Liquid Chromatography: Theory and Methodology

zyxwvutsrqp zyxwvutsr zyxwvutsrq zyxwvutsrqpon zyxwvutsrqp zyxwvutsr zyxw Anal. Chem. 1994,66, 500R-546R Liquid Chromatography: Theory and Methodology John G. Dorsey' Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 4522 1-0 172 William T. Cooper Department of Chemistry, Florida State University, Tallahassee, Florida 32306-3006 John F. Wheeler Department of Chemistry, f urman University, Greenville, South Carolina 296 13 Howard G. Barth E. I. du Pont de Nemours & Company, P.O. Box 80228, Central Research and Development Department, Experimental Station, Wilmington, Delaware 19880 Joe P. Foley Department of Chemistry, Villanova University, Villanova, Pennsylvania 19085-1699 - Review Contents Books, Reviews, and Symposia Proceedings Theory and Optimization Theory Optimization Data Analysis Figures of Merit and Peak Modeling Deconvolution and/or Quantitation Retention Behavior Peak Purity/Homogeneity Miscellaneous Normal Phase Reversed Phase Stationary Phases Mobile Phases Biopolymer Separations Column Packings Reversed Phase Ion Exchange Preparative Liquid Chromatography of Biopolymers Affinity Chromatography Reviews and Theoretical Models Affinity Supports Ligand Immobilization and Applications Ion Chromatography Mobile-Phase and Sample Effects Stationary Phases Suppressor Technology, Quantitation, and Detection Secondary Equilibria Reviews and General Theory Ion Pairing Micellar Liquid Chromatography (MLC) Miscellaneous Geometric and Optical Isomers Reviews New Chiral Stationary Phases Geometrical Isomers 500R 500R 502R 503R 504R 505R 510R 514R 517R 520R 524R Analytical Chemistry, Vol. 66, No. 12, June 15, 1994 Multidimensional Chromatography and Column Switching Preparative LC Reviews Theory /Optimization Packings/Columns/Hardware Selected Applications Pre- and Postcolumn Derivatizations Microcolumn and Open Tubular LC Trace Analysis Physiochemical Measurements Partition Coefficients/Hydrophobicity Measurements Ligand-Binding Measurements Thermodynamic Studies Kinetic Studies Conformational Studies 525R 527R 528R 531R 532R 533R This review covers fundamental developments in liquid chromatography during the period of approximately December 1991 through December 1993. As with the past two issues of the Fundamental Reviews, there are separate reviews on instrumentation and size exclusion chromatography; this review is of important developments in the chemistry of the separation process. The primary searching methods for this work have been CAS on-line and CA Selects. Each author has supplemented these with search methods of their own. This is not meant to be a comprehensive review of all published papers during this time period; rather, we have tried to select those papers which we feel are significant developments. We have largely restricted the covered material to the readily accessible English language literature. Comments and suggestions concerning this review are welcomed and encouraged and should be sent to the first author (J.G.D.). A. BOOKS, REVIEWS, AND SYMPOSIA PROCEEDINGS Anumber of books were published during this review period, but our search revealed no general books on the subject of 0003-2700/94/0366-0500$14.00/0 0 1994 American Chemical Society zyxwvutsrqponm John G. Dorsey is Professor of Chemistry and Chairman of the Analytical Division at the University of Cincinnati. He received his Ph.D. degree in analytical chemistry in 1979 at the University of Cincinnati and then spent ten years on the faculty at the University of Florida, where he received four departmental, college, and university teaching awards. He returnedto Cincinnati as Professor in 1989. His research interests are in the areas of fundamental liquid chromatography, analytical applications of micelles and organized media, flow injectionanalysis, capillary electrophoresis, and old Bordeaux wines. He has about 75 publications in these areas and serves on the Editorial Boards of six analytical and chromatographic journals. John is also a member of the Executive Committee of the ACS Subdivision of Chromatography and Separations Chemistry, has instructed short courses in liquid chromatographyfor pharmaceuticaland consumer products companies, and has organized chromatography symposia for the Pittsburgh Conference, EAS, FACSS, and American Chemical Society Meetings. John is the recipient of the 1993 Award for Distinguished Scientific Research from the University of Cincinnati and the 1993 Akron Section Award of the American Chemical Society. William T. Cooper is currently Associate Professor of Chemistry, Adjunct Professor of Oceanography, and Director of the Terrestrial Waters Institute (TWaIn) at Florida State University. He receiveda B.S. in chemistry from the University of Tennessee, Knoxville, and a Ph.D. in chemistry from Indiana University. While at Indiana he worked in the Biogeochemical Laboratories of Professor John Hayes, where he studied interactions between organic solutes and mineral surfaces using inverse gas and liquid chromatography techniques. Environmental biogeochemistry is the primary theme of his research, which includes development of two-dimensional separation methods for analyzing complex environmental and biological samples, inverse gas liquid chromatography studies of the surface chemistry of heterogeneous geological materials, characterization of the chemical composition of soil and sedimentary organic matter by NMR spectroscopy, and use of liquid chromatography and NMR in studies of microbial degradation and metabolism of toxic organic chemicals. John F. Wheeler is an Assistant Professor of Chemistry at Furman University in Greenville, SC. He received his B.S. degree in chemistry summa cum laude from Georgetown College in Kentucky in 1986, and his Ph.D. from the University of Cincinnati in 1990 under Distinguished Research Professor William R. Heineman. As a student at Cincinnati, he was awarded graduate research fellowships from the Analytical Division of the ACS, the Proctor & Gamble Co., and the University’s own Research Council and BiochemicalKhemistry Research Center. I n 1990 Dr. Wheeler joined with Dr. Dorsey’s research , group as a postdoctoral associate until accepting his current position at Furman in January 1992. Dr. Wheeler’s research interests include fundamental studies in protein separations using HPLC and HPCE, clinical applications of liquid separations, and electroanalyticalchemistry. He participated in the NATO Advanced Study Institute on Theoretical Advancements in Liquid Chromatography and Related Separation Techniques in 1991 and was an invited speaker in the recent FACSS Symposium, Separation Scientistsof the 2 1st Century. Since his recent arrival or national meetings regarding fundamental and applied studies of HPCE and HPLC, including invited presentations at the Frederick Conference on HPCE and HPLC, including invited presentations at the Frederick Conference on HPCE, the Pittsburgh Conference, and the Western Carolinas Chromatography Discussion Group. Dr. Wheeler serves as a regional consultant in the area of chemical separations and has instructed short courses on GUMS, HPLC, SEC, and HPCE. He is an active member of the Analytical Division of the American Chemical Society, the Society for Electroanalytical Chemistry, and the Council on Undergraduate Research. Joe P. Foley is an Associate Professor of Chemistry at Villanova University. He graduated Valedictorian from Centre College of Kentucky in 1978 with a B.S. in chemistry and chemical physics and received his Ph.D. in chemistry from the University of Florida in 1983. After a twoyear National Research Council Postdoctoral Fellowshipat the National Institute of Standards and Technology, he joined the faculty of Louisiana State University in August 1985 and continued there until accepting his appointment at Villanova in 1 199 1. Dr. Foley’s research interests are f in the fundamental and applied aspects of chemical separations, and he has published about 35 refereed papers and 6 book chapters pertaining to capillary electrophoresis, micellar electrokinetic chromatography, high-performance liquid chromatography, and supercritical fluid chromatography. A participant at the 1991 NATO Advanced Study Institute on Theoretical Advances in Chromatography and Related Separation Techniques, Dr. Foley has organized numerous scientific symposia for the American Chemical Society, the Pittsburgh Conference, and the Federation of Analytical Chemistry and Spectroscopy Societies (FACSS) and has served as a consultant for the health, pharmaceutical, chemical, and petroleum industries. He serves on the Editorial Boards of The Analyst and the Journal of Microcolumn Separations and is a member of Sigma Xi, the American Chemical Society, the Scientific Committee of the Frederick Conference on Capillary Electrophoresis,and the ExecutiveCommittee of the Chromatography Forum of the Delaware Valley. zyxw zyxwvutsrqpo ~ Howard G. Barth is a research associate of the Corporate Center for Analytical Sciences at the DuPont Experimental Station, Wilmington, DE. Before joining the DuPont Co. in 1988, he was a research scientist and group leader at Hercules Research Center. He received his B.A. (1969) and Ph.D. (1973) in analytical chemistry from Northeastern University. His specialities include polymer characterization, size exclusion chromatography, and high-performance liquid chromatography. He has published over 60 papers in these and related areas. Barth has also edited the book Modern M e w s of Particle Size Analysis (Wiley, 1984) and coedited Modern Methods of Polymer Characterization (Wiley, 1991). He has also edited five symposium volumes on polymer characterization published in the JournalofApplied Polymer Science. Barth was on the Instrumentation Advisory Panel of Analytical Chemistry and was Associate Editor of the Journal of Applied Polymer Science. He is cofounder and Chairman of the International Symposium on Polymer Analysis and Characterization. He has been appointed recently editor-in-the-chief of the Journal of Polymer Analysis and Characterization(Gordon and Breach Publishers) Barth is past Chairman of the Delaware Section of the American Chemical Society, where he presently serves as councilor. Dr. Barth is a member of the American Chemical Society divisions of Analytical Chemistry, Polymer Chemistry, and Polymeric Materials Science and Engineering, the Society of Plastics Engineers, and the Delaware Valley Chromatography Forum. He is also a Fellow of the American Institute of Chemists and a member of Sigma Xi. ?d, \ I I zyxwvutsrqpon . liquid chromatography. Books relevant to specific topics are referenced in those sections. A number of general reviews, tutorials, and perspectives were published during this review period. First, for work occurring prior to 1992, the previous Fundamental Review covered work from 1990 and 1991 (AZ). Ettre continued his interest in the history of chromatography, and a description of the work from 1941 to 1951 was published under the title, “1941-1951: TheGolden Decadeof Chromatography” (A2). Scott published a more general review and description of liquid chromatography (A3). Snyder published two general reviews, one on the theory of chromatography (A4) and the other on optimization of separations (A5). Poppe also published a general description of column liquid chromatography, including descriptions of instrumentation and chemistries (A6). zyxw Analytical Chemistry, Vol. 66, No. 12, June 15, 1994 SOW zyxwvutsr zyxwvuts Finally, Majors published another survey of the use of various liquid chromatographic modes of separation ( A 7 ) . From a survey of 1000 readers of LC-GC, with a 16.3% response rate, he found that reversed-phase columns were used by 47.2%of the respondents, and that represents a decrease in usage from the previous survey in 1987. Normal bonded phase usage increased to 13.5%,adsorption (liquid-solid) increased to 9.8%, and ion exchange remained steady at 16.6%. Size exclusion, chiral columns, and hydrophobic interaction modes showed smaller percentages of use. B. THEORY AND OPTIMIZATION Theory. With the exception of the reviews cited immediately below, we have excluded nearly all theoretical contributions that can logically be placed in another category elsewhere in this review (e.g., reversed phase, geometric and optical isomers, preparative, etc.), especially those contributions pertaining to chromatographic properties (efficiency, retention, and selectivity). Reviews. Bellot and Condoret reviewed the different ways to express adsorption isotherms, competitive or not, in reversedphase chromatography. Once these equilibria are described, powerful liquid chromatography models, based on continuity equations, can be employed to describe the phenomena encountered in the separation process of biomolecules (BZ). Lundell reviewed the options in the implementation of gradient theory for the optimization of peptide separations in RPLC, discussing approaches for calculating retention times and bandwidths from experimental data and comparing different kinds of extrapolation with interpolation. The study was aimed at finding the best compromise between number of experiments, accuracy of predictions, and simplicity of calculations ( B 2 ) . Scott discussed the roles of molecular interactions and molecular size in controlling separation, in the context of the preparation of the stationary phase (B3). Band dispersion is discussed in term of the multipath effect, longitudinal diffusion, and resistance to mass transfer; and the basic components of a modern LC chromatograph are examined. Finally, Volpe and Siouffi reviewed (38 references) some of the problems in gas, liquid, and supercritical fluid chromatography ( B 4 ) .The authors challenge that HPLC suffers from “the lack of a reliable model to predict the capacity factor for a wide range of solutes”, that retention mechanisms on some phases are not fully understood, and that diffusion coefficient data are scarce. General. A theory by Roachdescribing theoverlapofcircles in a two-dimensional plane was elegantly generalized by Davis to an n-dimensional space, where n is the number of orthogonal axes. This statistical theory of spot overlap is proposed for n-dimensional separations. Extensive computer simulations show that the theory describes overlap very well in threedimensional spaces and modestly well in four-dimensional spaces, when the number of components is large. The theory shows that the maximum number of spots per unit capacity and the maximum number of any kind of multiplet per unit capacity both decrease geometrically with increasing n (B5). Liapis and McCoy constructed a mathematical model of perfusion chromatography for column systems (B6). The model was developed to describe the dynamic behavior of single- and multicomponent adsorption in columns having perfusive adsorbent particles (the perfusive particles have a nonzero intraparticle fluid velocity), but was also found to be applicable to columns with “diffusive particles”, i.e., ones in which intraparticle fluid velocity is zero. For adsorption systems having relatively fast or infinitely fast interaction kinetics (Le., analyte-stationary-phase dynamics), the use of perfusive particles can potentially provide improved column performance. Later McCoy et al. used a mathematical model of binary perfusion chromatography (competitive adsorption involving two components) to simulate, study, and compare the separation of two components with perfusive and purely diffusive particles (B7). The superior separation efficiency obtained with the perfusive adsorbents was attributed to the intraparticle fluid flow which enhances intraparticle mass transport. Jandera and Guiochon investigated the dependence, in preparative HPLC, of band deformation and splitting on the volume and concentration of the sample, the composition of the mobile phase, and the column temperature. They showed that if a solvent with a higher elution strength than the mobile phase is used to dissolve poorly soluble samples, significant deformation of the band profiles occurs, especially when the column is overloaded; eventually, band splitting may take place. This behavior was observed in nonaqueous RPLC of cholesterol and other low-polarity analytes ( B 8 ) . Belenkii and co-workers described some peculiarities of zone migration and band broadening in the gradient RPLC of proteins with respect to membrane chromatography (B9). Blackwell and Carr developed an eluotropic series for the separation of Lewis bases on zirconium oxide (BZO). By increasing the precision of their HPLC system, Dreyer et al. demonstrated that steric exclusion effects can have a far greater influence on retention data than is commonly assumed (BZI ) . Gloeckner discussed the control of adsorption and solubility in gradient HPLC separation of copolymers from styrene and methacrylates, with an emphasis on the principles of sudden transition gradients ( B I 2 ) . Hsu and Chen reported that a conventional definition of column efficiency, N = tR2/u2,is inadequate for large-scale liquid chromatography with small equilibrium constants (linear adsorption isotherms) ( B I 3 ) . Liu and co-workers realized all the predicted advantages of high-temperature, open-tubular liquid chromatography with 50- and 100-pm-i.d. capillary columns (BZ4).At 200 OC, over one million plates were obtained for acetophenone on a 19.6 m X 50 pm column. Slais et al. suggested and verified that analytes can be separated in a capillary in which the phase adjacent to the internal surface of the capillary is allowed to flow and provided a mathematical description of retention under theseconditions (BZ5,BZ6). Finally, Wankat developed a method for “scaling” chromatographic systems (BI 7). Rules were presented, applicable to concentrated isocratic systems, gradient elution, and displacement chromatography, which enabled the pressure drop and the degree of resolution to be kept constant. Optimization. DeStefano and co-workers described RPLC method development based on column selectivity ( B l 8 ) . Gustavo-Gonzalez and Asuero applied the extended Hildebrand solubility parameter treatment to optimize the RPLC determination of pharmaceuticals (BZ9). Hamoir et al. constructed models for the prediction of initial chromato- zyxw zyxwvu 502R zyxwv zyxwvutsrqp zyxwvutsr Analytical Chemistty, Vol. 66,No. 12,June 15, 1994 zyxwvuts graphicconditions in pharmaceutical analysis by RPLC (B2O). Heinisch and co-workers described several prioritized criteria when searching for the best eluent strength using interpretive methods (B21). Iwaki et al. utilized FUMI, an information theory-based chromatographic response function, to select the naphthylethylurea chiral stationary phase that provides the best precision of peak shape and overlap (B22). Palasota and co-workers described the application of the venerable sequential simplex algorithm for improved separation of Lphenylalanine, L-tyrosine, and L-tryptophan in a constrained simplex mixture space (B23). Vanbel et al. discussed chemometric optimization in drug analysis, providing a critical evaluation of the quality criteria used to determine drug purity (B24). Wieling and co-workers performed robustness testing of an optimized RPLC system for the separation of six sulfonamides using the rules of error propagation (B25).Wilce et al. applied experimentally derived retention coefficients to the prediction of peptide retention times, focusing on myohemerythrin (B26). Computer- OrientedApplications. Computer simulations have begun to play an increasingly important role in the optimization of separations and in the understanding of retention phenomena. Bowman and co-workers compared two commercially available, semiautomated HPLC solvent optimization software packages and found that the resultant optimized chromatographic separation is dependent on a combination of the operator’s objective, the capability of the software system, and the appropriateness of the data input (B27, 8 2 8 ) . Several other reports using in-house (B29) or commercial packages (B30-834) appeared in this review period for the direct optimization of chromatographic conditions. One software package was in the form of an expert system, devoted to the selection of optimization criteria (B35). Compound- Oriented Applications. Over 30 applications were reported (RPLC except as noted), including the separation of phenolic compounds in water by linear gradient elution (B36);L-phenylalanine, L-tyrosine, and L-tryptophan (B23); free cy-amino acids (B37);biogenic amines with reference to molecular structure (B38);triacylglycerols (B39);chlorophenols using a Doehlert design (B40); 1lsubstituted phenols ( 8 4 1 ) ;carboxylic acids, sugars, glycerol, and ethanol in wines (B42);carotenoids (B43);catecholamines (B44); 1,2-diacylsn-glycero-3-phosphocholine and 1,2-diacyl-sn-glycer0-3phosphoethanolamine in bovine milk (B45);thiocarbamates (846);plastics additives (B47);nitrosamines (B48);peptides (B2, B49); salbutamol and its decomposition products (BSO); nitrite, nitrate, and phenylenediamine isomers (BSI); doxorubicin, epirubicin, and their metabolites (B52);anthracyclines and their metabolites (B53);gentamicin in calf tissues (B54); and (6R)- and (6s)-leucovorin (B55). and Fell on strategies for peak-purity assessment, which highlighted many of the algorithms that can be applied with diode-array detection. The relative merits of the individual techniques are discussed, and a rationale is developed for their application (C4). Figures of Merit and Peak Modeling. The exponentially modified Gaussian (EMG) continues to be the model of choice in analytical-scale chromatography, although the number of significant papers published in this area decreased substantially. Jeansonne and Foley reported improved equations designed for electronic measurement of peak width and asymmetry for the calculation of chromatographic figures of merit for ideal and skewed chromatographic peaks reported (C5). Wu and Wei and Grimalt and Olive debated the merits and validity of a chromatographic form of the log-normal function (C6, C7). Deconvolution and/or Quantitation. Gerritsen et al. systematically evaluated generalized rank annihilation factor analysis, iterative target transformation factor analysis, and residual bilinearization for the quantitative analysis of diodearray detection data, examining the effect of interferents, peak resolution, relative peak heights, chromatographic reproducibility, and background absorbanceson the quality of the results (C8). Li et al. showed how similarity transformations can be used in conjuction with the generalized rank annihilation method (GRAM) to convert complex eigenvalues and eigenvectors that appear frequently when the generalized eigenproblem is solved to real eigenvalues and eigenvectors, thereby permitting spectra and profiles of pure constituents to be estimated (C9). Cladera et al. successfully employed a nonlinear leastsquares method for the deconvolution of severely overlapped peaks. Their results were independent of retention time, peak shape, or degree of overlap, but were sensitive to high degrees of spectral similarity among overlap components and the requirement to know every coeluting component (CIO). Josefson and Tekenbergs-Hjelte described a similar partial least-squares approach to deconvolution that requires mixtures of known concentrations and compared it to a generalized rank annihilation method for calibration (C1I). Liang et al. reported a new method for the detection and resolution of two-component mixtures of drug isomers based on heuristic evolving latent projections (HELP) which proved to be superior to factor analysis and evolving factor analysis (CI2). Later they used HELP to quantitate and then avoid errors in the perdendicular drop algorithm for the singlewavelength chromatographic integration of closely eluting peaks (CI3). Single-wavelength quantification was good, provided that the wavelengths chosen were close to the peak maxima. Keller and Massart used evolving factor analysis (CI4 ) for the deconvolution of overlapping peaks, Nelson reported a method for the deconvolution and subsequent accurate determination of overlapping peak areas (CIS),and Tauler et al. reported the deconvolution and quantitation of unresolved mixtures with HPLC-diode array detection using a selfmodeling curve resolution method based on different factor analysis techniques: evolving factor analysis and rank annihilation (CI6).Schostack and Malinowski applied window factor analysis and matrix regression analysis to peak zyxwvu zyx zyxwvutsr zyxwvu zyxwvu zyxwvutsr C. DATA ANALYSIS A revised printing of Dyson’s excellent 1990 monograph on chromatographic integration methods was issued during this review period (CI), with the same coverage of topics as described in the previous review (C2). Jang and Brown provide 45 references in their review of methodsfor peakidentification by analysis of mass spectrometric and diode-array detector data in the HPLC separation of nucleicacid constituents (C3). Complementing this was a review (95 references) by Castledine Analytical Chemistry, Vol. 66,No. 12, June 15, 1994 503R deconvolution; their results did not exactly agree with the known concentrations, but did agree with those obtained by rank and matrix regression analysis ((217). Bahowick and Synovec evaluated the effects of retention time precision, adsorption isotherm linearity, and detector linearity on the qualitative and quantitative analysis using the sequential chromatogram ratio technique (C18). Precise peak alignment to within 0.8% of the baseline peak width was necessary for qualitative interpretation of the ratio chromatogram. A peak-shape analysis method, based on the scaled point-by-point difference of normalized peaks, was adapted as a diagnostic test for peak-shape change and as a means of correcting the ratio value of injected concentrations. Retention Behavior. Forgacs et al. used multivariate mathematical-statistical methods to compare the retention behavior of 16 ring-substituted aniline derivatives and 22 ringsubstituted phenol derivatives on porous graphitized carbon and octadecylsilica columns (C19-C22) and found that principal component analysis followed by two-dimensional nonlinear mapping is the most appropriate method for evaluation of large data matrices in RPLC. The high percentage of variance explained in the first principal component implies that unbuffered acetonitrile-water and methanol-water eluents have common elution characteristics, but with slightly different selectivities as manifested in their second principal component. Kaarsnaes and Lindblom employed PCA to investigate the retention characteristics of both analytes and separation media in hydrophobic interaction chromatography (HIC) (C23). Peak Purity/Homogeneity. This continued to be one of the more heavily investigated topics, primarily because of its importance in the pharmaceutical industry. Castledine and co-workers examined several multiwavelength approaches to the assessment of chromatographic peak purity, including absorbance ratios (C24) and correlation-based algorithms (C25), and utilized principal component analysis for the assessment of peak purity of the model drug sulfasalazine (C26). Fabre and Fell compared the efficiency of spectral suppression, absorbance ratio, and spectral overlay for peak purity with a model system of overlapping cephalosporins, cefotaxime and theophylline (C27), and found that the absorbance ratio was the least effective. Keller and co-workers also considered peak purity (C28), but their approach to data analysis was based on evolving factor analysis (EFA) ( C I 4 ) , with papers devoted to artifacts in EFA from heteroscedastic noise, nonzero or sloping baselines, and the scan time of the diode array detector (C29, C30); they also compared heuristic evolving latent projections and EFA methods (C31). Lincoln et al. compared the multivariate analysis of diodearray and mass spectrometric data for the assessment of chromatographic peak purity of drugs. When principle component analysis is used to estimate the number of components in a single chromatographic peak, diode-array data suffer from inherent limitations of sensitivity and spectral similarity for the minor components compared to mass spectral data (C32). Vandyke and Wentzell described some limitations of evolving principal component innovation analysis (C33). Szabo and Maguire compared four parameters as measures of peak purity with photodiode-array detection-conventional peak purity, average wavelength of a peak spectrum, peak area ratio, and peak absorbance ratios-and found that peak purity was the most sensitive index of coelution (C34). Miscellaneous. Li and Pardue developed and evaluated an error-compensating predictive data-processing method based on the analysis of data on the leading edge of a peak and its subsequent extrapolation to a steady-state response that would be observed if the system were saturated with sample (C35). Jarvis and Kalivas described a library searching algorithm that is relatively insensitive to impurities for the identification of components present in multicomponent samples (C36). Hsu et al. reported an efficient data reduction procedure based on the Kendrick mass scale for the on-line liquid chromatography-mass spectrometry identification of compound type of analytes in hydrocarbon mixtures (C37). Curran and McKean used cross correlation with digital techniques to improve the signal-to-noise ratio by up to 35 times in flow injection analysis and high-performance liquid chromatography (C38). D. NORMAL PHASE In this section we discuss analytical liquid chromatography systems in which the stationary phase is more polar than the mobile phase. This includes as stationary phases solid adsorbents, bonded and immobilized liquids, and chiral columns operated with nonaqueous, nonpolar, or moderately polar mobile phases. The term normal phase usually implies that adsorption-displacement processes control retention, and with some exceptions this convention is followed here. General and fundamental studies of retention in normal-phase LC, particularly those that address adsorption-displacement processes, will be reviewed first, followed by reviews of more specific aspects of liquid chromatography in the normal-phase mode. Zhong and Meunier used interference theory to describe competitive, nonequilibrium chromatographic behavior for two (01) and three (02) components. In the first article, these authors extended previously developed interference theory to include coupling of adsorption equilibria and diffusion. Interference through diffusion was shown to play a secondary role. In the second article, variations in flow velocity due to adsorption were taken into account. Interference between components occurred through equilibrium and rates of adsorption, as well as through these variations in flow velocity. Mingalyov et al. studied the fine structure of both normal- and reversed-phase modified silicas using nitroxide radicals (03). In the case of modified, normal-phase silicas, efficient destruction of stable “arch structures” in the bonded phase was noted when basic mobile phases were employed. Quantitative structure-retention relationships (QSRRs) of a series of 1,4-benzodiazepine test solutes were used to identify important