LC Theory Methodology

Liquid Chromatography: Theory and Methodology

John G. Dorsey* and William T. Cooper

Department of Chemistry, Florida State University, Tallahassee, Florida 32306-4390

Barbara A. Siles

Department of Chemistry, College of William and Mary, Williamsburg, Virginia 23187-8795

Joe P. Foley

Department of Chemistry, Villanova University, Villanova, Pennsylvania 19085-1699

Howard G. Barth

Central Research and Development Department, E. I. du Pont de Nemours & Company,P.O. Box 80228, Experimental Station, Wilmington, Delaware 19880

Review Contents

Books, Reviews, and Symposia Proceedings 592RTheory and Optimization 592RReviews 592RTheory 592ROptimization 594RData Analysis 595RReviews 595RGeneral Papers 595RPeak Purity/ Deconvolution 596RSolute Retention/ Stationary-PhaseCharacterization596RMiscellaneous 597RNormal Phase 597RReversed Phase 598RMobile-Phase Studies 599RStationary-Phase Studies 600RCapillary Electrochromatography 602RBiopolymer Separations 603RReviews 603RColumn Packing Materials 603RTemperature-Responsive Packing Materials 605RHydrophobic Interaction 605RReversed Phase 605RIon Exchange 606RSize Exclusion 608RPreparative Separations 608RAffinity Chromatography 608RReviews and Theoretical Models 608RAffinity Supports 609RAffinity Elution Conditions 610RSupport Activation: Methodology andCharacterization611RAffinity Ligand Immobilization, Characterization,and Applications611RIon Chromatography 613RMobile-Phase and Sample Effects 613RStationary Phases 614RSuppressor Technology, Quantitation, andDetection615RSecondary Chemical Equilibria 615RReviews 615RMicellar Liquid Chromatography 616RAcid

-

Base Equilibria 617RIon Pairing 618RMiscellaneous Complexation Equilibria 618ROptical and Positional Isomers 619RReviews 619RNew Chiral Stationary Phases 620ROther Studies 621RGeometric and Positional Isomers 622RMultidimensional Liquid Chromatography andColumn Switching622RPreparative LC 623RReviews 623RPackings/ Columns/ Hardware 623RTheory/ Optimization/ Methodology 624RSelected Applications 625RPre- and Postcolumn Derivatization 625RMicrocolumn and Open Tubular LC 626RTrace Analysis 628RPhysicochemical Measurements 629RLinear Free Energy Relationships/ Hydrophobicity/ Partition Coefficients629RAssociation and Stability Constants 630RThermodynamic Studies 631RKinetic Studies 631RConformational Studies 631RLiterature Cited 632R

This review covers fundamental developments in liquid chro-matography during the period of approximately October 1995through approximately October 1997. As with the past four issuesof the Fundamental Reviews, there are separate reviews oninstrumentation and on size exclusion chromatography;this reviewis of important developments in the

chemistry

of the separationprocess. The primary searching method for this work has been

CAS Online

, and each author has supplemented these with searchmethods of their own. There has been one small change in theorganization of this review; due to greatly increased interest, wehave added a section on

electrochromatography

, where the mobile

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phase is driven through a packed column by electroosmotic flow,rather than by a pressure drop. This method has the possibilityof greatly increasing peak capacity in LC separations, but thereis much work left before it becomes a routine tool.This is not meant to be a comprehensive review ofall publishedpapers during this time period; rather, we have tried to selectthose papers which we feel are significant developments. We havelargely restricted the covered material to the English languageliterature. Comments and suggestions concerning this review arewelcomed and encouraged and should be sent to the first author(J.G.D., e-mail, Dorsey@chem.fsu.edu). As readers are all aware,it becomes more and more difficult for any one person to stay intouch with the literature. Even with electronic search methods,some key papers are likely to be missed. To those authors whosework is omitted, we apologize.As a personal note, I have now led this review for five issues,which spans the entire decade of the 1990s. This will be my lastreview; it is time for new ideas and new energy. I thank you allfor your comments, suggestions, and support during this tenure.

BOOKS, REVIEWS, AND SYMPOSIA PROCEEDINGS

First, the previous Fundamental Review covered work occur-ring approximately from October 1993 through October 1995 andwas published during this review period (

A1

). Many otherspecialized reviews were published, and they will be cited duringthe respective sections of this review.Majors (

A2

) continued his useful surveys ofcolumn usage andnoted that reversed-phase columns account for 50% of newpurchases. Bidlingmeyer also discussed trends in column usageand development (

A3

). It is perhaps only a coincidence that bothauthors work for major column manufacturers!Several general, useful books were published during thisreview period. Snyder, Kirkland, and Glajch (

A4

) published thesecond edition of their highly popular book,

Practical HPLC Method Development

, and this is perhaps the most complete sourceofinformation on LC practice for the chromatography user. Neue(

