CHROMATOGRAPHY - Murdercube Purification... · gas}gas chromatography does not exist and ... retention mechanism is governed by interfacial ad- ... greater afRnity.

GrifRth OM (1986) Techniques of Preparative, Zonal,and Continuous Flow Ultracentrifugation; DS-468H.Palo Alto, CA: Spinco Division of Beckman Instruments.

Hsu HW (1981) In: Perry ES (ed.) Techniques of Chem-istry, vol. XVI: Separations by Centrifugal Phenomena.New York: Wiley.

Lavanchy AC and Keith EW (1979) Centrifugal separation.In: Grayson M and Eckroth D (eds) Encyclopedia ofChemical Technology, 3rd edn, vol. 5, pp. 194}233.New York: J Wiley.

Letki A, Moll RT and Shapiro L (1997) Centrifugalseparation. In: Ruthven DM (ed.) Encyclopedia ofSeparation Technology, pp. 251}299. New York:J Wiley.

Price CA (1982) Centrifugation in Density Gradients. NewYork: Academic Press.

Sheeler P (1981) Centrifugation in Biology and MedicalScience. New York: J Wiley.

Svedberg T and Peterson KO (1940) The Ultracentrifuge.Oxford: Clarendon Press.

CHROMATOGRAPHY

C. F. Poole, Wayne State University, Detroit, MI,USA

Copyright^ 2000 Academic Press

Introduction

Chromatography is the most widely used separationtechnique in chemical laboratories, where it is used inanalysis, isolation and puriRcation, and it is com-monly used in the chemical process industry as a com-ponent of small and large-scale production. In termsof scale, at one extreme minute quantities of less thana nanogram are separated and identiRed during anal-ysis, while at the other, hundreds of kilograms ofmaterial per hour are processed into reRned products.It is the versatility of chromatography in its manyvariants that is behind its ubiquitous status in separ-ation science, coupled with simplicity of approachand a reasonably well-developed framework in whichthe different chromatographic techniques operate.

Chromatography is essentially a physical methodof separation in which the components of a mixtureare separated by their distribution between twophases; one of these phases in the form of a porousbed, bulk liquid, layer or Rlm is generally immobile(stationary phase), while the other is a Suid (mobilephase) that percolates through or over the stationaryphase. A separation results from repeated sorp-tion/desorption events during the movement of thesample components along the stationary phase in thegeneral direction of mobile-phase migration. Usefulseparations require an adequate difference in thestrength of the physical interactions for the samplecomponents in the two phases, combined with a fa-vourable contribution from system transport proper-ties that control sample movement within andbetween phases. Several key factors are responsible,therefore, or act together, to produce an acceptable

separation. Individual compounds are distinguishedby their ability to participate in common intermolecu-lar interactions in the two phases, which can gener-ally be characterized by an equilibrium constant, andis thus a property predicted from chemical thermo-dynamics. Interactions are mainly physical in type orinvolve weak chemical bonds, for example dipole}dipole, hydrogen bond formation, charge transfer,etc., and reversible, since useful separations only re-sult if the compound spends some time in bothphases. During transport through or over the station-ary phase, differential transport phenomena,such as diffusion and Sow anisotropy (complexphenomena discussed later), result in dispersion ofsolute molecules around an average value, such thatthey occupy a Rnite distance along the stationaryphase in the direction of migration. The extent ofdispersion restricts the capacity of the chromato-graphic system to separate and, independent offavourable thermodynamic contributions to the sep-aration, there is a Rnite number of dispersed zonesthat can be accommodated in the separation. Conse-quently, the optimization of a chromatographic sep-aration depends on achieving favourable kineticfeatures if success is to be obtained.

The Family of ChromatographicTechniques

A convenient classiRcation of the chromatographictechniques can be made in terms of the phases em-ployed for the separation (Figure 1), with a furthersubdivision possible by the distribution process em-ployed. In addition, for practical utility transportprocesses in at least one phase must be reasonablyfast; for example, solid}solid chromatography, whichmay occur over geological time spans, is impracticalin the laboratory because of the slow migration of

40 I / CHROMATOGRAPHY / Derivatization

Figure 1 Family tree of chromatographic methods.

solutes through the crystal lattice. Two distinctphases are required to set up the distribution compon-ent of the separation mechanism, which explains whygas}gas chromatography does not exist andliquid}liquid separations are restricted to immisciblesolvents. When the mobile phase is a gas the station-ary phase can be a liquid or a solid and the separationtechniques are called gas}liquid chromatography(GLC) and gas}solid chromatography (GSC). Thesimple term GC encompasses both techniques but,unless otherwise speciRed, it usually means GLC sincethis is the most common arrangement. Separations inGLC occur because of differences in gas}liquidpartitioning and interfacial adsorption. In GSC theretention mechanism is governed by interfacial ad-sorption or size exclusion, if a solid of controlled poresize, such as a zeolite, is used as the stationary phase.When the mobile phase is a supercritical Suid (SFC)the stationary phase can be a liquid or a solid, and thedistribution process may be interfacial adsorption orabsorption.

When the mobile phase is a liquid the stationaryphase can be a solid (liquid}solid chromatography,LSC) with interfacial adsorption as the dominantdistribution process; a solid of controlled pore size(size exclusion chromatography, SEC), in which thedistribution constant is characteristic of the ratio ofthe solute size to the dimensions of the stationaryphase pore sizes; a solid with immobilized ionicgroups accessible to solutes in the mobile phase withelectrostatic interactions as the dominant distributionprocess (ion exchange chromatography or ionchromatography, IEC or IC); a solid with immobi-lized molecular recognition sites accessible to theanalyte in the mobile phase (afRnity chromatog-raphy, AC) in which the dominant distribution pro-

cess is the three-dimensional speciRcity of the molecu-lar interactions between the receptor and the analyte(a technique used in biotechnology); a porous solidcoated with a Rlm of immiscible liquid (liquid}liquidchromatography, LLC) in which the dominant distri-bution process is partitioning; or a solid with a sur-face containing organic groups attached to it bychemical bonds (bonded-phase chromatography,BPC) in which the dominant distribution processesare interfacial adsorption and partitioning.

Bonded phases in liquid chromatography are wide-ly used to tailor solid phases for different ap-plications, including LSC, SEC, IEC, IC and AC(Figure 2). Reversed-phase chromatography (RPC) isa particular form of bonded-phase chromatographyin which the mobile phase is more polar than thestationary phase (for most practical applications themobile phase is an aqueous solution). It is the mostpopular form of liquid chromatography because of itsbroad applicability to neutral compounds of widepolarity. In addition, by exploiting secondary chem-ical equilibria weak acids and bases can be separatedby pH control (ion suppression chromatography,ISC); ionic compounds by using ion pairing with anadditive of opposite charge (ion pair chromatogra-phy, IPC); and metal ions by the formation of neutralcomplexes (metal-complexation chromatography,MCC). By adding a surfactant to the mobile phase,micelles can be used to modify the overall distributionconstant (micellar liquid chromatography, MLC),and a totally aqueous buffered mobile phase anda decreasing ionic strength gradient can be used toseparate biopolymers with minimal disruption ofconformational structure (hydrophobic interactionchromatography, HIC). Bonded-phase chemistry isalso commonly employed to prepare stationary

Sepsci*21*TSK*Venkatachala=BGI / CHROMATOGRAPHY 41

Figure 2 Applications of bonded phases in LC.

phases with immobilized enantiomer-selective groupsfor the resolution of racemates by chiral chromatog-raphy.

The mobile phase can be transported through orover the stationary phase by application of externalpressure when the stationary phase is enclosed ina rigid container, or column. This is the ordinarymode of gas, supercritical Suid and liquid chromato-graphy. If the stationary phase is distributed as a thinlayer on a (usually) Sat support, such as a sheet ofglass or plastic, and the mobile phase is allowed toascend through the layer by capillary forces, then thismethod is referred to as planar or thin-layerchromatography (TLC). The fundamental basis of thedistribution mechanism between the mobile phaseand the stationary phase is identical to that describedfor column liquid chromatography, only the separ-ation format and transport mechanism for the mobilephase are different. TLC has largely supersededpaper chromatography (PC) in contemporary prac-tice. PC is mechanistically identical to TLC but, witha few exceptions, provides poorer separation charac-teristics. Bulk Sow of liquid mobile phases containingan electrolyte can also be transported through a col-umn by an electric Reld, through the process knownas electroosmosis. When a column packed with a sta-tionary phase is used this is called electrochromato-graphy, or since columns of capillary dimensionsare essential for this technique, capillary electro-chromatography (CEC). The distribution process forneutral solutes is independent of the transport pro-cess, and separations occur by the mechanisms in-dicated for liquid chromatography. Ionic surfactantscan form micelles as a continuous phase dispersedthroughout a buffer. In an electric Reld thesecharged micelles move with a different velocityor direction to the Sow of bulk electrolyte. Neutral

solutes can be separated, if their distribution constantbetween the micelles and buffer are different,by micellar electrokinetic chromatography (MEKC).The stationary phase in this case is referred to asa pseudo-stationary phase, since it is not stationary,but moves with a different velocity to the mobilephase. Ionic solutes in CEC and MEKC are inSuencedby the presence of the electric Reld and are separatedby a combination of chromatography and elec-trophoresis.

Mode of Zone Displacement

In nearly all chromatographic systems, transport ofsolute zones occurs entirely in the mobile phase.Transport is an essential component of the chromato-graphic system since the most common arrangementfor the experiment employs a sample inlet and a de-tector at opposite ends of the column, with sampleintroduction and detection occurring in the mobilephase (GC, SFC, LC, MEKC). In planar chromato-graphic systems (TLC, PC), sample introduction anddetection is performed in the stationary phase, but thedetection is of solute zones that have been trans-ported different distances by the mobile phase. InGC the movement of solute molecules from the sta-tionary to the mobile phase is controlled by the va-pour pressure of the solutes in the column, and isusually manipulated by varying temperature. At anoptimum temperature sample molecules will spendsome of their time in the mobile phase, where theywill be transported through the column, and sometime in the stationary phase, where they are dif-ferentiated by their capacity for intermolecular inter-actions with the stationary phase. Displacement ofsolute zones can be achieved in three distinct ways:frontal analysis, elution and displacement (Figure 3).


