Anal. Chem. 1990, 62,324 R-356 R (J22) ONen, D. M.; PeHln, M. J.; Calaway, W. F.; Young, C. E. In ref A4, 1988, PP 789-796. (J23) Sen, R.; Eagks, J.; Fairweather-Tat, S. J.; Portwood, D. E. Anal. h c . 1987, 24(12), 366-367. (J24) RBB, D. E.; Eagles, J.; Fairweather-Tait, S. J. J . Mlcronutr. Anal. 1987, 3(2), 107-1 17. (J25) Awrwal, S. K.; Kinter, Mlchael: Wllls. M. R.; Savory, J.; Herold, D. A. Anal. chsm.WEB, 67(10), 1099-1103. (J26) Wolf, W. R.; LaCrolx, D. E.; Kochansky,J. J . Micfonutr. Anal. 1988, 4(2), 145-154: (527) Fakbank, W. M., Jr. Nwl. Instr. Mthoab phys. Res. 1987, 407-414. (J28) Fakbenk, W. M., Jr.; LaBelle, R. D.; Keller, R. A.; Miller, C. M.; Poths, J.; Fearey, 8. L. In ref A5, 1989, pp 53-56. (529) S&weatert, E. A. Anatmt 1989, 114(3), 269-274. (J30) ollver, 8. M.; Bretscher, M. M.; Farrar, H., I V Appl. Radiat. h o t . 1989, 40(3), 199-208.
(J31) Wang. P.; Majldi, V.; Holcombe. J. A. Anal. Chem. 1989, 61(23). 2652-2658. (J32) Bass, D. A.; Holmbe. J. A. A M I . Chem. 1888, 60(8). 578-582. (J33) Igarashi, 0.; Ozima, M. ShRsuvyo Bun&/ 1987, 35(5), 256-290 (CAI091181:1624024). (534) Ma%, E.; Brachi M.; Criaud, A.; Foulllac, C.; Vuatar, F. D. C. R . Acad. Sci., Ser. 3 1987, 305(11), 989-974 (CA108[10]:86986j). (J35) Wacker, J. F. oeochimice Cosmochlm. Acta 1880, 1421-1433. (J36) Suszcynsky. D. M.; D’Angelo, N.; Merlino, R. L. Rev. Sci. Instrvm. 1988, 59, 1376-1379. (J37) Qeene, W. M.; Hertney, M. A.; Hess, D. W.; Oldham. W. G. Mater. Res. Soc. Symp. Proc., 117(PTo~essDiagn.: Mater., Combust.. Fusion) 1888, 61-65. (J38) Young, D. T.; Burch, J. L.; Marshall, J. A. Nucl. Inshum. Methods phvS. Res. 1989, 840-841(2), 750-754.
Liquid Chromatography: Theory and Methodology John G . Dorsey* Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221 -01 72
Joe P . Foley Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803
William T.Cooper Department of Chemistry, Florida State University, Tallahassee, Florida 32306
Robert A. Barford ERRC- USDA, 600 E . Mermaid Lane, Philadelphia, Pennsylvania 191 18
Howard G . B a r t h E. I. du Pont de Nemours & Company, P.O. Box 80228, Central Research & Development Department, Experimental Station, Wilmington, Delaware 19880
INTRODUCTION
This review covers fundamental developments in liquid chromatography durin the period of December 1987 through approximately Decemter 1989. This year for the first time there are separate reviews on instrumentation and size exclusion chromato raphy; this review is of important developments in the caemistry of the separation process. The primary searchin methods for this work have been CAS Online and CA Sefects. Each author has supplemented these with search methods of their own. This is not meant to be a comprehensive review of all published apers during this time period;rather, we have tried to select tiose 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 should be sent to the first author (J.G.D.).
A. BOOKS Where ossible in this section, reviews are cited along with the publisied book. A eneral book on liquid chromatography was authored by GilEert ( A I ) . A volume of the Wiley Chemical Analysis series devoted to modern LC was edited 324 R
by Brown and Hartwick (A2)and reviewed (A3). A book on practical LC originally in German was translated and updated (A4)and reviewed (A5). The fifth volume of Horvath’s series, High Performance Liquid Chromatography: Advances and Perspectives, appeared (A6) as did the 28th volume of the Advances in Chromatography series ( A n , and both were reviewed (A8, A9). A book devoted to method development was published (AIO) and reviewed ( A l l ) ,as was a book devoted to troubleshooting LC systems (A12, A13). Books devoted to individual techniques or applications of liquid chromatography are cited in the sections corresponding to those techniques.
B. REVIEWS AND SYMPOSIA PROCEEDINGS Several veneral reviews ap ared during this review period. A special issue of ChemicaEeviews was devoted to chromatography and had several papers devoted to liquid chromatography ( B l ) . Kirkland reviewed liquid-phase se aration techniques, includq LC (B2). Schoenmakersand M&olland gave an overview o method development in liquid chromatography (B3),and Majors reported the results of a surve of column usage in LC (B4). Ahuja reviewed trace and u% tratrace analysis in LC (B5) and the development of high-
0003-2700/90/0362-324R$09.50/0 0 1990 American Chemical Society
LIQUID CHROMATOGRAPHY John 0. D w n y Is Prafessw 01 Chemistry and Chairman 01 lhe Analytical Division at the University of Cincinnati. He received his Ph.0. degree in analytical chemistry in 1979. under 1.W. Gilbert at the University of Cincinnati. and then spent 10 years on the facuny 81 the University of Flwaa. He returned to Cincinnati in 1989. His research interests are in the areas 01 fundamental liquid chromatography. analytical applications of mi. c~llesand organized media. flow injection analysis. and old Bordeaux wines. He has about 50 publications in these areas. and serves on the editorial board 01 Anap;cal Letters. RioChromatcqaphy. and Jwmalof L;& Chrmtography.
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Joe P. FOIw is Assistant Rofessor of Chemistry at Louisiana State University. HB received his B.S. in Chemistry and chemical physics hom Centre College of Kentucky in 1978. After concludina a ninemonth Staff Assistant position at 6mtre. he began his graduate studies at the University of Florida in June 01 1979 and received his Ph.0. in Chemistry in 1983. He then accepted a two-year National Research Council Postdoctml Fsllowship at the National Ins1ilute of Standards and TeChnOlDgy (then NBS). He joined the faculty Of LSU in August of 1985. Dr. Foley'r research interests are in the fundamental and applied aspects of chemical separations, and he has over 18 pUblicatiOn5 8n the areas Of high-performance liquid chromatography. supercritical fluid extractionslchromatography. capillary zone electrophoresis. and micellar electrokinetic Chromatography. He has organized or chaired severai Symposia and is a member of the Division 01 Analytical Chemistry 01 the ACS and an Executive Cornminee member of the New Orleans Chromatography and Analytical DisCuSiion Group. Wllllam T. Coop., is currently Asmiate Profesw of Chemistry. Adjunct Professor of Oceanagraphy. and Associate Director of the Center for Biomedical a Toxicological Research at Florida State University. He r e ceived a B.S. in chemistry from the University 01 Tennessee. Knoxville. and a PII.0. in chemistry from Indiana University. While at Indiana he worked in the Biogeochemical t Laboratories 01 Professor John Hayes. where ha studied interactions between organic solutes and mineral surlaces using inverse gas and liquid chromatography techniques. He hes been a visiting scientist and consultant lrx the Waste l~olationBranch of the Earth Sciences Division. Oak Ridge (TN) National Laboratory. as well a3 an Air Force Summer FacuMy Research Fellow at the EnvirOniCS Laboratory, Tyndall AFB (FL). AS a technical consuitant to the Florida Department of Environmental Regulatbn. he has participated in the development 01 many of Florida's water quality regulations. Dr. Cooper has Served as Chairman of the Environmental Sciences Dkisim of the Florida Academy 01 Sciences and a9 President of the Tallahas~eeSubseCtimn 01 the American Chemical Society and was a cofounder and the first president Of the TallahasSee Chromatography Association. Environmental biogeochemistry is the primary theme 01 his research, which includes development of twodimensional Separation mthods for analyzing complex environmental and biological samples. inverse gas and liquid chromatography studies of the surface chemistry of heterogeneous. geological materials, characterization of the chemical Composition 01 soil and Sedimentary organic matter by NMR spenroscopy. and the use 01 liquid chromatography and NMR in studies 01 microbial degradation and m e tabolism of toxic organic chemicals He is a member 01 the American Geophysical Union and the Analytical. Environmental and Geochemistry Divisions 01 the American Chemical Society. While he has not won any major awards. he has shared some 01 the world's finer wines with his fellow coauthors.