molecular processes in HP-liquid affinity chromatography, HP-reversed-phase LC, and HP-normal-phase LC (04). QSRR equations derived for normal-phase LC confirmed the importance of specific dipolar and chargetransfer interactions. QSRR-based models emphasized the distinctly different retention mechanisms in the three LC modes. As always, several reports were noted that described the role of the mobile phase in normal-phase retention. Hsu and Cooper (05) used system peaks to study the interactions of zyxwvuts zyxwv 504R zyxwvutsrq zyxwvutsrq AnalyticalChemistiy, Vol. 66,No. 12,June 15, 1994 solvent modifiers with an aminopropyl bonded phase. These authors noted a 'breathing effect" in the bonded phase due to incorporation of polar modifiers. They also argued that at higher modifier contents where the bonded phase is completely saturated with modifier, a partition model appears to be more appropriate in describing retention than the adsorptiondisplacement model. Good correlation between the Snyder solvent-strength parameter and solvochromatic parameters (dipolarity/polarizability (T), H-bond acidity (CY),and H-bond basicity ( P ) ) was noted in another report (06). The A correction to the *-parameter was shown to be minor, but statistically significant. Finally, a useful graph was presented which allows proper choice of a binary solvent mixture when a known solvent strength is needed (07). Only a few descriptions of new bonded or immobilized stationary phases for normal-phase use were noted. The first successful normal-phase application of silica-immobilized bovine serum albumin (BSA) was reported, however (08). Multifunctional solutes with hydroxyl and carboxyl groups were used as test solutes, and various combinations of hexane, neat diethyl ether, and HC1-saturated diethyl ether used as mobile phases. Retention data were consistent with normalphase behavior. Carbowax 20M thermally immobilized on controlled-porosity glass or silica gel was shown to be an effective stationary phase for the separation of benzodiazepines when used with simple binary normal-phase solvent mixtures (09). Normal-phase separation of aromatic hydroxy compounds at elevated temperature demonstrated on an capillary, open-tubular cyanopropyl column was also reported (010). Conventional solid adsorbents still attract attention. A new unmodified silica that shows promise for use in separating polar and basic compounds was described by Kirkland and co-workers ( 0 11). This new, highly purified, low-acidity porous silica (type B) was compared to conventional type A silica, and the effects of sample type and loading on retention, column efficiency, and peak shape were documented. The authors point out the advantages of normal-phase liquidsolid chromatography in certain separations (e.g., positional isomers) and suggest that this new type B silica may be suitable for applications that were previously restricted to the reversedphase mode. The special properties of Florisil were described in two reports ( 0 1 2 , 0 1 3 ) . In both, log-log plots of retention parameters on Florisil and silica were used to identify the unique selectivities of Florisil and the influence of mobilephase modifiers on its adsorption properties. Several highly specific and selective normal-phase separations were described. The separation of chloro-added and chloro-substituted PAH was studied on a variety of commercially available bonded, normal-phase columns ( 0 1 4 ) . Cyanopropyl silica was shown to be suitable for group separations of both chloro-added and chloro-substituted PAH; aminopropyl silica showed sufficient selectivity for group separations of chloro-substituted PAH only. An electrondonating stationary phase, 2-( 1-pyrenyl)ethyl silica also showed high affinity for the chloro-substituted compounds. Cleanup methods for the chloro-substituted PAH in environmental matrices were also described. The difficult separation of perhydro PAH was accomplished with an extremely polar tetranitroimidofluoreno stationary phase and very nonpolar binary FC-77/trifluorochloroethane mobile phase ( 0 1 5 ) . A diol column was shown to be very effective in the prefractionation and semipreparative isolation of individual compounds in aroma extracts of plants and foodstuffs (016). This application of the diol column is particularly enhanced by the use of highly volatile mobilephase solvents, since, under these conditions, nonpolar compounds (e.g., hydrocarbons) are not retained but polar compounds are easily eluted. Finally, bare silica was used to accomplisha class separation of triacylglycerolsand cholesterol esters as their hydroperoxides ( 0 1 7 ) . The increased number of reports of optically active stationary phases used in the normal-phase mode attests to the growing popularity of this approach to chiral separations. Guiochon's group generated adsorption isotherms of (-)- and (+)-methyl mandelate on a 4-methylcellulose tribenzoate phase in hexane-2-propanol (018). It was concluded that a simple competitive adsorption isotherm could be used to model chiral separations where the recognition mechanism is not well understood and adsorption behavior is not ideal. Pirkle and Welch studied the effects of nonspecific adsorption processes on chiral recognition (019),particularly the role of polar functional groups not required for recognition. Thirteen chiral stationary phases prepared from P-cyclodextrin and containing 3,5-dimethylphenylcarbamateresidues were described in another report (020). Enantioselectivity is apparently enhanced by a high degree of carbamate substitution on the P-cyclodextrin. Three families of displacers were synthesized and tested on Pirkle-type naphthylalanine silica chiral phases in the normal-phase mode ( 0 2 1 ) . Each family consisted of a large number of homologs which were generated by varying the lengthof thealkyl tail section and which covered a wide range of adsorption strengths. A chiral separation of P-blockers on bare silica after derivatization with one of three chiral reagents was also described (022). Finally, we again include here developments in detector technology which are of unique importance to normal-phase LC. As in previous reviews, reports of nonaqueous electrochemical detection for use with normal-phase LC were noted. For example, postcolumn amperometric detection in normalphase solvents improved the detection of 1,25-dihydroxy vitamin D3 and 25-hydroxy- 16-ene-23-yne vitamin D3 1000fold over UV detection ( 0 2 3 ) . Two reports described new initiatives in coupling normal-phase LC and mass spectrometers. Van Leuken and Kwakkenbos showed that the addition of gaseous ammonia to a thermospray ion source followed by filament-on ionization provided chemical ionization mass spectra dominated by the [M + NH4]+ ions ( 0 2 4 ) . This approach is particularly advantageous for the normal-phase mode where other ionization methods have not proven entirely suitable. Lawrence interfaced a normal-phase packedcapillary LC column to a magnetic sector mass spectrometer using continuous-flow fast atom bombardment ( 0 2 5 ) . zyxwvutsrq zyxwvuts zyxwvut zyxwvut zy zyxwv E. REVERSED PHASE Reversed-phase liquid chromatography (RPLC) continues to be the most popular mode of analytical LC. This popularity in usage is reflected in the continuing research interest in this mode of separation. Advances are being made in better understanding of the molecular mechanism of retention, in better synthetic schemes for reproducible preparation of the zyxwvut Analytical Chemisrty, Vol. 66, No. 12, June 15, 1994 e 505R stationary phases, in the design of stationary phases with greater pH stability and less susceptibility to secondary retention processes such as those exhibited by basic amines, and in many, many new applications. A book by Szepesi appeared, with the title, How to Use Reverse Phase HPLC (EZ).Interestingly, this is one of the few appearances of the word 9eversen, almost universally “reversed” is the preferred form. Also, a special issue of the Journal of Chromatography appeared, comprised entirely of invited papers and devoted to the retention process in reversedphase LC ( E 2 ) . This issue is likely to become a landmark publication and is interesting in showing the diversity of opinion that still exists concerning the retention process. Three of the most important and general of these papers will be discussed next, and many of the other papers will be cited further below. It is now generally accepted that the nature of the silica is one of the larger variables in the synthesis of traditional reversed-phase stationary phases. As well as pore diameter and volume, such criteria as type and concentration of trace metal impurities and pretreatment procedures make a large difference, even when the exact same bonding chemistry is used. Cox reviewed silica structure, and especially the issues regarding the deleterious effects of residual silanol groups of reversed-phase stationary phases ( E 3 ) . Carr et al. published an extremely important and valuable paper discussing the driving forces for retention in RPLC ( E 4 ) . Using alkylbenzenes as test solutes, they showed that most of the free energy of retention arises from net attractive processes in the stationary phase, and not from net repulsive processes in the mobile phase! This is further evidence that the solvophobic theory is not an accurate description of the retention process. They also found that the free energy of transfer of a methylene group from the mobile to stationary phase is similar to the energy of transfer of a methylene group from the same mobile phase to pure bulk hexadecane, further supporting the partition model of retention. In the same paper they compared measured and computed activity coefficients and showed that the regular solution theory is a grossly inadequate model of interactions in water and hydroorganic mixtures. Tijssen et al. thoroughly reviewed the existing lattice models of the stationary phase, especially those of Martire, Dill, and Scheutjens ( E 5 ) . While there are subtle differences among the three models, they are all similar in their generalized treatment of the stationary phase, and they emphasize entropy effects which arise from the molecular organization of chains. There are also differences and controversies which exist, especially between the Dill and Scheutjens models. The Dill model has been extensively verified experimentally, and it will be interesting to watch attempts at experimental verification of the Scheutjens model. The remainder of this section is broadly divided into categories of stationary and mobile phases. This was done for convenience of organization only, and it should be stressed that chromatographic retention is a function of the thermodynamic difference in the chemical potentials between the two phases. The “solvophobic theory”, which is still widely cited, does not account for the effects of the stationary phase. It bases retention on association of two solute molecules in a single solvent rather than on the transfer of a solute from one solvent to another. While important historically in that it provided the first physicochemical description of the retention process, it should not be viewed as a current level of understanding. Stationary Phases. Five other highly valuable reviews of relevance to stationary-phase structure and organization appeared in the special issue of the Journal of Chromatography ( E 2 ) . Rutan and Harris reviewed electronic spectroscopic investigations of the stationary phase in RPLC (E6). These include fluorescence, UV-visible absorption, and diffuse reflectance methods which have given insight into stationaryphase chain organization, orientation, and polarity and into the solute microenvironment when associated with the stationary phase. Sentell discussed both N M R and ESR methods of investigating stationary-phase structure ( E 7 ) . These investigations give direct measures of stationary-phase chain organization, including solvation effects, and can easily show both temperature and temporal effects. Gilpin reviewed his group’s extensive work over the past 15 years using principally N M R and IR to investigate the organization and structure of the stationary phase (E8). Wheeler et al. discussed phase transitions in reversed-phase stationary phases ( E 9 ) .In addition toa general review, they discussed the role of computer simulations in analyzing transition behavior and reviewed analytical theory and molecular simulation approaches to chromatographic stationary phases. Jaroniec reviewed partition and displacement mechanisms in terms of phenomenological thermodynamics (EZO).He represents the formation of the solvent-surface stationary phase via a displacement mechanism and the distribution of the solute between the mobile and stationary phases via a partition mechanism. Solvation of the stationary phase by the mobile-phase solvent(s) has been studied since at least 1980, but continues to be of interest. This solvation effect affects solute selectivity, reequilibration time following a gradient elution, and stationary-phase organization among other effects. Schunk and Burke published a very thorough review of the solvation layer mixture in terms of the length of the bonded hydrocarbon moieties, the surface bonding density, the hydrogen-bonding water adsorption at the silica surface, and the enhanced concentration of organicsolvent from the mobile phase (EZI ) . Poppe extensively treated distribution isotherms in reversedphase systems, including single-component and binary isotherms for both sample and mobile-phase components ( E 1 2 ) . He discussed the treatment of adsorption in terms of surface excess and showed the relevance to determination of the void volume for predicting elution curves in preparative LC, for understanding system peaks, and for insight into the sorption process. Bliesner and Sentell used deuterium N M R as a probe for stationary-phasesolvation (EZ3,EZ4).They used a unique method of solvating the stationary phase at common operating pressures before the N M R experiments, and their data should have more relevance to actual LC conditions than previous work where solvation has occurred at ambient pressure. Cole and Dorsey discussed the effect of stationary-phase solvation on shape selectivity (EZ5).They found that small amounts of hexanol added to traditional mobile phases did not increase shape selectivity, suggesting that changes in the solvation of the stationary phase did not impart a significant change in the level of surface ordering. Miyabe and Suzuki measured zyxwvutsr zyxw zyxwvuts zyxwvutsrq 506R zyxwvutsrqp AnalyticalChemistry, Vol. 66, No. 12, June 15, 1994 zyx zyxwvutsrqpo zyxwvuts zyxwvutsrq zyxwvut zyxwvutsrq intraparticle diffusion and adsorption on ODS silica and found that the isosteric heat of adsorption and the activation energy of surface diffusion were constant regardless of the amount adsorbed, suggesting that ODS has an energetically homogeneous surface (E16). The surface diffusion coefficient of p-tert-octylphenol was found to be about lV-lO-’ cm2/s. Shape selectivity of stationary phases, especially as compared between monomeric and polymerically derivatized materials, continues to be of interest. Sander and Wise, who pioneered this understanding of the selectivity differences available from the two synthetic processes, published a very thorough review of selectivity effects for the separation of both planar and nonplanar solutes (EZ7).There were also four noteworthy original works during this review period. Epler et al. compared 60 (!) commercially available and 5 experimental stationary phases for the separation and recovery of seven carotenoid compounds (EZ8).They found that polymeric C- 18 phases generally provided better selectivity for difficult pairs than did monomeric phases. Van Heukelem et al. found that polymeric C-18 columns offered superior selectivity for structurally similar phytoplankton pigments than did monomeric C-18 columns (E19). Pyell et al. found similar improvements in selectivity with a polymeric column for tetrachlorodibenzo-p-dioxin(TCDD) isomers (E20). Garrido and Zapata reported improved separations of chlorophylls using a polymeric column ( E 2 I ) . The separation of highly basic solutes on silica-based reversed-phase stationary phases is troublesome because of strong interactions possiblewith any remaining hydroxyl sites. While no new approaches to the design and synthesis of basedeactivated columns were found during this review period, there were five reports of studies of existing columns and methodologies. Nicholls et al. compared base-deactivated and polymeric (resin-based) columns for the separation of anthracyclines and their metabolites (E22). They found that while the resin-based columns eliminate any strong interactions from residual hydroxyl sites, they were too retentive for the compounds of interest. Bogusz et al. compared the chromatographic properties of seven commercially available columns which are marketed as “base-deactivated” materials (E23). Using acetonitrile-based mobile phases, they found silanol effects to be small, but present, in all the columns examined, and they recommend that the useof amine modifiers as a competing base is still necessary. McCalley evaluated columns for the separation of tobacco alkaloids and found that a test procedure originally published by Engelhardt and co-workers, using pyridine as an additional probe compound, was helpful in selection of the best available column (E24). Li studied the silanol effect on the separation of metal complexes (E25), and Salamoun et al. described a cationexchange separation of choline and acetylcholine on the free shielded silanols of reversed-phasecolumns (E26). They found that these columns exhibited better pH stability and longer lifetimes than normal silica-based cation exchangers, and they also noted that acetylcholine is an effective and sensitive test compound for the measurement of adsorption by residual silanols. Pore size effects of the base silica also continued to be of interest. Northrop et al. studied the isocratic retention of a series of polystyrenes on a C-4, bimodal pore diameter column using two different binary mobile-phase systems (E27). Nagasawa et al. studied the effects of pore size by using octadecylsilanized porous glass (E28). As porous glass can be made with a very narrow range of pore size distribution and similar pore shape regardless of size, it provides a nice medium for study of pore size. They found that retention is inversely proportional to pore diameter, that is, the larger the pore diameter the shorter the retention of test probes. This is likely simply due to a reduction in surface area and resulting decrease in the volume phase ratio. Itoh et al. compared nonporous, 100- and 300-AC-18 silicas for the separation of various small molecules (E29). They found that steep gradients gave very rapid separation times on the nonporous material. Other studies of retention phenomena also appeared. Cole and Dorsey investigated the temperature dependence of retention using both van’t Hoff analysis and differential scanning calorimetry (E30). They noted phase transitions of reversed-phasestationary phases with bonding densities greater than 2.84 pmol/m2. They also measured thermodynamic constants for the transfer of solutes from the mobile to stationary phase and compared these values to previously reported values for bulk phasepartitioning. Their data showed that the retention process is not well modeled by bulk-phase oil-water partitioning, and they found that the entropic contribution to retention becomes more significant with respect to the enthalpic contribuition as the stationary phase bonding density increases. Buszewski and Kulpa investigated the influence of bonding density on the determination of the void volume (E31),and Jakus compared adsorption and partition models for the separation of polychlorinated biphenyls (E32). Tanaka et al. reviewed stationary-phase effects, especially selectivity issues, for silica-based reversed phases, polymerbased packings, and graphitized carbon packing materials (E33). Solvatochromic analysis of retention mechanisms did not receive as much interest as during previous review periods. Carr published a very thorough review of the use of the Kamlet-Taft scale for the study of chromatographic retention processes (E34). Vallat et al. used solvatochromic analysis to show that for lipopholicity determinations an octadecyl poly(viny1 alcohol) copolymer stationary phase has a closer resemblance to the 1-octanol-buffer system than does an octylsilane phase (E35). Park et al. used solvatochromic analysis to show that four different brands of cyano bonded phases which have similar bonding densities and which were all prepared from monofunctional cyanopropylsilane reagents have quite different hydrogen-bonding donor and acceptor strengths (E36). Park et al. also used solvatochromic analysis to compare the use of a C- 18 column and a bonded cyclodextrin column for the separation of small molecules (E37). While a separate section of this review deals with physicochemicalmeasurements, it is useful to cite here three reviews that have special relevance to reversed-phase LC. Kaliszan extensively reviewed quantitative structure-retention relationships, especially as applied to reversed-phase LC (E38). Lambert reviewed the modeling of oil-water partitioning and membrane permeation using RPLC (E39), and Dorsey and Khaledi reviewed hydrophobicity estimations by RPLC (E40). Analytical Chemistry, Vol. 66, No. 12, June 15, 1994 507R zyxwvutsrq Three reports appeared on the practical use of reversedphase columns for the adjustment of selectivity. DeStefano et al. discussed the use of C-8, phenyl, and cyano columns for the adjustment of band spacing (E41). Wells et al. described selectivity differences on seven different branched and linear alkyl bonded phases, but concluded that selectivity effects due to variation in the mobile phase dominated the results ( E 4 2 ) . Chen et al. discussed a very important problem in routine LC analysis, that of column equivalency (E43). They described a method for standardization of different C- 18 columns based on a linear retention equation and assuming that most of the difference in retention is from a difference in phase ratio. The synthetic process continues to generate much interest, both in the design of new synthetic schemes and also in the use of novel ligands. Pesek and Williamsen reviewed the synthesis and use of bonded liquid crystal materials for LC and noted that the separations appear to occur based on such molecular properties as length/breadth ratios and planarity ( E M ) . Scott and Simpson reviewed developments in bondedphase synthesis carried out at Birkbeck College, London (E45). In particular, they discuss the fluidized-bed method and describe a novel squalane type of phase where the long hydrocarbon chain is attached to the silica surface at a number of different points along the chain. This phase behaves as a short-chain phase and is shown to be suitable for the separation of biomolecules. In original publications, Wirth and Fatunmbi described a highly novel approach using a horizontal polymerization of mixed trifunctional silanes (E46). This approach was designed to combine the hydrolytic stability of selfassembled monolayers with the selectivity and reproducibility of monomeric stationary phases. Kirkland et al. described improved C- 18 materials prepared from diisobutyl-n-octadecylsilanes and showed that this material provides higher stability with low-pH mobile phases and with high temperatures (E47). Kibbey and Meyerhoff described the synthesis of a covalently bound tetraphenylporphyrin-silica gel stationary phase (E48)and showed its applicability for shape-selective separations of PAH compounds (E49). Kimata et al. described the synthesis of electron-acceptor and electron-donor phases (E50) and nitrophenylethylsilylated silica (E51)and showed their applicability for the separation of dibenzo-p-dioxins. Basiuk and Chuiko synthesized dioxopyrimidine bonded phases and showed their applicability for the separation of nucleic acid components ( E 5 2 ) . Restricted access packings have become popular since about the middle 1980s. These materials are dual-mechanism stationary phases which have a hydrophilic exterior, with hydrophobic pores so that biological samples can be directly injected without protein precipitation. The unique feature of these packings is that they prevent the access of matrix components such as proteins while selectively retaining the drug components and their metabolites. There were four reports of studies of new and existing materials during this review period. Kimata et al. compared a number of newly synthesized restricted access materials with two commercially available phases for retention and selectivity for various drugs (E53). Ge et al. showed that polypyrrole is an effective stationary phase for basic drugs in the presence of proteins ( E 5 4 ) . Specifically, they showed that proteins elute at the dead volume while high retention was observed for caffeine and theophylline. Beth et al. reported the use of a beaded, hyper-cross-linked microporous polystyrene material, which is functionalized with tris groups at the outer surface of the particles, and showed that it was useful for both sample cleanup and analytical separations (E55).Pompon et al. showed that the use of a precolumn of a restricted access material allows elimination of proteinaceous material and allows precise kinetics of the degradation of an oligonucleotide in cell culture to be measured without radiolabeling or sample preparation (E56). The use of the word “polymeric” to describe stationary phases prepared from di- and trifunctional silanes, while historic, has become confusing. The introduction several years ago of polymeric resin-based reversed-phase stationary phases has added a totally different meaning to polymeric stationary phases, and careful choice of descriptive words is necessary. As well as cross-linked resin materials, there is also much research interest in polymer coating of traditional silica or alumina particles. The interest in these phases is driven largely by the greater pH stability, and interest in these phases, as shown by new research reports, continues strong. Hanson et al. published two reviews of both the synthesis and retention properties of polymer-coated stationary phases (E57, E58). In terms of new reports, interest in the cross-linked resins was robust. Frechet’s group published two reports of the synthesis and characterization of macroporous polymeric rods as stationary-phase media and showed that these materials can be made with high permeability, allowing good flow properties (E59, ,5760). Hjerten et al. also described the synthesis of continuous polymer beds, which can be regarded as rods with channels, and showed that a refined synthetic process yields greater pH stability than an earlier method (E61). Hosoya and Frechet described a synthetic method for the preparation of monodisperse macroporous poly(styrenedivinylbenzene) particles in the 5-6-pm range and characterized their chromatographicand flow properties (E62). Hosoya et al. discussed the control of steric selectivity of polymer gels provided by diluents and cross-linking agents (E63). Hirayama et al. described the synthesis and characterization of new resin materials prepared from N,N-dialkylacrylamide (E64, E65) and from the suspension copolymerization of alkylvinyl ether with triethyleneglycol divinyl ether (E66). Jagodzinski et al. described the preparation of a 4-pm polystyrenedivinylbenzene resin, and its surface derivatization with cyano and diol groups (E67). Polymer-coated materials also generated continuing interest. Erler and Heublein described the synthesis and characterization of amino-functionalized materials derived from polybutadiene epoxide-coated silica (E68). Hanson et al. designed and synthesized novel reversed-phase materials of nonporous and porous polymethacrylate-coated silicas (E69). They showed that, by varying the hydrophobicity of the ‘polymer coating, selective unfolding of polypeptides can be achieved, allowing manipulation of the chromatographic profile. Forgacs compared the retention characteristics of 16 aniline derivatives on polyethylene-coated silica and a traditional C- 18 column and, by principal component analysis, showed that the retention behavior of the two columns was slightly different (E70). Hanggi and Marks discussed zy zyxwvuts zyxwvut 508R Analytical Chemistry, Vol. zyxwvutsrq 66,No. 12, June 15, 1994 zyxwvutsrqpo zyxwvutsrq reversed-phasematerials prepared by coating zirconia particles with polybutadiene and showed that these materials remain chromatographically stable even after exposure to 1 M NaOH at 100 OC (E7Z)! Garbow et al. combined structural characterization by solid-state 13CN M R and measurement of chromatographic performance to study various polybutadiene-coated alumina materials (E72). New to this review period is a substantial interest in the use and study of graphitized carbon reversed-phase stationary phases. Forgacs et al. were especially active, publishing five papers dealing with this subject (E73-E77). They investigated the influence of various physicochemical parameters on the retention of some ring-substituted phenols (E73, E74) and ring-substituted aniline derivatives(E75). They also compared the graphitized carbon column and a C-18 column for the separation of some monoamine oxidase inhibitory drugs (E76) and phenol derivatives (E77). Mada et al. also used a carbon column for the separation of unsaturated disaccharides (E78) and found that the elution order differed greatly from traditional silica bonded phases. Porous zirconia particles also received some interest. Yu and El Rassi synthesized microspherical zirconia particles and surface derivatized them with both monomeric and polymeric octadecylsilane reactions (E79). They found the surface coverage to be equivalent to octadecyl silica materials, and they compared the two modified zirconia materials for various separations. Wirth et al. derivatized porous zirconia with various ligands, including C- 18, carbohydrate, and Cibacron Blue F3GA groups, and found the pH stability of these materials to vary from pH 10.5 to pH 13 (E80). Mobile Phases. As reported in thestationary Phase section, there were a number of very important reviews dealing with the role of the mobile phase which were published in the special issue of the Journal of Chromatography ( E 2 ) . Tan and Carr published a very important paper discussing extrathermodynamic relationships, specifically the relationship between slopes (S)and intercepts of plots of In k'vs mobile-phase composition (E8Z). They show that the linear relationship which is often found betweens and In k',, the capacity factor in 100%water, can be an artifact that results from statistical considerations and recommend a new form of this plot for methods development, based on an average k'value. This paper should be read by all workers interested in mobile-phase effects and the development of "expertn systems. Valkd et al. also discussed the S parameter, albeit from a much more pragmatic viewpoint (E82). Snyder et al. developed a classification of solvents based on their dipolarity and