Figure 3 Mode of zone displacement in chromatography.

In frontal analysis, the mobile phase introduces thesample continuously onto the column (or the sampleis the mobile phase) until eventually the column issaturated with the sample and the component withthe lowest afRnity for the stationary phase isdisplaced from the column by sample components ofgreater afRnity. When the zone of pure compon-ent has completely exited the column it is followed bya mixture containing the next component, and so on.Frontal analysis can be used to obtain thermodyn-amic data from chromatographic measurements andto isolate a less strongly retained trace componentfrom a major component. However, quantitation foreach component in a mixture is difRcult, and atthe end of the experiment the column is contaminatedby the sample so that reuse requires stripping thesample from the column.

In displacement chromatography the sample is ap-plied to the column as a discrete band and a substance(or mobile-phase component) with a higher afRn-ity for the stationary phase than any of the samplecomponents is continuously passed through the col-umn. The displacer pushes sample components downthe column and, if the column is long enough,a steady state is reached. A succession of rectangularbands of pure components then exits the column.Each component displaces the component ahead of it,with the last and most strongly retained component

being forced along by the displacer. At the end of theseparation the displacer must be stripped from thecolumn if the column is to be reused. Displacementchromatography is used mainly in preparative andprocess chromatography, where high throughputs ofpure compounds can be obtained (note that the con-tact boundary between zones may not be discrete andthe collection of pure compounds may be restricted tothe central region of the displaced zones).

In elution chromatography the sample is applied tothe column as a discrete band and sample compo-nents are successively eluted from the column dilutedby mobile phase. The stationary and mobile phasesare normally at equilibrium prior to sample introduc-tion. The mobile phase must compete with the sta-tionary phase for the sample components; separationwill only occur if the distribution constants for thevarious components, resulting from the competition,are different. Elution chromatography is the mostconvenient method for analysis and is the most com-mon method of separation in GC, SFC, LC andMEKC. Development, a modiRcation of the elutionmode, is used in planar chromatography. Samples areapplied to the dry layer as compact spots or bandsand the layer subsequently contacted by the mobilephase, which ascends and moves the sample compo-nents to positions higher up the layer in the directionof mobile-phase Sow. The separation is (usually)stopped before the mobile phase reaches the oppositeedge of the layer and neither the eluent nor the samplecomponents exit the layer. The two processes can becompared; all components travel the same distanceand are separated in time using the elution mode incolumn chromatography, whereas all componentshave the same separation time and are separated inspace (migration position) in planar chromatographyusing the development mode.

Chromatogram

The information obtained from a chromatographicexperiment is contained in the chromatogram. Whenthe elution mode is used this consists of a plot of theconcentration or mass proRle of the sample compo-nents as a function of the Sow of the mobile phase oras a function of time. Typically the y-axis will bedetector response and the x-axis time or volume ofmobile phase in column chromatography or migra-tion distance in planar chromatography. The positionof each peak in the chromatogram is characteristic ofthe identity of the compound and the area under thepeak is a function of the concentration or amount ofeach compound. Peak widths in the chromatogramare controlled by solute-dependent kinetic factors,which in turn can be used to deduce values for


Figure 4 Calculation of the RF value in planar chromatography.ZX distance moved by the sample from the sample origin; Z0,distance between the solvent entry position and the sample origin;Zf, distance between the solvent entry position and the solventfront.

characteristic physical properties of either the soluteor the mobile and stationary phases.

The position of a peak in the chromatogram ismade up of two contributions: (1) the time (or vol-ume of mobile phase) required by a compound thatdoes not interact with the stationary phase to reachthe detector from the sample inlet, called the columnhold-up time or dead time; and (2) the time thatindividual compounds spend in the stationary phase(all compounds spend the same time in the mobilephase). The column hold-up time is a feature of theexperimental system and is not fundamentally relatedto solute properties. Because of this, retention time isnot a useful parameter for column comparisons.A more useful term is the retention factor (previouslyknown as the capacity factor), k, deRned as the ratioof the time the solute spends in the stationary phase tothe time it spends in the mobile phase. The ratio ofthe retention factors for two solutes is called theseparation factor, �, which by convention is alwaysexpressed with the larger retention factor in the nu-merator (�51). The separation factor expresses theease with which the chromatographic system canseparate two compounds, and is directly related tothe difference in free energy for the interactionsof the two compounds in the chromatographic sys-tem. It is a major optimization parameter, as we shallsee later. In planar chromatography retention is usu-ally expressed as the retardation factor, RF, equiva-

lent to the ratio of the distance migrated by the solutezone, ZX, to the distance moved by the solvent front,Zf!Z0, measured from the sample application posi-tion, (15RF50), as illustrated in Figure 4. Theplanar chromatographic retardation factor and thecolumn retention factor are simply related by k"(1!RF)/RF.

Peak Shape Models

For an ideal separation the peaks in the chromato-gram are usually considered to be Gaussian. This isa convenient, if not always accurate, model and peakasymmetry can arise from a variety of instrumentaland chromatographic sources. The most commontypes of peak distortion are skewness (the peak frontis sharper than the rear) and tailing (the rear of thepeak is elongated compared to the front). Althoughinstrumental sources of peak asymmetry should, ofcourse, be minimized, chromatographic sources can-not always be avoided. Curve Rtting by computeroffers the possibility of deconvoluting chromato-graphic peak proRles into their individual contribu-tions. The exponentially modiRed Gaussian function,obtained by the combination of a Gaussian functionwith an exponential decay function (that provides forthe asymmetry in the peak proRle), is often an accept-able description of chromatographic peaks in analyti-cal applications.

Chromatographic sources of peak asymmetry re-sult from mechanical effects, for example theformation of voids in the stationary-phase bed andexcessive extra-column volumes, and from isothermcharacteristics. Most of the theory of analyticalchromatographic separations is based on a linear iso-therm model where the compositions in the station-ary and mobile phases are proportional and charac-terized by a distribution constant that is independentof sample size and composition (Figure 5). The peaksresulting from a linear chromatography model aresymmetrical and can be characterized by a normaldistribution. The width of the chromatographic zoneis proportional to retention and can be obtained dir-ectly from peak shape considerations. The extent towhich the properties of the chromatographic systemcontribute to zone broadening (peak widths) is givenby the number of theoretical plates, N. For a normaldistribution this is equivalent to (tR/�t)

2 , where tR isthe retention time and �t is the peak standard devi-ation in time units. Simple algebraic manipulation ofthis formula permits calculation of N from the peakwidth at base or half-height, etc. For column com-parison purposes the height equivalent to a theoret-ical plate, H, equivalent to the column length dividedby N, is generally used.


Figure 5 Influence of isotherm type on peak shapes.

Nonlinear isotherms (nonlinear chromatography)result in the production of asymmetric peaks. Lang-muir isotherms are frequently observed for adsorp-tion interactions on surfaces with an energeticallyheterogeneous distribution of adsorption sites withincompatible association/dissociation rate constants.For sorbents with monolayer coverage, Langmuir-type isotherms result when solute}stationary phaseinteractions are strong compared with solute}soluteinteractions. Because the interactions between solutesare comparatively weak, the extent of sorption de-creases following monolayer formation, even thoughthe concentration in the mobile phase is increasing. Inthis case the concentration of the component in thestationary phase at equilibrium is no longer propor-tional to its concentration in the mobile phase and thepeak shape and retention time will depend on thesample composition and amount. Anti-Langmuirtype isotherms are more common in partition systemswhen solute}stationary phase interactions are rela-tively weak compared with solute}solute interac-tions, or where column overload results from theintroduction of large sample amounts. Such conditionsare common in preparative chromatography, whereeconomic considerations dictate that separations areoptimized for production rate and to minimize mo-bile phase consumption and operating costs.

Flow through Porous Media

For an understanding of zone dispersion in chromato-graphy, an appreciation of the mobile-phase linearvelocity through different porous media is im-

portant. Gases are highly compressible and an aver-age linear velocity for the column is used. Liquids canbe considered incompressible and the average andoutlet velocity should be about the same. Supercriti-cal Suids are often assumed to be incompressible forthe purpose of calculation, more for convenience thanreality, with local velocity changes reSecting changesin density along the column. For packed columnscontaining porous particles with Suid mobile phases,the Sow of mobile phase occurs predominantlythrough the interstitial spaces between the packingparticles and the mobile phase occupying the particlepore volume is largely stagnant. Slow solute dif-fusion through this stagnant volume of mobile phaseis a signiRcant cause of zone broadening for con-densed phases. The mobile-phase velocity for achromatographic system may be determined by divid-ing the column length by the retention time of anunretained and unexcluded solute from the pore vol-ume (average velocity) or the retention time of anunretained and excluded solute (interstitial velocity).

The mobile-phase Sow proRle and changes in localvelocity are products of the driving force used toinduce bulk Sow of mobile phase through theseparation system. These driving forces can beidentiRed as capillary, pneumatic or electroosmoticforces. Capillary forces are responsible for thetransport of the mobile phase in planar chromato-graphy (PC and TLC). These forces are generallyweak and result in a mobile-phase velocity thatdecreases with migration distance from the solventstarting position (Figure 6). Capillary forces areincapable of providing a sufRciently high velocity


Figure 6 Relationship between mobile-phase velocity and mi-gration distance for capillary-controlled and forced-flow develop-ment in planar chromatography. (Reproduced with permissionfrom Poole CF and Wilson ID (1997) Journal of PlanarChromatography 10: 332, copyright ^ Research Institute forMedicinal Plants).

Figure 7 Flow profile for an open tube and a packed column using pneumatic and electroosmotic driving forces.

to minimize zone broadening. This has a numberof consequences: zone brodening is largely domin-ated by diffusion; the useful development lengthfor PC is set by the range of acceptable mobile-phase velocities; separation times are increased;and the separation potential of PC is less than thatpredicted for a constant and optimum mobile-phasevelocity.