A5

) published a useful book on

HPLC Columns: Theory, Technol-ogy, and Practice

. This book is more academically oriented, butis still a highly useful resource for the practicing chromatographer.Sadek (

A6

) edited a unique book,

The HPLC Solvent Guide

, whichis quite likely the most complete volume of information everassembled on solvents and should find shelf space in virtuallyevery LC laboratory.Other books published during this period include

HPLC and CE: Principle and Practice

by Weston and Brown (

A7

) and threeedited, assembled texts,

High Performance Liquid Chromatogra- phy: Fundamental Principles and Practice

(

A8

),

High Performance Liquid Chromatography: Principles and Methods in Biotechnology

(

A9

), and

HPLC: Practical and Industrial Applications

(

A10

).Several other more specialized texts were also published, andwhere appropriate, they are referenced in the individual sectionslater in this review.Many symposia proceedings were published also, as iscustomary for many of the yearly chromatography meetings, andagain, individual papers from these are referenced in the individualsections. It has become much more common for these proceed-ings to be published as part ofthe open literature in journals suchas

Journal of Chromatography

, and it is not necessary to duplicatetheir references here.

THEORY AND OPTIMIZATION

Reviews.

With the exception of the reviews cited below, wehave excluded nearly all theoretical contributions that can logicallybe placed in another category elsewhere in this review (e.g.,reversed phase, optical and positional isomers, preparative, etc.),especially those contributions pertaining to chromatographicproperties (efficiency, retention, selectivity).Lochmuller et al. reviewed the advances in HPLC for theseparation of polymers and oligomers. Retention mechanisms,the dynamics of the partition chromatography process, evidencefor significant delays in polymer equilibration to mobile-phaseconditions, and effect of the temperature dependence of polymersolubility were discussed (

B1

). Conventional chromatographicbehavior was verified for the separation of polystyrene, poly-(methyl methacrylate), and poly(ethylene oxide) samples. Fabredefined and described the role of robustness testing in liquidchromatography and capillary electrophoresis. Although screen-ing designs may be sufficient to set method limits, responsesurface designs are more important in method transfer becausethey give a more comprehensive picture of the method (

B2

).Ooms discussed the ever-increasing role of temperature controlin liquid chromatography (

B3

). Many new specialty columns andchemistries require accurate control at sub- or superambienttemperatures, and conventional RPLC columns can sometimesshow favorable changes in retention or selectivity at temperaturesbeyond traditional limits.Davydov reviewed the determination of adsorption isothermsfrom solution and their use in chromatographic optimization; aswell as the effects of intermolecular interactions in mixture onindividual component adsorption isotherms (

B4

). Muldoon andStanker described the recent development and application of molecular imprinting polymer (MIP) technology for residueanalysis, reviewing the process of MIP synthesis, the basis foranalyte-MIP recognition, and current applications of this technol-ogy for residue analysis (

B5

). Pirogov et al. reviewed the simplexalgorithm as a tool for finding the optimal analytical conditionsand systematically discussed the advantages and disadvantages.The mathematical algorithm was described, its known modifica-tions were compared, and examples were given of the practicaloptimization of detergent conditions in ion chromatography (

B6

).

Theory.

Kanazawa et al. proposed a useful new chromatog-raphy concept in which the surface properties and resultingfunction of the HPLC stationary phase are controlled by externaltemperature. This method should be ideal for separations of biological and biomedical peptides and proteins using onlyaqueous mobile phases (

B1

). The influence of column temper-ature on the retention of steroids on the copolymer-modifiedstationary phases was more significant than in the case of homopolymer-modified columns (

B8

).Two research groups examined the effects of pressure onretention and selectivity. Ringo and Evans examined the role of pressure in separations where the primary mechanism for soluteretention is inclusion complexation (

B9

). Using the positionalisomers ofnitrophenol as model solutes and

β

-CD as the stationaryphase, pressure-induced decreases in solute retention factors

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ranging from

-

2 to

-

35% were observed experimentally forpressures from 40 to 340 bar. For this phase it appears that thepressure dependence of solute retention is due primarily to thepressure-induced dissociation ofthe cyclodextrin

-

solute complex(

B9

). Using a warfarin/

β

-CD system, they were able to measurepressure-induced perturbations in complexation equilibria todetermine the change in partial molar volume upon complexation(

B10

). McGuffin and Chen also measured a pressure dependenceon retention with a homologous series of derivatized fatty acids.They compared their results to a thermodynamic model derivedfrom regular solution theory, which suggested that state effectsalone are not sufficient to describe the measured change in soluteretention and that variations in interaction energy with densitymust also be considered. By using an extended van der Waalsrelation that better describes polar fluids, good agreement wasobserved (

B11

).Foley and Ahuja published without derivation a unifying theoryof separation that is valid for both neutral and charge analytes(