hydrogen-bondingacidity and basicity as measured by the Kamlet-Taft solvatochromic scheme (E83). They compared this classification with the well-known Snyder solvent triangle and found general similarity (E83). Jandera reviewed the correlation of retention and selectivity with interaction indexes and with lipophilic and polar structural indexes (E84). Smith also reviewed functional group contributions to retention (E85). Tchapla et al. discussed molecular interactions based on their extensive study of homologous series (E86).Park et al. discussed the utility of the UNIFAC activity coefficient determination method for understanding the magnitude of solute-solvent interactions (E87). They showed that while this method is not accurate enough to beuseful for quantitative determination of retention, it is useful in qualitatively explaining a variety of issues. Equations governing the relationship between log k' and the volume fraction of organic modifier generated considerable interest during this review period. As well as being of interest for optimization of separations and use in expert systems, these equations are also relevant for the calculation of log k',, the capacity factor in a mobile phase of 100% water. This value has use in hydrophobicity and biopartitioning estimation and is impossible to measure for most solutes as they are totally retained with this mobile phase. Hsieh and Dorsey showed that log k', values are best estimated by extrapolation of the solvatochromic E ~ ( 3 0 scale, ) rather than by a volume percent solvent measure (E88). The use of the S index, discussed earlier, also generated considerable interest. In a series of papers, Chen et al. discussed the effects of molecular structureon both log k', and t h e S index (E89-E92). SadlejSosnowska and Sledzinska measured capacity factors for five steroid hormones in both methanol and acetonitrile mobile phases and evaluated five models of chromatographic retention (E93). All workers in this area are again urged to see the paper by Tan and Carr (E8Z). Modeling and simulation of retention also generated considerable interest. Most of this interest is driven by the desire to generate reliable expert systems. Valko and Slegel investigated a number of molecular parameters for the prediction of retention of structurally unrelated series of drug molecules (E94). Lopes-Marques and Schoenmakers (E95) and Lewis et al. (E96, E97) in three papers appearing back to back described models to account for the simultaneous effects of pH and organic modifier concentration. Jakus and Jakus and Miertus described a new approach to theoretical evaluation of the Gibbs free energy of solvation and applied it to the prediction of retention in RPLC (E98, E99). Wang et al. described a method of predicting retention from the boiling point of homologs (EZ00).Woodburn et al. investigated the energetics of solute retention and correlated retention with solute topology (EIOZ, EZ02). Quantitative structure-retention relationships and linear solvation energy relationships also continued to generate interest. Kaliszan et al. used QSRR techniques to investigate the mechanism of retention of benzodiazepines on affinity, reversed-phase, and adsorption columns and showed that retention on the reversed-phase column was from a combination of dispersive and electrostatic intermolecular interactions between the solute and molecules of both stationary and mobile phases (E103). Roses and Bosch used a normalized ET parameter for the prediction of retention (EZ04), and Hu and Hao compared two methods of calculating hydrogen bond acidity and basicity values for use in the Kamlet-Taft solvatochromic equation (EZOS). Theuse of additives to themobile phase, both to dynamically block residual silanols and to add special selectivity to the separation, also continues to generate interest. There were two reports of studies of silanol masking; Nowicky et al. described the use of inorganic salts to improve peak shapes of alkaloids (EZOb),and Stoev and Uzunov studied various amine additives for the improvement of separation of two basiccompounds (EZ07). Aiken et al. added sodium dodecyl sulfate (SDS),at concentrations below the critical micelle zyxwvutsrq zyxwvutsr zyxw zyxwv zyx Analytical Chemistry, Vol. 66, No. 12, June 15, 1994 0 509R zyxwvuts zyxwvutsrq zyxwvutsrq zyxwvuts zyxwvutsrqp concentration, to a mobile phase to allow direct injection of serum for porphyrin analysis (E108).This causes the serum proteins to elute at the void volume, and limits of detection for this direct injection technique then averaged 0.06 pmol for seven different porphyrin compounds. Tsukahara et al. investigated the use of octane as an additive for the separation of metal-tetraporphyrin complexes (E109). They reported that the addition of octane changed the elution order, improved resolution, and reduced analysis time. Park et al. added calix[6]arene-p-sulfonate to a mobile phase for enhanced separation of some monosubstituted phenol isomers (E110). They reported that this additive decreased retention times but increased the selectivity between the isomers. Finally, Bernal et al. studied the use of methyl and ethyl acetates as organic modifiers for the separation of impurities from steroids (El11 ). There were several important thermodynamic studies during this review period. Alvarez-Zepeda et al. described a thermodynamic study of the differences between acetonitrilewater and methanol-water mobile phases (El12). They related the enthalpy and entropy of solute transfer to the solute partial molar excess quantities and found that while methanol solution chemistry is governed by solvent-solvent hydrogen bonding, entropic effects arising from the solvation of solute molecules by clusters of acetonitrile govern solute retention in the acetonitrile-water system. Cole et al. discussed the temperature dependence of retention and from van’t Hoff analysis concluded that the hydrophobic effect is not the driving force for retention except in highly aqueous, hydrogen-bonding solvents (El13). Wu and Wu investigated the temperature dependence of separation of C-25 epimers of steroidal sapogenins and found that separation of the epimers increases with a decreasein column temperature, with optimal resolution within the temperature range of 0-10 OC (E114).There is much accumulating evidence that low temperatures can enhance shape selectivity on both monomeric and polymeric stationary phases, and this is often an underutilized method of improving resolution for difficult separations. Salo et al. (El15) and Han et ai. (El16)also described the temperature dependence of retention of retinoates and alkaloids, respectively. Berthod et al. published two papers on the use of oil in water microemulsions for mobile phases in RPLC (El17, E118). They characterized efficiency and retention as a function of the microemulsion composition (El17)and showed applicability for rapid screening of illegal drugs in sports (El18). The characterization and description of selectivity also was of interest. Jaroniec and Gilpin described a combined partition-sorption mechanism for describing nonspecific selectivity (El19). Pietrogrande et al. used principal component analysis (PCA) and cluster analysis to study specific selectivity effects of different mobile and stationary phases for the separation of flavonoids, and the PCA showed that solvent type is the most significant factor in clustering chromatographic data (El20). West and Mowrey characterized the selectivities of 12 reversed-phase solvents, using three probe molecules and the 2-ketoalkane retention index system (E121).Sutcliffe and Corran compared the selectivity of RPLC, capillary electrophoresis, and micellar electrokinetic capillary chromatography for the separation of neurohypo510R AnalyiicalChemistry, Vol. 66, No. 12, June 15, 1994 physeal peptides and analogs (E122). A number of other interesting and useful fundamental papers also appeared during this review period. Moeckel et al. discussed the use of high-precision gross retention times of the n-alkanes pentane to heptadecane for determining column dead volume (E123).They reported that there was an apparent linear decrease with increasing carbon number, but if retentiondata arecorrected for poreexclusion, a uniform dead volume is obtained. Glavina and Cantwell studied the role of the compact part of the electrical double layer in the simultaneous sorption of different ions of the same charge on a RPLC stationary phase (El24). Wells and Clarkcontinued their studies of the hydrophobic substitutent contributions to retention (El25). Barman and Martire thoroughly studied the factors influencing retention and resolution of substituted alkylbenzenes (E126).Dreyer et al. showed that, with care, retention time precision and reproducibility can be reduced to a standard deviation of 0.001 (!) and showed that there is an apparently linear decrease in effective dead volume with increasing solute size, which they attribute to steric exclusion effects (E127).Klimes et al. studied the relationship between log k’ values and structure of a group of dipyridyl sulfides (EI28).Finally, Yamauchi showed that retention indexes of phenols can be used for prediction of retention and selectivities of mobile and stationary phases (El29). F. BIOPOLYMER SEPARATIONS Publications regarding biopolymer separations using HPLC have been insightful and prolific over the two years covered by this review, December 1991-December 1993. Roughly 100 abstracts regarding macromolecule separations were retrieved from CAS searches every two weeks during this period; as a result, it is possible to provide only a minute segment of the new information available in the present review. We have attempted to center our coverage on fundamental advances of general interest, since specific applications reviews are available elsewhere. Many of the articles discussed here are the result of papers presented at the International Symposium on High-Performance Liquid Chromatography of Proteins, Peptides and Polynucleotides. Manuscripts submitted from the 1 lth (1991, Washington, D.C.) and 12th (1992, Sydney, Australia) Meetings were published in special symposia volumes of the Journal of Chromatography (Fl, F2). Other important contributions were collected from symposiavolumes from the 15th (Basel) and 16th (Baltimore) International Symposia on Column Liquid Chromatography (HPLC ‘91 and HPLC ‘92, (F3, F 4 ) ) . A comprehensive review of advancements in the area of size exclusion chromatography is provided elsewhere in this issue. An excellent synopsis of peptide and protein separations is provided in the biannual Applications Review in this journal (F5).Oligonucleotide separations and fast protein separations using HPLC and HPCE were recently reviewed by Baba (F6)and Wu (F7), while a general overview of the application of HPLC for large-scale work was contributed by Jungbauer ( F 8 ) and Wisniewski (F9). A critical comparison of the denaturing effects of various chromatographic techniques for biopolymer separations was provided by Sadana (FlO),pH and buffer effects on peptide separations were discussed by Young and co-workers (FI1 ), and a summary of various approaches useful zyxwvutsrqpo in sample preparation for bioseparations was furnished by Majors and Hardy (F12). Reviews covering chromatographic methods and applications relating to food science in particular were published in a special volume of the Journal of Chromatography (F13). Column Packings. Investigations of new packing materials for biopolymer separations have commanded significant attention over the last two years. Because of the restrictive pH limitations and lengthy analysis times often imposed by conventional HPLC supports, interest continues in developing convenient alternatives. For example, Kirkland introduced a new superficially porous silica-based support for fast biopolymer HPLC with 30-nm porescontained within a l-mmthick outer layer on a spherical solid core. These Poroshell supports exhibit much improved mass-transfer characteristics and can be used for size exclusion separations or derivatized for rapid reversed-phase separations (FI4). Honda et al. examined the use of nonporous silica ultramicrospheres blended with polyethylene particles as reversed-phase composite packings that are useful for performing separations of proteins over a very broad range of molecular weight (F15).Hearn and co-workers investigated the use of C-l8-, Cibacron Blue-, carbohydrate-, and polybutadiene-modified porous zirconia particles as supports for macromolecule separations and found these phases to besuperior to silica with respect to pH stability, and superior to organic polymer-based supports with respect to mechanical rigidity (2716). Yu and El Rassi investigated the use of nonporous C- 18-coated zirconia for conventional HPLC solutes, peptides, and proteins (F17)and likewisefound the mechanical stability and extended pH range (2-12) associated with these phases to be particularly attractive. Conroy and co-workers reviewed the chromatographic properties of alumina-based stationary phases and contrasted them with silica supports (FI8),while Haky et al. thoroughly examined the comparative performance of reversed-phase aluminas for peptide separations (FI9). In summary, Haky and coauthors found that narrow-pore (11 nm) aluminas behave equal or superior to wide-pore (21 nm) phases, and phases consisting of fused microplatelets provide superior resolution to spherical aluminas. Ladisch et al. introduced the use of a novel continuous stationary phase in which yarns made from 10-20-mm phenylene phthmalamide are rolled and packed into conventional HPLC columns (F20). These phases are highly stable to both temperature and pH and were found to be particularly usefui for protein separations under high eluent velocities. King and Pinto studied nonporous quartz fibers as a novel media for protein purification (F2I). Although the short fibers investigated demonstrated somewhat limited sample capacity, the low operating pressures associated with these fibers permit exceptionally high linear velocities. The applications of perfusion (gigaporous) chromatographic media for macromolecule separations have been extended by Regnier’s group to include ion-exchange and immunoaffinity separations of proteins (F22, F23). Using a five-step synthesis and derivatization sequence, poly(styrenediviny1benzene)-based ”fimbriated” sorbents were prepared that exhibit excellent stability and high protein-loading capacity (F22). An extensive mathematical framework for perfusion chromatography was further advanced by Liapis and co-workers (F24, F25). According to this model, the improved efficiency associated with perfusive media is a result of the enhanced intraparticle mass transport associated with intraparticle fluid flow. Rodrigues et al. likewise examined the importance of intraparticle convection for gigaporous supports (F26), as did Carta and co-workers (F27). High-performance membrane chromatography (HPMC) is garnering extensive attention as suggested by the growing number of citations recovered for this review. Belenkii et al. extended conventional gradient HPLC theory to gradient HPMC for proteins (F28), while Cramer and co-workers examined the utility of an ion-exchange membrane system for both analytical and preparative-scale protein separations using gradient elution (F29). Frey et al. examined the separation efficiency for stacked membranes of poly(viny1 chloride) coated with a polyethylenimine anion exchanger and report that dispersion inside the membrane is the dominant band-broadening mechanism (F30). Tennikova and Svec considered several separation parameters using ion-exchange, hydrophobic interaction, and reversed-phase membranes and compared them with conventional HPLC columns; the convective flow associated with the membrane separations is found to significantly enhance the speed and efficiency of the former for protein purification (F31). Interest also continues in the use of hydroxyapatite (HAP) and related phases for selective protein separations. Crystalline porous and nonporous pyrophosphates of Mg, Ca, Sr, Mn, and Zr were prepared by Inoue and Ohtaki (F32). Although the authors found retention behavior similar to HAP for Ca and Sr phases, Mg, Mn, and Zr varied significantly, providing a new mode of selectivity for some separations. Both membrane proteins and water-soluble proteins were separated when complexed with sodium dodecyl sulfate using HAP phases according to Lundahl et al., who report a positive correlation between polypeptide chain length and elution order as a function of the number of SDS binding sites on the proteins (F33). Vola, Lombardi, and Mariani compared conventional HAP with a ceramic-based HAP for the purification of monoclonal antibodies and discovered the ceramic material to be much more mechanically stable, thus permitting higher flow rates with similar resolution to the conventional materials (F34). Similarly, Aoyoma and Chiba used spherical HAP beads to separate IgA and IgM monoclonal antibodies with quantitative recoveries (F35). In a final application, Kishino et al. demonstrated that HAP is useful as the analytical column in the determination of al-acid glycoprotein from serum, when coupled when an anion-exchange column for sample cleanup (F36). In other representative applications of new chromatographic phases, Hodges, Unger, and co-workers evaluated the use of porous and nonporous silica coated with polymethacrylate to produce stationary phases varying in their hydrophobic character. These supports were found to be convenient for manipulating the unfolding of polypeptides (F37)and proteins (F38) in order to selectively tailor the retention process. Kutsuna and co-workers developed a new synthetic procedure for producing a polymer-coated amino-bonded stationary phase that demonstrates improved stability for the separation of carbohydrates and nucleotides as compared with conventional amino-bonded silicas (F39). Choma and Dawidowicz published results in which Carbowax 20M-derivatized silica zyxwvut zyxw Analytical Chemistry, Vol. 66,No. 12, June 15, 1994 511R zyxwvutsrqpo zy zyxwvuts was used for normal-phase, reversed-phase, and size exclusion protein HPLC (F40),while Pochapskey and Gopen prepared a new stationary phase based on the covalent attachment of benzylsilane (which mimics phenylalanine) to silica. Such an approach is particularly useful for studying free energies of interaction of amino acid side chains using this so-called “mimic” stationary phase (MSP) (F41).Finally, Takenaka and co-workers used benzoic acid derivatives covalently attached to aminopropyl silica for the selective separation of oligonucleotides through an intercalative mechanism (F42). Reversed Phase. There is a tremendous body of scientific literature covering the separation of peptides using reversedphase HPLC supports. In an effort to improve the efficiency of method development and in light of the significant physicochemical information that can beobtained from RPLC, interest continues in the developing optimization strategies for predicting peptide retention. Lundell critically evaluated the use of gradient predictions for peptide separations and concluded that, with selection of a proper calibration gradients, gradient optimization is highly acccurate (F43). Lundell and Markides further illustrated the development of an optimization strategy for the separation of angiotensin from a complex matrix (F44), while Moritz and Simpson developed a continuous gradient elution method for capillary RPLC separations of peptides and proteins (F45). With regard to micellar RPLC, Kord and Khaledi examined the selectivity of various organic modifiers for the separation of amino acids and peptides and illustrated that the conventional Snyder classification of organic solvents is not directly applicable to micellar-based separations (F46). Hearn and co-workers successfully extended their previous work in peptide predictive scales by deriving amino acid group retention coefficients from peptide subsets related to the protein myohemerythrin under a variety of mobile-phase and stationary-phase conditions ( F 4 7 ) . Chabanet and Yvon proposed a new model for amino acid indexes in which the contribution of each residue to overall retention decreases as function of peptide length (F48). In contrast to previous approaches, a nonlinear multiple regression analysis was used to generate the experimental retention set, which provides improved predictive accuracy for longer chain peptides (>10 residues) in which secondary structure becomes more important. In assessing the influence of peptide-protein conformation on retention (and vice versa), Unger et al. developed a retention model for polypeptide and protein separations that reflects the contributions of stationary-phase chain mobility and steric effects in inducing conformational changes, as well as the hydrophobicity of the stationary phase itself (F49). Hearn et al. examined the thermodynamic behavior of bombesin, P-endorphin, and glucagon on (2-18 and C-4 over a large range of temperatures (F50)and suggest that the stationary-phase interactive surface contact area (5’)is dramatically altered between 50 and 60 “C, corresponding with disruption in the helical segments of these peptides. Funasaki and co-workers examined the retention of cyclic dipeptides including five diastereomeric pairs on reversed-phase supports and rationalized their retention using a holistic molecular surface area approach ( F 5 2 ) . Although such an approach requires considerable prior conformational knowledge, it is generally applicable to larger peptides as well. Houghten and co-workers examined the ability of proline (F52) and cysteine (F53) to induce secondary conformation in a series of polypeptide chains and report that the type of helical conformation induced by the C-18 stationary phase is related to the position of the amino acid substituent and the net hydrophobicity of the peptide. Of particular note in the proline study ( 5 2 ) , the antimicrobial activity of the model peptides investigated varied inversely with the reversed-phase retention time, which clearly demonstrates the efficacy of RPLC for modeling biological activity. Otvos et al. examined the retention behavior of a series of glycopeptides to which mono- and disaccharides were attached and discovered that decreased retention times were directly correlated to a higher extent of glycosylation (F54). This likely reflects induced conformational changes as well as the net increase in the hydrophilicity of the peptide chains. Reversed-phase HPLC for the separation of large nucleic acids is also commonly practiced, and similar to peptide separations, retention is found to be a function of chain length, base (residue) composition, and secondary structure of the molecule. For example, Huber et al. separated detritylated oligonucleotides using reversed-phase cross-linked poly(styrene4ivinylbenzene) phases with a particlediameter of 2.3 mm (F55). Treatment of the matrix with poly(viny1 alcohol) during polymerization of the support was found to improve the resolution of phosphorlyated species, which eluted based on sequence ratio in the order C C G C A < T. To better explain the retention of nucleic acids, Liautrad has developed a “multiple-point interaction theory” that expresses nucleic acid retention in terms of simultaneous solute interactions at several stationary-phase sites (F56). Both reversed-phase and hydrophobic interaction chromatography are widely applied to the separation of proteins. The relative contributions of partitioning vs adsorption have not been easily understood for protein RPLC, although studies in this area have been prolific for some time. In some of the most recent work, Koyama et al. considered column length and mobile-phase composition over a broad range of solute molecular weights and conclude that the partition model is better supported by their experimental data, although the influence of partitioning on separation is limited under steep gradient conditions (F57). Often superior chromatographic resolution for proteins is provided using acidic mobile phases (e.g., 0.1% TFA). Since the silica surface is hydrolytically sensitive, column instability is frequently observed. Kirkland et al. demonstrated the efficacy of their monomerically derivatized C-8 phases (in which silane isopropyl groups provide steric protection of the underlying silica) for the separation of wheat proteins (F58).In other work, Olson and Gehant investigated ultrafast reversed-phase and ion-exchange HPLC for the separation of proteins derived through recombinant DNA technology using columns packed with a gigaporous (400-nm pore) stationary phase (F59). This application punctuates the future direction of HPLC for expedient, on-line HPLC analysis in the biotechnology sector. Karsanas and Lindblom used multivariate analysis to map the selectivity of hydrophobic interaction media (F60). The HIC retention order for seven proteins was compared with literature data on hydrophobicity indexes, surface charge, etc., to assess the important factors in retention; for phenyl and butyl gels in particular, both hydrophobicity and surface charge zyxwvutsrqpo 512R 9 zyxwvutsrq Analytical Chemistty, Vol. 66,No. 12, June 15, 1994 zyxwvutsrq zyxwvutsrqp zyxwvutsrqp zy zyxwvu (or lack of surface charge) were found to be important in elution, whereas other HIC media were much less susceptible to surface charge. In a unique practical application, Geng and Chang used HIC successfully as a means to separate denaturing agents from proteins such that protein refolding is facilitated in the HIC environment (F61). Bertrand and co-workers examined the separation of a fungal proteinase using C-18 supports that were coated with poly(oxyeth1ene)type nonionic surfactants (e.g., Brij-76). The use of this surfactant coating was found to provide a dynamically prepared, stable HIC phase, and no surfactant was required in the mobile phase to achieve reproducible separations (F62). In contrast, Peterson and Wolf used low levels (0.05%) of sodium dodecyl sulfate that was included in the chromatographic mobile phase to improve resolution for the separation of soybean proteins (F63). Mhatre and Krull interfaced fastprotein HIC with low-angle light scattering as an elegant approach to separate and characterize four proteins in less than 5 min using a fast mobile-phase gradient (F64). The casual reader is referred to a submission by Pennings et al. which provides a general overview of the development of HIC and reviews specific applications to the purification of plant proteins (F65). Ion Exchange. Advances in stationary-phase matrices for high-performance ion-exchange (HPIEC) separations of proteins and oligonucleotides have been significant in the last two years. Guerrier et al. examined the cation-exchange chromatography of a series of basic peptides using a recently introduced cationic support (Zephyr) in which dextran is used to produce a highly hydrated polymer layer to prevent mixedmode retention (F66). The utility of such a support was demonstrated by the linear relationship observed for peptide retention vs peptide charge (+1 to +7), which may greatly facilitate the prediction of peptide retention based on structure. Rongen, Adler, and Scouten have critically evaluated the retentivity, resolution, and load capacity of the Zephyr cationexchange materials vs other commerically available HPIEC supports and find them to compare very satisfactorily for preparative applications in particular (F67). Etoh and coworkers observed significant differences in protein retentivity for strongly anionic HPIEC resins using dimethylaminomethylstyrene-dimethacrylate polymers vs chloromethylstyrene-dimethacrylate polymers, which further demonstrates the influence of the polymer matrix in affecting separation (F68). In addition, Phillips and co-workers investigated the performance of a new line of commercially available HPIEC supports based on polymethacrylate gels derivatized with strong anion-exchange and weak cation-exchange functionalities (F69). Blackwell and Carr examined protein retention on their base-modified porous zirconium oxide supports and describe retention as a combination of both cation-exchange and ligand-exchange interactions for these media (F70). By appjying an eluotropic strength scale for some 30 Lewis bases developed for carboxylic acid separations, these authors were able to study the elution conditions and relative ion-exchange and ligand-exchange retention contributions for a broad range of protein analytes. In an interesting kinetics study, Johansson et al. used compressed, nonporous agarose beads for ionexchange separations of papain-proteolyzed fungal cellulases (F71).Separations were achieved in less than 1 min with this matrix, allowing real-time determination of proteolytic cleavage. Theoretical and empirical treatments for optimizing ionexchange separations of proteins and peptides have been usefully extended during the review period. Stahlberg, Jonsson, and Horvath expanded their current theoretical model for protein HPIEC (F72) to consider the effects of van der Waals interactions between proteins and the stationary phase under the influence of high salt concentrations (F73). Data simulated for retention as a function of ionic strength (log k’ vs 1/Z1l2) from their combined equation for Coulombic and van der Waals interactions is in good agreement with experimentally derived data for several proteins. A general treatment of the selectivity effects of the displacing salt in peptide/protein separations on conventional IEC supports was provided by Malmquist and Lundell, who used a chemometricsbased approach on a large chromatographic data set to demonstrate that the primary effect of the displacing salt is nonspecific;thus, retention relates more to changes in effective gradient slope than to selectivity effects imposed by the counterion (F74). Nonetheless, Hearn’s group examined bandwidths associated with protein separations using different tentacle-type ion exchangers and showed that the relative chromatographic effects of different displacer salts can also be a function of the specific commercial phase used for separation (F75). Wu and Walters studied the effects of stationary-phase ligand density for isocratic protein HPIEC using cation-exchange-based chromatographic silicas (F76). As expected from Regnier’s earlier model, retention as governed by the number of binding sites on the protein surface (2) directly corresponds with the density of the stationaryphase ion-exchange groups. In two multifaceted chromatographic approaches for ion-exchange analysis, Lundell and Markides developed an optimization strategy for the twodimensional separation of peptides using a combined IECreversed-phase system in which a step gradient is used for HPIEC with a fast linear gradient for RPLC (F77). Meanwhile, Hodges’ group demonstrated that strong cationexchange media could be used in a mixed-mode approach for peptide separations in which both hydrophilic and ion-exchange interactions are employed for selectivity (F78). By varying the mobile-phase concentration of acetonitrile, column selectivity is effectively altered as hydrophilic interactions become much more significant in the more nonpolar