Pneumatic transport of the mobile phase is com-monly employed in column chromatography. Themobile phase is pressurized externally to the column(a simple high pressure cylinder with regulator inthe case of a gas, or a mechanical pump for liquids).The pressure gradient across the column provides thedriving force to overcome the resistance to Sow pre-

sented by the stationary phase and the rest of thesystem. In LC, Darcy’s law relates the properties ofthe mobile phase, characteristic features of the col-umn, and the external pressure required to obtaina useful mobile-phase velocity. This law can be statedas:

u"�PK0d2P/�L [1]

where u is the mobile phase velocity, �P is the pres-sure drop across the column, K0 is the column per-meability, dP is the average particle diameter, � is themobile phase viscosity, and L is the column length.Since a minimum value for u is required for accept-able column performance and separation times, andthe available column pressure drop is constrained toan upper limit by material and safety considerations,then there is a Rnite limit to the range of permissibled2

P/L values that can be used. Thus a compromisemust be accepted between separation time and ef-Rciency, which results in an upper limit to the numberof theoretical plates that can be obtained for fastseparations or the use of long separation times whenvery large numbers of theoretical plates are requiredfor a separation.

Bulk liquid Sow under electrophoretic conditionsoccurs by electroosmosis. At the column wall or par-ticle surface (packed columns) an electrical doublelayer results from the adsorption of ions from themobile phase or dissociation of surface functionalgroups. An excess of counterions is present in thedouble layer in comparison with the bulk liquid andin the presence of an electric Reld shearing of thesolution occurs only within the very thin diffusepart of the double layer, transporting the mobilephase through the column with a nearly perfect plugproRle (Figure 7). The velocity of the bulk liquid Sow


Figure 8 Representation of flow anisotropy in a packed col-umn.

Figure 9 Representation of resistance to mass transfer in themobile and stationary phases. The dashed line represents theequilibrium position and the solid line the actual position ofthe solute zones.

is given by:

u"��E/4�� [2]

where � is the solution dielectric constant, � is the zetapotential (potential at the boundary between thecharged surface and the start of the diffuse partof the double layer), and E is the electric Reldstrength. Note that there is no explicit dependence onthe particle size and column length, which limit thetotal efRciency of columns when the Sow is pneu-matically driven. The column length and column in-ternal diameter, however, cannot be treated as inde-pendent variables in MEKC and CEC, but are relatedthrough Joule heating of the electrolyte and its ef-fect on the mobile-phase Sow proRle. Heat is gener-ated homogeneously throughout the electrolyte butthe temperature variation across the column diameteris parabolic. Radial temperature gradients betweenthe centre of the tube and the column wall causezone broadening resulting from sample diffusionand solvent density and viscosity differences inthe direction of Sow.

Zone Broadening

Rate theory attempts to explain the kinetic contribu-tion to zone broadening in column chromatographyas the sum of three main contributions: Sow ani-sotropy (eddy diffusion), axial diffusion (longi-tudinal diffusion), and resistance to mass trans-fer. Flow anisotropy is illustrated in Figure 8. Whena sample band migrates through a packed bed, theindividual Sow paths must diverge to navigatearound the particles such that individual Sow streamsare of unequal lengths. These variations in Sowdirection and rate lead to zone broadening thatshould depend only on the particle size and homogen-eity of the column packing. Flow anisotropy can be

minimized by using particles of small diameter witha narrow particle size distribution in columns witha high and homogeneous packing density. For open-tubular columns, Sow anisotropy is not a contribu-ting factor since the streamlines have no obstacles intheir way to cause disruption of the sample proRle.

Axial diffusion is the natural tendency of sol-ute molecules in the mobile phase to redistributethemselves by diffusion from a region of high con-centration to one of lower concentration. Its contri-bution to zone broadening depends on the solutediffusion coefRcient in the mobile phase andthe column residence time. Diffusion of solutemolecules occurs in all directions but only the compo-nents in the plane of mobile-phase migration contrib-utes to the peak proRle observed in the chromatogram.

Resistance to mass transfer in either the stationaryor mobile phases is a consequence of the fact thatmass transfer in the chromatographic system is notinstantaneous and equilibrium may not be achievedunder normal separation conditions. Consequently,the solute concentration proRle in the stationaryphase is always slightly behind the equilibrium posi-tion and the mobile-phase proRle is similarly slightlyin advance of the equilibrium position (Figure 9). The


Figure 10 van Deemter plot of the column plate height as a function of the mobile-phase velocity. The solid line represents theexperimental results and the broken lines the theoretical contribution from flow anisotropy (A), axial diffusion (B/u) and resistance tomass transfer (Cu).

resultant peak observed at the column exit isbroadened about its zone centre, which is locatedwhere it would have been for instantaneous equilib-rium, provided that the degree of nonequilibrium issmall. Contributions from resistance to mass transferare rather complicated but depend on the columnresidence time, mobile-phase velocity, stationary-phase Rlm thickness, the particle size for packedcolumns, the solute diffusion coefRcients inthe mobile and stationary phases, and the columninternal diameter.

The relationship between zone broadening (col-umn plate height) and the mobile-phase velocity isgiven by the hyperbolic plot known as a van Deemtercurve (Figure 10). The solid line represents the ex-perimentally observed results and the dotted lines thecontributions from Sow anisotropy (A term), axialdiffusion (B/u) and resistance to mass transfer(Cu). In this generic plot we see that there is anoptimum velocity at which a particular chromato-graphic system provides maximum efRciency (aminimum column plate height). The position of thisoptimum velocity and the general curvature of theplot strongly depend on the characteristics of thechromatographic system, as shown by the valuesgiven in Table 1.

Gas Chromatography

Gases of low viscosity with favourable solute dif-fusivity, such as hydrogen and helium, are commonlyused as mobile phases in GC. For these gases the

minimum in the plate height occurs at a high opti-mum mobile-phase velocity, resulting in efRcientand fast separations. At these high mobile-phase vel-ocities the contribution from axial diffusion tothe column plate height is minimized. For thin-Rlmcolumns, resistance to mass transfer in the mobilephase is the main cause of zone broadening, while forthick-Rlm columns resistance to mass transfer in thestationary phase is equally important. Since dif-fusion in gases is relatively favourable, the columninternal diameters required to maintain an acceptablecontribution from resistance to mass transfer in themobile phase offer little difRculty in practice.For supercritical Suids, solute diffusivity is not asfavourable as for gases and in the case of liquids mustbe considered unfavourable. The unfavourable slowoptimum mobile-phase velocity in SFC (in practiceopen-tubular columns are operated at 10 or moretimes the optimum velocity to obtain an acceptableseparation time) requires signiRcantly smaller inter-nal diameter capillary columns than those needed forGC to minimize resistance to mass transfer in themobile phase. At mobile-phase velocities used in prac-tice the contribution of axial diffusion to the col-umn plate height is negligible compared with thecontribution of resistance to mass transfer in the mo-bile and stationary phases. For fast, high efRciencyseparations, column internal diameters (100 �m arerequired and much smaller diameters are preferred.As densities and solute diffusivity become moreliquid-like, column dimensions for reasonable


Table 1 Characteristic values for column parameters related to zone broadening

Parameter Mobile phase

Gas Supercritical fluid Liquid

Diffusion coefficient (m2 s�1) 10�1 10�4}10�3 10�5

Density (g cm�3) 10�3 0.3}0.8 1Viscosity (P) 10�4 10�4}10�3 10�2

Column length (m)Packed 1}5 0.1}1 0.05}1Open-tubular 10}100 5}25

Column internal diameter (mm)Packed 2}4 0.3}5 0.3}5Open-tubular 0.1}0.7 0.02}0.1 (0.01

Average particle diameter (�m) 100}200 3}20 3}10Column inlet pressure (atm) (10 (600 (400Optimum velocity (cm s�1)

Packed 5}15 0.4}0.8 0.1}0.3Open-tubular 10}100 0.1}0.5

Minimum plate height (mm)Packed 0.5}2 0.1}0.6 0.06}0.30Open-tubular 0.03}0.8 0.01}0.05 '0.02

Typical system efficiency (N)Packed 103}104 104}8�104 5�103}5�104

Open-tubular 104}106 104}105

Phase ratioPacked 4}200Open-tubular 15}500

performance start to approach values similar to thosefor LC and are not easily attained experimentally.Slow diffusion in liquids means that axial dif-fusion is generally insigniRcant but mass transferin the mobile phase is also reduced, requiring col-umns of very small internal diameter, preferably(10 �m, which are impractical for general laborat-ory use. Packed columns dominate the practice of LCwhile open-tubular columns are equally dominant inthe practice of GC, with both column types usedin SFC.

Packed columns in GC are prepared from compar-atively coarse particles of a narrow size distributionand coated with a thin homogeneous Rlm of liquid forhigh performance. The relatively large particle sizeand short column lengths are dictated by the limitedpressure drop employed for column operation. Forthin-Rlm columns, resistance to mass transfer in themobile and stationary phases is the main cause ofzone broadening with a contribution from Sow ani-sotropy. For thick-Rlm columns, resistance to masstransfer in the stationary phase tends to dominate.The intrinsic efRciencies of open-tubular columnsand packed columns of similar phase ratio arecomparable, but because the two column types dif-fer greatly in their relative permeability at a Rxedcolumn pressure drop, much longer open-tubular col-umns can be used. Thus, packed GC columns are

seldom more than 5 m long while columns withlengths from 10 to 100 m are commonly usedin open-tubular column GC, resulting in a 100-foldincrease in the total number of theoretical platesavailable. In general, packed columns are usedin GC for those applications that are not easilyperformed by open-tubular columns, for exampleseparations that require a large amount of stationaryphase for the analysis of very volatile mixtures, orwhere stationary phases are incompatible with col-umn fabrication, preparative and process-scale GC,etc.