resolution liquid chromatography ( 8 6 ) ~ The proceedings of the 12th International Symposium on Column Liquid Chromatography appeared (87).Symposia on specialized techniques are not reported here, rather the individual papers are cited in the relevant sections.
C. THEORY AND OPTIMIZATION Theory
With the exception of the reviews cited immediately below, we have excluded nearly all theoretical Contributions that can
Robert A. Barlord is Research Leader at the Eastern Regional Research Center 01 the Agricultural Research Sewice-United States Department of Agriculture. located in Philadelphia. where he Conducts research: (1) to ascertain Structures of biopolymerS including proteins and polysaccharides. determine factor3 that perturb there structures, and relate the structures to functional. biochemical, and nutritional properties of agricultural prcducts: (2) to develop new technologies. in support of USDA's regulatory branch. for BxpedBioUSly detecting chemical residues and their metabolites in animal products. A graduate of Temple University he has autha& or coauthored over 105 icientilic publications. contrihuled five baok chapters. and coedited the books. Melhcds lor hotdo A m i y s k and MBcromOlecular Interectiom and Food Colloid SIabilW. He is an active member of the Analytical and the Agricuitural and Food Chemistry Divisions Of the ACS and the Chromatography Forum of the Delaware Valley. He has organized numerous symposia for these Organizations and was Chairman of the Sixth International Symposium on Column Liquid Chromatography and General Chairman of The 1981 Meeting of the Federation of Analytical Chemistry and Applied Spectroscopy Societies. He has received the Chromatography Forum 01 the Delaware Valley Award and the Pennsylvania Chapter 01 the American Institute of Chemists Honor Scroll. Howard 0. Barth is a member of the r e Search staff 01 the Analyiical Division of the Central Research 6. Development Department at DU Pant Experimental Statimn. WiL minston. DE. Before joining the Du Pont Company in 1988. he was a &arch scientist and group leader at Hercule~Research * ' .. Center. He received his B.A. (1969) and Ph.0. (1973) in analytical Chemistry from Northeastern Univeristy. He is a frequent lecturer at short courses sponsored by the ACS Polymeric Materials Science and Engineering Division. His specialties include polymer characterizatlon. size exclusion chromatography, and high-performance liquid chromatography. He has published over 4 5 papers m these and relaled areas and has a130 edited a book. Modern Melhcds 01 ParWle Size Analvsis. published by Wiley. and two symposium voIumes on polymer characterizatimn. Barth we5 on the lnstr~mentationAdvisory Panel of AnaIyticcaIChemishy and was Associate Ednor of the Jowml Of Applied Polymer Science He is cofounder of the lntemational Symposium on Polymer Analysis and Characterization and is past Chairman of the Delaware Section 01 the ACS. Or. Barth is a member of the ACS divisions 01 Analytical Chemistry. Polymer Chemistry. and Polymeric Materials Science and Engineering. and the AAAS. and the Delaware Valley Chmmatograohv Forum. He is also a Fellow of the American Institute of Chemists. ~
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logically he placed in another category (e.g. reversed phase, geometric and optical isomers, preparative, etc.), especially those contributions pertaining to chromatographic properties (efficiency, retention, and selectivity) of a specific separation mude. Reviews. The dynamics of separation was reviewed by Weber and Carr, with an emphasis on the determination of separation efficiency, solute dispersion, and retention ( C l ) . A variety of separation modes were reviewed, including affinity chromatography (C2, C3), immunoaffinity chromatography (C4),chiral separations (see Geometric and Optical Isomers), hydrophobic interaction chromatography (CS), nonlinear chromatography (CS), high-performance displacement chromatography (C7), spacer displacement chromatography (CS), and micellar LC (see Secondary Equilibria). Several aspects of reversed-phase chromatography were reviewed, including retention mechanisms (C9), retention indices (CIO),the retention behavior of large polycyclic aromatic hydrocarbons ( C I l )and sulfur and sulfur-containingcompounds (CIZ),and polymer-based packing materials (C13). Other reviews not specific to a given separation mode included one on column design (C14),dynamically modified silica (C15),and chromatographic data systems (CI6). Resolution/Peak Overlap. One of the more difficult challenges faced by chromatugraphers is the accurate and unambiguous description of the separation between (or overlap of) two adjacent peaks. Although the conventional resolution parameter defined years ago works well for Gaussian peaks, ANALYTICAL CHEMISTRY. VOL. 62. NO. 12. JUNE 15. 1990
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it is inconvenient in practice since for overlapping peaks of unequal area or peak height it cannot be directly correlated with errors of integration algorithms (perpendicular drop, tangent skim) or peak purity at selected cutpoints. Moreover, in addition to the difficulties arising when aks are of unequal size, real chromato raphic peaks are sel om truly Gaussian, and this greatly r uces the accuracy or precision with which peak overlap can be defined if not precluding it altogether. Among other ualities, the ideal resolution parameter should be as follows: Ti) measurable directly from the chromato am, even on overlapping peaks; (ii) applicable to any peak s ape; (iii) invariant with respect to chromatographic time and concentration scales; (iv) commutative with respect to peak identity; (iv) directly correlated with errors of integration and peak purity at selected cutpoints; (v) and normalizable to an appropriate range. Although none of the parameters reported during this review period appear to have all of these desirable features, they do possess one or more of them and therefore warrant the attention of the chromatographic community. El Fallah and Martin (C17)reported a new index of performance of the chromatographic separation between two adjacent peaks, the discrimination factor do. Values of do range between 0 and 1, depending on their relative peak heights separation. The discrimination factor re uires no assumptions regarding peak sha e and is measure8 directly from the chromatogram. A modi&ed or effective peak capacity was defined in terms of constant do, and I t and (1, were compared with conventional peak capacity and R Starting from the classical resolution function, Schoenmagers et al. (C18) derived modified expressions that correct for large variation in peak area and peak asymmetry. As a consequence of these modifications, each peak of an overlapped pair has its own unique value for resolution. Good correlation with relative peak overlap, another measure of separation, is usually obtained, although the modified resolution ex ressions are relatively complex and thus somewhat tegous to use. Strasters and co-workers recently reviewed a number of different expresaions for the characterization of peak overlap for skewed peaks of varying peak heights including classical resolution, resolution based on the individual peak widths, resolution based on the first and second moments, etc. as well as those discussed above (C19). More recently, Dose and Guiochon (C20)reported a normalized measure of overlap, R, that appears to be a natural measure of the power of preparative chromato aphy to enrich two components of a solution. R also has t e above-mentioned properties of invariance and commutativity and requires no assumption regarding peak shape. Gradient Elution. Martin (C21) reexamined the basic inteval equations used to predict solute retention in gradient elution and found that if the retention equation is set up with variables expressed in terms of inlet and outlet conditions rather than conditions within the column, considerable simplification of the equation can be achieved, articularly if the mobile phase components are not retaineb: If one or more components is retained, the elution time of a solute increases. If only the strong component is retained and has a linear isotherm (constant capacity fador k Q,the increase in elution time using a linear solvent gradient is given by k'&/2. In a similar study, Markowski and Golkiewicz derived and experimentally verified a general equation for the retention of a solute eluted by stepwise gradient elution (C22). Boehm and Martire (C23) derived a simpler, more general theory describing homopolymer fractionation by HPLC with gradient elution. The theory addresses the significant effects that sample concentration has on the retention behavior of high molecular weight compounds. The resulting expresaions confirm the feasibility of separating flexible oligomers and homo olymers. Verzele et al. investigated the influence of particye size on peak dispersion in the gradient elution reversed-phase chromatography of phenones and proteins (C24). Miscellaneous. Foley et al. observed that negative deviations in the flow rate from set values are an everyday Occurrence in HPLC and developed a theory to explain these systematic errors in flow rate for pure and binary mobile phases (CW). About half of the observed error-which ranged from 2 to 12% de endin on the pressure, mobile phase composition, and so vent elivery system-was explained by nonideal mobile and stationary phase behavior. An approach to correct for these nonidealities and a method for measuring