mobile phases. The development of HPIEC for the separation of oligonucleotides continues to receive moderate attention. Lloyd, Warner, and Kennedy examined quarternized polyethylenimine-coated anionic exchange media for the separation of poly(rA) and poly(rC) oligomers produced by alkaline hydrolysis (F79). As anticipated, wide-pore (400 nm) silicas were found to be superior to smaller pore (100 nm) silicas for improving resolution of longer oligomers. Herpich and Krauss studied volatile mobile phases of ammonium carbonate and ammonium bicarbonate for rapid preparative separations of nucleic acids using a strong anion-exchange resin (F80). Vaerman et al. developed a predictive equation for DNA oligomers which allows the calculation of a theoretical NaCl concentration for elution based on oligomer base sequence (F81). Also, Pehlivan et al. were successfulin achieving ligand- zyxwvu AnaEytical Chemistry, Vol. 66,No. 12, June 15, 1994 * 513R zyxwvutsrqpon exchange separations of nucleosides and nucleic bases using Ni2+- and Cu2+-loaded glyoximated diaminosporopollenin phases (F82). In a direct comparison using antisense DNA oligomers, however, Cohen et al. evaluated slab and capillary gel electrophoresis in conjunction with reversed-phase and ion-exchange separations and demonstrated that these conventional chromatographic separations generally provide inferior resolution vs electrophoretic approaches for oligomers more than 20 bases long (F83). Preparative Liquid Chromatography of Biopolymers. With the growing number of commercially produced recombinant products, it is essential that more efficient and convenient methods be established for biopolymer purification; thus, efforts to improve the theoretical framework and reduce optimization time for preparatory separations have been the focus of many excellent contributions during the review period. Cretier and co-workers examined the injection conditions associated with maximum recovery for preparatory HPLC using computer simulation of a binary mixture and note that the optimum injection concentration is an increasing function of the column’s plate efficiency (F84). Knowledge of this correlation is important when scaling up analytical separations to the preparatory scale, where column efficiencies are typically not as large. Guiochon et al. also studied optimum conditions for purification as a function of column length and efficiency, but reaffirm that for gradient elution of proteins in particular, the traditional relationships of column efficiency and resolution may not be valid since retention is largely independent of column length (F8.5). As they point out, wide, short columns can be used just as effectively as long, slender columns of the same total volume to achieve maximal resolution with minimal sample dilution and solvent consumption. In a separate contribution, El Fallah and Guiochon measured the protein band profiles of lysozyme on a reversed-phase column and compared them with profiles calculated using a Craig implementation of the equilibrium-dispersive model (F86). Excellent agreement was obtained for this comparison, in contrast to the profiles calculated using a conventional Langmuir isotherm. Cox and Snyder considered the relationship between the mobile-phase gradient and solute-solute displacement for reversed-phase protein separations and found that the saturation capacity of the column as well as the purity and recovery of the products is largely unaffected by the gradient slope. Thus, chromatographic conditions that permit maximal protein adsorption (to maximize the effects of displacement) accompanied by a steep gradient slope facilitate an optimal degree of productivity from the column (F87). Bellot and Condoret studied chromatographic selectivity as a function of the loading factor for preparative ion-exchange separations of proteins and also illustrated the theoretical implications of solute displacement interactions associated with overloading the column (F88). Mao, Prince, and Hearn presented their own mathematical model for determining the optimal flow rate, effluent concentration, and loading volume for affinity and ion-exchange preparatory separations that invokes computer simulation of the nonlinear adsorption isotherms and analysis of a minimal laboratory data set (F89). In two process-scale contributions, Jungbauer et al. successfully demonstrated the scaleup of hydrophobic interaction chro- matography for a recombinant protein by maintaining a constant height/diameter ratio for the separation column (F90), while Prouty reviewed plant-scale separations using ion-exchange and affinity chromatography for recombinant protein separations (F91). Preparatory applications based upon displacement HPLC (HPDC) are indeed gaining significant attention from researchers. During the review period, Frenz provided an excellent synopsis of this technique and discussed the current problems associated with its use (F92),and Cramer illustrated the practical considerations in developing displacement methods for proteins in particular (F93). In addition, Hodges and co-workers discussed the current use of solute displacement chromatography (SDC) for the purification of synthetically produced peptides (F94). Jen and Pinto established a systematic approach based on the stoichiometric displacement model for selecting (or designing) polymeric displacers for use in protein separations (F9.5). Their simulations indicate that heterogeneous displacers may be just as useful as homogeneous displacers as long as each of the components has higher affinities for the stationary phase than the sample. Cramer’s group demonstrated the efficacy of dextran-based polyelectrolytes as nontoxic, high-affinity displacers for proteins separated on cation- and anion-exchange supports, which showed good experimental agreement with simulations obtained using a steric mass action ion-exchange model (F96). A heterogeneous mixture of several carboxymethyldextrans was further invoked by Torres and Peterson as spacer displacers of varying column affinity for protein separations utilizing anion exchange (F97). Hossain and Do developed a comprehensive model for reversible and irreversible protein denaturation with respect to its influence on the displacement profiles obtained from chromatographic separation (F98). Their simulations suggest that smaller particle sizes, shorter columns, and a slower velocity are effective in achieving better productivity for protein separations. In a final application, Waterborg and Robertson applied SDC to the separation of DNA restriction fragments of closely related size (F99).While neither agarose gel electrophoresis nor analytical ion-exchange HPLC was able to resolve these species, the implementation of solute displacement on nonporous ion-exchange media resulted in separation-purification of several hundred micrograms of product in less than 30 min. zyxwvutsrq 514R Analytical Chemistry, Vol. 66, No. 12, June 15, 1994 G. AFFINITY CHROMATOGRAPHY Reviews and Theoretical Models. A large number of excellent reviews and theoretical treatises pertaining to affinity separations have appeared during the latest review cycle. Among the most useful reviews, Liapis discussed the mechanisms of adsorption in affinity processes and the need for appropriate mathematical models to describe adsorptiondesorption and the mechanisms of mass transfer ( G I ) . VidalMadjar and Jaulmes reviewed quantitative aspects of highperformance affinity chromatography in which isocratic elution is used to study the molecular interactions between biochemical species (G2). Lowe summarized the affinity HPLC purification of industrially significant proteins (G3) and discussed the synthesis and performance of affinity adsorbents for purifying injectable pharmaceutical proteins (G4, G5). Phillips provided a comprehensive review of the zyxwvutsrqpo zyxwvuts zyxwvutsrqp zyxwvutsrqp zyxwvu zyxwvutsr history, concepts, and techniques of affinity and immunoaffinity separations (Gb), and Kasai reviewed the extensive affinity literature associated with trypsin and related enzymes (G7). Kobata and Endo reviewed the use of immobilized lectin columns for oligosaccharide analysis (G8), and an extensive review of boronate affinity chromatography was provided by Shyamali et al. (G9). Immobilized metal ion affinity chromatography (IMAC) was reviewed extensively by Porath and co-workers (GIO),Wang andco-workers ( G I I ) , Yip and Hutchens (GIZ), and Mrabet (G13). McCoy reviewed much of the progress in the theoretical framework for affinity separations and presented a general theory for column affinity chromatography. This treatment assumes a nonlinear adsorption isotherm and rate expression and is applied to several special cases based on affinity separation (G14). Galan et al. examined the influence of the stationary-phase particle diameter on the adsorption equilibria and mass transfer for affinity separations of L-asparaginase (G15).Mobile-phase ionic strength was considered over an entire range of particle sizes (55-135 mm) and is found to be more important on the equilibria of smaller particles, whereas the rate of diffusion of the protein in the larger particles is on the same order as for free diffusion in solution. Massom and Jarrett examined porosity effects for oligonucleotide separations on a DNA-silica affinity matrix and suggest that 30-50-nm pores provide optimal coupling efficiencies and capacity for these separations (G16). Weak affinity chromatography (HPLAC) was studied by Wikstrom and Ohlson, who developed a computer simulation for assessing the advantages of this technique relative to conventional affinity separations (G17). Experimental data are presented as a function of particle size, concentration of the adsorbent ligand, and peak capacity. Yamamato and Sano developed a rapid mathematical approach for predicting the productivity rate (P) for affinity separations as a function of the mobile-phase linear velocity (m)by measuring breakthrough curves for several different sizes of porous and nonporous chromatographic media (G18). This approach may be particularly useful in the scale-upof affinity operations using mechanically rigid chromatographic media not restricted by pressure considerations. In a newly developing area, Tsao et al. developed a general multicomponent rate model for radial flow affinity chromatography which accounts for radial dispersion, external mass transfer, and intraparticle diffusion (G19). Affinity Supports. The most significant driving force for the development of new affinity matrices over the last two years relates to improvements in the productivity of these supports and their potential for scale-up. Ryu and co-workers critically compared several different commercial matrices for the affinity purification of protein C including cross-linked agarose, poly(methy1 acrylic), cellulose, and poly(viny1alcohol) polymers (G20). After pressure tolerance, coupling efficiency, and the kinetics of adsorption-desorption were examined, cellulose was reported to be somewhat superior in regard to its overall productivity. Thalhamer’s group investigated the use of nitrocellulose paper as a solid-phase matrix for immunoaffinity chromatography (G2I). By grinding frozen paper into very fine particles which were separated based on size, a new chromatographic support is generated that is exceptionally inexpensive and convenient to use. In another study, Fisher et al. discussed the synthesis and application of polymeric microspheres (0.4-2.7 mm) based on a methacrylate polymer for protein purification using affinity ligands (G22). Interest in the use of membranes for affinity purification also continues, as Bailon and co-workers investigated using receptor affinity chromatography based on hollow fibers (MRAC) (G23). These supports are found to be much more productive than conventional gels owing to their much superior mass-transfer properties. In related work, Nachman considered the kinetics of hollow fiber membrane-based immunoaffinity supports and demonstrated that the adsorption kinetics, rather than mass transfer, become the limiting feature of the membrane-based separations (G24). Wear et al. examined the chemical functionalization and application of a new matrix for membrane affinity separations based on a commercially available poly(ether-urethane-urea) fiber exhibiting a high internal surface area (G2.5). In addition, Langlotz and Kroner examined the coupling conditions for protein A and IgG to an epoxy-activated membrane for protein affinity separations (G26). Meanwhile, Miller and co-workers developed a new recombinant protein A matrix for radial affinity chromatography that can be used with high flow rates without generating significant back pressure (G27). In a potentially important application to process analysis, Chase and Draeger illustrated that expanded beds may be useful in the affinity purification of materials containing significant amounts of particulates (e.g., broken cells) (G28). Chromatographic performance is not diminished in these beds vs conventional packed beds operated with the same mass of stationary phase and at the same flow rate. Further, Lowe et al. developed a process-scale approach for affinity separations based on a perfluorocarbon emulsion reactor (G29). In this procedure, a polymeric fluorosurfactant is prederivatized with the affinity ligand and then emulsified in a perfluorocarbon oil. Sedimentation is used as the separation mechanism, which is accomplished in a four-stage reactor unit that permits adsorption, washing, elution, and reequilibration to be carried out continuously. Ligand Immobilization and Applications. A great number of interesting affinity chemistries have been investigated during the current review cycle. While it is impossible to provide a comprehensive list in this report, several representative examples are discussed briefly. Scouten and co-workers investigated the useof three new aliphatic boronates as ligands for affinity separations using catechols to assess their overall utility (G30). Although the binding affinities werequantitative in some cases, the overall stability of these ligands was somewhat limited. Kitigawa achieved maximal loading of phenylboronic acid groups for bioamine and nucleoside separations using the multiple reaction sites generated by polyamines chemically bonded to a polymeric support (G31). Wu et al. used histidine bonded to silica as a pseudobioaffinity ligand for the fractionation of human IgG (G32). Two histidine immobilization chemistries (direct vs carbodiimide coupling) were compared with respect to their binding capacity and chromatographic performance, and the influence of the initial silica type was studied. Vidal-Madjar and coworkers investigated the chromatographic properties of benzamidine polymers deposited on porous silica for trypsin zyxwvutsr Analytical Chemistry, Vol. 66, No. 12, June 15, 1994 515R zyxwvutsrqpo zyxwvutsrq loading (G33), while Wheatley and Schmidt studied the immobilization of proteins on an epoxy-activated affinity support as a function of ammonium sulfate concentration (0.4 M-2.5 M) (G34). From their data for 13 proteins, these authors propose a kinetic model for complexation which assumes the presence of a noncovalent protein-support equilibrium that is strongly influenced by salt-induced hydrophobic interaction. Mohan and Lyddiatt discussed the effects of ligand location within particles for the productivity of a silicabased solid-phase matrix for immunoaffinity chromatography (G35). Productivity was found to be directly associated with the ligand load applied, which was optimized for human IgG immobilization at approximately 3 mg/mL. Petkov et al. considered the importance of the orientation of immobilized protein in affinity separations and presented two approaches which use the carbohydrate moiety as the binding domain (G36). In contrast, Matsumoto et al. studied the immobilization of proteins for affinity supports using methyl vinyl ethermaleic anhydride copolymer, which permits attachment of the ligands through their amino groups to form stable amide bonds (G37). Ngo and Katter discussed the preparation and use of a new synthetic ligand for purifying immunoglobulin G which can be used over a significantly broader range of solvent conditions than protein A or protein G (G38), while Takitani and co-workers extended their previous work by describing the preparation of an andochymotrypsin-diol-silica for the affinity purification of peptides containing aromatic amino acids (G39). Li and Geng prepared a high-performance affinity packing for the purification of a-amylase based on the attachment of starch to a silica substrate using an epoxysilane (G40). Takagi et al. investigated the efficient coupling of DNA to a macroporous silica support using a psoralen derivative (G41). Their procedure permits a large amount of DNA to be loaded onto an affinity column, thereby making this matrix useful for studying drug-DNA binding interactions by weak (reversible) affinity chromatography. Yashima et al. reported the binding of three nucleic acid analogs to porous silica which give selective separations of oligonuceotides based on complementary hydrogen bonding between the nucleic bases of the stationary phase and the adsorbent (G42). In a similar application, Inaki and co-workers immobililized deoxyadenosine on silica gel as an HPLC phase for the separation of oligonucleotides, which they also attributed to base-pairing interactions (G43). Finally, Ostrowski and Bornsztyk described the purification of DNA-binding proteins using a tandem DNA-affinity column in which several distinct DNAaffinity modules are connected in series (G44). Using such an approach, it is possible to adsorb proteins that bind to DNA nonspecifically prior to adsorption of the particular proteins of interest. Several excellent reviews in the area of immobilized metal ion affinity chromatography were mentioned above (GI 0G13). In addition, a large body of work was published during the review period involving new IMAC chemistries, a sampling of which is provided here. Zachariou and Hearn introduced a potentially important new mode of selectivity for IMAC using 8-hydroxyquinoline as the chelating agent for nonconventional metal ions including Fe3+, AP+, Ca2+, and Yb3+ (G45). These supports exhibit preferential specificity for oxygen-rich groups including aspartic and glutamic amino acid residues, as opposed to the histidine, tryptophan, and cysteine interactions observed with Cu2+andNi2+. Extension of this workusing 0-phospherine as thechelating agent likewise imparted an additional mode of selectivity using hard Lewis metal ions (G46). In a separate study, Porath’s group investigated the chromatographic behavior of phosphorylated amino acids, peptides, and proteins and their nonphosphorylated counterparts of identical structure on an Fe3+ IMAC support (G47). This work further illustrates that the phosphate groups as well as the carboxylic acid and aromatic amino acid groups exhibit significant interaction with Fe3+IMAC ligands. Smidl, Plicka, and Kleinmann synthesized two sorbents for IMAC based on a commercially available, hydrophilic methacrylatematrix (G48). Iminodiacetic acid was covalently attached to the surface of the two matrices using spacers of different length, and direct chromatographic comparison demonstrated that the longer spacer arm for Cu2+promotes better resolution by preventing undesirable interactions between the protein and support. Peptides synthesized from solid-phase supports can be conveniently purified using conventional IMAC supports provided that histidine, tryptophan, or cysteine is not a part of the sequence. Selective interactions between the metal ion and the unprotected, unprotonated a-amino group permit an effective separation step as described by Lindeberg and co-workers (G49). Further treatment of the specific nature of the amino acid-M2+ interactions is provided by Anderson and Sulkowski (G50). In a unique application, Hutchens and Yip investigated the use of synthetic, metal-binding peptides as biomimetic stationary phases useful in the study of biologically important metal-ion interaction and transfer events (G51). By permanently immobilizing selected peptides in agarose, their metal-ion interactions were conveniently evaluated; these phases were also shown to be analytically useful for the isolation of metal-ion specific proteins from plasma. Immunoaffinity chromatography is a significant subset of affinity chromatography that continues to receive much attention. Although the reader is referred elsewhere for the innumerable applications of the technique, several fundamentally attractive new approaches have been recently developed. For example, both Nilsson, Hakanson, and Mattiasson (G52)and Cassidy, Janis, and Regnier (G53) have developed sequential addition competitive binding immunoassays based on the use immunoaffinity chromatographic supports. Hage, Thomas, and Beck have also demonstrated the advantages of this approach and describe a mathematical equation based on nonlinear adsorption to account for the calibration response obtained using this technique (G54). Although positive affinity methods in which the antigen of interest is chromatographically retained continue to be the most popular approach for purification, negative affinity modes for selective removal of impurities also areavailable. Wheatley demonstrated the use of multiple ligand immunoaffinity chromatography for the removal of albumin and transferrin from IgG (G55). Unlike the positive affinity approach, only a single chromatographic step is required for protein purification. Using a similar approach, Riggin, Sportsman, and Regnier developed a immunochromatographic application in which antibodies are used to selectively adsorb a preselected zyxwvuts zyx zyxwvutsrq zyxwvutsr zyxwvuts 516R AnalyticalChemistry, Vol. 66, No. 12, June 15, 1994 zyxwvutsrq zyxwvut zyxwv zyxw protein from the sample; however, subsequent analysis is carried out on the remaining antigens not absorbed from the sample using size exclusion (or other) chromatographies (G56). By comparing the chromatographic profiles with and without immobilized antibody, a rapid and convenient method for analyzing cross-reacting variants is established. In a tandem column format, Flurer and Novotny used dual-column immunoaffinity chromatography to remove selected antigens (e.g., albumin, transferrin) from human plasma prior to plasma profiling by RPLC (G57). Proteins selectively removed in this manner were subsequently available for quantitation following desorption from the immunoaffinity support. McConnell and Anderson developed an improved method for the determination of fibrinogen in plasma using a methacrylate-based HPIAC column to reduce nonspecific adsorption (G58). Heparin interference was not observed in this format, and an increased linear range vs conventional approaches was observed. In a final example, Rule and Heoion used coupled columns to develop an effective method for the on-line identification of drugs from urine (G59). A high-performance protein G immunoaffinity column connected in-line with a reversed-phase column permits selective extraction and focusing of the drug of interest prior to detection by mass spectral analysis. H. ION CHROMATOGRAPHY Ion chromatography (IC) continues to generate much interest, both in research directed toward improving the technology and especially into new applications. Except for those reports devoted exclusively to the development of one or the other of the methodologies, we will not distinguish between suppressed and nonsuppressed techniques. During the period of this review, there have been advances made in the design and synthesis of ion-exchange stationary phases, in the understanding of the retention process, in the development of better mobile phases for certain applications, and in new detection techniques. Except for perhaps the area of stationary-phase design and characterization, there appears to be less fundamental activity in IC compared with previous review periods. This may signifythe maturing of the technique, with most energies now devoted to new applications rather than studies of the separation process. A book and several reviews of IC appeared during this review period. Haddad and Jackson published a very thorough book on principles and applications of IC ( H I ) , and the International Ion Chromatography Sympsium, held in 1991, was published as a special issue of the Journal of Chromatography ( H 2 ) . Individual papers from that issue will be cited at the appropriate place in this section. Dasgupta published a nice tutorial on IC, focusing on developments over the past five years ( H 3 ) ,and Walton published a thorough chapter on ion-exchange chromatography, including ionexclusion,ion-pairing, and ligand-exchange mechanisms ( H 4 ) . Several more specific reviews were published, including works on speciation of trace metals (HS),a review of EPA-regulated methods (H6), a review of environmental chemical characterization ( H 7 ) , applications in clinical research ( H 8 ) , and two reviews of the use of IC for food analysis (H9, HIO).A discussion of expert systems for the selection of chromatographic conditions in ion chromatography also appeared ( H I ] ) ,as did a paper describing practical experiences with on-line I C for process monitoring (H12). While this review deals primarily with the chemistry of the separation process, rather than with particular applications of the technique, a few especially interesting applications of IC appeared during this period, and these are included to show the breadth of application of IC and that this technique is being used for obtaining physicochemical information, as well as for more traditional quantitative analysis. Two reports from different groups discussed the use of IC for the determination of inorganic and organic ions present in snow and ice cores (HI 3, HI 4), with one of the reports dealing with Antarctic samples collected during the Italian Antarctic Expedition of 1987-1988 ( H I 3 ) . The use of gas-phase IC for the separation and measurement of both small and large carbon clusters, including fullerenes was reported (H15, H16). Ion chromatography was also used for the measurement of physical constants and studies of reaction mechanisms. Ye and Schuler reported a study of intramolecular charge transfer in polyhalogenated systems (HI 7). Three reports appeared describing methods to measure stability constants: of divalent cations with tartaricacid ( H I 8 ) ,metal ions with polycarboxylic acids (H19), and dissociation constants of various aromatic carboxylic acids (H2O). A study was also reported of the investigation of the lithium isotope effect in water-dimethyl sulfoxide mixed-solvent systems ( H 2 I ) . Two reports of physicochemical measurements with more chromatographic relevance also appeared. A mathematical method for the evaluation of distribution coefficients (&) in the M"+/Na+ exchange system was reported, which allows the numerical evaluation of Kd values as a function of exchanging ion and/or total electrolyte concentration (iY22). A method for the quantitation and measurement of equivalent conductance of unidentified analytes by suppressed IC with conductivity detection was also reported (H23). They reported accuracy of concentration and equivalent conductance of better than 90% for monoprotic acids, but results for polyprotic acids were less satisfactory. Regardless, this method should prove quite useful when faced with mixtures of unknown solutes. Mobile-Phase and Sample Effects. We have arbitrarily broken the IC review broadly into mobile-phase and stationary-phase sections. This has been done merely for convenience of organization, and it should be kept in mind that retention, for any chromatographic process, is controlled by the difference in the chemical potentials of the solute between the two phases. Two studies of system peaks in IC appeared during this review period. Yamamoto et al. described a new method for determining the adsorption isotherm of undissociated eluent acid from the capacity factor of the system peak (H24). Michigamiand Yamamoto further discussed the system peak from an IC system of an ODS column coated with cetyltrimethylammonium bromide and trtimellitate (pH 4.0) as eluent (H25). Lee and Hoffman described the effect of injection solvent on peak height in IC and found, unsurprisingly, that when the injection solvent is stronger than the mobile phase, the peak is broadened, while a sharper peak is obtained when a weaker injection solvent is used (H26). Sample treatment of strongly acidic and basic samples generated interest during this review period. Jackson and Jones described a hollow-fiber, membrane-based sample zy zyxwvutsrqpon zyxwvut zyxw Analytical Chemistry, Vol. 66,No. 12, June 15, 1994 517R zyxwvutsrqpo zyxw preparation device for the pretreatment of acidic samples prior to analysis by IC (H27). They reported that a device consisting of aminated DuPont Nafion fiber immersed in a counteriondonating solution effectively neutralized samples with a pH as low as 1. Two separate reports used electrochemical methods for the neutralization of excess acid or base in samples. Siriraks and Stillian described the development of a highcapacity electrochemical membrane suppressor which electrolyzes water to create the acid or base required for neutralization and permits contamination free acid-base neutralizations of concentrated samples (H28). Haddad et al. described an off-line electrodialysis method for the neutralization of strongly alkaline samples (H29). The simultaneous separation of anions and cations, and the separation of ions of different charge, are still problematic. New approaches to both problems were published during this review period. For the simultaneous separation of anions and cations, Saari-Nordhaus described a dual-column approach using an eluent that contains both anion- and cation-driving ions (H30). This allows the simultaneous separation of anions and cations with one injection, one pump, a switching valve, and one detector. IeGras described a method for the simultaneous separation of anions and divalent cations, using EDTA to complex the divalent cations (H31). Umile and Huber described a dual-column approach to the separation of inorganic and organic anions (H32). Because of the large differences in distribution coefficients between the two different types of anions, two columns with widely differing capacity were used. The method was automated and applied to the separation of anions from a drinking water sample. The choice and study of mobile-phase ions continues to generate interest. Gadam et al. investigated the nonlinear adsorption properties of dextran-based polyelectrolytes in anion- and cation-exchange chromatography systems and discussed the implications with respect to their ability to act as efficient displacers in ion-exchange displacement sytems (H33). Slais and Fried1 described a method for the optimization of the mobile-phase composition with a pH gradient of approximately five units (H34). Haldna published a method for determining the concentrations of OH-, H C O j , and C032adsorbed onto the stationary phase when using carbonate eluents (H3.5). Daignault and Rillema published an interesting study describing