Liquid Chromatography

The intrinsic efRciency per unit length of packedcolumns in LC increases as the particle diameter isreduced. It can also be increased by using solvents oflow viscosity, which result in smaller contributions tothe column plate height from resistance to mass trans-fer and Sow anisotropy. Operation at low mobile-phase velocities compared to GC further minimizesthe contributions from resistance to mass transfer inthe mobile phase at the expense of longer separationtimes. The pressure drop required to maintain a con-stant mobile-phase velocity is proportional to theratio of the column length to the particle diametersquared. Since the available operating pressure isRnite, the column length must be reduced as the


Table 2 Achievable theoretical plate numbers in HPLC andCEC

Particle size HPLC CEC(�m)

Length Plates/ Length Plates/(cm) column (cm) column

5 5 55 000 50 115 0003 25 45 000 50 170 0001.5 10 30 000 50 250 000

Column pressure drop"400 atm for HPLC and the field strength(30 kV in CEC for operation at the minimum point in the vanDeemter plot.

particle diameter is decreased. Consequently, mostseparations in LC are performed with a total of about5000}20000 theoretical plates that is largely inde-pendent of the particle size. However, since the reten-tion time at a constant (optimum) mobile-phase velo-city is proportional to the column length, this arbit-rary Rxed number of plates is made available ina shorter time for shorter columns packed with small-er diameter particles. Thus the principal virtue ofusing particles of a small diameter is that they permita reduction in the separation time for those separ-ations that do not require a large number of theoret-ical plates.

Conventional column diameters in analytical LC at3}5 mm are comparatively large so as to minimizezone broadening from extracolumn effects inearlier instrument designs and have become the defacto standard dimensions, even though instrumentcapabilities have improved over time. Smallerdiameter columns have been explored to reducemobile-phase consumption (which is proportionalto the square of the column radius) and to enhancemass detection through reduction in peak volumes,but offer no improvement in the intrinsic columnefRciency, except perhaps for columns with alow column diameter-to-particle size ratio. Capillarycolumns of 0.1 to 0.5 mm internal diameterpacked with 3}10 �m particles can be used inrelatively long lengths for the separation of complexmixtures, where a large number of theoreticalplates is required. Such columns probably minimizethe contribution form Sow anisotropy while at thesame time providing a better mechanism for the dissi-pation of heat caused by the viscous drag of themobile phase moving through the packed bed. Theoperation of these columns is still pressure-limitedand separation times an order of magnitude greaterthan for GC have to be accepted as the price for highefRciency.

The enhancement of intraparticular mass transportis particularly important for the rapid separation ofbiopolymers, whose diffusion coefRcients areperhaps 100-fold smaller than those of low molecularweight compounds in typical mobile phases used inLC. Also, the high surface area porous packings usedfor small molecules may be too retentive for bio-polymers with a signiRcant capacity for multisite in-teractions. For these compounds short columnspacked with 1.5 and 2 �m pellicular or porous par-ticles are used for fast separations. Longer columnscontaining perfusive particles of a large size withlarge diameter through-pores to promote convectivetransport can also be used for fast separations. Per-fusive particles are also used for the preparative-scaleseparation of biopolymers.

Supercritical Fluid Chromatography

In SFC, mobile-phase modiRcation of the stationaryphase and its dependence on Suid density, togetherwith the variation of Suid density along the length ofthe column, result in additional sources of zonebroadening that cannot be treated in an exact way.Packed columns used in SFC are identical in type tothose used in LC. When separations can be achievedwith a modest number of theoretical plates (up toabout 80 000), then packed columns provide muchfaster separations, perhaps up to an order of magni-tude, than open-tubular columns, which are generallypreferred when very large numbers of theoreticalplates are required.

Systems with Electroosmotic Flow

Plug Sow in CEC results in a smaller contribution tothe plate height from Sow anisotropy and transaxialdiffusion compared with pressure-driven columnliquid chromatography, while contributions to theplate height that are Sow-proRle-independent are thesame. The absence of a pressure drop in electroos-motically driven systems provides the necessary con-ditions to achieve a larger total number of theoreticalplates in CEC in a reasonable time through the use ofsmaller particles and longer columns (see Table 2 andFigure 11). Under normal operating conditions CECcolumns have the potential to provide column platenumbers 5}10 times higher than LC columns. Ulti-mately the performance in CEC is limited by Jouleheating, which causes additional zone broadeningand restricts applications of CEC to the use of micro-columns, since columns with a small internal dia-meter ((100 �m) are required for efRcient heatdissipation. The dominant cause of zonebroadening in MEKC is axial diffusion, with signi-Rcant contributions from slow sorption}desorptionkinetics between the analyte and micelles and elec-trophoretic dispersion arising from the polydispersity


Figure 11 Separation of aromatic compounds by CEC ona 50 cm�50 �m i.d. fused silica capillary column packed with1.5 �m spherical octadecylsiloxane-bonded silica gel with 70%(v/v) acetonitrile buffer as mobile phase, temperature 253C, andfield strength 30 kV.

Figure 12 Variation of the average plate height as a function of the solvent front migration distance for conventional and highperformance silica gel layers with capillary-controlled and forced-flow development. (Reproduced with permission from Poole CF andPoole SK (1997) Journal of Chromatography A 703: 573, copyright ^ Elsevier Science B.V.)

of micelle sizes. Resistance to mass transfer in themobile phase is minimized by the capillary dimen-sions of the column and the small size and homogene-ous distribution of the micelles throughout the mobilephase combined with the near-perfect plug Sow of themobile phase. Thermal dispersion, as described forCEC, is an additional potential source of zone

broadening resulting from radial temperature gradi-ents. Separations in MEKC are typically carried outwith between 100 000 and 500 000 theoreticalplates. Adsorption of solutes on the column wall cangreatly reduce the potential column efRciencyand experimental conditions should be optimized tominimize these contributions whenever possible.

Planar Chromatography

The consequence of the suboptimal mobile-phasevelocity in planar chromatography obtained by capil-lary-controlled Sow is that zone broadening is domin-ated by diffusion. Since the mobile-phase velocityvaries approximately quadratically with migrationdistance, solutes are forced to migrate through regionsof different local efRciency and the plate heightfor the layer must be expressed by an average value(Figure 12). Each solute in the chromatogram experi-ences only those theoretical plates over which it mi-grates, with solutes close to the sample applicationpoint experiencing very few theoretical plates andthose close to the solvent front experiencing up to anupper limit of about 5000. High performance layers,with a nominal average particle size of about 5 �m,provide more compact zones than coarser particles,provided that the solvent front migration distancedoes not exceed about 5}6 cm; beyond this pointzone broadening exceeds the rate of zone centreseparation. When the development length is opti-mized the separation performance of conventionallayers (average particle size about 10 �m) is not very


Figure 13 Plot of the reduced plate height (H/dP) against thereduced mobile-phase velocity (udP/DM) for a high performanceand a conventional TLC layer using forced-flow developmentsuperimposed on a curve for an ideal LC column. (Reproducedwith permission from Fernando WPN and Poole CF (1991) Jour-nal of Planar Chromatography 4: 278, copyright ^ ResearchInstitute for Medicinal Plants.)

Figure 14 Separation of polycyclic aromatic hydrocarbons byforced-flow TLC with online detection (elution mode). A silica gelhigh performance layer, migration distance 18 cm, with hexane asthe mobile phase (0.07 cm s�1) was used for the separation.(Reproduced with permission from Poole CF and Poole SK (1994)Analytical Chemistry 66: 27A, copyright ^ American ChemicalSociety).

different from that of the high performancelayers; the primary virtue of the latter is that a shortermigration distance is required to achieve a given ef-Rciency, resulting in faster separations and more com-pact zones that are easier to detect by scanning den-sitometry. The minimum in the average plate heightunder capillary-controlled conditions is alwaysgreater than the minimum observed for forced-Sowdevelopment, indicating that under capillary-control-led Sow conditions the optimum potential perfor-mance is currently never realized in full. Under for-ced-Sow conditions the minimum in the plate heightis both higher and moved to a lower velocitycompared with values anticipated for a column in LC,(Figure 13). Also, at increasing values of the mobile-phase velocity, the plate height for the layer increasesmore rapidly than is observed for a column. At thehigher mobile-phase velocities obtainable by forced-Sow development, resistance to mass transfer is anorder of magnitude more signiRcant for layers thanfor columns. The large value for resistance to masstransfer for the layers may be due to restricted dif-fusion within the porous particles or is a product ofheterogeneous kinetic sorption on the sorbent and thebinder added to layers to stabilize their structure. Theconsequences for forced-Sow TLC are that separ-ations will be slower than for columns and fast separ-ations at high Sow rates will be much less efR-

cient than for columns, although in terms of totalefRciency and separation speed the possibilitiesfor forced-Sow development are signiRcantly betterthan those of capillary-controlled separations(Figure 14).

Separation Quality

The general object of a chromatographic separationis to obtain an acceptable separation (resolution)


Figure 15 Influence of the separation factor (�) and the reten-tion factor (k) on the resolution of two closely eluting peaks incolumn chromatography. (Reproduced with permission fromPoole CF and Poole SK (1991) Chromatography Today, p. 31,copyright ^ Elsevier Science B.V.)

between all components of interest in a mixture with-in the shortest possible time. The resolution betweentwo peaks in a chromatogram depends on how wellthe peak maxima are separated and how wide the twopeaks are. This can be expressed numerically by theratio of the separation of the two peak maximadivided by the average peak widths at their base.Baseline separation of the peaks is achieved at a res-olution of about 1.5 but a value of 1.0, representingabout 94% peak separation, is taken as an adequategoal for components that are difRcult to separate.Resolution is also simply related to the properties ofthe chromatographic system. For this purpose it isconvenient to consider a simple model of a three-component mixture in which the optimum columnlength is dictated by the number of theoretical platesrequired to separate the two components that aremost difRcult to separate, and the total separationtime is dictated by the time required for the last peakto elute from the column. The resolution of the twopeaks that are most difRcult to separate is thenrelated to the column variables by:

RS"(�N/2)�[(�!1)/(�#1)]�kAV/(1#kAV) [3]

where kAV is the average value of the retention factorfor the two peaks, or in an approximate form by:

RS"(�N/4)�[(�!1)/�]�k2/(1#k2) [4]

for peaks with approximately equal base widths inwhich the elution order of the peaks is k2'k1.