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the instrumental contribution to flow rate error were also described. Martire (CHI derived equations based on Darcy's law for the spatial and temporal density distribution functions, aver e densities, and column profiles of the mobile phase fluidygas, liquid, or supercritical), and the observed (column averaged) capacity factor(s) and column profiles of solute componentts). Gemmel et al. reported a chromatographic separation utilizing sequentially a gas, supercritical fluid, and liquidlike fluid as the mobile phase (C27). Two distinctly different approaches, the Craig distribution model and the numerical solution of the mass balance equation, were used by Czok and Guiochon (C28) to simulate the movement of bands through a chromatographic column and were found to be equivalent. The physical significanceof each approach was also discussed. Normally in LC the assumption is made that any deviations from idealit due to interactions between solute(s), injection solvent, animobile phase can be neglected. Khachik and co-workers showed that such effects can be si ificant in the separation of carotenoids, depending on the re ative solubility of the carotenoids in the injection and eluting solvents and on initial injection solvent/elution solvent interactions at the top of the column (C29). Engelhardt and co-workers reported the dependence of retention and peak broadenin of natural and synthetic polymers on sample size (C30). f'eak broadening was markedly affected by sample size on nonporous material, and any advantages in reduced peak broadening gained by using nonporous materials were lost as sample size exceeded about 100 pg. As noted in the previous review, there has been mounting evidence that no one single compound can serve as a voidvolume marker in LC, since 'every molecule may have its own characteristic void volume". In spite of this possibility, work has continued in this area, albeit to a much lesser extent (C31, C32). Finally, a new separation mode coined "isotope dilution liquid chromatography" (IDLC) was introduced by Banerjee (C33).
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Optimization
Reviews. The overview of contemporary method development rovided by Schoenmakers and Mulholland (C34) discussej in lo ical sequence a number of im ortant topics, including the &finition of the separation pro lem, selection of the most suitable chromatographic method, method development expert systems, selectivity and system optimization, and method validation. Reviews by Berridge (C35, C36) described the role of chemometrics in method development, focusing on the problems of method development, approaches to separation optimization, and sequential and/or iterative experimental desi s. Mant and Hodges reviewed the optimization of pepti% separations, providing a thorough explanation of the following procedure they recommended (i) selection of the required separation mode(s); (ii) assessment of the performance characteristics of the columns to be used; (iii) selection of the initial mobile phase conditions, utilizing any information obtained from the standards and any knowledge of the structure, charge, polarity, or other chromatographically relevant ph sical properties; and (iv) optimization of gradient rate a d f l o w rate to provide maximum R, in the minimum time period (C37). Finally, Jandera reviewed the methods for calculating retention volumes and bandwidths for mobile phase gradients designed to change solvent strength, selectivity, or both (C38).These calculations facilitate the prediction of optimum binary and ternary gradients. Original Papers. Sekulic and Haddad examined the effects of peak tailing on computer optimization procedures (C39, C40). Peak tailing, if not accounted for, results in the inaccurate estimation of peak overla which in turn results in poor optimization. By measuring t e relative area overlap of two tailed peaks and relating (translating) this to the resolution value corresponding to two Gaussian peaks with the same relative area overlap, resolution-based criteria previously employed can be used for the optimization of the separation of tailed peaks. Ghriit and Snyder (C41) discussed several pertinent aspects of the o timization of gradient elution for small or large molecules y computer simulation, including potential sources of error and ways to minimize
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them, the classification of samples into one of three roups according to their response to a change in gradient confitions, and the optimization of separations in which band spacing depends on gradient conditions. Grushka et al. measured the retention and selectivity surfaces (plots of k'or a as a function of pH, methanol content, or temperature) of deoxyribonucleotides in reversed-phase chromatography and found little interdependence of pH, methanol content, or temperature (C42).As a consequence, several ap roaches to optimization are possible, including one based on e prediction of k as a function of pH, temperature, and mobile phase composition for subsequent o timization of a or R via window diagrams, etc. De Galan an8co-workers discussed the determination of starting conditions for modifier optimization (C43)and su gested that for or anic modifier optimization in reversed-p ase chromatograp y, all the necessary information (isocratic k ' estimates in MeOH-water, THF-water, and ACN-water) can be obtained from a single water-methanol gradient run and then used for accurate mobile phase optimization. They subse u,ently reported a method for correcting inaccurate isocratic9k estimates (C44). Several papers focused on the optimization of preparative LC. Gareil and Rosset showed that in the optimization of preparative LC it is important to recognize whether a given preparative chromatogram has been obtained under volumeor mass-overload conditions or both (C45).That way the Optimization of in'ection conditions, stationary phase particle size, and/or mokile phase flow rate can roceed in a straightforward manner. Ghodbane and Guioc on employed a simplex alForithm and a procedure previously described for the simulation of elution profiles of binar mixtures to optimize the production rate (C46).VariabLs considered included mobile hase velocity, sample size, column length, and average partic e size. Mobile phase velocity and sample size were optimized in stage one, whereas column length and aver e particle size were considered in stage two, as well as the trae-offs between the roduction rate and yield. They also used a semiideal mo el of chromatography to establish idelines for redicting the optimum extent of overloadin v47)and to &date the influence of the sample amount an8 volume of a binary mixture on the production rate (C48). Although the production rate cannot be optimized for both components simultaneously, in general the best results were obtained when low volume, concentrated samples were employed. Rittenhouse developed a computer simulation using a fixed database of five reversed- hase columns, three or anic solvents, and nine compoun s in order to illustrate t&,e o erational fundamentals of LC (C49).DAgostino et al. empEyed the commercial software package CHEOPS for the optimization of mobile phase composition in the gradient elution reversed-phase chromatographic separation of amino acids and steroids (C50).Hodges et al. wrote an interactive computer simulation program called Pro Digest-LC for the separation of peptide and protein digests by size-exclusion,ionexchange, or reversed-phase chromatograph (C51). Miscellaneous. As evidenced b the let ora of optimization criteria/response functions puglishe8during this period, optimization, like beauty, appears to be in the eyes of the beholder (C.52459). Numerous papers utilizing chemometrics were re orted, includin the simplex algorithm (C60,C61), factori3designs (C62, !63), 01 expert systems (C64-C71)as their "strategy" for optimization. Several noteworth applications were also published (C72-C76).
