by sedimentation,elutriation, and centrifugal air techniques.
Column back pressure: Seehead pres-sure.
Column chromatography: Any form of chromatography 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 capillary in CE used to perform the liquid-phaseseparation.
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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 partitioning mechanism.
Cross-linking: During the process of copolymerization 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 by the amount of the monomer added to thereaction. For example, divinylbenzene is a typical cross-linking agent for the produc-tion of polystyrene ion-exchange resins.The swelling and diffusion characteristicsof a resin are governed by its degree of
cross-linking.Cyclodextrins: Cyclic oligomers of sev-eralD-( )-glucopyranose units used in chi-ral HPLC and CE separations; popularones are named-, -, and -cyclodex-trins; they have a truncated cone shape, a relatively hydrophobic cavity, and primary and secondary hydroxyl groups at theirends; they separate on the basis of differen-tial inclusion of enantiomers; modiedcyclodextrins with derivatized hydroxylgroups also are used for selectivity modi-cation.
DDDead volume ( V M): The columndead
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 asV M. Thesystemdead volume includes theadditional volume in the tubing that con-nects the injector and detector to the col-umn. The system dead volume usually is
approximated by injecting a small, essen-tially unretained species. Uracil, acetoneand thiourea are most commonly usedspecies in reversed-phase chromatography.See alsoadjusted retention volume,holdup volume, andvoid volume.
DEAE:Seediethylaminoethyl.Degassing: The process of removing dis-
solved gas from the mobile phase before orduring use. Dissolved gas may come out of solution in the detector cell and causebaseline spikes and noise. Dissolved air can
affect detectors such as electrochemical (by reaction) or uorescence (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 vacuumask) or on-line evacuation from a tubemade of a gas-permeable substance such aspolytetrauoroethylene (PTFE).
Denaturing HPLC: Using reversed-phaseHPLC to investigate genetic mutations by the 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 packing material primarily used for low-pressure
biochromatography; 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 ( D M or D S): A fun-damental parameter of a molecule in gas,solution (D M), or the stationary phase(D S). Expressed in square centimeters persecond.D M 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 105 cm2/s.
Diol phase: A hydrophilic phase that isuseful in normal and reversed phase. It is a diol 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) ( K c):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,K c is used when the con-centration in the stationary phase isexpressed per unit volume of the phase(V R V M K cV S). In a solid stationary phase,K g is used and is expressed permass (weight) of the dry solid phase. Inadsorption chromatography with a well-characterized adsorbent of known surfacearea, the concentration in the stationary phase is expressed per unit surface area.
D M: Seediffusion coefficient.d p: See particle size.D S: Seediffusion coefficient.Dwell time: The time equivalent to
dwell volume; determined by the product
of ow rate and the dwell volume.Dwell volume: The volume between thepoint of mixing of solvents (usually in themixing chamber or at the proportioning valves 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 voumes 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 dynamicoating is to generate a new stationary phase 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 protonate
silanols and negate their effect. Anotherexample is coating the phase with a hydrophilic polymeric material to preventadsorption of proteins.
EEE: Seeseparation impedance.
: Seeinterparticle porosity.Eddy dispersion (diffusion) term ( ):
The A term in the van Deemter equation.It is the contribution to plate height fromthe heterogeneity in axial velocities as a
result of the particle size and geometry of the packing, as well as wall effects; A
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2 d p, where is an empirical column con-stant. Typical values offor well-packedcolumns are 0.8–1.0. Some theories of chromatography indicate a velocity-depen-dent contribution to the height equivalentto a theoretical plate (HETP) from thisprocess. Also known as eddy diffusion,ow-heterogeneity induced broadening,and the multipath term. See alsovanDeemter equation.
e: Seeinterstitial 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 ow cell in UV detection, thislength is shorter than the capillary length.
Effective plate height ( H eff ): The col-umn length divided by the effective platenumber.
Effective theoretical plates ( N eff ): Also
called the effective plate number by IUPAC. The true number of plates in a column, because it corrects theoreticalplates for dead volume.N eff 16[(t R /w b)2], wheret R is the adjustedretention time andw b is the bandwidth of the peak (see Figure 2). It is a better gureof merit than simple plate number forcomparing devices of very different geome-tries and phase ratios.
Efficiency ( N or H ): A measure typically determined by the number of theoreticalplates (N ) calculated from the equationN 16(V R /w b)2 16 (t R /w b)2, wherew bis the peak width measured at the base (seeFigure 2). If the peak width is measured athalf height, the following equation is used:N 5.545 (V R /w h)2. The plate height(H ) or HETP is determined byH L/N .The efficiency of asymmetric peaks is bet-ter determined from the peak centroid andvariance by mathematical analysis of thepeak shape. See alsoFoley–Dorsey equa-tion.
Effluent: The mobile phase leaving the
column; same aseluate.i: Seeintraparticle porosity.
Electroendosmotic ow: Seeelectro-osmotic ow.