B12

). A “master resolution equation (MRE)” that is universallyapplicable to HPLC, capillary zone electrophoresis, capillaryelectrochromatography, and micellar electrokinetic chromatog-raphy was described; the MRE reduces to equations previouslyderived by others for each of the specific separation modes whenthe appropriate constraints are employed. Thede et al. derivedanalytical peak shape equations for first-order reversible reactionsoccurring in a chromatography reactor by treating the reversiblereactions as consecutive reactions with alternating products. Forsmall to medium conversions, the correspondence between theseequations and a numerical solution of the partial differentialequation was sufficiently close that the latter could be substitutedin fitting procedures for the determination ofrate constants (

B13

).Li and Carr reported the accuracy of various approximateempirical methods for estimating the diffusion coefficients of alkylbenzenes and alkylphenones in acetonitrile (ACN)/ water andmethanol (MeOH)/ water solvent systems and then developed anovel empirical modification of the Wilke

-

Chang equation inorder to more accurately estimate these solute diffusion coef-ficients (

B14

). The errors of the proposed correlations were nogreater than 10%for both ACN/ water and MeOH/ water systems,which represented a 2

-

3-fold better acuracy than those of theother two correlations. The authors recommended the use ofthismodified Wilke

-

Chang equation with the above homologousseries ofsolutes for the evaluation ofcolumn performance in RPLC(

B14

).Cantwell et al. published three papers concerned with intra-particle sorption rate and liquid chromatographic band broadeningin porous polymer packings. In the first paper, they proposed amethod by which an experimentally measured sorption

-

ratecurve for a solute on a chromatography sorbent may be used toaccurately predict the plate height and peak shape contributionsofintraparticle sorption (

B15

). In the second paper, they focusedexclusively on macroporous poly(styrene

-

divinylbenzene) sor-bents (e.g., PRP-1) and found that slow intraparticle processesare the major cause of band broadening of naphthalene on PRP-1(

B16

). In their third paper, they reported that the cause of slowintraparticle mass-transfer rates is the slow rate of diffusion intothe polymer matrix (

B17

). Farnan et al. investigated boththeoretically and experimentally the effect of intraparticle mass-transfer resistances on the peak shape at flow velocities currentlyused in high-speed protein chromatography. The peak asymmetryof the protein bands was quantified by the difference betweenthe first statistical moment and the retention volume of the peak apex rather than the peak skewness because the latter was muchmore difficult to determine. Ageneral method for the evaluationof the mass-transfer characteristics of a given chromatographysorbent from the variation in peak asymmetry with reducedvelocity was then introduced (

B18

).Metting and Coenegracht found that the predictive results of a well-designed neural network were better or comparable to thoseobtained with linear and nonlinear regression models (

B19

). Topredict and understand the profiles oflarge-size system peaks andvacancy bands in liquid chromatography, Guiochon et al. derivedan analytical solution for the ideal model ofchromatography. Thissolution provided a more profound explanation of the formationofthe large system peaks than previous studies (

B20

). Later theyused an equilibrium-dispersive model to calculate band profilesfrom the experimentally measured column efficiency and isothermdata (

B21

). Guan-Sajonz and Guiochon showed that the pressureused to pack a column can have an dramatic effect on theperformance characteristics of the column. As the packingpressure was increased, the mass of packing material inside eachcolumn increased. The column external porosity, determined byan inverse size exclusion chromatography method, decreased asthe packing pressure increased, while the internal porosityremained constant. Interestingly, the retention factor and thecolumn efficiency of each compound increased linearly withincreasing packing (

B22

).Trathnigg et al. studied the chromatographic behavior of low-molecular-weight poly(ethylene glycol) (PEG) and its mono-anddimethyl ethers in the isocratic mode of reversed-phase liquidabsorption chromatography on a C18-modified silica column(

B23

). Experimental data were interpreted according to simplemolecular statistical theory based on the model of a rigid-rodmolecule, which is capable of being adsorbed inside the homo-geneous layers near the pore walls. This approach allowed thedetermination ofthe free energies oftransfer ofthe repeating unit(oxyethylene group) and the two types of end groups (methoxyand hydroxy groups) from a mixed eluents into a bonded C18phase. The effective thickness ofthe bonded layer was estimatedas well as the critical point of adsorption for poly(oxyethylene),which is ofimportance for separation ofmacromolecules by usingthe crititical-condition mode of liquid chromatography (

B23

).Chen and Horvath compared both theoretically and experi-mentally two modes of on-column UV absorption detection withpacked capillaries in liquid chromatography. The efficiency of the chromatography system was the same using either detectionmode, and the two modes of on-column detection gave equallyreproducible results in both qualitative and quantitative analyses.The sensitivity of detection in the second mode, however, was



2

T

(1

+

k

′

) times that observed in the first mode for a peak havinga retention factor of

k

′

in isocratic elution under otherwise identicalconditions (

B24

). Vailaya and Horvath used a simplified versionof the solvophobic theory to reexamine a large set of retentiondata with nonpolar and weakly polar eluites in reversed-phasechromatography (RPC) to test certain predictions by the theoryand to clarify further the roles of the mobile and the stationary

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