Integration/Statistical Moments. Papas and Tougas (02) evaluated the accuracy of peak deconvolution algorithms used by commercial integrators (tangent skim and perpendicular drop) for Gaussian and exponentially modified Gaussian (EMG) overla ping peaks. Using multiple linear regression, the authors eveloped equations that accurately redict errors of the deconvolution algorithms as determined y numerical simulation. The equations require knowledge of the resolution (as defined conventionally), variance ratio, area ratio, peak asymmetry, and peak height ratio. Eikens and Carr (03)used a nonstochastic (error propagation) approach to evaluate and derive equations that predict the uncertainty in the measurement of statistical moments (including peak area) calculated via the simmation method. To obtain the best precision, they recommend the use of narrow integration windows, symmetric about the first centroid and just wide enough to encompass the whole peak. For an accuracy and precision of 0.1% in the measurement of peak area, a minimum sampling rate sampling rate of 80 points/a is re uired. An alternative method of statistical moment calcuqation based on experimentally measured raphical peak parameters (height, width, and asymmetr8 and the assumption of a Gaussian or EMG peak shape was presented by Jeansonne and Foley (04)and compared with the conventional summation approach. Provided the peak shape assumption is valid, chromatographic peak characterization using this method is superior to the summation method and is particularly advantageous where peaks are asymmetric, noisy, overlapping, or data limited (insufficient sampling rate). Peak Modeling. According to Papas (DI), current evidence suggests that "the EMG is the best model proposed so far to mimic real chromatographic data." Naish and Hartwell provided additional support for the EMG by showing its accuracy for typical isocratic LC peaks under a wide ran e of conditions (D5).In contrast, Olive et al. evaluated t e effectiveness of several mathematical models (Gaussian, lognormal, amma, Weibul, and two modified Gaussians) for peak deconvoyution in GC and LC using a wide varriety of experimental conditions and instrumental designs and found the log-normal function to best fit their data set (06). Numerical Differentiation. Although the utility of differentiation is widely appreciated in spectroscopy, in chromatography it has been exploited to a much lesser extent, partly because this differentiation feature is not routinely included with the detector, integrator, or chromatographic data stations. Fasanmade and Fell discussed various applications of time domain differentiation in HPLC (07). Grushka and Atamna illustrated the utility of the second derivative to estimate integration limits and/or cut-points for severely overlapped Gaussian peaks (08). Chemometric Approaches. Only a few examples are noted here since an entire review is devoted to chemometrics elsewhere in this issue. Delaney et al. employed cluster analysis and principle component analysis (PCA) to classify nine columns into three groups of differing chromatographic behavior (09);PCA was useful in identifying key test compounds for the columns. Lochmuller et al. employed PCA and
D. DATA ANALYSIS
the reference run is defined as the run with the hi hest peak count and that the use of two or more channels al ows peaks to be identified. Vandeginste et al. (012) evaluated two approaches for the deconvolution/quantitation of overla ping peaks using HPLC/photodiode array data: self-mo eling curve resolution, and the matching of observed spectra with a linear combination of pure spectra obtained from a library. The effects of concentration,resolution, and spectral similarity on the accuracy of interative target testing factor analysis for peak tracking and/or deconvolutionwere reported by Strasters et al. (013) and Seaton and Fell (014).A partial least squares (PLS) calibration model was developed by Oehman et al. (015)in which the number of X matrix variables was reduced considerably by using the scores from PCA as the X variables prior to a PLS calibration. The reduced data handles outliers
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Reviews. Undoubtedly one of the most valuable contributions to this entire section was Papas' excellent critical review of chromatographic data systems (01).Spanning 3 decades, this review covered the following broad areas: characterization and resolution of overlapping peaks, noise and filtering, evolution of peak-sensing devices, and evaluation techniques and results. It shows that although chromatographic data systems have evolved significantly over the years, they still have a number of significant shortcomings, including but not limited to their inability to accurately measure (deconvolute) peak areas for partially overlapping peaks or their inabilit to detect coelution for heavily overlapped peaks with no visitle shoulder.
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just as well as the nonreduced data and, moreover, gives smoother and more monotone curves for both prediction and outlier rejection. Ebel and Mueck employed principal component analysis from HPLC/diode array data to detect unresolved chromatographic peaks and evaluated the method according to the spectral similarity, degree of resolution, and absorbance ratios (D16).In a similar study, Gemperline and Hamelton (DI7)showed that resolution, spectral similarity, and relative concentration ratios affect the limit of detection of the minor component in an overlap d peak and developed a method for estimating the net ana&cal signal due to the minor component. The estimate is accurate over a wide range of resolution and allows one to judge, using knowledge of experimental error, whether or not the minor component will be detectable. Strasters et al. (D18)reviewed three multivariate techniques-multicomponent analysis, target factor analysis, and interative target transformationffactor analysis-for eak deconvolution in the context of optimization. Final&, Reich applied a newly developed pattern orithm based on KNN (kth nearest neighbor) recognition to detect pea s with a signal-to-noise ratio down to unity
(D19).