the effect of the nature of the mobile-phase cation on anion retention (H36). They found that, in general, retention times of anions decreased as the size of the metal ion increased. Jackson and Bowser evaluated a number of eluents which are suitable for use with simultaneous conductivity and direct UV detection (H37). They found that octanesulfonate-borate was perhaps the most versatile of the eluents investigated, as it had good separation selectivity and gave no system peaks and chloride responsecould be eliminated when direct UV detection was used. Both borate-gluconate and carbonate-hydrogen carbonate also proved to be very useful screening eluents for use with direct UV and nonsuppressed and suppressed conductivity detection, respectively. Retention models of solutes in ion chromatography are still of interest. Yamamoto et al. described a revised model for a priori estimations of analyte capacity factor and peak intensity when using multiple eluents (H38). Hirayama and Kuwamoto described a numerical analysis of elution behaviors of substituted benzoate anions (H39). They divided the substituent effect into three terms, a linear free energy relationship term including the anion-exchange constants and the dissociation constants of the sample anions, a steric effect term, and a positional effect correlation term. Stationary Phases. The development of new stationary phases and the characterization of existing phases generated much interest during this review period. Three reports appeared describing the use of conductive materials as stationary phases. Nagaoka et al. reported using a microporous glassy carbon powder which adsorbs ionic substances They found that as the by electrochemical charging (H40). potential applied to the stationary phase was decreased, retention times of divalent ions increased. In a second report, they coated the carbon with immiscible crown ether solutions or polyaniline films to improve selectivity (H41). The use of polyaniline stationary phases was also studied by Syed and Dinesan (H42). They prepared polyaniline resins by a onestep redox polymerization of aniline with persulfate and used the resins for binary and ternary separations. The use of silica as a substrate for preparing an ion-exchange phase continues to be very popular. Polymer coating of bare silica followed with derivatization to form ion exchange groups is one approach to the design of new phases. Kurganov et al. prepared a novel type of ion exchanger by multipoint covalent binding of polystyrene chains onto the surface of porous silica followed by polymer analogous modification of the bonded They prepared both anion and cation exchangers layer (H43). and applied them to the separation of nucleotides and proteins. Huhn et al. prepared stationary phases by polymer coating of silica gel with either polystyrene or poly(glycidy1methacrylate) and then sulfonating the polymer ( H 4 4 ) . This gave a lowcapacity, strong-acid cation exchanger which they then applied to the separation of metal ions in tap and mineral water and grape juice samples. Dynamic coating of hydrophobic derivatized silicas is also of use for preparing new stationary phases. These methods are useful when the need for an ion-exchange separation is sudden and infrequent. While these dynamic methods are attractive, great care must be exercised with temperature control and other chromatographic conditions, as this will affect the amount of adsorption of the active material onto the support and then the day-to-day reproducibility. Janos et al., in two reports, described the preparation and characterization of a cation exchange phase by permanently coating aC-l8columnwithdodecylsulfate(H45,H46).Theyshowed the utility of these phases for the separation of both transition and heavy metal ions. Macka et al. described the separation of some platinum(I1) complexes by an ionic strength gradient on an ion-exchange phase prepared by coating a C- 18 column with octanesulfonate (H47). They reported only a slight baseline shift during the phosphate gradient, even at 210 nm, and reported an equilibration time of 3 min between runs was sufficient to give retention time reproducibility better than 1%. Lamb et al. published two reports of the use of macrocycles for dynamic coating of hydrophobic silicas (H48, H49). They showed that crown ethers, adsorbed onto the surface, can be complexed with alkali metal cations to form anion-exchange sites (H48). They also used cryptand macrocycles for the same purpose (H49). Hu et al. used a charged zyxwvut z 518R Analytical Chemistry, Vol. 66, No. 12, June 15, 1994 zyxwvutsrq zyxwvu zwitterionic stationary phase with pure water as the mobile phase (H50). They found that, for inorganic analytes, anions and their countercations always eluted together and that the retention time of inorganic solutes is only dependent on the anions. Jones, Challenger, and Hill reported investigations of dye-coated columns for chelation exchange IC (H51, H52). They found this system worked well for the determination of trace metals in high ionic strength media, such as concentrated brines and seawaters. The use of inorganic substrates other than silica continues to be of interest. Blackwell and Carr reported an eluotropic scale of mobile-phase strength for a variety of Lewis bases for ligand-exchange-ion exchange chromatography on zirconia (H53). Yaoet al. described thesynthesisand characterization of a tin(1V) vanadopyrophosphate microcrystalline ion exchanger (H54).It was found to have a relatively high ionexchange capacity (3.17 pequiv/g) and they showed its applicability for the separation of amino acids. Many other studies of silica-based ion-exchange phases were also reported during this review period. Erler and Hublein described the preparation and characterization of amino-functionalized phases derived from polybutadiene epoxide-coated silica (H55). Kiessling and Mack studied the alkali stability of tentacle-type silica phases and found it to be generally better than traditional silica-based phases (H56). Nesterenko described the use of silica bonded L-hydroxyproline for the separation of some inorganic anions (H57). Suhara et al. reported a novel synthesis for diethylamino-derivatized silica and its characterization (H58).They first deposited 1,3,5,7-tetramethylcyclotetrasiloxaneon the silica surface and catalytically polymerized it. An anionic ion exchanger containing diethylamino groups was synthesized by hydrosilylation of allyl glycidyl ether followed by treatment with diethylamine. They found this phase to be stable to washes with alkaline solution. Saari-Nordhaus and Anderson reported the use of a mixed-mode, reversed-phase-ion-exchange phase for the simultaneous separation of inorganic anions and carboxylic acids (H59). Nesterenko reported the use of secondary and tertiary amino groups for the preparation of silica-based anion-exchange columns (H60). Ryan and Glennon described the use of a biochelating material and dextran-coated silica for trace metal preconcentration before IC separation ( H 6 l ) . The preparation and characterization of novel polymerbased materials also continues to be of interest. These materials generally offer improved pH stability, but often lower pressure limits and stricter solvent limitations. Kumagai and Inoue compared poly(hydroxymethacry1ate) and poly(vinyl alcohol) gels as base materials for anion exchangers (H62). They found that hydrophobicity, T-T interactions, and base material nature could be used to improve the characteristics of anion-exchange resins. Caruel et al. investigated the degree of cross-linking and size on the resolution and purity of polyols separated by ligand exchange (H63). Brodda et al. pyrolytically degraded IC resins and analyzed the gaseous products (H64). While they did not speculate how this process might be used to study ion-exchange stationary phases, similar studies have been performed on reversed-phase materials, giving insight into synthetic methods of commercial materials. Fujiwara et al. described the preparation of an anion-exchange resin from the reaction of chloromethylated polystyrene with tris(2,6-dimethyoxypheny1)phosphine and found it possessed high selectivity for noble metal ions such as gold(II1) and platinum(1V) (H65). SaariNordhaus et al. compared a hydroxyethyl methacrylate-based anion exchange with quaternary amine functional groups with the traditional agglomerated pellicular-based anion exchangers commonly used for chemically suppressed IC systems (H66). They found the new material had the proper selectivity for the separation of common inorganic anions. Letourneur et al. described the derivatization and characterization of nonporous polystyrene beads which were highly substituted with phosphate groups, grafted via spacers on hydroxylated polystyrene (H67). They found these materials gave ion-exchange properties which are different from those of the usual stationary phases containing carboxymethyl or sulfopropyl groups. Tennikova et al. prepared copolymer beads of glycidyl methacrylate-ethylene dimethacrylate with hydrophobic alkyl chains and hydrogen sulfonate groups (H68). Ludwig described applications of a pellicular anion-exchange resin, prepared by hydrophobically coating a quaternized latex on a monodisperse, nonporous resin (H69). H e found that the low-capacity material was ideal for single column IC, with both conductivity and indirect UV detection. Morris and Fritz studied cation separations on carboxylic acid resins where the exchange group is on the cross-linking benzene ring of the resin or on a short spacer arm from the ring (H70). There was also one report of the use of a restricted access phase for ion chromatography. These stationary phases have been studied and used in reversed-phase chromatography for several years and allow direct injection of proteinaceous materials without column clogging. Buchberger et al. described the use of a C-8 reversed-phase restricted access column for its applicability to ion interaction separation of anions (H71).They found that this method allowed the separation of some physiologically important anions in serum samples without prior removal of protein. Martin and Giacofei compared six different commercially available columns for the ultratrace determination of anions in high-purity water (H72). Comparisons were made on the basis of chloride retention, resolution of fluoride and acetate, column efficiency for nitrate and sulfate peaks, capacity, and run time. They found four of the six columns acceptable, although no single column met every requirement. Suppressor Technology, Quantitation,and Detection. New detection schemes for IC are not explicitly covered in this review; the reader is referred to the LC Equipment and Instrumentation review also in this issue. We will include, however, a few reports dealing with detection aspects of IC. Pessoa et al. reexamined the nonlinearity of calibration in the determination of anions with suppressed conductivity detection (H73). They found curvature for nitrate, bromide, and sulfate calibrations using plots of log-normalized peak area vs log anion concentration. They also discussed several procedures to avoid having large errors in analyzing samples over a wide range of concentrations. Blo et al. described a dynamic method for the preparation of diluted standards for trace analysis (H74). Their system uses a diffusion cell and a capillary tube of known dimensions which continuously provides large volumes of highly diluted standards. They zyxwvutsr zyxwvutsr Analyfcal Chemistty, Vol. 66,No. 12, June 75, 1994 519R zyxwvutsrq reported that the cell was able to work for nearly a month before the source amount decreased by 1%. Hayakawa et al. discussed the changing signal intensity for nonsuppressed conductometric detection when using a carboxylic acid eluent of low pH (H7.5). They showed that sample peak intensity is enhanced prior to the system peak and is suppressed after it. Berglund et al. published two reports on two-dimensional conductometric detection in IC (H76, H77). Following the detector of a conventional NaOH eluent suppressed IC system, a constant concentration of NaOH is introduced by an in-line microelectodialytic NaOH generator, and this is followed by a second conductivity detector (H76). The second detector background is typically maintained at a level of 20-30 pS/cm (corresponding approximately to 0.1 mM NaOH), and the detector output is the same as what would be observed in a single-column mode with a low concentration of NaOH eluent. The system provides qualitative information about peak purity and eluite pKa and, together the twodetector outputs, provides detectabilities at the microgram per liter level for fully dissociated to very weak acids. They also described a system for postsuppressor conversion of eluite acids to a salt (H77). They reported sensitivity to very weak acids is far better than detection in the suppressed mode, and the two-dimensional information allows estimation of the pK, of unknown eluites. Dolgonosov and Krachak described a highly selective method for the determination of ammonium ions using the suppressor as a postcolumn reactor ( H 7 8 ) . They reported limits of detection of 0.1 ppm ammonium ion in water in the presence of 100-fold amounts of alkali metals and inorganic anions. utilization in designing separations. Yi and co-workers reviewed the analysis of metal ions by HPLC (15). Werner reviewed methods for the assay of nucleotides, nucleosides, and nucleobases in biological samples, with emphasis on capillary electrophoretic, reversed-phase, and ion-pair reversedphase techniques (Id). Advantages and limitations of ionpair reversed-phase chromatography are discussed with examples from biochemistry and clinical chemistry. Ion Pairing. The number of fundamental studies of ionpairing separations appeared to decrease somewhat during this review period. Abidi observed a linear relationship between log k of phosphatidic acids or their methyl esters and total number of carbon atoms of tetraalkylammonium phosphates, except for tetrabutylammonium phosphate (17). Dolan provided a brief tutorial on baseline and peak-splitting problems (18). Fornstedt examined peak distortion effects due to large system peaks in ion-pair adsorption chromatography. Because large system peaks contain big concentration deviations of the mobile-phase components compared to the bulk eluent, analyte peaks may be severely distorted upon coelution with them. An example of such analyte peak distortions is given for the analysis of suramin, and guidelines are given for avoiding these effects (19). Later, Fornstedt and Westerlund showed that such distortion can occur even when analyte and system peaks are well separated (110).In general, analyte peaks were more easily deformed by counterion system peaks than by co-ion system peaks. Additional guidelines were reported. Geissler and Vaneldik developed an ion-pair chromatographic procedure using an acetonitrile gradient for the simultaneous detection (down to ppb) of a series of nitrogensulfur oxides including nitrilotrisulfonate (NTS), imidodisulfonate (IDS), hydroxylaminedisulfonate (HADS), N-nitrosohydroxylamine-N-sulfonate(NHAS), hydroxylaminesulfonate (HAMS), aminosulfonate (SA), and the standard ions NOz-, NO3-, and Sod2-. The method allows anions with charges from -3 to -1 to be separated on the same column in one analytical run (ZIZ). Kord and Khaledi examined the effects of organic solvent and surfactant concentration on efficiency, elution strength, and selectivity in MLC and IPC (ZZ 2 ) ,thus providing an interesting comparison-see micellar LC section for details. Narkiewiczmichalek combined the fundamental assumptions of previous electrostatic theories based on a one-site occupancy model with modern theories of surfactant and solute adsorption based on a multisite models and was able to explain experimental findings previously classified as exceptions (113). Ohtsuka et al. examined 2-(2-pyridylazo)-5- [N-(sulfoalky1)aminolphenol derivatives as a precolumn chelating agent for trace metal determination by ion-pair RPLC (114). Otu and co-workers investigated the separation of gold cyanide complexes from other metal cyano complexes using eluents based on tetrabutylammonium hydroxide and acetonitrile. By varying the concentration of inorganic anions (carbonate or perchlorate), interference from base metal and silver cyanide complexes on gold cyanide separation could be eliminated z zyxwvutsrqpo I. SECONDARY EQUILIBRIA The investigation and utilization of secondary chemical equilibria (SCE) such acid-base, complexation, ion-pairing, and solute-micelle associations for modern LC continued in this review period, with many reports concerned with the simultaneous use of two or more phenomena. Reviews and General Theory. Medina Hernandez and Garcia Alvarez-Coque provided a thorough review ( 5 5 references) of solute-mobile phase and solute-stationary phase interactions in MLC (ZI),discussing both electrostatic and hydrophobic interactions as well as the dependence of retention on pH, ionic strength, eluent modifiers, and solvent strength. Timerbaev and Petrukhin reviewed the retention mechanism of anionic chelates in ion-pair reversed-phase chromatography (12). More recently, Timerbaev and Bonn reviewed some significant advances in the theoretical aspects and practical applications of "complexation ion chromatography", including separations based on ion pairing (13). The retention behavior and separation mechanism of noncomplexed and complexed metal analytes were discussed from the point of view of basic coordination chemistry (stability of metal complexes, effective charge of metal atom, ligand complexing ability, etc.). Various metal complexation IC techniques and their common features were evaluated relative to other methods for the analyses of transition and heavy metal ions. Karcher and Krull reviewed (40 references) the use of in situ, multidentate complexing eluents for the HPLC determination of metal species (14)and described the mathematics of complexation equilibria and its zy zyxwvu zyxwvutsr 520R Analytical Chemistry, Vol. 66,No. 12, June 15, 1994 (1Z.5). In an attempt to reduce the reequilibration time needed between gradient runs, Patthy investigated five pairing ions of different size (and sorption kinetics), viz., trifluoroacetate, heptafluorobutyrate, octylsulfonate, dodecyl sulfate, and tetrabutylammonium, each at two different concentrations, and three types of gradients (acetronitrile, salt, and mixed gradients). By use of common nucleotides and some primary amines as analytes, chromatographic conditionswere identified in which one to three column volumes of solvent A were sufficient for reequilibration between gradient runs, although a general solution to slow reequilibration could not be found (116). Pettersson and Gioeli reported the direct separation of enantiomeric amines using a chiral counterion, (-)-2,3: 4,6,-di-O-isopropylidene-2-keto-~-gulonic acid ((-)-DIKGA) dissolved in polar mobile phases, water-methanol or 2-propanol-acetonitrile. High enantioselecitivites (1 -2-1.7) were obtained for metoprolol, oxprenolol, remoxipride, mefloquine, and p-hydroxyephedrine (117). Shelver et al. developed a mathematical model to explain the effect of amine type, amine concentration, hydrogen ion concentration, and methanol concentration in the analysis of bilirubin (118). In one of the more novel IPC studies, Hu and co-workers loaded an ODS column with “strong/strong” charged zwitterionic species and then used pure water as the mobile phase for the separation of inorganic solutes and organic zwitterionic solutes. Inorganic anions (analytes) and their countercations were always observed to elute together, with the retention times dependent only on the anionic species. Ion pairing between the analyte anions and their countercations, followed by the simultaneous electrostatic attraction and repulsion interactions between the zwitterionic charged stationary phase and ion pair, appears to explain these results, as well as the retention behavior of organic zwitterionic analytes (119 ) . Timerbaev et al. compared ion-exchange and ion-pair RPLC methods for the separation of metal 4-(2-pyridylazo)resorcinol complexes (120). Van Leuken and co-workers described the performance of heptafluorobutanoic acid, trideca fluoroheptanoic acid, and nonadecafluorodecanoic acid as volatile ion-pairing agents for the analysis of mixtures of amino acids and their corresponding amides using thermospray mass spectrometric detection. Retention times using nonadecafluorodecanoic acid were comparable to those obtained with n-dodecanesulfonic acid, and the repeatability of the tests and the calibration graphs was good. Mass spectrometric sensitivity was greatly improved by postcolumn addition of trifluoroacetic acid and ionization with gaseous ammonia with one negative charge (125). Finally, Zou and co-workers correlated the retention of 16 phenylamine- and naphthylaminesulfonic acids in RP-IPC with n-octanol-water partition coefficients (log P) (126), reported the retention behavior of aromatic sulfonic acids as a function of methanol and acetonitrile content (127), and examined the combined effect on retention of organic modifier concentration and column temperature (128) or organic modifier and ion-pair reagent zyxwvu zyxwvu (121). (129). Applications. Brouwer et al. developed an on-line trace enrichment/column liquid chromatographic (LC) method for the determination of aromatic sulfonic acids in surface water. The sulfonic acids are preconcentrated as ion pairs on a polymer-based PLRP-S precolumn and on-line desorbed to a PLRP-S analytical column using an aqueous acetonitrile gradient. The influences of counterion concentration, pH, and ionic strength on the retention of the sulfonic acids on the analytical column were reported (130). Jen and co-workers reported methods for the determination of metal ions such as Pb(II), Ni(II), Cd(II), Fe(II), and Cu(I1) (131), as well as Cr(II1) and Cr(V1) (132), after formation of their EDTA complexes. Other IPC applications included the following: the separation of molecular species of phosphatidylserine (133); the direct serum injection analysis of porphyrins using SDS below its cmc (134);separation and indirect detection of amino acids (135);separation of characteristic amino acids of collagen hydrolysate, Le., proline, hydroxyproline, lysine, and hydroxylysine (136); separation of papaverine drug congeners, namely, papaverine ( l ) , moxaverine (2), drotaverine (3), and ethaverine (4) hydrochlorides (137);determination of several barbiturates and analgesic drugs (138) using the sodium salt of 1-decanesulfonic acid; 10 biogenic amines in beers, i.e., histamine, tyramine, serotonin, 6-phenylethylamine, tryptamine, cadaverine, putrescine, agmatine, spermine, and spermidine (139); pyridinium cross-links in both serum and synovial fluid samples using an aqueous 10 mM pentafluoropropionic acid mobile phase and fluorescent detection (140); aluminum in natural waters (141); transformation products of dehydro-tascorbic acid (142);unphosphorylated thiaminerelated compounds (143); epimeric UDP-sugars (144); aminoglycoside antibiotics (145) by LC/EC/MS; cephapirin and desacetylcephapirin in milk (146); arsenic species using an alkaline aqueous mobile phase and a PS-DVB column (147); N-nitrosodimethylamine and its metabolites (148);and sugars at alkaline pH and pulsed electrochemical detection (149). Micellar Liquid Chromatography (MLC). Fundamental Studies. Bailey and Cassidy measured column efficiency as a function of the concentration of sodium dodecyl sulfate (00.3 mol/L), in the presence and absenceof small concentrations of 1-propanol (0-0.8 mol/L) (150). Column efficiencies showed distinct maxima in the micellar region of SDS at a concentration near 0.01 mol/L, which became broad in the presence of 1-propanol. Together with other results they concluded that the low efficiencies observed in MLC are not due to mass-transfer effects related to surfactant loading on the reversed phase and that the addition of alcohol improves mass transfer kinetics of some phenomenon in the mobile phase. Using micellar mobile phases consisting of SDS, HTAB, or Brij-35 over a wide range of surfactant concentrations, zy zyxwvutsr zyxwvut zyxwvutsr zyxwvu Zhao and co-workers utilized RP-IPC with ICP-MS detection for the determination of gold-based antiarthritis drugs and their metabolites (122) and cisplatin and its possible metabolites (123). Watkins and Gorrod optimized mobilephase pH, pairing-ion concentration, secondary-ion concentration, and percentage organic modifier for the separation, detection, identification, and quantitation of the isomeric 1Nand 3N-oxide metabolites of metoprine, pyrimethamine, and trimethoprim (124). Zhang et al. employed thermodynamic and numerical treatments combined with Gouy-Chapman theory to show that the effect of salt concentration ( C ) on the retention factor in RP-IPC can be expressed as In k’ = A + B In C(s), where B is related to the charge number of the solute and A is determined by both electrostatic and nonelectrostatic interactions and can be linearly correlated with logarithm of the capacity factor in RP-HPLC for the solute Analyticel Chemistry, Vol. 66,No. 12, June 15, 1994 521R zyxwvutsr zyxwvuts zyxwvutsrq zyxwvuts zyxwv zyxwvutsrq Gonzalez et al. measured solute-micelle association constants of several polycyclic aromatic hydrocarbons, compared the structural features of the compounds with their capacity factors, and correlated their octanol-water coefficients with hydrophobicity as indicated by MLC (151). RodriguezDelgado and co-workers correlated retention data of polycyclic aromatic hydrocarbons with several molecular descriptors for SDS, HTAB, and Brij-35 micellar systems (152). Garcia et al. used alcohols as modifiers to optimize the separation selectivity of benzene and naphthalene derivatives on a C- 18 column (153). Torres-Lapasio and co-workers modeled the retention behavior of catecholamines, amino acids, and phenols and other aromatic compounds in MLC with mobile phases containing an alcohol. The function l/k’= A p + B4 + C&J D,where p and 4 are surfactant and alcohol concentration, proved to be satisfactory (154). Hu et al. discussed the retention mechanism involved in enantiomeric separation of binaphthyl compounds with micellar bile salt mobile phases. Experimental data support the existence of a heterogeneous C- 18 phase comprised of solvated and micelle-adsorbed portions, the latter of which is responsible for the enantioselectivity (155). Khaledi’s research group contributed significantly to our understanding of MLC. Kord and Khaledi provided an interesting comparison of MLC with ion-pair chromatography (IPC), in which they examined the effects of organic solvent and surfactant concentration on efficiency, elution strength, and selectivity in MLC and IPC. Although selectivity varies with surfactant concentration in both, the overall variation in selectivity and elution order was more pronounced in MLC. Iterative regression optimization designs work better in MLC because retention behavior is more regular and reproducible, and more accurately modeled. Whereas the separation of a mixture of amino acids and peptides requires an organic solvent gradient in IPC, isocratic conditions suffice in MLC and are generally better than IPC separations. Efficiencies of MLC and IPC were found to be comparable. They also compared the effect of solvent strength on selectivity in MLC and reversed-phase liquid chromatography. In contrast to RPLC, in which hydrophobic (methylene) selectivity decreases monotonically with increasing organic solvent, in MLC there is usually no direct relationship (156). As a result, selectivities frequently increase as the concentration of organic modifier (Le., solvent strength) is increased. When selectivity in MLC is observed to decrease with an increase in organic solvent, increasing the micelle concentration can frequently provide a simultaneous enhancement of selectivity and reduction in retention. Solvent selectivity principles based on Snyder’s “selectivity triangle” are generally not applicable in MLC; e.g., the selectivity provided by 2-propanol, acetonitrile, and tetrahydrofuran (different solvent groups) was very similar, whereas that provided by 2-propanol and butanol (same solvent group) was very different (157). Finally, Strasters et al. utilized an iterative regression optimization strategy for the simultaneous optimization of the concentration of surfactant, 2-propanol, and pH for the separation of amino acids and peptides (158). Later they reported a revised model for the prediction of retention and reviewed appropriate optimization strategies for the separation of ionizable compounds (159). + 522R Analytical Chemistry, Vol. 66,No. 12, June 15, 1994 Applications. After determining the effects of different amounts of several organic solvents, Bonet-Doming0 and coworkers reported a method for the determination of diuretics in pharmaceutical preparations using 0.05 M SDS and 3% propanol) (160). They later useda 0.07 M SDS-OS%pentanol mobile phase to determine amiloride, bendroflumethiazide, chlorthalidone, spironolactone, and triamterene in tablets and reported the changes that occurred in the solute-micelle binding constants and the water-stationary phase partition coefficients upon the addition of pentanol (161). Carretero et al. reported a rapid method for the determination of banned drugs in sport (including stimulants, anabolic steroids, and diuretics) using a hydroorganic SDS mobile phase (162). Retention parameters of the drugs were established, as was the effect of an organic modifier on retention. Amin and co-workers exploited the stable, nonquenching environment of (SDS) micelles and determined steroids as low as 100 pg in urine by MLC with sensitized terbium fluorescence detection; the micellar environment facilitated an efficient Forster energy transfer from steroid donors to terbium ion acceptors (163). Haupt and co-workers used MLC with an ai-acid-glycoprotein column to monitor ( R ) - and ( S ) naproxen in human liver microsomes (164). Finally, Aiken and co-workers used a surfactant-containing eluent for the analysis of porphyrins by direct serum injection (134). Miscellaneous. Acid-Base Equilibria. Several researchers studied the effects of acid-base equilibria in conjunction with other chromatographic properties. Although pH effects on chromatographic separations are usually very well understood (qualitatively at least), there are nevertheless some oftenoverlooked subtleties which can be exploited for improved selectivity and resolution. Gonzalez and co-workers published two tutorials on the evaluation of ionization constants of drugs in hydroorganic solutions by RPLC (165,166). Chaminade et al. also reported an efficient RPLC method for measuring the pKa values of six chlorinated phenols (167). Finally, Sereda and co-workers examined the effect of a-amino group on peptide retention behavior in RPLC and determined pKa values of the a-amino group of 19 different N-terminal amino acid residues (168). Rohrer and Olechno demonstrated that, under the elution conditions required to separate the deuterated glucoses on an anion-exchange column, monosaccharide retention time is linearly related to pK, and used the relationship to estimate the pK, of other deuterated glucose compounds (169). Zoest et al. employed a 3 X 3 factorial design in the study of the chromatographic behavior of 13 protonated amines in RPLC using sodium dodecyl sulfateas the pairing ion. The maximum retention factors were correlated to the pKa, hydrophobicity, andconnectivityindexofthesolute,as well as the concentration of acetonitrile and tetrabutylammonium (TBA) in the mobile phase (170). p H . Several groups reexamined the effect of pH on the retention of ionizable (ionogenic) compounds, primarily for purposes of optimization via computer simulations or expert systems. Most of the retention models were based on previously reported phenomenological models. Lewis and co-workers described computer simulation software that predicts RPLC separation of monoprotic ionizable substances as a function of mobile-phase pH. z zyxwvutsrq zyxwv zyxwvutsrqp zyxwvutsrqpo zy zy zyxwvutsrqpo Experimental data from runs at three different pH values (constant organic modifier) are used to estimate values for the solute pK, and the capacity factors (k’) of the ionized and nonionized forms of each ionizable solute. Experimental requirements for the accurate (<f4%) prediction of selectivity as a function of pH are discussed, and the reliability of the software is demonstrated for mixtures of substituted benzoic acids, substituted anilines, and catecholamine-related compounds (171). They then examined the optimization of separation by the simultaneous variation of pH and solvent strength (7%B ) for acidic (substituted benzoic acids) and basic samples (substituted anilines) (172). With the exception of one compound (3,5-dimethylaniline) in one particular pH range ( 3 . 