Column Chromatography

To a reasonable approximation, the three contribu-tions to resolution (efRciency, selectivity andtime) can be treated independently and optimizedseparately. Resolution increases only as the squareroot of N, so although the inSuence of efRciencyis the most predictable parameter in the resolutionequation, it is also the most limited. In practice allseparations have to be made in the range N"103}106

(Table 1). For GC this full range is available, so thatincreasing the column length or, better, reducing thecolumn internal diameter of an open-tubular columnat a constant length (separation time is proportionalto column length), is often an effective strategy.For LC only a modest number of theoretical platescan be obtained in a reasonable time. In this case thegeneral approach is to use the maximum availablevalue for N and optimize resolution by changing theother variables. SFC is an intermediate case in whichthe general strategy depends on whether the Suid ismore gas-like or liquid-like.

The separation factor determines the ability of thechromatographic system to differentiate betweenthe two components based on the difference intheir thermodynamic interactions with the mobileand stationary phases. When �"1 a separation isimpossible but, as can be seen from Figure 15, onlya small increase in � above unity is required to im-prove resolution considerably. At comparativelylarge values of �, resolution is little inSuenced byfurther changes; indeed, separations in which �'2are easy to achieve. Selectivity optimization is thegeneral approach to improve resolution in LC, wherea wide range of mobile and stationary phases areavailable to choose from and a wide range of dif-ferent retention mechanisms can be employed. Em-pirical or statistically based experimental approachesto selectivity optimization are often used because ofa lack of formal knowledge of exact retention mech-anisms for computer-aided calculations. Althoughpowerful, selectivity optimization in LC can bea time-consuming process. The ease of achievinga separation by selectivity optimization can be


Table 3 Factors affecting resolution in column chromatorgraphy

Value of N needed forRS"1 at k"3 for differentvalues of �

Value of N needed for RS"1 atdifferent k values for �"1.05 and1.10

� N k �"1.05 �"1.10

1.005 1 150 000 0.1 853 780 234 2601.01 290 000 0.2 254 020 69 7001.02 74 000 0.5 63 500 17 4201.05 12 500 1.0 28 220 7 7401.10 3 400 2.0 15 880 4 3601.20 1 020 5.0 10 160 2 7901.50 260 10.0 8 540 2 3402.00 110 20.0 7 780 2 130

Figure 16 Separation of aromatic compounds by MEKC usinga 65 cm (effective length 50 cm)�50 �m i.d. fused silica capillaryand a mobile phase containing 30 mmol L�1 sodium dodecylsulfate and 50 mmol L�1 sodium phosphate/100 mmol L�1 so-dium borate buffer (pH"7) at a field strength of 15 kV. (Repro-duced with permission from Terabe S (1989) Trends in AnalyticalChemistry 8: 129, copyright ^ Elsevier Science B.V.)

illustrated by the data in Table 3, which indicate thenumber of theoretical plates required for a separ-ation. These data can be compared to the data inTable 1, which indicates the number of theoreticalplates available for different chromatographicsystems. This is a clear indication of the need forselectivity optimization in LC and SFC, and the morerelaxed constraints for GC.

Resolution will initially increase rapidly with reten-tion, starting at k"0, as shown in Figure 15. By thetime k reaches a value around 5, further increases inretention result in only small changes in resolution.The optimum resolution range for most separationsoccurs for k between 2 and 10. Higher values ofk result in long separation times with little concomi-tant improvement in resolution, but they may benecessary to provide sufRcient separation spaceto contain all the peaks in the chromatogram.

The separation time is given by:

tR"(H/u)�16R2S�[�/(�!1)2]�(k2#1)3/k2

2) [5]

If the separation time (tR) is to be minimized, then theacceptable resolution should not be set too high(RS"1); the separation factor should be maximizedfor the most difRcult pair to separate; the reten-tion factor should be minimized (k"1}5) for themost difRcult pair to separate; and the columnshould be operated at the minimum value for theplate height corresponding to the optimum mobile-phase velocity.

Micellar Electrokinetic Chromatography

The resolution equation for MEKC is identical toeqns [3] and [4] but contains an additional term,(tM/tMC)/[1#(tM/tMC)k1], to account for the limitedelution range (all solutes must elute between the re-tention time of an unretained solute, tM, and a solute

totally retained by the micelles, tMC; see Figure 16).The intrinsic efRciency of MEKC is much higherthan column liquid chromatography, and optimiza-tion of the separation factor depends on a differ-ent set of parameters (changing surfactant type,use of additives, etc). Large values of the retentionfactor are unfavourable for obtaining high resolutionsince the additional term added to the resolutionequation tends to zero at high k values. The optimumvalue of k for maximum resolution is around 0.8}5,corresponding to (tM/tMC)1/2. The retention factor isusually optimized by changing the surfactant concen-tration.


For a single development under capillary-controlledSow conditions the TLC analogue of the generalresolution equation for column chromatography canbe expressed in approximate form as:

RS"[(N1RF2)1/2/4]�[(k1/k2)!1)]�(1!RF2) [6]

where N1 is the maximum number of theoreticalplates available corresponding to the solventfront position. The use of N1RF2 is only a rough


Figure 17 Variation of the resolution of two closely migratingzones as a function of the RF value for the faster moving zone.(Reproduced with permission from Poole CF and Poole SK (1991)Chromatography Today, p. 669, copyright ^ Elsevier ScienceB.V.)

Figure 18 Temperature programmed separation of fragrance compounds by GC on a 30 m�0.25 mm i.d. fused silica open-tubularcolumn coated with DB-1, film thickness 0.25 �m, helium carrier gas 25 cm s�1 and temperature program 403C (1 min isothermal) then40}2903C at 53C min�1. (Reproduced with permission from J&W, copyright ^ J&W Scientific Inc.)

approximation for the number of theoretical platesthat a particular zone has migrated across. Relativelysmall changes in selectivity have enormous impact onthe ease of obtaining a given separation in TLC, sincethe total number of theoretical plates available fora separation is never very large. Separations in TLCare fairly easy when RF2!RF1'0.1 and very dif-Rcult or impossible for RF2!RF140.05 in the regionof the optimum RF value for the separation. Max-imum resolution is obtained at an RF value of about0.3 and does not change much in the RF range of 0.2to 0.5, as can be seen in Figure 17. Resolution is zerofor compounds that are retained at the origin ormigrate with the solvent front.

General Elution Problem

Constant separation conditions, for example isother-mal operation in GC and isocratic elution in LC,are unsuitable for separating samples containingcomponents with a wide retention range. Employingaverage separation conditions will result in a poorseparation of early-eluting peaks, poor detectabilityof late-eluting peaks, and excessively long separationtimes. In GC there is an approximately exponentialrelationship between retention time and soluteboiling point under isothermal conditions. For mix-tures with a boiling point range 'c. 1003C it isimpossible to identify a compromise temperature thatwill provide an acceptable separation. The solution inthis case is to use temperature programming, Sowprogramming, or both. Temperature programming isthe most common and usually involves a continuouslinear increase in temperature with time, althoughother programme proRles are possible, including seg-mented programmes incorporating isothermal peri-ods. The reduction in separation time, increase inpeak capacity, and nearly constant peak widths ob-tained are illustrated by the separation in Figure 18.The general elution problem in LC is solved usingsolvent-strength gradients. Here, the composition ofthe mobile phase is changed as a function of time.Binary or ternary solvent mixtures are commonlyused as the mobile phase in which the relative com-position of the strong solvent (that solvent with thecapability of reducing retention the most) is increasedover time. In SFC it is usual to programme the den-sity, mobile-phase composition or temperature asa single factor, but it is also possible for some combi-nation of parameters to be changed simultaneously.The goal remains the same, as indicated by the


Figure 19 Separation of Triton X-114 by SFC using pro-grammed elution on a 10 cm�2 mm i.d. column packed with3 �m octadecylsiloxane-bondedsilica gel at 1703C with UV detec-tion. (A) Carbon dioxide/methanol (2#0.125) mL min�1 at210 bar; (B) as for (A) with pressure programmed form 130 to375 bar over 8 min; and (C) using a mobile-phase compositiongradient from 0.025 to 0.4 mL min�1 methanol over 8 min at 210bar. (Reproduced with permission from Giorgetti A, Pericles N,Widmer HM, Anton K and Datwyler P (1989) Journal of Chromato-graphic Science 27: 318, copyright � Preston Publications, Inc.)

Figure 20 Separation of the 3,5-dinitrobenzoyl esters ofpoly(ethylene glycol) 400 by (A) a single conventional develop-ment and (B) by incremental multiple development with a step-wise gradient of methanol, acetonitrile and dichloromethane over15 developments (Reproduced with permission from Poole CF,Poole SK and Belay MT (1993) Journal of Planar Chromatogra-phy 6: 438, copyright � Research Institute for Medicinal Plants.)

density- and composition-programmed separation ofoligomers in Figure 19.