9
E. NORMAL PHASE This year’s review of li uid chromatography introduces a separate section on normJ-phase LC, which includes liquidsolid adsorption chromatography. This c e is a recognition of the growing use of bonded stationary p ases in the normal-phase mode. Here, “normal phase” is used in the conventional sense to mean systems in which the stationary phase is more polar than the mobile phase. Bonded phases appear to be slowly replacing traditional solids such as silica and alumina as the sorbents of choice for use in normal-phase LC, althou h silica and alumina still find widespread use. Funiamental studies of liquid-solid adsorption chromatography (LSC) continue to appear. Martire extended earlier treatments of liquid chromatography with bonded phases to include LSC with binary mobile phases and both hom eneous ( E l )and heterogeneous (E21surfaces. In both cases,%rtire’s unified theory of adsorption chromatography, which is based on statistical thermodynamics and a mean-field lattice model, indicates that the natural-state variables of the mobile phase are ita reduced tern rature and density. Mixed mobile phases in LSC were also tE subject of several studies that used the Jaroniec equation to investigate solute-solvent interactions. Borowko and Jaroniec (E3) identified competitive adsorption between solute and solvent as being the most significant fador that influences the solute distribution coefficient, and the shape of this dependence was controlled by solute-solvent interactions. Oscik-Mendyk and co-workers concluded that the type of molecular associations in the bulk mobile phase were independent of the nature of the stationary phase (E4) and confiied the formation of multicomponent solvates (E5). Borowko proposed a simple method for calculating retention of solutes with complex shapes based on what has been termed the quasi-chemical theory of LSC (E61and demonstrated that this approach can be used to predict isomer separations. The role of the mobile phase in liquid solid chromatogra hy with silica and alumina was the subject of numerous stuies. Park and Carr (ET)reexamined the solvent strength (to) scales for silica and alumina using linear solvation energy relationships (LSER). The LSER analysis was based on solvent dipolarity (**I, hydrogen bond acidity (a),and basicity (8) parameters which were obtained from solvatochromic characteristics of the solvents. Regression of solvent stren th values with those solvatochromic parameters confirmed tfat the silica surface is essentially acidic, with no si nificant basicity, while the alumina surface is primarily basicsut does exhibit significant acidity. The surfaces appeared to have a proximately equal dipolar character. Gazdag and co-workers 88)demonstrated that the selectivity of steroid separations could be improved by varying class P (polar,non-self-hydrogen bonding) modifiers in the mobile phase. Variations of class N (nonpolar) and class AB (amphoteric, self-h drogen solvents were not as effective in improving8erectivity. They o showed that the elution order of these steroids was a function of the t pe of class P solvent used. Palamareva and Palamarev (ESfdescribed a microcomputer program that could be used to calculate solvent strength (eo), localization (m),and polarity (P?parameters of a number of normal-phase
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solvent mixtures. The authors included a nomogram that expressed solvent strengths of several mixtures as a function of the volume percentage of the polar solvent modifier. Adam et al. (EIO)demonstrated the surprisin result that some organic amine radiopharmaceuticalscould%e better separated on a silica normal-phase column using a reversed-phase-type solvent com r i d of acetonitrile and 4 mM dibasic ammonium phosphate Euffer than when a CI8 column was used. Cyano- and amino-derivatized silicas remain the most popular bonded normal stationary phases, and several studies attempted to define their retention characteristics relative to bare silica. Oestman and Colmsjo ( E l l ) used a series of unsubstituted and alkylated benzenes and polycyclic aromatic hydrocarbons (PAH) to compare silica with aminopropyl- and cyanopropylsilica. These authors noted a decreased effect of methyl and mono-n-alkyl substitution on retention with the amino phase relative to bare silica, which they attributed to increased selectivity for r electrons of the amino phase. Weaker retention of PAH on the cyano phase relative to silica and differences in the effecta of alkyl substitution on retention were interpreted as an indication that cyano groups of the bonded phase, and not residual silanols, were the primary adsorption sites. Pietrogrande and co-workers (E12)carried out a similar study using benzodiazepines, a pharmaceutically important class of molecules. These authors monitored the change in retention that occurred with increased amounts of polar solvents (2-propanol and ethyl acetate) added to a hexane-based mobile phase. They interpreted their data by using the Soczewinski-Snyder displacement model and concluded that the aminopropyl phase was significantly stron er than either the cyanopropyl phase or bare silica. The s o concluded that, at least for these molecules, residual si anols were the primary adsorption sites on the cyanopropyl phase, not cyano groups. The apparently contradictory results of these two studies continue the controversy that has existed for some time regarding the exact nature of adsorption sites in cyanopropyl columns. Ando, Nakayama, and Hara studied the retention of fatsoluble vitamins in silica (E13),aminopropyl (E14)and cyanopropyl columns (E14). With ethyl acetate and tetrahydrofuran as the polar mobile phase modifier, the amino phase was more retentive than bare silica, which was in turn more retentive than the cyano phase. With 2-propanol as the modifier, however, the cyano phase was more retentive than silica, confirming previous studies which indicate that 2propanol is a strongly localizing solvent when used with silica. Interest’ ly, these workers observed that the best and thuxighest resolution for these compoun s were shapes obtained with a silica/Bpropanol system, su gesting that mixed retention mechanisms might be involved when using bonded-phase columns. Smith and Cooper used a different approach in their comparison of aminopropyl, anopropyl, and 1,Zdihydroxypropyl propyl ether (diol) b o n x d phases (E15).They calculated extended solubility parameters for these phases and then, using the acid, base, and orientation parameters, positioned them on a stationary phase selectivity triangle. The positions of the cyano and amino phases on Smith and Cooper’s stationary phase triangle were in agreement with previous predictions, but an ether linkage in the diol phase ap arently neutralizes some of ita expected acidity. Smith antCooper calculated solubility parameters by using both partition and displacement models and concluded that only the displacement model gave meaningful results, and then only when the retention of nonl&ing solutes was considered. Their results are reflective of the current status of what is known about retention in bonded normal-phase columns. While the SOCzewinski-Snyder displacement model is a useful means of evaluating retention data, it does not as yet provide a sufficiently predictive capability. There is particular disagreement about whether the cyano phase has ita own unique character or behaves only as a deactivated silica. Pharr and co-workers introduced two new normal bonded phases that were used to separate PAHs and PCBs (E16). These two phases, (trich1oroacetamide)propyl and (trichloroethoxy)propyl, ap ear to offer increased charge transfer and hydrogen bonding c!aracteristics over fluorine-containing phases. In an interesting marriage of electrochemistry and chromatogra hy, Yao et al. (E17)described the coupling of an electric fiepd to amino and silica normal-phase LC columns.
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Since the compounds and mobile phases in their study were not ionic, the electric field did not induce electrophoretic migration. The authors attributed increases in chromatographic efficiency that they observed to orientation effects induced by the electric field. The electric field apparently influences the probability of hydrogen bond formation between polar solutes and the stationary phase through these orientation effects. The unique ability of silica to separate positional isomers was demonstrated by Tan and Brzuskiewicz (EM),who compared normal- and reverse-phase separations of tocopherol and tocotrienol isomers. Aqueous reversed-phase,nonaqueous reversed-phase (NARP), and cyano, amino, and silica normal- hase systems were tested. A silica column used with a 99:l\exane/propanol solvent mixture was the on1 system capable of resolving all isomers of these two types orvitamin E group compounds. Isomer separationswere also the subject of a paper by Walczak et al. (El9),who studied the separation of a series of ositional and configurational isomers of 4- and 4'-substituter!chalcones of the general formula XC6H4CH= CHCOCBH,Y. Standard normal hases (cyano, amino, diol and nitro) were compared against 1;ive ! charge-transfer phases: n-propyl picryl ether; 3-(2,4-dinitroanilino)propyl;3,5-dinitrobenzoamidopropyl; tetrachlorophthalimidopropyl;and caffeine. Using fador analysis to interpret their results, these workers concluded that the separation of substitutional isomers on charge-transfer phases did not differ substantially from that on standard phases, but the separation of the E-s-cis and 2-s-cis chalcone isomers was much better on the charge-transfer stationary phases. Mobile phase effects in normal- hase chiral separations were the subject of two studies. Afanya and Taylor (E201 demonstrated that alcoholic modifiers greatly reduce the resolution of chiral esters of amino acids and surmised that the alcoholic moiety interfered with hydrogen-bonding chiral recognition. Aprotic modifiers such as dichloroethane and 1,1,2-trichloro~uoroethanegave superior results. In a similar study, Macaudiere and co-workers (E21) showed that the retention order of chiral *-acid N-(DNB) derivatives of aminoamides reversed upon changing from hexane-ethanol to hexane-chloroform or hexane-methylene chloride mobile phases. The authors suggest that either solvation of the solute or a change in the conformation of the chiral stationary phase was responsible the observed inversion.