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 ow ( v eo ): Bulk ow of
solvent within capillary caused by presenceof zeta potential (electric charge) at thecapillary walls and absence of ow resis-
symmetrical peak;V R Ft R , whereF isthe ow rate andt R is the retention time of the 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 separation of ion-exchange resins that requirea particularly narrow size range, such asamino acid resins. The technique involvesthe upward ow 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 silica gels sometimes are sized by elutriation
Endcapping: A technique used toremove silica gel silanol groups that may remain after reaction with a large silylatingagent such as octadecyltrichlorosilane. The
column is said to be endcapped when a small silylating reagent (such as trimethyl-chlorosilane or dichlorodimethylsilane) isused to bond residual silanol groups on a silica-gel–based 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.
Endtting: The tting at the end of thecolumn that permits connection to theinjector or detector. Most HPLC endt-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 containing immobilized enzyme on the inner wallis coupled with a CZE capillary; an enzy-matic reaction occurs with the injected
sample, and the products and unreactedsubstances enter the separation capillary;used to improve separations, detection, oranalyte preconcentration.
T: Seetotal porosity.Exchange capacity: Seeion-exchange
capacity.Excluded volume: Seeinterstitial vol-
ume.Exclusion chromatography: Seeion-
exclusion chromatography andsteric exclusion 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, electroosmoticow may be toward anode or cathode andcontributes to overall retention in CE tech-niques.
Electrophoresis: The movement of sam-ple ions under the inuence of an appliedvoltage.
Electrophoretic mobility ( ): 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 inversely proportional to solution viscosity and theion’s 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 liquid–solidor adsorption chromatography. In normal-phase chromatography, a nonpolar solventsuch as pentane would be at the low end of the 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 weak and acetonitrile strong. Thus, when devel-oping a method or running a gradient, aneluotropic series is useful for selecting sol-vents. See alsoSnyder o.
Elute: To chromatograph by elutionchromatography. The termelute is pre-ferred overdevelop,which was used inolder nomenclature.
Elution: The process of passing mobile
phase through the column to transportsolutes down a column.Elution chromatography: The most
commonly used chromatographic methodin which a sample is applied to the head of the column as a narrow zone and individ-ual analytes are separated and eluted fromthe end of the column. Compare withdis-
placement chromatography and frontal analysis.
Elution volume ( V R): Refers to the vol-ume of mobile phase necessary to elute a
solute from a column. It is the volumefrom the point of injection to the volumeat maximum concentration (apex) for a
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volume, called the exclusion volume. Many SEC packings are known by their exclusionlimit. For example, a 105 column of porous silica gel will exclude any com-pounds with a molecular weight greaterthan 100,000, based on a polystyrene cali-bration standard.
Exclusion volume ( V 0, V ei): 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 modied Gaussian peak: An asymmetric peak resulting from passing a 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 for
the Foley–Dorsey equations. See alsoFoley–Dorsey equation.Extracolumn effects: The total band
broadening 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, endttings, 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 theextracolumneffects.
FFF: See ow rate.
: See ow resistance parameter.Fast LC: Use of HPLC of short columns(1.5–7 cm) with conventional inner diam-
eters (2–6 mm) packed with small particles(3- or 5- m d p). Separation times in therange of minutes, or even seconds, arecommon.
Fast protein LC (FPLC): A termed coinedto cover the specic use of HPLC for sepa-rating proteins. Generally, glass columns,moderate pressure, and sphericalmicrobeads are used for FPLC.
Flash chromatography: A very fast form
of classic LC used by synthetic organicchemists for rapid purication. Performed
primarily in the normal-phase mode,sometimes with reversed-phase chromatog-raphy.
Flow rate ( F ): The volumetric rate of ow of a mobile phase through an LC col-umn. Typical ow rates are 1–2 mL/minfor a conventional 4.6-mm i.d. HPLCcolumn.
Flow resistance parameter ( ):
d p2/B o, whereB o is permeability. See also permeability.
Fluoro phase: One of a family of aliphatic and aromatic reversed-phasematerials in which a substantial fraction of the bonded phase is uorinated. Some-times called uorous phases or peruorophases. Typically these phases have differ-ent selectivities than hydrocarbon phases.
Foley–Dorsey equation: A correction of the plate count and retention time for peak tailing 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: Seecapillary zone electrophoresis.
Frit: The porous element at either end of a 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-tting. 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 rst-eluted compound, the third-least-sorbedcompound with the rst 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 as frontal analysis.
Fronting: Peak shape in which the frontpart of the peak (before the apex) in a chromatogram tapers in advance of theremainder of the peak; that is, the front isless steep than the rear. The peak has anasymmetric distribution with a leading edge. The asymmetry factor for a fronting peak 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 using poorly packed columns.
GG: The obstruction or tortuosity factor.
Molecular diffusing term. See alsotortuos-ity.
Gaussian curve: A standard error curve,based on a mathematical function, that is asymmetrical, bell-shaped band or peak.
Most chromatographic theory assumes a Gaussian 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 C max exp[ (t t R )2/2 2].
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 dened as a colloidal dispersioof a solid and liquid in which the solid isthe continuous phase.
Gel-ltration chromatography (GFC): Also called aqueous size-exclusion chro-matography. Performed with aqueousmobile phases. Generally refers to molecular size separation performed on soft gelssuch as polydextrans, but analysts also canuse highly cross-linked polymers, silica gels, and other porous media. Most gel-ltration separations involve biopolymers
and 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 aq