Solvent-strength gradients in TLC are usually dis-continuous and achieved through the use of uni-dimensional multiple development. This is accom-panied by zone refocusing resulting in a larger zonecapacity and easier-to-detect separated zones. All uni-dimensional multiple development techniques em-ploy successive repeated development of the layer inthe same direction with removal of the mobile phasebetween developments. Each time the solvent fronttraverses the sample zone it compresses the zone inthe direction of development because the mobilephase contacts the bottom edge of the sample zoneRrst where the sample molecules then start to moveforward before those molecules ahead of the solventfront. Once the solvent front has reached beyond thezone, the refocused zone migrates and is broadenedby diffusion in the usual way. When optimized, itis possible to migrate a zone a considerable distancewithout signiRcant zone broadening beyond that ob-served for the Rrst development. If the solvent com-position is varied for all, or some, of the developmentsteps during multiple development, then solventstrength gradients of different shapes can be pro-duced. With increasing solvent-strength gradients it isusually necessary to scan the separation at a numberof intermediate development steps corresponding tothe development at which different componentsof interest are separated, since in later developmentsthese zones may be merged again because ofthe limited zone capacity in TLC. Alternatively,


Figure 21 Separation of the major deoxyribonucleosides andtheir 5�-monophosphate esters by multidimensional LC-LC. Thefirst column is a strong anion exchange column and the seconda reversed-phase column. The unseparated nucleosides, A, areswitched to the second column after which the 5�-monophosphateesters, B to D are separated on the IEC column and the parentdeoxyribonucleosides, E to H, are separated on the RPC column.(Reproduced with permission from Sagliano N, Hsu SH, FloydTR, Raglione TV and Hartwick RA (1985) Journal of Chromato-graphic Science 23: 238, copyright � Preston Publication, Inc.)

incremental multiple development can be used witha decreasing solvent-strength gradient. In this case,the Rrst development distance is the shortest andemploys the strongest solvent composition, whilesubsequent developments are longer and employmobile-phase compositions of decreasing solventstrength. The Rnal development step is the longestand usually corresponds to the maximum useful de-velopment length for the layer and employs theweakest mobile phase. In this way sample compo-nents migrate in each development until the strengthof the mobile phase declines to a level at which someof the sample zones are immobile, while less retainedzones continue to be separated in subsequent devel-opment steps, affording the separation of themixture as a single chromatogram (Figure 20). In-cremental multiple development with a decreasingsolvent-strength gradient is easily automated.

Multidimensional and MultimodalChromatography

The analysis of complex mixtures requires a verylarge peak capacity since the probability of peakoverlap increases with the number of compoundsrequiring separation. Multidimensional and multi-modal chromatographic systems provide a betterroute to achieving high peak capacities than ispossible with single-column systems. The necessarycharacteristic of these systems is that the dominantretention mechanism should be different for eachdimension. Other uses of multidimensional andmultimodal chromatography include trace enrich-ment, matrix simpliRcation, increased samplethroughput, and as an alternative to gradient elutionin LC.

Multidimensional column chromatography in-volves the separation of a sample by using two ormore columns in series where the individual columnsdiffer in their capacity and/or selectivity. Multi-modal separations involve two or more chromato-graphic methods in series, for example, the onlinecoupling of LC and GC (LC-GC) or SFC and GC(SFC-GC). Both methods involve the transfer of thewhole or part of the eluent from the Rrst column toanother via some suitable interface. The function ofthe interface is to ensure compatibility in terms ofSow, solvent strength and column capacity. The de-sign requirements and ease of coupling differsigniRcantly for the different chromatographicmodes. Coupling GC-GC, SFC-GC, SFC-SFC, LC-LC, LC-GC and LC-TLC are routine and other com-binations such as SFC-TLC, SFC-LC and GC-TLChave been described in the literature. Trace enrich-

ment and sample clean-up on short pre-columns isRnding increasing use in the automated determina-tions of drugs in biological Suids and crop protectionagents in water by LC-LC. Figure 21 illustrates theseparation of a mixture of deoxyribonucleosides andtheir 5�-monophosphate esters using LC-LC with ananion exchange column and a reversed-phase columnconnected in series by a microvolume valve interface.The neutral deoxyribonucleosides are switched asa single peak for separation on the reversed-phasecolumn while the phosphate esters are resolved by theanion exchange column. The separation time remainsacceptable since both separations are performed al-most simultaneously. TLC-TLC is commonly calledtwo-dimensional TLC and is a widely used qualitativemethod of analysis. It is very easily performed byplacing a sample at the corner of the layer and devel-oping the plate in the normal way, evaporating thesolvent, turning the plate through a right angle anddeveloping the plate a second time at 903 to the Rrstdevelopment. If adequately optimized this is a very


powerful separation method, but more frequentlythan not, solvents of different composition areused for the two developments employing retentionmechanisms that differ in intensity rather thankind, and the zones are only dispersed around thediagonal between the two development directionsand not uniformly over the whole layer.

Mode Selection

Chromatography provides many different ap-proaches for the separation of mixtures. There aremany instances where the same mixture can be ad-equately separated by more than one approach. Inthis section we will take a mechanistic look at howsolutes are separated by the common chromato-graphic techniques to provide some guidelines formethod suitability.

If the only consideration were efRciency andspeed, then GC would be the preferred technique. Inpractice, GC is restricted to thermally stable com-pounds with a signiRcant vapour pressure at the tem-perature required for their separation. The uppertemperature limit for common GC stationary phasesis 200}4003C. Few compounds with a molecularweight greater than 1000 Da have sufRcient va-pour pressure to be separated in this temperaturerange, and many low molecular weight compoundsare known to be labile at temperatures required fortheir vaporization. Derivatization techniques extendthe scope of GC to otherwise labile compounds byforming thermally stable derivatives, often with in-creased volatility, and by tagging compounds withspeciRc groups that simplify trace analysis using oneof the selective and sensitive group or element-selec-tive detectors available for GC.

Under typical conditions the mobile phase in GCbehaves essentially as an ideal gas and does not con-tribute to selectivity. To vary selectivity either thetemperature is changed or a new stationary phase(column) is employed for the separation. Temper-ature and separation time are closely connected inGC. The range over which temperature can be variedis usually short and will likely provide only a smallchange in selectivity, but because of the large numberof theoretical plates available for a separation in GC,this may be sufRcient to provide adequate resolu-tion. Provided that stationary phases that differin their relative capacity for intermolecular interac-tions are selected, then larger changes in selectivitycan be anticipated by stationary-phase optimization.In modern column technology the most versatilegroup of stationary phases are the poly(siloxanes),which can be represented by the basic structure}(R2SiO)n}, in which the type and relative amount of

individual substituents can be varied to create thedesired variation in selectivity (R"methyl, phenyl,3,3,3-triSuoropropyl, cyanoethyl, Suorine-contain-ing alcohol, etc.) Special phases in which R containsa chiral centre or a liquid-crystalline unit are used toseparate enantiomers and geometric isomers. Othercommon stationary phase include hydrocarbons,poly(phenyl ethers), poly(esters) and poly(ethyleneglycols), although many of these phases are restrictedto packed column applications because of difR-culties in either coating or immobilizing them on thewalls of fused-silica capillaries, favoured for themanufacture of open-tubular columns. The solvationparameter model provides a reliable systematized ap-proach for selectivity optimization and the predictionof retention in GLC. For GSC the stationary phase isusually silica, alumina, graphitized carbon, organicpolymer or zeolite porous particles (packed columns);or a thin layer dispersed over the inner surface ofa capillary column with an open passageway downthe centre (porous layer open-tubular column, orPLOT column). These materials are used to separateinorganic gases, volatile halocarbon compounds, lowmolecular weight hydrocarbons and, in particular,geometric and isotopic isomers.

LC and GC should be considered as complement-ary techniques. Since the only sample requirement forLC is that the sample has reasonable solubility insome solvent suitable for the separation, and sinceseparations by LC are commonly carried out close toroom temperature, thermal stability is not generallyan issue. The large number of separation mechanismseasily exploited in the liquid phase provides a highlevel of Sexibility for selectivity optimization. In gen-eral, many applications of LC can be categorized asthose for which GC is unsuited and includes applica-tions to high molecular weight synthetic polymers,biopolymers, ionic compounds and many thermallylabile compounds of chemical interest.

Mode selection within LC is quite complicatedbecause of the number of possible separation mech-anisms that can be exploited, as illustrated in Fig-ure 22. Preliminary information on the molecularweight range of the sample, relative solubility in or-ganic solvents and water, and whether or not thesample is ionic, can be used as a starting point toarrive at a suitable retention mechanism for a separ-ation. The molecular weight cutoff at 2000 in-dicated in Figure 22 is quite arbitrary and reSects thefact that size exclusion packings are readily availablefor the separation of higher molecular weight solutes,although size exclusion is not used exclusively toseparate high molecular weight compounds becauseof its limited peak capacity. Wide-pore packingmaterials allow polymers with a molecular weight


Figure 22 Selection of the separation mechanism in LC based on the criteria of sample molecular weight, solubility and conductivity.(Reproduced with permission from Poole CF and Poole SK (1991) Chromatography Today, p. 455, copyright ^ Elsevier Science B.V.)

exceeding 2000 to be separated by conventional sorp-tion and ion exchange mechanisms.

Liquid}solid chromatography (LSC) is character-ized by the use of an inorganic oxide or chemicallybonded stationary phase with polar functional groupsand a nonaqueous mobile phase consisting of one ormore polar organic solvents diluted to the desiredsolvent strength with a weak solvent, such as hexane.A characteristic of these systems is the formation ofan adsorbed layer of mobile-phase molecules at thesurface of the stationary phase with a compositionthat is related to the mobile-phase composition butgenerally not identical to it. Retention is essentiallydetermined by the balance of interactions the soluteexperiences in the mobile phase and its competitionwith mobile-phase molecules for adsorption sites at

the surface of the stationary phase. The position andtype of polar functional groups and their availabilityfor interaction with discrete immobile adsorptionsites is responsible for selectivity differences whensilica or alumina are used as stationary phases. Theability of LSC to separate geometric isomers has beenattributed to the lock}key type steric Rtting of solutemolecules with the discrete adsorption sites on thesilica surface.

Reversed-phase liquid chromatography (RPC) ischaracterized by the use of a stationary phase that isless polar than the mobile phase. A chemicallybonded sorbent or a porous polymer could be used asthis stationary phase, while for most practical ap-plications the mobile phase contains water as one ofits major components. RPC is ideally suited to the


separation of polar molecules that are either insolublein organic solvents or bind too strongly to inorganicoxide adsorbents for normal elution. RPC employingacidic, low ionic strength eluents is a widely estab-lished technique for the puriRcation and characteriza-tion of biopolymers. Other favourable attributes in-clude the possibility of simultaneous separation ofneutral and ionic solutes; rapid equilibrium betweenphases facilitating the use of gradient elution; and themanipulation of secondary chemical equilibria in themobile phase (e.g. ion suppression, ion pair forma-tion, metal complexation and micelle formation) tooptimize separation selectivity in addition to vari-ation in solvent type and composition of the mobilephase. A large number of chemically bonded station-ary phases of different chain length, polarity andbonding density are available to complement mobile-phase optimization strategies. About 70% of all sep-arations performed in modern LC are by RPC, whichgives an indication of its Sexibility, applicability andease of use. The main driving force for retention inRPC is solute size because of the high cohesive energyof the mobile phase compared to the stationaryphase, with solute polar interactions, particularly sol-ute hydrogen bond basicity, reducing retention. TheseRndings strongly reSect the properties of water,which is the most cohesive of the solvents normallyused in LC, as well as a strong hydrogen bond acid.