F. REVERSED PHASE In a recent survey of column usage, reversed-phase columns were reported used by 57% of the respondents (FI).This popularity in usa e is reflected in the continued research mterest in this m d e of separation. Advances are being made in better understandings of the molecular mechanism of retention, in better synthetic schemes for reproducible preparation of the 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. Dorsey and Dill (F2) reviewed the current understanding of the molecular mechanism of retention of reversed-phase chromatography. Statistical mechanical theory initially devel0 d by Dill (F3) proposes that retention can be described by e chemistry of contacts of the solute with the stationary and mobile phases and by the partial orderin of the stationary phase chains. Dorsey and Dill (F2)comparefthese predictions as well as previous theories of retention with available experimental data and, in general, found ood agreement with the theory of Dill. The "solvophobic tfieory", 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. Hence the solvophobic theory does not take cognizance of the interactions of the solute with the second "solvent", the cavity in the stationary phase, it takes into account only the cavity in the mobile phase. Stationary Phases. Several important reviews of the preparation of reversed-phase materials were published during this review period. The preparation and characterization of bonded phases have been exhaustively reviewed by Sander and Wise (F4). They discussed both the types of bonded
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phase li ands and the effects of substrate parameters such as parti8e size, shape, and pore diameter on the performance of the resulting materials. Van den Driest et al. (F5)more restrictively reviewed octadecyl bonded phases on silica gel su ports, and Dietrich et al. (3'6) reviewed the preparation of %onded phases on silica gel and discussed the importance of the preparation of the silica substrate. Nawrocki and Buszewski (F7)reviewed the influence of the silica surface chemistry and structure on the properties, structure, and coverage of alkyl-bonded phases. There were several advances made both in the method of preparation of the stationary phases and in the types of appended groups. The poor pH stability of most columns is perhaps the greatest weakness of the commercially available stationary phases. Kirkland et al. (F8)described the synthesis of two new types of bonded phases that exhibit significantly im roved pH stability. The first class uses bifunctional (or bidktate-type) silanes, which contain one reactive site on each of two silicon atoms of the silane. These two silicon atoms are connected by a bridging group, such as -0- or -(CH )"-. The second type contains one or two bulky groups, such as isopropyl or tert-butyl, on the silicon atom of the silane. Apparent1 these groups provide steric protection of the Si0-Si bonrfa ainst acid hydrolysis and allow the use of low pH mobile plases. Sentell et ai. (F9)reported the use of ultrasound to drive the reaction between silica and the reactive silane. Compared to the traditional reflux reactions, this allows independent control of the temperature of the reaction, and synthesis of materials at a tem erature of 3O C was reported to be especially efficacious. &ey reported C18bonding densities as great as 4.07 pmol/m2 of silica surface, compared to values of 2.5-3.0 pmol(m2 for commercially available columns. These high bonding densities should also rovide significant protection of the stationary phase against ydrolysis by extremes of pH. Khong and Simpson (FlO)reported a novel synthetic method based on fluidized bed technology. They successfully fluidized silica particles as small as 7 pm and reported the bonding and subsequent endcappin of these materials. They cited advantages of in situ removaf of fines and hi h batchto-batch reproducibility. Mant and Hodges (F1I) iescribed an on-line silanization rocedure for the restoration of deteriorated analytical anisemipreparative reversed phase columns. Pumping a mixture of a 2:l molar ratio of pyridine to monochlorodimethylalkylsilane in dichloromethane through the deteriorated column produced excellent restoration. Secondary retention processes in reversed-phase separations were also of significant interest. Any stron interaction of the solute such as hydrogen bonding to resitual hydroxyl sites or complexation with trace metals present on the surface can lead to tailing and irreproducible separations. Sadek et al. (F12)investi ated the influence of trace metals on retention pretreatment of the silica with acid improves and showed chromatographic behavior of the derivatized material. They noted that the difference does not seem to be from the removal of possible direct solute/metal interaction, but rather from the decreased numbers of metal hydroxides and the attenuated influence of metals on the surface silanol groups. Two new methods were ro osed for monitoring surface silanols. Khurana and Ho (hydescribed a titration method using sodium hydroxide, with the stationary phase materials slurried in a medium of 10% aqueous salt. While this rovides a quantitative measure of the hydroxyl sites, Mant an Hodges (F14)proposed a chromatographic test to monitor their activity. Using a series of four synthetic peptide standards with net charges from +1to +4 to show the presence and extent of free silanols, they showed that the resulting chromatograms would also demonstrate what changes should be made to the mobile hase to minimize ionic interactions. It would be highly &sirable for the manufacturers of stationary phases to adopt a test such as this as part of their column characterization. Eisenbeiss (F15)studied the effect of the support materials on chromatographic pro erties and argued that the abnormal retention effects cannot sufficientlyexplained by unreaded silanol groups. Rather, Eisenbeiss proposes that a nonhomoenergetic silica surface is responsible. Whatever the cause of the secondary retention effects, a very practical goal is modification of either the mobile or stationary phase to eliminate the effect. Stadalius et al. (F16)
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proposed both the use of less acidic columns and the optimization of the mobile phase. The showed that narrower, more symmetric peaks are obtainedrby using a pH between 2.5 and 3.5, with hi her buffer concentrations, with potassium salts instead of s ium, and by adding amine modifiers such as triethylamine or dimethyloctylamine. Freiser (Fl7)showed that the nature of the silane, the type of silica, and the chemistry of endcapping all influenced chromatographic behavior. Hartwick’s group (F184’20)investigated the stabilization of reversed-phasematerials and in the process described two useful techni ues of studying the stationary phase materials. They found tYiat the silica employed for bonding, the degree of coverage to which the phase is bonded, the chain length or bulkiness of the alkyl moiety on the silane, and the reactivity of the silane were all im ortant considerations for stability (F19). Using diffuse regctance FTIR, they found that trifluoroacetic acid caused a rapid partial loss of bonded phase over about one day, followed by a more gradual loss over the next several days (F18). They also described a new techni ue for analysis of short alkylsilane bonded phases using hydroauoric acid digestion and headspace analysis of the digestion products by open tubular GC (F20). Mobility and solvation of the bonded alkyl chains were also investi ated. Miller et al. (F21)used electron spin resonance under !ynamic mobile phase conditions to obtain the solvation ition of the bonded phase at various locales in the bon ed phase, configuration of the bonded phase, and solvent flow effects. They further found changes in the motional freedom of the bonded surface as a function of mobile phase composition and elution time and demonstrated chain stiffening with the introduction of water to the mobile phase. Claessens et al. (F22) studied stationary phases prepared from both monofunctional and difunctional silanes by NMR and described changes in the phases over time as being from loss of silane, gain in silanol content, and rearrangements of the silica-to-silane bonding. Surprisingly, they found hases prepared from monofunctional silanes to be more stab6 than phases of the same silane chain len h repared from trifunctional silanes. Pfleiderer et al. $237 used spin-lattice relaxation times determined by NMR to correlate chain motion with retention of paracelsin peptides. They studied chain lengths from C, to C18 and found the freedom of movement of the alkyl chains was highest for C6 to C8 