0 - 4 3 , plate numbers of 12 000-20 000 were observed for each sample over a wide pH range, in contrast to the findings of Schoenmakers et al. (173). Schoenmakers and co-workers also considered the variables of pH and solvent composition for the optimum separation of ionogenic (ionizable) substances. Using the retention data of several acidic, basic, and neutral solutes, with methanol as the organic modifier, they developed accurate retention models based on a unified formalism that is applicable to all ionogenic solutes (174) . They then reported an interpretive optimization procedure that takes into account the effect of pH on efficiency (platecount) and peakshape (asymmetry) as well as retention (173). Finally they described an expert system (IONICS) for the simultaneous optimization of pH and organic-modifier concentration. A genetic algorithm is employed instead of classical nonlinear least-squares methods for the fitting of the experimental data to the model (175). Morvai and co-workers described the buffer and pH dependence of the retention of phenylthiocarbamylamino acids in RPLC (176). Slais showed that, in certain cases, gradient elution needed for the separation of complex samples can be achieved without sophisticated gradient instrumentation. Anionic species and ampholytes can be separated via pH gradients generated by the reaction of two or more buffer components added to the sample with two or more ionizable groupson thestationaryphase (177). Rodgerset al. examined the separation of ionizable compounds using MLC, derived and verified a retention model for the simultaneous optimization of pH and micelleconcentration, and discussed appropriate strategies (159). Vervoort et al. compared two approaches for the RPLC analysis of 32 basic drugs differing in basicity, polarity, and number and type of nitrogen atoms (178). In the first, the effect of mobile-phaseadditives and buffers on the performance of a single conventional column was determined and optimized. Although tertiary and quaternary amines can both be used as silanol blockers, the latter proved to be aggressive toward silica-based stationary phases. In the second approach, eight different columns designed especially for the separation of basic drugs were evaluated; results were highly variable. Cyclodextrin-Mediated Separations. The number of separations using some type of cyclodextrin as a mobile-phase additive appears to have stabilized. Most of these papers are covered in the section on geometrical and optical isomers. Vazquez and co-workers examined the interaction between aflatoxins and P-cyclodextrin (179). Anigbogu et al. measured the formation constants for P-cyclodextrin complexes of anthracene and pyrene using RPLC (180); the ana1yte:CD stoichiometry was found to be 1:l and 1:2, respectively, although pyrene complexation was not observed until tertbutyl alcohol or cyclopentanol was added to the mobile phase (1% (v/v)). In a follow-up paper, Husain et al. measured the influence of a series of tetrabutyl comodifiers (with different functional groups) on the retention behavior of the 8-cyclodextrin-pyrene complex (181) in hydroorganic mobile phases with 59% methanol. Other Complexation Equilibria. Timerbaevet al. developed a mathematical equilibrium model for describing the RPLC behavior of metal chelates (182) which takes into account possible chemical reactions accompanying the chromatographic process for metal chelates, i.e., dissociation, hydrolysis, adduct formation, etc. Basicmodel predictions wereconfirmed experimentally using metal dithiocarbamates and dithiophosphates. They then developed and compared a number of multiparametric retention models for metal di-n-alkyldithiophosphates in RPLC (183). The most meaningful one ( R = 0.976) was log k’ = 0.716 (0.236 f O . O l O ) n ( C ) -(0.040 f 0.018)E(n)-(0.048 f 0.003)C + (0.029 f O.OlO)Z, where n(c) is the carbon number, E(n) is the orbital electronegativity of themetal atom, Cis thevolume concentration of theorganic modifier, and Z is the parameter of proton-donating ability of the mobile phase. Chellquist and Searle reported an HPLC method for measuring complex formation equilibria by competitive chelation (184). Chen and co-workersdeveloped and evaluated a novel and sensitive method for the study of carbamate formation equilibria of amino acids and peptides in aqueous solution using RPLC with cetyltrimethylammonium bromide (185). N o correlation was found between the formation constant and the ammonium pKa of the 1 1 L-a-amino acids and peptides. Ichinoki et al. examined the effect of column temperature on separation and determination of dithiocarbamate chelates by reversed-phase HPLC (186). Blackwell and Carr described the ligand-exchange chromatography of free amino acids and proteins on porous microparticulate zirconium oxide (187) and also compared ion- and ligand-exchange chromatography of proteins on same using organic and inorganic Lewis base eluents (188). Toma and Silva described the elution behavior of N-heterocyclic derivatives of mixed ruthenium(I1)-sulfoxide complexes in RPLC (189). Ohtsuka et al. characterized 2-(2-pyridylazo)5- [N-(sulfoalkyl)amino] phenol derivatives for their potentioal as precolumn chelating agents for trace metal determination by IPC with spectrophotometric detection (114). Sarzanini et al. studied the ion-interaction chromatography of metal ions complexed with Plasmocorinth B dye (190), while Hao et al. examined the retention behavior of a-hydroxyisobutyric acid complexes of thorium(1V) and uranyl ion in RPLC (191). Miura examined the effect of tetraalkylammonium salt on the retention of metal chelates in RPLC (192). Kim et al. studied the elution behavior of palladium(I1)-isonitrosoethylacetoacetate imine chelates in RPLC (193). Chellquist and Dicken investigated the effect of mobile-phase modifiers on the simultaneous LC elution of a gadolinium complex and free ligand (194). Zachariou et al. reported o-phosphoserine as a new chelating ligand for use with hard Lewis metal ions in the immobilized-metal affinity chromatography of proteins + Analytical Chemistry. Vol. 66,No. 12, June 15, 1994 523R zyxwvutsrqpon zyxwvu zyx zyxw (195). Kuroda et al. described the ion-interaction separation of nitrilotriacetato complexes of the rare earth elements with postcolumn reaction detection (196). Other applications included the following separations (using RPLC mode, except as noted): metal-8-quinolinethiol complexes (197);alkylamine enantiomers using helical nickel(I1) complexes as mobile-phase additives (198);metal ions after in situ complexation with (*)-trans- 1,2-diaminecyclohexaneN,N,N’,N’-tetraacetic acid (199);metal ions as picolinaldehyde 4-phenyl-3-thiosemicrbazonechelates (1100);aromatic amines by high-performance ligand-exchange chromatography (HPLEC) with copper(II), nickel(II), and zinc(I1) loaded chelating silica gel columns (1101); copper(II), nickel(II), palladium( 11), and oxovanadium(1V) complexes of tetradentate Schiff bases derived from stilbenediamines (1102); hydroxy acids as their copper(I1) complexes by reversed-phase liquid chromatography with UV detection (1103);platinum(11), rhodium(III), palladium(II), osmium(IV), nickel(II), and cobalt(I1) complexes (1104); isomers of cobalt(II1) complexes on a column packed with a clay-ruthenium(I1) complex adduct (1105); and metal ions using EDTA as complexing agent in the mobile phase (131, 1106). J. GEOMETRIC AND OPTICAL ISOMERS Our literature search turned up over 400 original papers on optical isomer separations. It is beyond the scope of this review to include all of these references. Fortunately, numerous reviews were published which will alert the reader to most of the articles that we were forced to exclude. Reviews. Two of the reviews published during this period had a fairly broad perspective. Siret et al. offer practical guidelines for the choice and use of chiral stationary phases in liquid chromatography ( J I ) . After summarizing the state of the art, the authors present their recommendations concerning the selection of the best of among approximately 70 commercially available chiral stationary phases (CSPs), with particular emphasis on the selection of brush-type chiral phases according to solute structure. Taylor and Maher also provided a broad, practical perspective ( J 2 ) . Other reviews of chiral separations were more specific or less comprehensive in their coverage. Aboul-Enein presented an overview of various cellulose-based CSPs (27 references) which are commercially available for direct separation of drug racemates (JS),with a brief discussion of the advantages, disadvantages, and mechanisms of resolution. Perrin and Pirkle reviewed (56 references) commercially available brushtype chiral selectors for the direct resolution of enantiomers ( J 4 ) . Caude et al. described chiral stationary phases derived from tyrosine (J.5). Noctor and Wainer summarized (27 references) the results of an interesting approach to alter retention and enantioselectivity on a human serum albuminbased CSPs: the addition to the mobile phase of known HSA binders such as warfarin, ibuprofen, and tryptophan. Although in some instances the “displacement” mechanism reduced retention and enantioselectivity, in others they were increased due to allosteric interactions ( J 6 ) . Finally, Bourque and Krull reviewed a solid-phase derivatization approach for amines based on immobilized isocyanates containing a 3,Sdinitrophenyl moiety. These reagents are useful for Pirkle-type recognition of stereochemical composition of amines and related nucleophiles (J7). Chen and Lin reviewed the synthesis, characterization, and chiral recognition ability of optically active polyurethanes containing a coumarin dimer component (J8). Chen and Shiao did likewise for CSPs derived from (-)-anti head-to-head coumarin dimer: preparation, characterization, and chiral recognition ability (J9). Gallinella et al. summarized the enantiomeric separation of amino alcohols on dinitrobenzoyldiaminocyclohexane chiral stationary phases (110). Gasparrini and co-workers reviewed the chromatographic separations of trans- l ,2-diaminocyclohexane derivatives (J1l ) . Iwaki and co-workers reviewed the optimization of naphthylethylurea multiple-bonded chiral stationary phases for optical resolution of enantiomeric amino acid derivatives (JI 2). Li and Purdy reviewed cyclodextrins and their applications in analytical chemistry (513), and Krull and co-workers described chiral drug analysis in biofluids by liquid chromatography (514). Vandenbosch et al. described HPLC methods for the chiral separation of /%adrenergic blocking agents (J15), whereas Vinal1 focused on the use of chiral chromatography in the agrochemical industry (516). Finally, Wainer reviewed the impact of new HPLC-CSP technology on the study of stereoselective pharmacokinetics (JI 7 , 518). Four compound-oriented reviews pertinent to geometric and other structural isomers were published during this period. Wiseet al. described thedetermination of polycyclic aromatic hydrocarbons by HPLC (519),summarizing their experience in column selection, approaches to analyzing complex PAH mixtures, and the accurate quantitation of PAHs in environmental samples. Smith reviewed the HPLC analysis of oxysterols of current biochemical and biomedical interest (J20). Lesellier et al. reviewed the analysis of carotenoids by HPLC and supercritical fluid chromatography (SFC) ( J 2 1 ) , including method selection as a function of pigment type (xanthophylls, catotenes, cis-trans isomers), and the effects of the mobile phase (specific interactions, hydrogen bonding) and the stationary phase (nature and type of linkage: monofunctional or polyfunctional, end capping of residual silanols) on solute retention, based on differences in the chemical structures of the pigments. Finally, Herbreteau critically the HPLC methodologies employed for sugar analysis (J22). New Chiral Stationary Phases. Fewer new CSPs were synthesized since the last review, joining the legions of CSPs already developed. The question “Do we really need such a multitude of CSPs or can we design twenty or fewer that have broad applicability?” that was asked in the previous two reviews still does not appear to have been definitively answered. Instead, several apparently new CSPs have been reported. Arlt et al. described two new diastereomeric methacryloylamides that provide good enantioselectivity, can be obtained in good yield in kilogram quantities, and can be polymerized on stationary phases as well as transformed into cross-linked bead polymers for use in preparative separations (J23). Birkinshaw and Taylor developed a family of cyclic peptides varying in ring size and functionality and achieved enantiomeric separations of 3,s-dinitrobenzoyl esters and propylamides of various amino acids as chiral selectors (J24). Gasparrini et al. separated eight nonsteroidal antiinflammatory zyxwvutsr zyxwvutsrqp 524R AnalyticalChemisfty, Vol. 66, No. 12, June 15, 1994 zyxwvutsrq zyxwvuts zyxwvutsrqp agents (derivatives of arylpropionic acids) as l-naphthalenemethylamides on a chiral stationary phase containing the N,N’3,5-dinitrobenzoyl derivative of ( 1R,2R)-diaminocyclohexane as a chiral selector (J2.5). Hargitai et al. prepared and evaluated 13 3,s-dimethylphenyl carbamoylated 0-cyclodextrin stationary phases for normal-phase HPLC separation of enantiomers (526). All materials were prepared by three different methods and contained 3,5-dimethylphenylcarbamate residues. They considered the influence of different spacers, the amount of immobilized carbamoylated 0-CD, and the degree of substitution on 8-CD on the enantioselectivity of the CSPs (more substitution provided greater enantioselectivity). Hyun et al. investigated the chromatographic separation of the stereoisomers of the N-(3,5-dinitrobenzoyl) derivatives of 15 dipeptide methyl esters and 9 dipeptide alkyl esters on three different CSPs derived from N-acylated a-arylalkylamines, two of which contained second stereogenic centers (J27). Li and Purdy developed several multiple-interaction CSPs based on a-,8-, or y-cyclodextrin or a derivative to which other functional groups have been added (J28). In addition to the usual inclusion interaction, these CSPs possess aromatic groups capable of T-T interactions, polar hydroxy groups capable of H-bonding with the analyte, and bulky nonpolar groups that provide steric repulsion. Lin and Yang synthesized and evaluated a series of four CSPs containing carboxylic acid via amide linkage to y-aminopropylsilanized silica gel (J34). Lastly, Oi et al. also prepared seven chiral stationary phases by bonding axially dissymmetric 2’substituted- 1,l’-binaphthyl-2-carboxylic acids to aminoalkylsilanized silica gels through an amide linkage and investigated the effect of the 2’-substituents (CN, COOH, CONH2, CONHEt, CONEt2, and OC&) (J3.5). The CSPs possessing a 2’-carboxyl substituent showed the best performance and efficiently discriminated several enantiomeric amino acids, amines, and alcohols as their 3,5-dinitrophenyl derivatives, and biaryls bearing 2,2’-polar substituents using normal-phase HPLC. Oliveros et al. prepared and evaluated seven new CSPs obtained from (27)-phenylalanine derivatives, (R)-Mosher’s acid, and (5’)-naproxen and obtained the best results when the chiral entity bonded to y-aminopropyl silica gel was 3 3 dimethylanilido-, 3,S-dimethylbenzoyl-, or 3J-dimethoxybenzoyl-(27)-phenylalanine and -(S)-naproxen (J36). They also prepared several chiral triethoxysilanes from acidic or basic chiral compounds and bonded them onto silica gel (J37). Yang and Lin prepared a series of 12 chiral stationary phases with arylcarbamoyl derivatives of amino acids bonded either ionically or covalently to (3-aminopropyl) triethoxysilanemodified silica gel (538). These CSPs generally provided recognition ability for the separation of enantiomeric amide derivatives of amino acids, amino alcohols, amines, and acids, with theionic CSPs bearing (8-phenylcarbamoyl-(27)- or -(I?)phenylglycine showing the best chiral resolution performance for all four types of enantiomeric solutes. A chiral recognition model of these CSPs for enantiomer separations was proposed. Yan et al. resolved a-amino acid enantiomers by ligandexchange chromatography on a new chiral packing in which the chiral ligands were linked onto a hydrophilic polymeric matrix through long spacers (539). GeometricalIsomers. Relative to enantiomers, geometrical isomer separations received much less attention by researchers. Nevertheless, several noteworthy papers were published. A number of researchers examined the separation of fullerenes (J40-546),some with novel combinations of mobile and stationary phases (J47, J48). Others have examined the separation of taxol and related diterpenoids (J49);taxol (J50); taxanes (J.51) on columns developed specifically for them; paclitaxel (taxol(R)) in human plasma (J.52); polycyclic aromatic hydrocarbons (J.53); polychlorinated dibenzo-pdioxins (3.54); isomeric conjugated fatty acids (J.55);carotenoids and their oxidation products (J56);imipramine and desmethylimipramine (J.57);alkylphenols (by pK,) (J58);and iodo amino acids (J59). zyx zyxwvutsr zyxwvutsrqp zyxwvutsrq 2,4-bis[carbamoyl(alkyl)methylamino]-6-chloro-s-triazine (J29). Mano et al. reported two new CSPs. One was based on conalbumin-conjugated silica gel, which showed good stability for the separation of racemic azelastine, an antiallergic drug, as a new chiral stationary phase for high-performance liquid chromatography (530). The other was based on flavoprotein, a glycoprotein present in chicken egg white, and provided baseline separations of acidic (ketoprofen), weakly acidic (warfarin), and neutral (benzoin) compounds (J3J). The latter phase showed good stability to the variation of pH and organic solvents, which greatly influence the retention factors and enantioselectivities for several model drugs. Marle et al. prepared a new CSP based on a glycoprotein (cellulase, CBH I) isolated from a culture filtrate of a fungus, Trichoderma reesei, and resolved enantiomers of acidic and basic drugs (532). Compared with other similar chiral phases, high enantioselectivity was obtained for /%blocking agents despite low capacity factors, and no significant decrease in enantioselectivity was observed after daily use over a period of 4 months with varying mobile-phase compositions. Retention and enantioselectivity of amines increased with increasing pH of the mobile phase, and vice versa for the acids. The addition of organic solvents to the mobile phase decreased the retention, but increased the enantioselectivity for the analytes. Ohkubo et al. examined copper(I1) complexes of Schiff bases of chiral amino alcohols as chiral ligand-exchange stationary phases (J33);direct separation of a large number of amino alcohol, amine, amino acid, and hydroxy acid enantiomers was accomplished using octadecylsilanized silica coated with the binuclear copper(I1) complex of N-salicylidene-(R)-2amino- 1,l -bis(2-butoxy-5-tert-butylphenyl)-3-phenyl1-propanol (1) and water or aqueous-rganic eluents containing copper(I1) ion as mobile phases. Oi and Miyano also synthesized a CSP derived from (27)- [ 101paracyclophane- 13- zyxwv zyxwvutsrqp K. MULTIDIMENSIONAL CHROMATOGRAPHY AND COLUMN SWITCHING As in previous Fundamental Reviews, the term “multidimensional chromatography”will be restricted to those systems in which the second or any subsequent columns are operated in a mode different from the first column. That is, true multidimensional chromatography involves a change in retention mechanisms between columns. We include here multidimensional coupling of liquid and gas chromatography because of the unique power and growing popularity of this combination. Column switching implies multiple-column Analytical Chemistry, Vol. 66,No. 12, June 15, 1994 525R systems without changes in retention mechanisms and is often used for sample enrichment and/or cleanup. Only on-line multidimensional and column switching techniques will be reviewed here because of their greater accuracy and precision, decreased analysis times, and amenability to automation. Several reviews appeared detailing applications of multidimensional chromatography and column switching in various fields. van Zoonen discussed the applicability of different LC-LC and LC-GC combinations in pesticide residue analysis (K1).The principles of coupled gradient HPLC and gel permeation for polymer analysis were described by Kilz ( K 2 ) . Column switching techniques in drug analysis were reviewed ( K 3 ) , and two specific examples used to emphasize the importance of a quality assurance program for coupled column chromatography in this field ( K 4 ) . Grob presented a general overview of two-dimensional HPLC-capillary GC (K.5).The advantages of coupling flow injection analysis and HPLC were also reviewed ( K 6 ) . On-line, directly coupled multidimensional liquid chromatography normally requires the use of mobile phases compatible to some extent with both columns. Some of the most straightforward and useful combinations include achiral/ chiral, size exclusion (SEC)/reversed-phase, and ion-exchange (IEC)/reversed-phase. An achiral reversed phase column is frequently used to separate an enantiomeric pair from other matrix components, and the pair is then transferred to a chiral column for complete separation. Examples of this technique include enantiomeric separations of the drugs ketoprofen (K7) and pimobendan ( K 8 ) in human plasma and a new calcium blocker in dog plasma (K9).A similar technique utilized an affinity precolumn containing anhydrochymotrypsin immobilized on diol-silica to trap specific peptides, which were then separated on an RPLC analytical column (KIO).The coupling of size exclusion and reversed-phase columns was demonstrated in the analysis of cyclopentenyl cytosine and its active metabolite cyclopentenyl cytosine 5’-triphosphate (Kl I). The simultaneous determination of clobazam, diazepam, and their main metabolites in plasma was also accomplished in this way ( K 1 2 ) . A powerful combination of size exclusion/ reversed-phase two-dimensional chromatography and electrospray ionization/triple-stage quadrupole mass spectrometry was described by Seta et al. ( K 1 3 ) . Coupled ion-exchange/ reversed-phase columns were used in drug screenings (K14, K1.5) and in the analysis of small peptides (K16). In the latter work, a linear gradient elution in the R P column was optimized for speed and resolution. In a related but unique combination, size exclusion and weak anion-exchange columns were coupled for the determination of human proinsulin fusion protein produced by Escherichia coli (K17). Another powerful if somewhat more difficult combination that appears to be growing in popularity is electrophoresis and RPLC. Both acidic and basic analytes in plasma samples were determined by zone electrophoretic sample treatment (ZEST) coupled on-line with reversed-phase columns (K18). ZEST/RPLC was also applied to environmental analyses ( K l 9 ) . Jorgenson’s group (K20)described three combinations suitable for the analysis of complex biological samples, each which included capillary zone electrophoresis (CZE); SEC/ CZE, RPLC/CZE, and CZE/fast capillary zone electrophoresis (FCZE). Several other interesting two-dimensional combinations were described. The determination of creatine with direct serum injection was accomplished with a Pinkerton internal surface reversed-phase (ISRP) column coupled to a conventional RP (2-18 column (K21). The ISRP column retains smaller analytes while large biomolecules are washed to waste; separation of target analytes occurs in the C-18 column. The group separation properties of an aminopropyl column were combined with the high shape selectivity of a porous graphite column for the analysis of PCBs, PCDD/Fs, PAHs, and related compounds in environmental matrices (K22). The normalphase separation of phospholipids was improved by introducing limited solvophobic retention through the series coupling of a silica column and variable lengths of an octadecylsilica column (K23). Finally, multimicro-HPLC was introduced by Voroshilova and Kaiser (K24). This technique involves the use of two packed fused silica microcolumns of different selectivities serially coupled and placed inside a tubular oven. Overall selectivity of thecoupled column combination is altered by stepwise movement of the tube oven to heat a different portion of the polar or nonpolar column bed. The result is an HPLC system with physically changeable selectivity that is particularly suitable for identifying peak overlap. In an analogous technique, selectivity tuning in a system of coupled columns of different retention characteristics was accomplished by changing the flow rate in the second column (K2.5). Flow rates necessary for optimum resolution were calculated from capacity factors measured on the individual columns. Column switching without change in retention mode can be a useful approach for separating complex mixtures. Dapoxetine and its mono- and didesmethyl metabolites in human plasma were determined by first separating them from late-eluting endogenous substances with a C-8 precolumn, then front-cutting the fraction containing the analytes, and transferring them to another C-8 column for quantitative separation (K26). Low molecular mass organic and inorganic anions were determined simultaneously using column switching and a combination of medium- and high-capacity anionexchange columns (K27). On-line sample enrichment and cleanup using column switching techniques has become virtually a routine method. Some of the more novel combinations and their applications include the following: enrichment of organophosphorus pesticides on XAD-2 resin followed by RPLC separation (K28); concentration of pesticides by electrodialysis and separation by RPLC (K29);and dialysis and immunoaffinity isolation of aflatoxin M1 in milk samples (KSO). A major increase in the popularity of direct, on-line coupling of LC and G C columns was also noted. Numerous reports of applications of this truly multidimensional technique appeared in the last two years. However, only fundamental developments in the technology, particularly interface technology, will be reviewed here. The use of open-tubular traps for phase switching in RPLC-GC was investigated ( K 3 1 ) . Analytes in the water-methanol mobile phase were sorbed onto the stationary phase of an open-tubular column, and the aqueous mobile phase was then removed by purging with nitrogen. The analytes were desorbed onto the capillary GC column with hexane. A coupled LC-GC system was described which uses a modified multidimensional gas chromatography zyxwvu zyxwvuts zyxwvutsr zyxwvutsrqpo zyxwvutsrq 526R Analytical Chemistry, Vol. zyxwvutsrq 66,No. 12, June 15, 1994 zyxwvutsrqpo switching valve (K32). The system also featured cryogenic focusing, solvent exclusion from the analytical G C column, and independent carrier gas suppliesto the uncoated precolumn and analytical column. An on-line solvent evaporator was used to reconcentrate solutes and exchange solvent in the eluent of an LC column; the reconcentrated solutes were then transferred to an analytical G C column (K33). Mixing in the sample loop of a loop-type interface was studied by Grob and co-workers (K34). Finally, an interface for LC-capillary supercritical fluid chromatography was described (K35).The interface employs solvent evaporation, after which analytes are transferred to and deposited on an impactor interface. Pressure programming of the COZmobile phase then elutes the analytes onto the capillary column. L. PREPARATIVE LC Reviews. Colin ( L l ) reviewed column overload, column efficiency, packing materials, and economic aspects of largescale HPLC. A comprehensive review on the design and operation of preparative HPLC systems for protein and peptide purification was reported by Wisniewski (L2). The fundamentals of preparative HPLC were discussed by Szepesy and Bekasy (L3), Gottschall (L4-L6), Belenguer (L7), and Samsonov (La). Bansal (L9) reviewed analytical and preparative HPLC applications with emphasis on proteins. Largescale LC with a focus on food components was covered by Kaneko (LIO). In a chapter on HPLC, Poppe (LI I) discussed preparative HPLC methodologies. A book on preparative, and process-scale LC, derived from an intensive short course, describes the state-of-the-art developments in these areas (Llla). Theory/Optimization. Guiochon and co-workers (LI 2) studied factors affecting the separation and loading capacity of proteins in preparative gradient HPLC with emphasis on column length. Jandera and Guiochon (LI 3) presented a model to help explain band profile deformation when a solvent with a elution strength higher than the mobile phase is used to dissolve poorly soluble samples in preparative HPLC. Ma and Guiochon (L14) studied steep boundaries of bands of single components and binary mixtures in nonlinear LC. Adsorption isotherms and overloaded elution profiles of phenyln-alkanes on porous carbon were measured by frontal analysis by Diack and Guiochon (LI5). Frontal analysis was also used to determine the axial dispersion coefficient of enantiomers on microcrystalline cellulose triacetate (1516). Adsorption isotherms of lysozyme in reversed-phase preparative LC were measured by Guiochon et al. (LI7). Felinger and Guiochon (1518)studied the change in pressure drop during large-scale LC of viscous samples and determined that the pressure drop becomes significant only when the injected concentration is very high. Cramer (LI9) reviewed the practical considerations in developingdisplacement chromatographic methods for protein purification. Gerstner and Cramer (L20) found that heparin could be used as an efficient and nontoxic displacer of proteins in anion-exchange systems. These workers (L2I) also studied the effect of induced salt gradients when using protamine displacers for cation-exchange chromatography of proteins. Cramer and co-workers (L22) investigated the nonlinear adsorption properties of dextran-based polyelectrolyte dis- placers in anion- and cation-exchange chromatography. Cox and Snyder (L23) reported on the influence of gradient parameters on displacement effects in preparative LC of proteins. Guidelines were given for the design of reversedphase gradients for the purification of proteins in which solutesolute interactions are maximized. Cox (L24a) studied the mass-overloaded separation of insulins using reversed-phase gradient elution. Bahowick and Synovec (L24b) used a sequential chromatogram ratio technique to characterize and correct for retention time variation, peak-shape change due to concentrations exceeding the adsorption isotherm linear range, and detector nonlinearity. Hsu and Chen (L24c) described a general procedure for estimating the number of theoretical plates in preparative LC. Jen and Pinto (L25) established a theoretical framework for the selection and design of polymeric displacers for displacement chromatography of proteins. Torres and Peterson (L26) discussed the purification of complex protein mixtures by ion-exchange displacement chromatography using spacer displacers. Hearn et al. (L27) presented a method for determining optimum operating parameters for protein purification using LC. Flow rate and effluent concentration at which the adsorption stage is terminated are optimized to give maximum production rate. Bellot and Condoret (L28) presented a theoretical study of ion-exchange preparative chromatography of a two-protein mixture. They based their studies on a stoichiometric displacement model for the basic interactions between proteins and the stationary phase. Barwe and Wichmann (L29) used displacement chromatography on strongly acidic cation-exchange resins for the preparative separation of amino acids. Waterborg and Robertson (L30) used solute-displacement ion-exchange HPLC for the purification of DNA restriction fragments. Huang and Jin (1531) investigated an operating strategy for reversed-phase displacement chromatography using ion-pair partitioning. This approach was applied to a mixture of nucleotides. Fulton et al. (L32) compared cation-exchange and reversed-phase perfusion LC for preparative purification of a peptide. The cation-exchange column had a significantly higher loading capacity than the reversed-phase column. Inoue and Ohtaki (1533) studied the influence of chromatographic conditions on the binding capacities of hydroxyapatite for globular proteins. Jungbauer et al. (L34) described displacement effects in preparative ion-exchange chromatography of human erythrocyte superoxide dismutase. Camacho-Torralba and coworkers (L35) designed and synthesized displacers for use with Pirkle-type naphthylalanine silica stationary phases operating in the normal-phase mode. The displacers contain a a-acid head, an H-bonding center, and a solubility adjusting tail. Meyer (L36) studied peak purity, yield, and throughput in preparative LC of cyclohexanone-cyclopentanone selfdisplacement chromatography. Cretier and co-workers (L37) used simulated chromatograms, based on the Craig model, to study theeffect ofinjection conditions on overload gradient elution separation in LC. In subsequent reports, this group also applied this simulation algorithm to study the influence of column efficiency at isocraticconditions (L38)and optimum injection concentration zyxwvutsrqpon zyxwvut zyxw Analytical Chemistry, Vol. 66,No. 12, June 15, 1994 527R zyxwvutsrqp zyxwvutsrq under gradient elution (L39). Gariel (L40) experimentally evaluated simple nonlinear models for predicting sample size in preparative LC. Kawai et al. (L41, ,542) proposed a method for measuring the adsorption isotherm and mass-transfer coefficients using an analytical column. These data were used to scale up to preparative separations. Wang et al. (L43) presented a computer-assisted method for optimizing semipreparative reversed-phase HPLC of aldicarb pesticides. Row and Lee (L44) determined the effect of sample size on peak shape in preparative reversed-phase HPLC of pyrimidines and thymines. Katti (L45)presented an approach for isolating minor components in multicomponent mixtures utilizing displacement effects. Hatanaka and Ishida (L46)described a method for continuous separation of a multicomponent system by combining multiple LC columns. Kaminski (L47) reported on a test for determining the degree of distortion of the flow profile in preparative LC. This group (L48) also studied the use of infinite diameter columns for preparative LC separations. Tsao and co-workers (L49) reviewed strategies for largescale LC separations by gradient elution. A detailed model also was presented for predicting band elution, gradient profile, and other operating parameters. Wankat (L50)developed a method for scaling chromatographic systems which is applicable for gradient elution and displacement chromatography. Hu et al. (1551) developed a model to describe preparative LC with a step gradient. This group (L52) used a simulation model to investigate a number of chromatographic parameters on the shape and spreading of the band profile. Whitely et al. (1553)used a rate model to study nonequilibrium adsorption-desorption kinetic behavior in preparative LC. Carta and Stringfield (L54)described a model to predict overload effects in gradient elution from isocratic elution data. Berninger et al. (1555)reported on a model for simulation of reaction and nonequilibrium dynamics in multicomponent fixed-bed adsorption processes. This model can be used for complex protein separations and scaling with respect to flow rate, particle size, and feed concentration. Packings/Columns/Hardware. Johansson (L56)reviewed methods to study leakage of components from separation media used for protein purification. Perry and Szczerba (L57) studied the effect overloading conditions as a function of particle shape. Matsuda et al. (L58) evaluated the use of an ion-exchange resin, TSKgel Super Q-Toyopearl 650, for preparative separation of proteins. Wingren et al. (1559) evaluated the use of Superdex 200 as a support for liquidliquid partition chromatography of proteins in an aqueous described two-phase (PEG-dextran) system. Yang et al. (MO) the use of cotton as a stationary phase for protein separations. King and Pinto (L61)evaluated the application of nonporous, silica-based fibers with anion-exchange functionality for preparative LC of proteins. HiLoad columns for preparative purification of proteins and peptides was reported by Stauder (L62). Bokihara et al. (L63)reviewed preparative separations of proteins and peptides using Parcosil, Parcomer, and Titanosphere packings. Preparative HPLC systems were described by Verillon and Claverie (L64) and Common (1565). Mahar et al. (L66) reported on a valving device for precise gradient control for large-scale LC. Lawing et al. (L67) presented a procedure for packing annular-expansion preparative HPLC columns. A continuous rotating annular chromatograph was used by Takahashi and Goto (L68) for protein separation. Selected Applications. Specific applications for preparative LC have been reported for azetidinyl methyl sulfinates (L69), antimetabolic liponucleotides (L70), bacitracin (L71), biologically active amines (L72), carbohydrates (L73), carotenoids (L74),cis-and trans-chlordene (L75),crudeoils (L76), cyclosporine (L77),dextrin (L78), enantiomers (L79-L87), erucic acid (L88),fluorescein (L89),fullerenes (L90-L105), insulin (L106), metallothionein (L107, L108), monosialogangliosides (L109), nikkomycin (LI IO), oligonucleotides (1511I , LI 12), oligosaccharides (L113), peptides (L114), phospholipids (L115-L117), plasmid DNA (L118, L119), polyamicacids (L120),prostaglandins (L121),proteins (L122LI24),vitamin D (L125, L126), and zinc chlorophyll (L127). zyxwv zyxwvutsr zyxwvut 528R Analytical Chemistty, Vol. 66, No. 12, June 15, 1994 M. PRE- AND POSTCOLUMN DERIVATIZATIONS This section of the review will first list relevant review articles and fundamental studies and will then group articles according to solute type or functional group. The searches revealed literally hundreds of articles published during this two-year period. Only those of a more fundamental nature will be included here. Derivatization is a necessary evil for many chromatographic analyses to add a detectable group, or even a more sensitive detectable group, to the solute(s) of interest. Chemistries have been developed for virtually every type of compound, and advances are now coming in the design of faster reactions, more selective reactions, and the synthesis of highly sensitive derivatizing agents. Somewhat surprisingly, most interest in this review period is still in precolumn schemes. Postcolumn derivatization offers inherent advantages over precolumn methods in terms of precision, ease of automation, and the lack of necessity for a single reaction pathway. However, these schemes require more care in the initial design to minimize extracolumn band broadening, and they require reasonably fast reaction kinetics. Many reactions developed initially for flow injection analysis should be easily adaptable to postcolumn reaction detection for liquid chromatography, and the fundamental studies of reactor designs for one area should be of use for the other. Hull et al. ( M I ) reviewed dispersion phenomena in flow injection systems, which is relevant for postcolumn reactors, and discussed both predictive and descriptive models. No general reviews of the derivatization process were found in the search for this period; however, several reviews specific to a particular derivatization method or class of solute were published. Krull et al. published two reviews, one on silicabased solid-phase reagents for on-line and off-line derivatization ( M Z ) ,and another on the use of complexing agents for separation and detection of metal species ( M 3 ) . Other reviews included a review of the fundamental concepts of postcolumn derivatization used in EPA method 53 1.1 and other published methods for the analysis of N-methylcarbamates ( M 4 ) , the use of liquid chromatography in combination with immobilized enzymes and electrochemical detection (MS), biochemical postcolumn reaction detection in LC, including the use of enzymes, binding proteins, and antibodies ( M 6 ) ,derivatization zyxwvutsrq zyxwvu methods for determination of amino acids (M7), the use of 4-(bromomethyl)-7-methoxycoumarin and analogs as deriva- tization agents (M8),and the use of fluorogenic reagents for the derivatization of catecholamines and related compounds (M9). This review period saw further interest in the use of solidphase derivatizing agents. The use of these agents in place of solution-phase reactions has certain advantages, including in some cases the ability to filter and reuse the derivatizing agent. Krull’s group continued their strong interest in this area (MIO-MId). Bourque et al. studied the effect of crosslinking, surface area, and porous nature of modified poly(styrene-divinylbenzene) on the stability of highly reactive acylating reagents toward high concentrations of aqueous base (M10). Zhou et al. immobilized a 3,5-dinitrobenzoyl group on silica and used it as a reagent for the aminolysis of (2-4-8 amines ( M I ] ) . Fisher and Bourque later used this same reagent immobilized on a polymer for the derivatization and determination of amphetamine in urine (M17). Szulc and Krull combined several polymer-bound o-nitrobenzophenone reagents containing different detector-sensitive tags in the same reactor for the on-line derivatization of amines (M12). They found that formation of multiple derivatives allowed numerous opportunities for quantitation from the same injection and also allowed improved identification from the retention times of the multiple derivatives. Zhou et al. studied solid-phase derivatization of amino acids and peptides with a 9-fluoreneacetyl- (FA-) tagged polymeric reagent and showed the feasibility of solid-phase derivatization for peptide mapping ( M 1 3 ) . Zhang et al. compared two silica reagents based on a 4-hydroxy-3-nitrobenzoylbackbone with 9-fluorenylmethoxycarbonyl (FMOC) and FA tags for the derivatization of primary and secondary amines (M14). They found generally better performance with the FMOC-tagged silica. Zhou et al. also reported the use of resin-based FA groups for on-line precolumn derivatization in direct plasma injection (M15). Bourque and Krull also described the preparation of polymeric reagents for the derivatization with UV tags of weak nucleophiles, such as alcohols, thiols, and phenols (M16). Finally, Jedrzejczak et al. described the synthesis of polystyrene-bound mixed carbonic-indole-2-carboxylicanhydride which was shown to be effective for in situ N-acylation of n-butylamine (M18). The use of postcolumn photolysis for the formation of fluorophores or chromophores or to increase sensitivity continues to generate interest. This method, where applicable, is simple, as generally no chemical agents are added. The photons are the derivatizing agent. Krull’s group was also active in this area. Dou and Krull described work aimed at identifying the photochemical products of amino acids, peptides and proteins, especially those which are electroactive (M19). Krull and Dou also published a more tutorial article on the determination of peptides and proteins using on-line postcolumn photolytic derivatization ( M 2 0 ) . Schaefer also described the determination of a novel 4-quinolone antibiotic using postcolumn photolysis ( M 2 1 ) . H e reported that, compared with fluorescence detection alone, the derivatization step increased the intensity about 80-fold and significantly improved the selectivity. Immobilized enzymes would seem to offer an excellent means of producing postcolumn reactions; unfortunately, the concentrations of organic solvents that are generally necessary for separation by either normal- or reversed-phase chromatography rapidly denature the enzymes. In spite of this limitation, much work has been devoted over the years to the development of separations with mobile-phase conditions that are compatible with continued enzyme activity. Yamato et al. described a selective detection system for acetyl-coenzyme A (M22). The immobilized enzyme reactor (IMER) used phosphotransacetylase and had a substrate specificity sufficient to determine acetyl-coA. Kiba et al. used an IMER of tyrosinase for the selective determination of L-DOPA and L-tyrosine (M23). Maes et al. developed an IMER selective for the determination of 6-D-glucan oligo- and polysaccharides (M24). Immobilized cellulase converted the eluting oligomers to 6-D-glucose, which was oxidized by immobilized glucose oxidase, and the HzOz produced was measured with an electrochemical detector. Two other interesting fundamental schemes appeared during this review period. Zamir described the derivatization of saturated long-chain fatty acids with phenacyl bromide in nonionic micelles (M25). The use of micelles in this reaction overcame solubility difficulties with the long-chain fatty acids. Irth et al., in a highly novel work, described a postcolumn on-line immunochemical detection system (M26). The effluent from a reversed-phase column was mixed with fluorescein-labeled antibodies in a mixing union. Antigenic analytes react with the antibodies to form strongly fluorescent immune complexes, and then free antibodies are removed prior to fluorescence detection by passage through a small column packed with an antigen-bound support. They reported that the system tolerates up to 95% methanol or 45% acetonitrile, and they reported detection limits of 200 fmol for digoxin and 50 fmol for digoxigenin. During this review period there was a greatly increased interest in the pre- and postcolumn derivatization of metal ions and other inorganic species. Various transition metals were determined by derivatization with EDTA (M27), (A)trans- 1,2-diaminecyclohexane-N,N,Nf,N’tetraacetic acid (DCTA) (M28), 8-quinolinol-5-sulfonic acid (M29, M30), picolinaldehyde 4-phenyl-3-thiosemicarbazone(M31), 242pyridy1azo)-5-[N-(sulfoalkyl)amino]phenol (M32),2-(5-nitro2-pyridylazo)-5-(dimethylamino)benzoicacid ( M U ) ,aspartic acid 0-hydroxamate (M34),and 2-(6-methyl-2-benzothizolylazo)-5-(diethylamino)phenol (M35). Rare earth elements were derivatized with xylenol orange and cetylpyridinium bromide (M36) and 4-(2-thazolylazo)resorcinol (M37). Organotin compounds were derivatized postcolumn with morin (M38),and selenium was determined in coal after derivatizing with four different agents to form 2,1,3-benzoselenadiazoles (M39). Inorganic anionic species were also of interest, and here the chromatographic separation modes were a mixture of reversed-phase and ion chromatography. Iodide was converted postcolumn to 4-iodo-2,6-dimethylphenol (M40),chloride in biological fluids was determined as pentafluorobenzyl chloride (M41), nitrites in water were derivatized precolumn with 4-methyl-7-aminocoumarin(M42), nitrite and nitrate were converted to 1-nitro-2,4,6-trimethoxybenzene (M43),sulfide zyxwvu zy zyxw Analytical Chemistry, Vol. 66,No. 12, June 15, 1994 0 52QR zyxwvutsrqp zyxwv zyxwvutsrq zyxwvutsrqpon in an aqueous matrix was derivatized precolumn with 2-iodo1-methylpyridinium chloride ( M 4 4 ) ,cyanide was determined postcolumn with o-phthalaldehyde or naphthalenedicarboxaldehyde (M45),and cyanide in red blood cells was derivatized with 2,3-naphthalenedialdehyde(M46). Amines are likely the most heavily studied group for chemical derivatization because of their biological, industrial, and environmental importance. There are many classic reactions, and virtually every detection technique has been applied to the analysis of these compounds. There was a great deal of activity in this area during this review period, and for convenience, we have grouped these reports approximately by detection technique. Chemiluminescence continues to be studied, and there were reports of new methods for electrochemiluminescence detection of primary amines using tris(bipyridine)ruthenium(III) after derivatization with divinyl sulfone (M47),and of peroxyoxalate chemiluminescence detection of primary and secondary amines using luminarins 1 and 2 as labeling reagents (M48). Kouwatli et al. also compared the chemiluminescence detection using luminarin 1 with traditional fluorescent reactions ( M 4 9 ) . There were two reports of electrochemical tagging reactions for amine compounds. Cox et al. reported ferrocene tagging of amines, amino acids, and peptides (M50),and Zambonin et ai. described the determination of y-aminobutyric acid and various polyamines by electrochemical detection after derivatization with o-phthalaldehyde-@-mercaptoethanol(M51). New derivatizing agents which yield fluorescent products for amines were also introduced. These include 6-methoxy2-methylsulfonylquinoline-4-carbonylchloride ( M 5 2 ) ,three different oxazole compounds (M53),2 4 1-pyreny1)ethylchloroformate (M54),two chloroisothiocyanatoquinolines ( M U ) , 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate ( M 5 6 ) , and sodium benzoxazole-2-sulfonate ( M 5 7 ) . Because of the volume of samples analyzed by some laboratories, automated methods of derivatization continue to be of interest. Worthen and Liu discussed the automatic precolumn derivatization and separation of primary and secondary amino acids in plasma ( M 5 8 ) and Haynes et al. discussed applications of automated amino acid analysis using 9-fluorenylmethyl chloroformate as the tagging agent ( M 5 9 ) . Jansen et al. discussed the advantages and limitations of precolumn derivatization of amino acids with dabsyl chloride (M60),and Alaiz et al. used diethyl ethoxymethylenemalonate to generate amino acid derivatives with UV detectable groups at 280 nm ( M 6 1 ) . Several more specific amine-containing compounds were also of interest. Newcomb discussed the advantages and disadvantages of postcolumn derivatization with fluorescamine for detection of peptides ( M 6 2 ) ,Tellier et al. described a new tyrosine-specific precolumn fluorogenic derivatization method ( M 6 3 ) ,and Dave et al. described a postcolumn derivatization procedure for opioid peptides (M64). Catecholamines continued to generate new analytical methods. Jeon et al. described the determination of catecholamines, their precursors, and their metabolites in urine and plasma by postcolumn derivatization by chemical oxidation and derivatization with meso- 1,2-diphenylethylenediamine (M65). Descombes and Haerdi used 9-fluorenylmethyl chloroformate for derivatization of catecholamines and reported detection limits 17-350 times better than with electrochemical detection (M66). Ramos and Mike described an electrochemical reaction system which causes the basecatalyzed tautomerization of the oxidation products of catecholamines t o give highly fluorescent trihydroxyindole derivatives ( M 6 7 ) . Finally, Zheng et al. described a precolumn method for the derivatization of N-nitrosamines with 4-(2-phthalimidyl)benzoyl chloride (M68). After amines, the functional group that received the most interest in new derivatization schemes is the carboxylic acid group. Here the most interest was in the description of new fluorescent tags. These include thienylpyridine compounds (M69, M70), 3-(bromoacetyl)-7-methoxycoumarin( M 7 1 ) , 3-(bromomethyl)-7-methoxy1,4-benzoxazin-2-one ( M 7 2 ) , 2-(2,3-anthracenedicarboximido)ethyltrifluoromethanesulfonate ( M 7 3 ) , 4-(5,6-dimethoxy-2-benzimidazoyl) benzohydrazide ( M 7 4 ) , N-(bromoacetyl)-N’-[5-(dimethylamino)napthalene- 1-sulfonyl]piperazine (M75),and 2-(bromoacetyl)6-methoxynaphthalene ( M 7 6 ) . The derivatization and determination of several specific carboxylic acids was also described. In the interest of space, only the analyte will be listed; interested readers should then see the original article for the derivatizing group and reaction conditions. These include ascorbic acid ( M 7 7 ) and dehydroascorbic acid ( M 7 8 ) , methylmalonic acid (M79, M80), and hyaluronic acid ( M 8 1 ) . There were also three reports of the derivatization of fatty acids (M82-M84). The detection of saccharides also received alot of attention during this review period. Rakotomanga et al. described a simplex optimization of the precolumn phenylisocyanate derivatization of monosaccharides and found a major and stable derivative was formed under the optimal conditions, as compared to the several products which are often formed and result in several chromatographic peaks from each reducing sugar (M85). Zhang et al. published a very interesting paper describing the derivatization of oligosaccharides with N,N(2,4-dinitrophenyl)octylamine ( M 8 6 ) . This added a UV chromophore, added a hydrophobic tail to the solutes to make them more easily separated by reversed-phase LC, and also stabilized the negative ion species formed during analysis by fast atom bombardment mass spectrometry. A very efficient derivatization process! Other derivatizations include the use of 2-naphthoylimidazole ( M 8 7 ) , ethanolamine-boric acid ( M 8 8 ) ,purpald (4-amino-3-hydrazino-5-mercapto1,2,4-triazole) ( M 8 9 ) , thymol in concentrated sulfuric acid ( M 9 0 ) , hydrolysis with hydrochloric acid followed with derivatization with p-aminobenzoic acid hydrazide ( M 9 0 ) , and I-@methoxyphenyl)-3-methyl-5-pyrazolone( M 9 1 ) . There were three reports of new agents or methods for the detection of alcohols. These include 2-(2-(azidocarbony1)ethyl]-3-methyl-l,4-naphthoquinone,which adds both a fluorescent and an electrochemically active tag ( M 9 2 ) , 6-methoxy-2-methylsulfonylquinoline-4-carbonylchloride ( M 9 3 ) ,and 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole (M94). There were also three reports dealing specifically with the derivitization of nucleosides. These were all precolumn reactions to add fluorescent tags, and the reagents were 2-[5- zyxwvuts 530R Analytical Chemistty, Vot. 66, No. 12, June 15, 1994 zyxwvuts zyxwvutsrqpon zyxwvuts zyxwvut zyxwvutsrqp zyxwv (chlorocarbonyl)-2-oxazolyl]-5,6-methylenedioxybenzofuran (M95, M96), and phenylglyoxal (M97). New reactions and reagents were also reported for a variety of other compounds or functional groups. In the interest of space, only the analyte group will be mentioned; interested readers should then see the original article for the derivatizing group and conditions. These include hydroxysteroids (M98), corticosteroids (M99), ecdysteroids, (MIOO), S-(carboxymethyl)-L-cysteine and metabolites ( M l O I ) , biotin (MIO2), metoprolol (MIO3), monohydroxy compounds (MI O4), carbonyl compounds (MIO5),a-dicarbonyl compounds (MIO6), prostaglandins (MIO7),triacylglycerolhydroperoxides (MI O8), aldehydes (MIO9),glutathione (MIlO),thiols (MI1I,MI 12), penicillins and precursors (MI I3), N-methylcarbamate pesticides (MI 14), benzoylecgonine and ecgonine (MI 1 3 , conjugated dienes (MI 16-MI 18), 5-hydroxyindoles (MI I9), nitroaromatic, nitrate ester, and nitramine explosives (MI20), 6-mercaptopurine (MIZI),hydrazine (MI22),and cytokinins (MI23). N. MICROCOLUMN AND OPEN TUBULAR LC The proliferation of reports on liquid chromatography in small-diameter tubes has blurred to some extent the terminology used in previous reviews. To simplify the discussion of advances in this field, “microbore” will refer to columns with internal diameters between 0.5 and 2.0 mm, and the term “microcolumn” will be reserved for columns of less than 0.5mm i.d., whether they be packed or open tubular (OT). As in the 1992 Fundamental Review, microbore LC will not be specifically addressed, since this field has matured to the point that few fundamental developments appear in the literature. However, it should be noted that the practical advantages of these columns have led to their almost routine use. Reduced solvent flow rates in microbore columns also give them unique advantages over conventional LC columns in two-dimensional chromatography, and many citations in the Multidimensional LC and Column Switching section of this review include discussions of them. Fundamental studies of microcolumn LC included an evaluation of the quantitative reliability and long-term stability of a reversed-phase microcolumn system ( N I ) . Excellent stability was observed over an 8-month period, and quantitative reliability was excellent when external standard calibration and automated sample injection techniques were used. In another study of efficiency in open tubular LC, the impact of polydispersity in tube diameters was demonstrated using the Taylor-Aris and Golay theories (N2). Polydispersity was shown to be the dominant parameter controlling column performance when OT columns are operated at maximum efficiency. The use of elevated temperatures and temperature programming with microcolumns received considerable attention during the past two years. A study of extracolumn band broadening in split, high-temperature OT LC pointed out the impact of coldpoints in pre-and postcolumn connecting tubes ( N 3 ) . Liu et al. confirmed the advantages of elevated (100200 “C) temperatures in open tubular reversed-phase LC ( N 4 ) and reported on both normal- and reversed-phase open tubular separations of PAH (N.5). McGuffin and co-workers developed a temperature-programmed microcolumn LC/MS system as an alternative to conventional solvent-programmed LC/MS (N6). A specially modified oven permitted linear temperature programming between 40 and 100 O C at ramp rates of 0.1-0.5 OC/min. The system was used to separate with complete resolution saturated and unsaturated fatty acids in fish oil dietary supplements. Moore and Synovec introduced axial thermal gradient (ATG) micro LC, in which heated solvent is introduced into a microbore column at ambient temperature ( N 7 ) . The column acts as a heat sink, and flow programming the mobile phase raises the average column temperature with time. ATG LC has the potential to reduce recycle times relative to conventional radial column heating. Preparation of surface coatings for open tubular microcolumns was the subject of several reports. Goehlin and Larsson (N8) used four poly(organosi1oxanes) to coat narrowbore (5-15-pm id.) open tubular columns and studied their behavior in the regular cross-linked state and after swelling with n-heptane. A considerable improvement in efficiency was noted after swelling. Poppe and co-workers coated similar sized fused silica capillaries with polyacrylate films containing a lauryl functionality (N9). Thick (1 pm) films were obtained after photopolymerization. The lauryl functional groups resulted in considerable retention of polar compounds. Improved efficiency after swelling with nonpolar solvents was also noted. Deactivation of fused silica open tubular columns with poly(dimethylvinylsi1oxane) before coating and polymerization with another poly(dimethylvinylsi1oxane) layer was shown to improve 50-pm columns, but no increase in efficiency was noted for 10-pm columns (NlO). Thin layers of silica gel were deposited on the walls of 50 pm fused silica tubes using a hydrolytic polycondensation and gelling of tetraethyl orthosilicate (NI I). The silica gel was then bonded at high temperature with octadecylsilane. Other novel microcolumn stationary phases included C-60 buckministerfullerenes extracted from soot and packed into a 0.53-mm4.d. fused silica column ( N I 2 ) . The C-60 material exhibited unique shape selectivity for PAH. Enantiomeric separations comparable to those achieved on conventional columns were obtained with 320-pm4.d. fused silica columns slurry packed with bovine serum albumin adsorbed on silica gel and covalently bound L-dinitrobenzoylphenylglycine( N I 3 ) . Carbon- and octadecylsilica-packed microcolumns were evaluated for PAH separations, and the carbon sorbent column proved superior for several separations ( N I 4 ) . A bile acid derivative, 3- [(3-~holamidopropyl) dimethylaminol- l-propanesulfonate, was dynamically coated on silica gel and used in both enantiomeric (NI5) and ion chromatography (NI6) microcolumn separations. Finally, a simple laboratory method for preparing packed microcolumns was presented (NI 7). The method was also used to prepare columns after first coating the tube walls with a 0.4-pm film of immobilized Carbowax 20M; the coated columns showed a slight increase in efficiency, but their long-term stability was lower. New microcolumn techniques included the introduction of multimicro-HPLC by Voroshilova and Kaiser (NI8). This technique involves the use of two packed fused silica microcolumns of different selectivities serially coupled and placed inside a tubular oven. Selectivity of the coupled column combination is altered by stepwise movement of the tube oven to heat a different portion of the polar or nonpolar column Analytlcel Chemlstty, Vol. 66, No. 12, June 15, 1994 531R zyxw zyxwvutsr zyxwvutsrqpo zyxw bed. The result is an HPLC system with physically changeable selectivity and which is particularly suitable for identifying peakoverlapping. Parallel-current open tubular LC, in which both the mobile and retentive phases in an open tubular column move in the same direction, was also introduced (N19, N20). The retentive phase adjacent to the tube walls moves at a slower rate than the mobile phase at the center of the tube. The method was tested with a reversed-phase combination of water and cyclohexanol using phenol and hydroquinone as test solutes. Finally, on-column focusing was shown to be an effective approach for overcoming the reduced detectability exhibited by many microcolumn systems, particularly for earlyeluting peaks ( N 2 I ) . The rigorous demands of microcolumn LC for sensitive detectors and minimization of extracolumn band broadening make instrumental development an area of active research. Most of the work in this area during the past two years has apparently focused on detectors, although a microflow processor compatible with most conventional pumping systems was described ( N 2 2 ) . This microflow processor consists of a static micromixer and microsplitter with fixed split ratio and can convert milliliter per minute flows to microliter per minute, or even submicroliter per minute. Mass spectrometers are the most powerful of all chromatographic detectors, and their use with LC microcolumns has received considerable attention. A 320-pm-i.d. packed microcolumn was directly coupled to the source of quadrupole MS with an SFC-frit interface for the analysis of pentafluorobenzyl derivatives of all-trans- and 13-cis-retinoic acids (N23). Negative chemical ionization conditions were obtained by coaxial introduction of ammonia as the reagent gas. An aerosol generator interface was described for use with a commercially available particle beam unit for coupling microcolumns and a quadrupole MS ( N 2 4 ) . D20 was used as a reagent in microcolumn LC-electrospray ionizationquadrupole MS to induce peak shifts in mass spectra and determine the number of heteroatoms carrying H atoms (N25). Because of their inherently small size and high sensitivity for selected compounds, electrochemical detectors are attractive for use with microcolumns. Jorgenson’s group developed a rapid-scan electrochemical detector that uses a 9-pm carbon fiber as the working electrode ( N 2 6 ) . The detector has high sensitivity, and a large dynamic range, and can provide two-dimensional resolving power when coupled with a microcolumn separation. Another small-volume (< 1 nL) electrochemical detector was described that exhibited detection limits for OPA-derivatized amino acids that were below 1 fmol (N27). This detector can be used with either open tubular LC or capillary electrophoresis columns. Finally, a miniaturized flow cell for a conductivity detector was fabricated and used in the microcolumn ion chromatography separation of inorganic ions (N28). Other microcolumn LC detectors described include a flamebased thermionic GC detector adapted for LC microcolumns (N29) and used in the determination of organophosphorus compounds (N30). An FT-IR microscope and spectrometer were coupled to an LC microcolumn via an electrospray nebulizer (N31). Column effluent is disintegrated into charged droplets at the electrospray nozzle, solvent in the droplets is evaporated by a nitrogen sheath gas, and the separated components are deposited on a zinc-selenide plate for FT-IR analysis. Increasing sensitivity of UV detection in hightemperature microcolumn LC was the subject of another report (N32). The use of a Z-shaped microflow cell as well as optical fiber bundles was evaluated and found to yield an increase in sensitivity with only a small decrease in efficiency. Finally, a double-beam laser absorption system with thermal lens enhancement was developed (N33). The thermal lens enhancement yielded a 10-fold increase in sensitivity when compared to normal absorption measurements with the same circuitry. 