Ion exchange chromatography (IEC) is used for theseparation of ions or substances easily ionized bymanipulation of pH. Stationary phases are character-ized as weak or strong ion exchangers based on theextent of ionization of the immobile ionic centres,and as anion or cation exchangers based on thecharge type associated with the ionic centres. Thus,sulfonic acid groups are strong, and carboxylic acidgroups are weak, cation exchangers. Most of themetal cations in the Periodic Table have been separ-ated by IEC with acids or complexing agents as elu-ents. In clinical laboratories ion exchange has longbeen employed as the basis for the routine, automatedseparation of amino acids and other physiologicallyimportant amines involved in metabolic disordersand to sequence the structure of biopolymers. Soft,nondenaturing, ion exchange gels are widely used inthe large-scale isolation, puriRcation and separationof peptides, proteins, nucleosides and other biologicalpolymers. Metal-loaded ion exchangers and anionexchange chromatography of complexed carbohy-drates are well-established separation techniques incarbohydrate chemistry. The combination of pellicu-lar ion exchange columns of low capacity, low con-centration eluents with a high afRnity for the ionexchange packing, and universal, online detectionwith a Sow-through conductivity detector revolution-

ized the analysis of inorganic and organic ions inindustrial and environmental laboratories. As well aselectrostatic interactions, retention in IEC is in-Suenced by hydrophobic sorptive interactionsbetween the sample and stationary phase similarto those in RPC, and size and ionic exclusioneffects. Resolution is optimized by choice of themobile-phase counterion, the ionic strength, pH,temperature, Sow rate, and addition of organicmodiRers.

In size exclusion chromatography (SEC) retentiondifferences are controlled by the extent to whichsample components can diffuse through the porestructure of the stationary phase, as indicated by theratio of sample molecular dimensions to the distribu-tion of stationary-phase pore size diameters. Since noseparation will result under conditions where thesample is completely excluded from the pore volumeor can completely permeate the pore volume, the zonecapacity of SEC is small compared with that of theother LC techniques. The separation time is predict-able for all separations, corresponding (ideally) toa volume of eluent equivalent to the column voidvolume. No solvent optimization beyond Rndinga solvent for the sample that is compatible with thestationary phase is required. For synthetic polymersthis can result in the use of exotic solvents and hightemperatures. SEC is a powerful exploratory methodfor the separation of unknown samples, since it pro-vides an overall view of sample composition withina predictable time, and is also commonly employed insample fractionation to isolate components belongingto a deRned molecular size range. Analytical separ-ations employ small particles of rigid, polymeric orsilica-based gels of controlled pore size to separatesamples of different molecular size and to obtainaverage molecular weights and molecular weight dis-tribution information for polymers.

Fundamentally the retention mechanisms for LCand TLC are identical. TLC is selected over LC whenadvantage can be taken of the attributes of employinga planar format for the separation. Examples includewhen a large number of samples requiring minimumsample preparation are to be separated, when post-chromatographic reactions are usually required fordetection, or if sample integrity is in question. The useof a disposable stationary phase for TLC allowssample clean-up and separation to be performed si-multaneously. Reasons for preferring LC over TLCare its greater separation capacity for mixtures con-taining more components than can be adequatelyresolved by TLC; a wider range of stationary phasesare available for methods development; a wider selec-tion of detection techniques exist; and automation forunattended operation is more straightforward.


The retention mechanism for MEKC stronglyresembles that of RPC with two importantdifferences. Surfactants used to generate thepseudo-stationary phases provide a different typeof sorption environment to solvated chemicallybonded phases and, therefore, different selectiv-ity. The intrinsic efRciency of MEKC is signiRcantlygreater than that of LC and enhances resolution,although the peak capacity is lower owing to theRnite migration window for MEKC. A signiRcantnumber of RPC-type applications are now performedby MEKC, indicating that the method can competefavourably with RPC for some separations. MEKC isinherently a microcolumn technique, providing ad-vantages in coupling to other chromatographic sys-tems and for the analysis of samples only available insmall amounts. Disadvantages include sample intro-duction problems, limited dynamic sample concen-tration range, and poor limits of detection for traceanalysis (because of the very small sample sizes in-volved). Selectivity optimization is determined largelyby the choice of surfactant and the use of mobile- andstationary-phase additives.

Supercritical Suids have solvating properties thatare intermediate between those of gases and liquids.In addition, supercritical Suids are compressible sothat their density and solvating power can be variedby changing external parameters, such as pressureand temperature. This feature is unique to supercriti-cal Suids and represents a major approach to selectiv-ity optimization. Temperature not only affectsdensity, but may also inSuence the vapour pressure oflow molecular weight solutes, promoting some GC-like character to the retention mechanism. The mostcommon mobile phase is carbon dioxide, a relativelynonpolar Suid. More-polar Suids, such as water, am-monia or methanol, tend to have unfavourable criti-cal constants or are highly corrosive to column orinstrument components, limiting their use. Mixedmobile phases can be used to vary selectivity, such ascarbon dioxide}methanol mixtures, but miscibilityproblems and high critical constants for the mixedmobile phases may restrict the range of propertiesavailable. SFC can provide faster separations thanLC, but it is more restricted than LC in the choice ofmobile phases and retention mechanisms to vary sel-ectivity. SFC is compatible with most detection op-tions available for both GC and LC. All practicalapplications of SFC occur signiRcantly above ambienttemperature, which is unsuitable for the separation ofsome thermally labile compounds and most bio-polymers. Supercritical Suids such as carbon dioxideare unable to mask active sites on typical columnpackings, resulting in unsatisfactory separations ofpolar compounds owing to adsorption, which pro-

duces unacceptable peak shapes and poor resolution.However, SFC Rnds applications in many areaswhere GC and LC are unsatisfactory, for examplein the separation of middle molecular weightcompounds, low molecular weight synthetic poly-mers, fats and oils, enantiomers, and organometalliccompounds.

Instrumentation

Modern chromatographic methods are instrumentaltechniques in which the optimal conditions for theseparation are set and varied by electromechanicaldevices controlled by a computer external to the col-umn or layer. Separations are largely automated withimportant features of the instrumentation being con-trol of the Sow and composition of the mobile phase,provision of an inlet system for sample introduction,column temperature control, online detection tomonitor the separation, and display and archiving ofthe results. Instrument requirements differ signif-icantly according to the needs of the method em-ployed. Unattended operation is usually possible byautomated sample storage or preparation devices fortime-sequenced sample introduction.

Gas Chromatography

For GC a supply of gases in the form of pressurizedcylinders is required for the carrier gas and perhapsalso for the detector, for operating pneumatic valves,and for providing automatic cool-down by openingthe oven door. To minimize contamination, high pu-rity gases are used combined with additional puriRca-tion devices. Each cylinder is Rtted with a two-stagepressure regulator for coarse pressure and Sow con-trol. Fine tuning is achieved using metering valves orby electronic pressure control combining electro-mechanical devices with sensors to compensate auto-matically for changes in ambient conditions. The col-umn oven is generally a forced air circulation thermo-stat heated resistively and capable of maintaininga constant temperature or of being programmed overtime. The detector and sample inlet are generallythermostated separately in insulated metal blocksheated by cartridge heaters. The most commonmethod of introducing samples into a GC inlet is bymeans of a microsyringe (pyrolysis, headspace andthermal desorption devices can be considered special-ized sample inlets). For packed-column injectiona small portion of (liquid) sample is introducedby microsyringe through a silicone septum intoa glass liner or the front portion of the column,which is heated and continuously swept by carriergas. The low sample capacity and carrier gas Sow


rates characteristic of narrow-bore open-tubular col-umns require more sophisticated sample-introductiontechniques based on sample splitting or solvent elim-ination and refocusing mechanisms.

The principal methods of detection are varied, con-veniently grouped under the headings of gas-phaseionization devices, bulk physical property detectors,optical detection and electrochemical devices. FurtherclassiRcation is possible based on the nature of thedetector response } universal, selective or speciRc.The Same ionization detector and thermal conductiv-ity detector are examples of (near) universal de-tectors; the Same photometric detector, thermionicionization detector and atomic emission detectorare element-selective detectors; and the photoioniz-ation detector and electron capture detector are struc-ture-selective detectors. GC coupling to massspectrometry and IR spectroscopy is straightforwardand widely utilized for automated structure iden-tiRcation as well as detection. Detection in thegas phase is a favourable process and GC detectorsare among the most sensitive and versatile byvirtue of the range of mechanisms that can beexploited.

Liquid Chromatography

Modern LC employs columns with small particlesizes and high packing density requiring high pres-sures for operation at useful mobile-phase velocities.Syringe-type or single- or multiple-head reciprocatingpiston pumps are commonly used to provide the oper-ating pressures needed in conRgurations that dependon the design of the solvent-delivery system. A singlepump is sufRcient for isocratic operation. A singlepump and electronically operated proportioningvalves can be used for continuous variation of themobile-phase composition (gradient elution) or, al-ternatively, independent pumps in parallel (com-monly two) are used to pump different solventsinto a mixing chamber. Between the pump andsample inlet may be a series of devices (check valves,pulse dampers, mixing chambers, Sow controllers,pressure transducers, etc.) that correct or monitorpump output to ensure that a homogeneous, pulselessliquid Sow is delivered to the column at a knownpressure and volumetric Sow rate. These devices maybe operated independently of the pump or in a feed-back network that continuously updates the pumpoutput. Mobile-phase components are stored in reser-voir bottles with provision for solvent degassing, ifthis is required for normal pump and detector opera-tion. Loop-injection valves situated close to the headof the column are universally used for sample intro-duction. This allows a known volume of sample to be

withdrawn at ambient conditions, equivalent to thevolume of the injection loop, and then inserted intothe fully pressurized mobile-phase Sow by a simplerotation of the valve to change the mobile-phase Sowpaths. Although most separations are performed atroom temperature, either the column alone or thewhole solvent-delivery system may be thermostatedto a higher temperature when this is desirable orrequired for the separation. The separation ismonitored continuously on the low pressure side ofthe column using several bulk physical property,photometric, or electrochemical detectors Rtted withmicrovolume Sow cells.