chains. They also noted that retention of the peptides was maximum where alkyl group motility was least. The effect of bondin density of the alkyl chains was also studied with respect to fwth retention and selectivity. Many re rta still appear in the literature using carbon content from C P N analysis as the descriptor of phase loading, and most commercial columns are described in this manner. It should be stressed, ain, however, that this is not the relevant parameter a n d x t these re rk are often uninterpretable. As Unger et al. (F24) pointeEut early in the history of bonded phases, carbon content alone is often misleading because of differences in the surface area of the original silica, which results in different surface densities of the bonded alkyl groups. Carbon content is only useful if reported with the surface area of the bare silica. Manufacturers should be strongly urged to also report the surface area of the unbonded silica. Sentell and Dorsey (F25, F26)studied both partitioning and selectivity as a function of C1 bonding density over the range of 1.6-4.1 wrnol/m2. They found a maximum in partition coefficient at about 3.1 pmol/m2; above this chain density, creation of a solute-sized cavity in the stationary phase becomes energetically expensive due to enhanced chain ordering (F25). Actual chroma aphic retention was found to plateau at this chain density ue to compensating changes in the partition coefficient and stationary phase volume. They also studied both phenyl, or shape, selectivity and methylene selectivity over this same range of bonding densities (3’26). While methylene selectivity remained approximatelyconstant, phenyl selectivity was found to increase linearly with increasing chain density. These studies may explain the differences in selectivity found between stationary phases prepared from monofunctional and trifunctional silanes. Lork and Unger (F27).also studied retention as a function of chain density and chain length and found similar plateaus in retention. This plateau was found to occur in the range of
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2.3-3.2 pmol/m2, depending on the chain length and the solute. Buszewsh et d. ( F a ) studied the recovery of typtophan and its metabolites from human urine as a function of the bonding density of materials used for solid phase extraction. They found maximum recovery was obtained on a material prepared with monofunctional silanes and with a high bonding density of about 3.8 pmol/m2. With all of the variables that can affect the performance of commercial columns, reliable, useful means of evaluating similarities and differences in their properties are extremely valuable. Two important steps toward this goal were made during this review period. Delaney et al. (F29)described a chemometric approach to classifying stationary phases based on cluster analysis and principle component analysis. They found that columns could be grouped into three general groups displaying similar chromatographic behavior. Park et al. (F30) used solvatochromic studies to investigate four different bonded phases in each of two mobile phases. They showed that des ite the well-proven accuracy of the solvatochromic linear sogation energy method for predicting and correlating octanol water partition coefficients and water solubilities,the metho is limited to only certain types of bonded phases. Polycyclic aromatic hydrocarbons (PAH) have been extremely popular for probing stationary phase interactions. The solutes are easily detectable and are generally planar, and their size and breadth to width can be easily varied to robe selectivity effects. Jinno et al. (F31,F32) have addefto these studies by using peropyrene-type PAHs to probe various types of bonded phases and used various large PAHs to study stationary phases prepared from trifunctional silanes. The use of the word “polymeric” to describe stationary hases prepared from di- and trifunctional silanes, while toric, is becoming confusing. The introduction several years eric resin based reversed phase stationary phases :%etotally different meaning to polymeric stationary phases, and careful choice of descriptive words is necessary. Pietrzyk (F33) reviewed the use of macroporous organic polymers as stationary phases for liquid chromatography. Lee (F34)described the chromatographic evaluation of lar e pore and nonporous polymer resin phases and compared t em in efficiency, selectivity, and load capacity to a commercially available small pore material. The derivatization of these phases with pendant C groups has also been achieved in the last few years to give a p% stable stationary phase with retention characteristics similar to the traditional silica based materials. Dawkins et al. (F35) described the development of a rigid, macroporous polyacrylamide based packing with pendant C1 groups and compared the retention properties both to silica-$ased materials and to a polystyrene-based stationary phase. Tanaka et al. (F36) examined and compared olymer-based materials of polystyrenedivinylbenzene,popYml methacrylate, and esterified poly(viny1 alcohol). They found that under optimum conditions, materials with alkyl backbones showed performance comparable with silica-based materials. Yasukawa et al. (F37) described the development of a C18bonded vinyl alcohol copolymer gel stationary phase and compared the performance to commercial olystyrene and silica-based materials. The new material l s p l a ed excellent tolerance toward acidic, alkaline, and bufferdeluents, with little shrinkage or swelling. This material also showed excellent efficiency and no secondary retention processes with basic substances. Ohtsu et al. (F38)reported the development of silicone polymer coated silica gels modified with C or C groups. These materials were prepared by coating the surface of totally porous silica gel with a homogeneous silicone polymer film and then modifying the polymer with the ndant groups. They showed that these materials retained tK“,advantages of silica-based materials while showing strong resistance to pH extremes. The study of stationary phases with groups other than C8 or C continues to be of much interest. Smith and Miller (F39) compared three commercial cyano phases and found considerable differences among the three. Szabo et al. (F40, F41) thoroughly studied the preparation and retention characteristics of different phenyl hases, including phenylmethyl, phenylpropyl, diphenyl, angtriphenyl. The materials were all somewhat different and were also compared to a C18 phase. Krafft et al. (F42)prepared two perfluorinated stationary phases on silica gel and compared the retention properties
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against a traditional c18 material with a perfluoro- and a hydrogenated series of compounds. The transfer energy per CF2 group was found to be about 3 times higher on the perfluonnated stationary phase, but the transfer energy for a CH2 group was the same on either phase. This leads to significant increases in retention and se aration factors for perfluoro compounds on the fluorinate$ phases. Dohtsu et al. (F43) repared silica materials modified with anthracene and phtAde-containing groups and used these stationary phases for the separation of mononucleotides and pentadecamer oligonucleotides. They proposed that the intercalator-nucleic base stacking interactions were important for the separations obtained and proposed directions for the further, development of such stationary phases. Den and Lee (F44) prepared several oli omeric or polymeric 1,B-disilyloctane phases on silica anC f compared the retention for aromatics and aliphatic8 with a traditional c18 column. They also pre ared a re resentative oligomeric 1,8-disilyloctanephase wigthe safroi ligand as the terminal group. Apfel et al. (F45) desi ned and synthesized a stationary phase in which theophyhne residues were covalently bonded to a silica support throu h a c8 hydrocarbon linkage. This phase offered improve$ resolution in the separation of aromatic carboxylic acids over that obtained with conventional reversed-phase materials. They further correlated the capacity factors for a series of ring-substituted benzoic acids with the complexation constants with theophylline reported for these compounds in bulk solution. Mobile Phases and Selectivity. In what may prove to be a hi hly useful stud , Cole and Dorsey (F46) investigated reequilbration time fol2bwing gradient elution reversed-phase separations. They found that the reequilibration time can be drastically reduced by addin 3% n-propyl alcohol to both the strong and weak solvent. {his provides a constant composition of propanol during the solvent gradient and a much more robust stationary phase solvation. They