0. TRACE ANALYSIS The term “trace analysis” does not really have an absolute definition, but rather implies that a method produces sensitivities and/or selectivities beyond those of a “routine” analytical technique. With regard to liquid chromatography, trace analysis usually relies on (1) some sort of sample enrichment, ( 2 ) derivatization to produce a response in a specific and sensitive LC detector, or (3) the use of a highly sensitive or selective detector not commonly associated with liquid chromatography. Fundamental developments in these areas are discussed in several other sections of this review, and thus, as in previous Fundamental Reviews, only a selective sampling of trace analysis by liquid chromatography is presented here. The coupling of high-performance LC methods with element-specific analyzers to measure trace metal concentrations received considerable attention during the past two years. Inductively coupled plasma mass spectrometry (ICP-MS) appears to be particularly suitable for such analyses. Vela and Caruso reviewed the current state of ICP-MS used with LC and supercritical fluid chromatography and noted that methods have been developed for characterizing metal speciation for As, Hg, Sn, and Cr (01). Important factors related to the introduction of LC effluent into the plasma were also discussed by workers from the same laboratory ( 0 2 ) . Mason and Storms ( 0 3 )used size exclusion HPLC coupled with ICPMS to identify two Cd-inducible, metal-binding proteins in marine polychaetes. Ion chromatography and ICP atomic emission spectroscopy were combined to determine tin and tributyltin in environmental samples with high backgrounds of Ca, Fe, Na, and K ( 0 4 ) , as well as selected elements in refractory metals ( 0 5 ) . A thermospray interface was described that improved transport efficiency in the coupling of HPLC and flow injection analysis with flame atomic absorption spectrometry (06). The thermospray interface allowed a conventional flame burner to be used without any modification. One of the more interesting applications of LC for trace analysis was described by Nickles et al. ( 0 7 ) ,who measured subnanocurie activities of positron emission tomography (PET) tracer metabolites. In this system, a reversed-phase LC column was coupled to a BC-400 plastic scintillator flow cell sandwiched between two large y-detectors. The LC separation was necessary to distinguish the free drug input from metabolite production, and the flow cell design was optimized so as to minimize loss of chromatographic resolution. Developments in thermospray (TSP) interface technology has made LC/MS an almost routine technique for the analysis of organics, and one that is particularly applicable for trace zyxw zyxwvut zyxwv 532R Analytical Chemistry, Vol. 66, No. 12, June 15, 1994 zyxwvutsr zyxwvut zyxwvutsrqp zyxwvuts zyxwvutsrqp zyxwvuts zyxwvut analysis. For example, an LC/TSP/MS analysis of phenylurea herbicides was described which additionally combined on-line, precolumn enrichment on PLRP-S columns or cartridges containing C 1 8-loaded membrane disks (08). Enrichment and analysis of 50-mL surface and tap water samples yielded detection limits in the 5-20 ng/L range for the 15 herbicides tested. An increased interest in on-line concentration with column switching was noted for both organics and inorganics. The difficult problem of concentrating and separating polar organics such as pesticides was addressed in several reports. Techniques included an automated method using on-line enrichment and UV diode-array (09)or TSP/MS (010) detection. Coupled analytical reversed-phase columns were applied to the analysis of a single target analyte using direct injection of 770-rL water samples (011). Coquart and Hennion demonstrated that porous graphite was a superior sorbent than PRP polymer columns for very polar, watersoluble organic compounds ( 0 1 2 ) . The porous graphite precolumn was coupled on-line to a similar analytical column for the direct analysis of several phenolic compounds. These same workers also described (013) an on-line procedure for the direct analysis of polar mono-, di-, and methylchloroanilines that employed two enrichment columns, a polymer PRP column followed by a cation-exchange sorbent. A wateracetonitrile gradient combined with electrochemical detection produced chloroaniline detection limits below 39 ng/L in river water samples. Two reports described the use of on-line trace enrichment after dialysis of a sample mixture to remove interfering components. The antiviral drug l-(0-D-arabinofuranosy1)5-( l-propyny1)uracil and its metabolite 5-propynluracil were determined in plasma by on-line preconcentration on an ODS column followed by isocratic separation on an analytical ODS column ( 0 1 4 ) . Plasma samples were first treated with monochloroacetic acid and then dialyzed to reduce proteinbinding effects. A similar approach was used to measure pholcodine in plasma and employed enrichment on a weak cation-exchange column, separation on a cyano column, and fluorescence detection after dialysis of the plasma sample (015). Other on-line enrichment and LC separation methods for organics included the use of a microbore concentration column for phenobarbital in serum (016) and the development of a new enrichment column selective for polyaromatic hydrocarbons (017). The PAH-selective enrichment column was a donor-acceptor complex immobilized on silica gel and was combined with an ODS column, gradient elution, and wavelength-programmed fluorescence detection to measure various PAH in edible oils and fats at concentrations of 1-10 rg/kg. Finally, on-column concentration on a microbore column followed by thermal gradient elution was shown to particularly effective for the direct analysis of highly retained compounds such as high molecular weight PAH (018). On-line enrichment coupled to HPLC separation was not restricted to organics. Several reports described combinations of ion-exchange, ODS ion-pairing, or chelation enrichment columns coupled to ion chromatography or ion-pairing reversed-phase chromatography separations. Silica gel impregnated with Aliquat 336 and Titan Yellow proved effective as a chelating concentration column for several metal ions and was coupled to IC ( 0 1 9 ) . The determination of Cu(II), Al(III), and Fe(II1) as their 8-hydroxyquinolinate complexes was accomplished by enrichment and separation on C- 18 reversed-phase columns ( 0 2 0 ) . In an interesting twist to this theme, lanthanide impurities such as Sm, Gd, Eu, and Dy in reactor-grade uranium were determined by combining a semipreparative reversed-phase column that removed the U matrix with an analytical cation-exchange column that concentrated and separated the lanthanides (021). Highefficiency denudders that concentrate atmospheric SO2 were coupled to an ion chromatograph to yield detection limits on the order of 0.5 parts per trillion ( 0 2 2 ) . Selective derivatizations still appear to be a popular approach for trace analysis by LC. While most are intended to enhance detectability of target analytes, some reports described derivatizations that improved enrichment or separation. For example, enantiomeric separation of secondary amino acids was accomplished after precolumn derivatization with 9-fluorenylmethyl chloroformate and separation on a cyclodextrin bonded phase ( 0 2 3 ) . Separation and detection of C-4-C-9 carboxylic acids was improved after precolumn derivatization with 24trophenylhydrazine ( 0 2 4 ) ;detection limits of 50 pg/L were reported. The speciation of Hg was characterized by precolumn derivatization with dithizone and reversed-phase HPLC ( 0 2 5 ) . Ion-exchange and ion-pairing reversed-phase HPLC of heavy metals complexed with 4-(2pyridy1azo)resorcinol was described ( 0 2 6 ) . Reactions to enhance fluorescence detectability included precolumn derivatization of nitrosamines (027)and postcolumn derivatization of a variety of trace metals that normally do not fluoresce ( 0 2 8 ) . A direct LC analysis of the organotin pesticides fentin, cyhexatin, and fenbutatin was described that also employed fluorescing derivatives ( 0 2 9 ) . The pesticides were separated on a normal-phase cyanopropyl column, then photoconverted by UV irradiation, and complexed with morin before fluorescence detection. Finally, one of the instrumental constraints to LC trace analysis was addressed by Saha and Giese, who described a two-injector approach when carryover from injection to injection is a problem (030). P. PHYSIOCHEMICAL MEASUREMENTS This section deals with the use of HPLC for measuring thermodynamic, kinetic, adsorption, partitioning, association, ligand binding, and conformational properties of solutes. Thermodynamics, as related to separation mechanisms, is also covered. Readers interested in this topic may want to consult the corresponding section in the Size Exclusion Chromatography Fundamental Review article of this issue for related studies. PartitionCoeffcientdHydrophobicityMeasurements. The determination of partition coefficients (log P),usually related to octanol-water systems, and hydrophobicity parameters using reversed-phase HPLC continues to be a useful approach for characterizing potentially biologically active compounds with regards to quantitative structure-activity relationships (QSAR). Because of the connectivity between log P and the log of the capacity factor (log k’), reversed-phase HPLC is Anatytical Chemistty, Vol. 66,No. 12, June 15, 1994 5331 zyxwvutsr zyxwvuts zyxwvutsr zyxwvu a rapid method of estimating partition coefficients for QSAR studies. Hsieh and Dorsey (PI) discussed the extrapolation of k’ of solutes to 100%water using different methods and compared these data to measured values. Prediction of log k’, from retention data as a function of both mobile and stationary phases was described. Solvatochromic analysis was used to deconvolute stationary- and mobile-phase effects. Dave et al. (P2) investigated Snyder’s theory of linear gradient elution to predict the starting isocratic reversed-phase conditions for opioid peptides. Errors in this model were attributed to a mixed mechanism of retention involving solvophobic and silanophilic interactions leading to nonlinear relationships between log k’ and mobile-phase composition. Valko and Slegel (P3) described a chromatographic index to measure the lipophilic character of compounds in reversed-phase LC. The parameter is defined as the organic phase concentration in the mobile phase which is required for log k’ = 0. The value is characteristic of the compound and depends only on the type of organic modifier, pH, and temperature, and is independent on column type. Zou et al. ( P 4 )presented a method for characterizing solute hydrophobicity and electrostatic interactions using ion-pair reversed-phase LC. This approach was applied to dipeptides (P5)and phenylamine- and naphthylaminesulfonic acids (P6). This group (P7) also used multivariable regression analysis to predict logp values. Using reversed-phase HPLC, Belsner et al. (P8)found a linear relationship of log k’, to log P (at pH 7.4) for a series of nitrogen bases. Moeller et al. (P9) discussed the prediction of partition coefficients by a groupcontribution method (UNIFAC). Ford et al. (PIO)developed a rapid microscale method for the direct determination of log P i n which the solute concentration in both phases is measured by HPLC. Kaliszan and co-workers (PIJ) studied the use of a deactivated alkyl-bonded silica and lecithin-bonded propylamino silica columns for QSAR of organic bases including phenothiazine derivatives. They also compared results obtained on a reversed-phasecolumn, an aminopropyl stationary phase, and an aminopropyl phase coated with melanin ( P I 2 ) . Quantitative structure-retention relationships were compared for a set of 1,4-benzodiazepines analyzed on a human serum albumin-based chiral stationary phase, reversed-phase silica packing, and a graphitized carbon stationary phase (PI 3, PI 4 ) . Lipophilicity measurements were conducted by Nowotnik et al. (P15) on a cross-linked polystyrene packing, C-18derivatized cross-linked polystyrene packing, and a C-&coated silica packing, and by Kalman et al. ( P I 6 ) on C-8, C-18, and C-30 reversed-phase columns and a quaternary amino column using peptides as solutes. Pirkle and co-workers ( P I 7 ) used chiral stationary phases to study the reversed-phase behavior of homologous series of racemic solutes. Hydrophobicity parameters of proteins were measured on hydrophobic interaction columns (PI8),pyrazine and amino acids on a polybutadiene-coated alumina packing (PI 9 ) , phenolic derivatives on a porous graphitized carbon packing (P20), and soil humates on aminopropyl silica and alumina-containing immobilized humic acid (P21). Micellar liquid chromatography was used to study the hydrophobicities of nonsteroidal anti-inflammatory drugs (P22), sulfonamides (P23), and polycyclic aromatic hydrocarbons (P24). Micellar electrokinetic capillary chromatography was employed to determine the partition coefficients of catechols to SDS micelles, which was used as a model for drug permeability through lipid membranes (P25). MedinaHernandez et al. (P26)presented an interesting study in which they were able to correlate partition coefficients of diuretics, determined by micellar LC, and the sites of action within the nephron. Thus, the site of action of those diuretics having a lower retention is the proximal tubule and the loop of Henle; for those diuretics of intermediate retention, the action site is the distal tubule; and for those diuretics with increased retention, the site of action is the collecting duct. Funasaki et al. (P27) correlated log k’ with log P of cyclic dipeptides, including diastereomers, on a reversed-phase packing. Yamagami and Takao (P28-P31) published a series of papers on the use of reversed-phase HPLC to determine hydrophobicity parameters of pyrazine derivatives (P28), heteroaromatic compounds and their derivatives (P29, P30), and substituted benzyl N,N-dimethylcarbamates ( P 3 I ) . Woodburn and co-workers (P32)correlated log k’of polycyclic aromatic hydrocarbons, alkylbenzenes, and halobenzenes with log P, and hydrophobic surface area. Rekker et al. (P33)compared log Pvalues and hydrophobic fragment constants of polymethyl and polychloro derivatives of benzene and biphenyl with HPLC data. They also reported that normal-phase silica packings may provide substantial information on nonhydrophobic binding of aromatic ring systems. Wells and Clark (P34) used reversed-phase HPLC to determine hydrophobic substituent contributions of substituted benzanilides and benzamides. Sanchez-Moyano and co-workers (P35) studied the partition behavior of ringsubstituted anilines in reversed-phase HPLC and compared these results to data obtained from classical bulk-phase partitioning. Meluch and Lloyd (P36) compared partition coefficients of alcohol-water-cellulose acetate systems obtained from equilibrium sorption experiments to results from LC. In general, the LC values were smaller than the equilibrium distribution coefficients, which was attributed to the fact that equilibrium was not attained in the chromatographic column. Cardamone and Puri (P37) determined the surface hydrophobicity of proteins using equilibrium binding studies to an apolar fluorescent dye. These authors found no correlation between the dye-binding studies and results from reversedphase HPLC. The dye-based measurement of surface hydrophobicity appears to be the most appropriate means for assessing proteins to reflect their overall three-dimensional structure in solution. Reversed-phase HPLC was used to measure the solubility and hydrophobic properties of cyclodextrins (P38) and nitro polycyclic aromatic hydrocarbons (P39);the hyrophobicty of triporphyrin ethers (P40),analogs of arabinosylcytosine ( P 4 2 ) , azoles (P42), antihypoxia drugs (P43), and 1,Cbenzodiazepines ( P 4 4 ) ;partition coefficients of a radiosensitizer (P45), P-adrenoceptor antagonists (P46), pesticides (P47), and nifedipine (P48); and QSAR of dipyridyl sulfides (P49), hemisuccinyl derivatives of a-hydroxyprogesterone (P50), benzothiazines (P51),and triazines (P52). zyxwvu 534R Analytical Chemistry, Vol. 66, No. 12, June 15, 1994 zyxwvutsrq zyxwvutsr zyxwvu Ligand-BindingMeasurements. Wainer’s group (P53,P54) studied the behavior of drug-binding interactions on a immobilized human serum albumin chiral stationary phase and determined the enantioselectivity of ligand binding to immobilized albumin stationary phases (P55). Shibukawa et al. (P56) developed an on-line high-performance frontal analysis HPLC system for the stereoselective determination of unbound ketoprofen enantiomers and discussed its advantages over the ultrafiltration method. These investigators (P57) also reported on high-performance frontal microanalysis for the simultaneous determination of unbound and total carbamazepine concentrations in protein-binding equilibrium experiments. Loun and Hage (P58)used an HPLC affinity column containing human serum albumin to study the binding of thyroxine at the warfarin and indole sites of human serum albumin. Serum protein-binding studies using HPLC were reported for mexiletine (P59) and remacemide (P60). Pezzuto et al. (P61) described an HPLC system for the detection of compounds capable of binding to DNA. Backes and Rychnovsky (P62) used affinity columns, prepared by injecting sterols into a C-18 reversed-phase column, to measure the interaction of polyene macrolide antifungal agents with sterols. By comparing the elutionvolume of a polyene injected onto a sterol-free column with that of the polyene injected onto the sterol-doped column, an association constant was determined for the polyene-sterol complex. Konishi and coworkers (P63) studied the interaction between cyclodextrins and drugs with an immobilized cyclodextrin column and a reversed-phase column. Association constants were estimated by frontal analysis, and from k’ on the reversed-phase column in the presence of cyclodextrins. The stoichiometry and apparent formation constants of anthracene and pyrene @-cyclodextrincomplexes were determined by Anigbogu (P64) by reversed-phase HPLC. An HPLC affinity column containing immobilized modified GM1 was used to study the binding of human brain lectin in comparison with other carbohydrate-binding proteins (P65). Katoh et al. (P66) described a method for the analysis of the binding properties of lectin with fluorogenic sugar chains, in which the amount of free sugar chains, isolated by membrane ultrafiltration, is determined by HPLC. Thermodynamic Studies. Hanai et al. (P67) studied quantitative structure-retention relations in reversed-phase HPLC using values calculated from computational chemistry. Lee and co-workers (P68) investigated the effect of solvent and temperature on the reversed-phase retention behavior of phenols. Equations for predicting retention were derived by using hydrophobic substituent constants and the sum of Hammett’s constant and Taft’s steric constant. Rozylo and Janicka (P69) used a thermodynamic description of liquidsolid adsorption chromatography to predict analytical separations. The sorption mechanism for alkylbenzenes on a silvercontaining stationary phase was described by Rodionov et al. (P70) which enabled the optimization of isomeric alkylbenzenes. Karlsson and co-workers (P71) studied chiral discrimination of phenolic 2-dipropylaminotetralin derivatives by ion-pair chromatography. These authors reported surface areas, dipole moments, binding energies, and relative steric energies. Ching et al. (P72) used a cellulose chiral stationary phase to characterize the enantiomer selectivity of @-blockers. This group (P73) calculated capacity factors and enthalpy of adsorption from retention data of essential fatty acids separated by reversed-phase HPLC. Martire et al. (P74) described a thermodynamic approach for evaluating the relative contributions of the stationary and mobile phases to solute retention in reversed-phase HPLC. Economou et al. (P75) used HPLC to measure infinite dilution activity coefficients of benzene, toluene, p-xylene, and cumene in a reversed-phase system. Hearn and co-workers (P76) studied the thermodynamic behavior of peptides in reversedphase HPLC. Relative changes in entropy and enthalpy were determined which provided insight into the factors involved with the stabilization of secondary structure and the mechanism of interaction of peptides with hydrophobic surfaces. Salo et al. (P77) investigated the effect of temperature on the retention of retinoates in reversed-phase HPLC and studied the influence of molecular structure on standard enthalpy change. The elution behavior and enthalpy change of metal chelates of a pyrazolone derivative in reversed-phase HPLC was reported by Kang (P78). Jakus (P79) presented a new approach for the theoretical evaluation of Gibbs free energy of solvation as applied for estimating retention in reversed-phase HPLC. Suet al. (P80) studied the effects of temperature and eluant pH on the retention behavior of N-acetylglucosamine and di-N-acetylchitobiose on silica-immobilized hen egg white lysozyme in which binding enthalpy and entropy were measured. Woodburn et al. (P81) estimated the enthalpy and entropy changes associated with retention of nonpolar solutes on reversedphase packings. Kloubek (P82) studied the relation between the specific capacity factor and adsorption energy of aromatic hydrocarbons, which allows prediction of retention. Enthalpy of adsorption, Gibbs free energy, and activity coefficient data were used to study interaction between food nutrients and poly(viny1 acetate) immobilized on silica support (P83). Interaction between aflatoxins and 8-cyclodextrins was studied using reversed-phase HPLC in which enthalpy and entropy changes associated with the interaction of the reversed-phase support and complexed solutes were measured (P84). Hearn and co-workers (P85)presented analytical solutions for a mathematical model describing dynamic adsorption processes of proteins onto nonporous particles. Place et al. (P86)examined the adsorption of human serum albumin on reversed-phase packings using frontal analysis. Guiochon’s group measured the adsorption isotherms of cholesterol and related compounds on reversed-phase packings (P87) and methyl mandelate enantiomers on 4-methylcellulose tribenzoate coated on silica (P88). Daniels (P89) described a pseudophase LC technique to study mediated adsorption of a series of alkyl 4-hydroxybenzoates and poly(oxyethy1ene) nonyl phenyl ether onto solid surfaces. Mediated adsorption was increased by increasing the alkyl residue of the 4-hydroxybenzoic acid ester. Lu (P90) and Xu et al. (P91) used reversed-phase HPLC to determine dissociation constants of weak bases and acids. Gonzalez (P92) presented guidelines for determining acidity constants of drugs by HPLC. HPLC was used to determine pKa values from capacity factors of chlorinated phenols (P93). HPLC was used to determine the stability constants of magnesium adenosine triphosphate and magnesium adenosine zyxwv Analytical Chemlstty, Vol. 86, No. 72, June 15, 1994 535R zyxwvutsrqpo zyxwvut zyx zyxwvu zyxw diphosphate (P94),crown ethers and actinomycin with cations (P95),perrhenate complexes of citrate, tartrate, and oxalate (P96),and iron complexes with lactate, gluconate, and citrate (P97). Braco et al. (P98, P99) determined the self-association of gramicidin A with HPLC. Lemque and colleagues (P100) studied self-association of /3-lactoglobulin B in solution and in the adsorbed state on an anion-exchange column. Yeo and Rabenstein (P101) described a method for characterizing the kinetics and equilibrium of thiol-disulfide interchange reactions of the disulfide bonds in arginine vasopressin and oxytocin and related peptides using reversed-phase HPLC. Chau and Nelson (P102) used HPLC to measure the equilibrium constant between glutathione and dithiothreitol. Weber and co-workers (P103) developed a chromatographic method for the determination of equilibrium constants for the carbamate formation reaction from amino acids and peptides in aqueous solution. Ye and Schuler (P104) used HPLC to study intramolecular charge transfer in polyhalogenated systems. Kinetic Studies. Using HPLC, Nemirovskii et al. (P105) studied the metabolic kinetics of furan and benzene aldehydes in vivo. The rate constants and equilibrium constants of ligandexchange reactions between bis(alky1xanthato)palladiumand bis(N,N-dialkyldithiocarbamato)palladium determined by use of HPLC was reported (P106). Reversed-phase HPLC was used to measure the reaction rate constants of quenching agents with singlet 0 to study the quenching effect on magenta azomethine dyes (P107). UV flash photolysis followed by HPLC analysis of products was used to study partial photooxidation of 4-chlorophenol (P108) and phenol and p-benzoquinone (P109). Pirkle et al. (PI10)used chiral stationary phases to study the kinetics stereoisomer interconversion of atropisomers. A reversed-phase method was described for determining the kinetics of biotinylation of specific residues of peptides (PI1I ) . HPLC was used to investigate kinetics of base-catalyzed epimerization of [Co(acac)s(Me3en)] C104 (PI 12), interconversions of stereoisomers of thymidine glycol (PI 13), isomerization of eugenol (P114),and isomerization of N-acyl 5’O-trityl-2’-O-aroylnucleosides (PI15). Stability and degradation kinetics were reported for morphine (P116),etomidate (PI17 ) ,indomethacin (P118), norfloxacin (P119),famotidine (P120),pancuronium hydrobromide (interesting name) (P121), methylfolates (P122),and 5-halogene2’-deoxyuridines (P123). Hydrolysis kinetics measured with HPLC was reported for aromatic nitrogen mustards (P124), metal ion-catalyzed hydrolysis of ampicillin (P125),proteolysis (P126),tinidazole (PI27),metronidazole phosphate (P128),3-Oacyl oxazepam (P129),ethylene glycol butyl ether ester of triclopyr (P130), and epoxides of naphthalene (P131). Conformational Studies. Hearn and co-workers reviewed the use of HPLC to study protein folding (P132) and studied the conformational interconversions and relative rates of chromatographic relaxation of bombesin, glucagon, and P-endorphin (P133). Reversed-phase HPLC was used by Buettner et al. (P134) to investigate the ability of proline to perturb the secondary structures of peptides induced at waterlipid interfaces and the induced conformation of polyproline peptides. This group (P135) also used HPLC to study selfinduced conformational effects of peptide analogs. They found that strong peptide-peptide interactions occurred through interchain hydrophobic forces, which resulted in a presenting face to the C-18 group, consisting primarily of lysine residues. Watson and Kenney (P136) reported that multiple reversedphase peaks observed for recombinant human platelet-derived growth factor were caused by conformers of isomeric structures arising from the presence of proline-proline bonds within the primary structure of the protein. Benedek (P137, P138) studied the conformational changes and unfolding kinetics of brain-derived neurotrophic factor during reversed-phase HPLC with the use of an on-line right-angle light-scattering detector. Monger and Olliff (PI39) investigated the effect of mobile-phase composition and HPLC conditions on the reversed-phase behavior of &endorphin in terms of its conformational changes. 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