Common detection principles are UV absorbance,Suorescence, refractive index and amperometry.Coupling to MS and IR spectroscopy is becomingmore common, as is online coupling to nuclear mag-netic resonance (NMR) spectrometers. Detection isa more difRcult aspect in the condensed phaseand neither the variety nor operating characteristicsof LC detectors compare favourably with GC de-tectors, although they allow a wide range of sampletypes to be analysed routinely. Special materials areused in the fabrication of biocompatible and cor-rosion-resistant instruments for the separation of bio-polymers and for ion chromatography. Individualpumps can handle solvent delivery requirementsover a decade range or so of Sow rates. The diversityof column diameters used in modern LC for analysisand preparative-scale applications demands Sowrates that vary from a few �L per minute to tensof litres per minute. Consequently, instrumentsare designed for efRcient operation within a par-ticular application range and are not universalwith respect to column selection. Furthermore,analytical detectors tend to be designed with sensitiv-ity as the main concern and preparative-scale de-tectors for capacity, such that the two are generallynot interchangeable even when the same detectionprinciple is employed. For preparative-scale worksome form of automated sample fraction collection isnecessary and economy of operation may dictate in-corporation of an integrated mobile-phase recyclefeature.

Supercritical Fluid Chromatography

Instrumentation for SFC is a hybrid of componentsused in GC and LC modiRed to meet the requirementsof operation with a compressible Suid. The mobilephase is typically carbon dioxide (with or withoutmodiRer) contained in a pressurized cylinder and de-livered to the pump in liquid form. Syringe pumps orcooled reciprocating piston pumps modiRed for pres-sure control are commonly used. A high precision


pressure transducer is installed between the pumpand sample inlet for programming the inlet pressureor Suid density during the course of a separation.Simultaneous measurement of the column temper-ature and pressure control allows constant density ordensity programming under computer control if theappropriate isotherms are known or can be approxi-mated. Two pumps are generally used to generatemobile-phase composition gradients comprisingliquid carbon dioxide and an organic solvent. Loop-injection valves similar to LC are the most convenientdevices for sample introduction. The column ovenis usually a forced air circulation thermostat similarto those used in GC. The full range of Same-based detectors used in GC can be used with onlyslight reoptimization as well as the main optical de-tectors used in LC, after modiRcation for high pres-sure operation. A unique feature of the chromato-graph is a restrictor required to maintain constantdensity along the column and to control the linearvelocity of the Suid through the column. OriRce-typerestrictors are usually placed between the column anddetector for Same-based detectors and back-pressureregulators after the detector Sow cell for opticaldetectors.

MEKC and CEC

MEKC and CEC employ the same instruments asused for capillary electrophoresis with the addition ofoverpressure capability for the buffer reservoirswhen used for CEC. The separation capillary is ter-minated in two buffer reservoirs containing thehigh voltage electrodes that provide the electric Reldto generate the Sow of mobile phase. The bufferreservoirs can be moved into place pneumatically andsequenced automatically to introduce a sample vialfor sample introduction or a run buffer vial forseparation. The column area is thermostated to main-tain a constant temperature. A miniaturized opticaldetector positioned between the buffer reservoirsis commonly used for on-column detection. Someform of interlock mechanism is used to prevent oper-ator exposure to the high voltages, up to 30}50 kV,typically used. A high level of automation is achievedunder computer control and unattended operation isgenerally possible.


The total automation of sample application,chromatogram development and in situ quantitationin planar chromatography has proved difRcult.Instead the individual procedures are automated, re-quiring operator intervention to move the layer fromone operation to the next. Samples are typically ap-

plied to the layer as spots or narrow bands usinglow volume dosimeters or spray-on techniques.Application volume, method, location and samplesequence are automated for unattended operation.The chromatogram is obtained by manual develop-ment in a number of development chambers of dif-ferent design, or can be automated such that theconditioning of the layer, the selected solvents for thedevelopment, and the development length arepreselected and controlled through the use of sensors.For multiple-development techniques the layer can bealternately developed, dried, new solvent introducedand the process repeated with changes in the develop-ment length and mobile-phase composition for any orall the programmed development steps. Apparatusfor forced-Sow development is also available andresembles a liquid chromatograph with the columnreplaced by the layer sandwiched between a rigidsupport and a polymeric membrane in an over-pressure development chamber to allow externalpressure to be used to create the desired mobile-phasevelocity.

After development the chromatogram is recordedusing scanning or video densitometry. The uniquefeature compared with detection in columnchromatography is that the separation is recorded inspace rather than time while in the presence of thestationary phase. The common forms of detection areoptical methods based on UV}visible absorption andSuorescence. In mechanical scanning the layer ismoved on a translation stage under a slit projectingthe image of the monochromatic light source on thelayer surface and the light reSected from the surfacemonitored continuously with a photodiode or similardevice. Substances that absorb the light producea proportional decrease in the intensity of the reSec-ted light that can be related to the amount of samplepresent (for Suorescence there is a proportionate in-crease in the amount of light emitted at a wavelengththat is longer than the absorbed wavelength). Elec-tronic scanning is not as well developed but involvesuniformly illuminating the whole layer and imagingthe plate surface onto a video camera, or similardevice, to capture and integrate the static image of theabsorbing zones.

Conclusion

Many of the important developments in chromato-graphy have already been made, yet the techniquecontinues to evolve by the introduction of new mater-ials that extend the scope of existing methods andthrough Rnding new applications. General applica-tions are dominated by the techniques of gaschromatography and column liquid chromatography,


which are the most mature in terms of their evolu-tionary development, although it is widely recognizedthat column liquid chromatography still lacks a sensi-tive and universal detector for general applications.This void may be Rlled by mass spectrometry, whichhas made great strides in the last few years towardsthis goal based on particle-beam interfaces and atmo-spheric ionization techniques coupled with the devel-opment of low cost mass separators. By comparison,thin-layer chromatography and supercritical Suidchromatography have become recognized as tech-niques with niche applications and are unlikely tosupplant gas and column liquid chromatography asthe dominant chromatographic methods used in ana-lytical laboratories. The microcolumn techniques ofcapillary electrophoresis, micellar electrokineticchromatography, and capillary electrochromatogra-phy have quickly established themselves as usefullaboratory methods and are likely to become of in-creasing importance as they complete their evolution-ary cycle. In particular, the infant capillary electro-chromatography has the potential to replace columnliquid chromatography from many of its traditionalseparation roles, but has yet to reach a state of devel-opment to be considered as a routine laboratory tech-nique.

The only thing that is certain about science is un-certainty. Although chromatographic methods arelikely to dominate separation science for the Rrst partof the twenty-Rrst century, it would be a foolishperson who predicts their form, continuing develop-ment, and main applications. Throughout the historyof chromatography general approaches have had toadapt to changing needs brought about by dramaticshifts in the focus on different types of applications,and this has a signiRcant impact on the relative im-portance of the various techniques. However,chromatography should be considered as an holistic

approach to separations, and will be better under-stood and correctly employed if we abandon thecurrent trend to compartmentalize the techniquebased on specialization in individual subject areas.

See Colour Plate 3.

Further Reading

Berger TA (1995) Packed Column Supercritical FluidChromatography. Cambridge: Royal Society of Chem-istry.

Braithwaite A and Smith FJ (1996) ChromatographicMethods. London: Blackie Academic & Professional.

Giddings JC (1991) UniTed Separation Science. New York:Wiley-Interscience.

Guiochon G and Guilleman CL (1988) Quantitative GasChromatography for Laboratory Analysis and On-LineProcess Control. Amsterdam: Elsevier.

Guiochon G, Shirazi SG and Katti AM (1994) Funda-mentals of Preparative and Nonlinear Chromatography.Boston: Academic Press.

Heftmann E (1992) Chromatography, Parts A and B. Am-sterdam: Elsevier.

Jennings W, Mittlefehldt E and Stremple P (1997)Analytical Gas Chromatography. San Diego: AcademicPress.

Lee ML, Yang FJ and Bartle KD (1984) Open TubularColumn Gas Chromatography. Theory and Practice.New York: Wiley-Interscience.

Li SFY (1992) Capillary Electrophoresis. Principles, Prac-tice and Applications. Amsterdam: Elsevier.

Poole CF and Poole SK (1991) Chromatography Today.Amsterdam: Elsevier.

Robards K, Haddad PR and Jackson PE (1994) Principlesand Practice of Modern Chromatographic Methods.London: Academic Press.

Sherma J and Fried B (1997) Handbook of Thin-LayerChromatography. New York: Marcell Dekker.

Snyder LR, Kirkland JJ and Glajch JL (1997) PracticalHPLC Method Development. New York: J Wiley.

CRYSTALLIZATION

H. J. M. Kramer and G. M. van Rosmalen,Delft University of Technology, Delft, The Netherlands

Copyright^ 2000 Academic Press

Introduction

Crystallization from solution is a separation tech-nique where a solid phase is separated from a motherliquor. In contrast to other separation processes,

however, the dispersed phase consisting of numeroussolid particles also forms the Rnal product, that has tomeet the required product speciRcations. Crystalliza-tion can thus also be seen as a technique to obtainsolid products, where the crystallization process hasto be carefully controlled in order to meet the ever-increasing demands of the customer on particleproperties like particle size distribution, crystalshape, degree of agglomeration, caking behaviourand purity. Since the particles must also be easily

64 I / CRYSTALLIZATION / Derivatization

CHROMATOGRAPHY - Murdercube Purification... · gas}gas chromatography does not exist and ... retention mechanism is governed by interfacial ad- ... greater afRnity.

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