further investigated reequilibration time as a function of bonding density of the alkyl chains and found a maximum in reequilibration time at a bonding density of about 2.9 pmol/m2, in agreement with earlier partitioning studies. During this review period there have been many studies correlating solute and solvent properties with retention. These include solute-solvent interaction free ener ies and their correlation with mean field statistical thermo$ynamic theory (F47), solubility concepts (F48),solvatochromic parameters of solutes and solvents (F49-F51), enthalpy-entropy compensation measurements (F52), electrostatic effecta (F53,F54), and intramolecular hydrogen bonding effects (F55). Smith and Burr (F56F58)described retention predictions based on the molecular structure of the anal@ and retention indexes based on the alkyl aryl ketone scale, and Smith (F59) ublished a review of retention indexes in reversed-phase LC. bo usz and Aderjan (F60) re orted the use of a retention in&x scale based on 1-nitroal anes and compared it to the alkyl aryl ketone scale. Further descriptions of retention were based on predictions from retention of homologous series (8’61) and from group contributions to hydrophobicity (F62-F65). Solvent stren and selectivity also received considerable interest during review period. Synder et al. (3’66) showed both from a literature review and from new data that most samples exhibit significant c e8 in band spacing based on solvent strength optimization etzer and Biggs (2767) compared the elution stren hs of 11common LC solvents using a marker set of olycyc ic aromatic hydrocarbons. With the exception of TH!’, they found that solvent strength correlated with an observed red shift in the fluorescence maximum of the solutes in each solvent. For THF, they found the pure solvent and blended mixtures behaved quite differently. West (Fss)found that the solvent selectivity triangle concept failed to group solvents accord’ to selectivity for resolving aromatic compounds in reversed3ase LC. West further found that selectivity differences between solvents classified in the same group often exceed those for solvents classified in different groups. Jandera (F69)continued his theoretical and experimental investigations of selectivity, and described methods for the characterization of selectivity of oligomeric series. Jandera found that the effectiveness of gradient elution for the separation of oligomeric series depends strongly on their structure.
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Petrovic and Lomic (F70) used experimental capacity factors and calculated activity coefficients of the solutes in the mobile phase to study selectivit in reversed-phase LC. They concluded that a change in iondin density of the alkyl chains on the stationary phase does not ead to a change in CH2group selectivity. Pauls and McCoy (F71) investigated the effects of mobile phase composition on olefin group selectivity (the ratio of capacity factors for the n-alkane and 1-olefin of equal carbon number) for nonaqueous reversed-phase LC. They found that solvents containing alcohols demonstrated low olefin roup selectivitiescompared to those containing acetonitrile an8 that methylene selectivity was insensitive to the nature of the mobile phase. Dondi et al. (F72) investigated the selectivity pro erties of methanol, acetonitrile, and THF as organic moxifiers in the reversed-phase separation of flavonoid compounds. Temperature effects were also investigated for control of solute selectivity. Sander and Wise (F73) used polycyclic aromatic hydrocarbons to investigate shape selectivity of stationary phases prepared from both monofunctional and trifunctional silanes as a function of temperature. Column selectivity was found to vary continuously with temperature, regardless of the type of stationary phase, and shape reco nition ability of all phases was found to be hi hest at SU-!I ambient temperatures. They further propose! a model for these selectivity chan es based on the morphology of the bonded phase. Tchapfa et al. (F74) investigated the role of temperature in the behavior of a homologous series and, from van’t Hoff plots, found a critical number of carbon atoms of the homologous alkyl chain. This critical carbon number agreed with their previous findin s from discontinuities of plots of log k’vs carbon number. khey noted that this phenomenon is independent of the homologous series studied, of the solvent composition, and of the monofunctional or difunctional nature of the bonded phase, but it did de end on the length of the bonded alkyl group. All of the ogserved effects corroborate the idea that there is a penetration of the solute alkyl chain into the bonded layer. Martin et al. (F75) compared the retention mechanisms of homologous series and triglycerides in nonaqueous reversed-phase LC and also reported discontinuities in plots of log k vs carbon number as well as entropy and enthalpy of transfer vs carbon number. Hennion and Rosset (F76) extensively studied the continuin problem and controversy of void volume determination anfchoice of experimental robes for reversed-phase LC. They compared experimentafmeasurementswith calculated values from adsorption isotherms of the organic modifier and water and concluded that with a few UV detectable solutes it is possible to measure the void volume for any mobile phase composition of acetonitrile-water or methanol-water. Lau and Simpson (F77) studied a homologous series of alkanol modifiers to methanol-water mixtures to give equivalent separations on different reversed-phase columns. They noted that these methods compensate for the wide variations in separation characteristics they observed from columns from different manufacturers and even on a batch to batch basis from a given manufacturer. Hoffman et al. (F78)performed a highly useful and practical study of the distortion and multiplication of peaks that occur when solutes are dissolved and injected in a solvent that is significantly stronger than the mobile phase. They found that both solvent strength and the injected volume affect peak shape and they presented a qualitative interpretation of the phenomenon. Doehl (F79) investigated the chromatographic and spectroscopic properties of dissolved oxygen in mixtures of water and acetonitrile. Doehl showed that chromatographic properties of solvated oxygen can lead to UV absorbing peaks and proposed the use of this phenomenon as a measure of the efficiency of degassing techniques and for the determination of oxygen-dependent fluorescence quenching. Hansen et al. (F80-F82) continued their investigations into the use of dynamically modified silica to generate reversedphase stationary phases. They found that with the dynamic modification technique, basic drugs could be separated with virtually no peak tailing or other adverse effects commonly seen with bonded phase materials. These im rovements were found even when compared to bonded pIases especially deactivated for separating basic drugs and even if competing
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bases were added to the mobile phase.
G. BIOPOLYMER SEPARATIONS Liquid chromato raphy has been referred to as a 'compulsory ste in &e purification of proteins prepared with biotechnologidprocesses (GI). This statement is indicative of the monumental im act that chromatography has made in modern biopolymer ciemistry. Indeed, the literature search for this review uncovered a proximately eight thousand citations of the use of HPLC &r protein separations during the last two years. Thursts were in uses of HPLC for protein analysis, for micropreparative isolation of biologically active macromolecules, and for preparative scale separations. Primary meetings that focus on new developments are the International S posia on Hi h Performance Liquid Chromatography of E t e i n s , Peptises and Polynucleotides and the International Symposia on Column Liquid Chromatography. Both alternate between the United States and Europe and their proceedings, which are usually published in special journal volumes, are compendia of information on the topic (G2-G4). Reversed-Phase Chromatography
Adsorption Mechansisms. It has been established that the interactions between proteins and surfaces increase with increasing h drophobicity of the surface (G5, G6). Moreover, only a smalrportion of the rotein need enter the interface for adsor tion to occur. Afsorption then proceeds spontaneously t rough rearrangement of the protein chains (G5). Examination of lysoz e with a fluorescence spectrophotometer coupled to a S L C revealed that adsorption to alkyl silica was accom anied by a red shift of the emission maximum that was foBowed by blue shift (G7). The first shift was indicative of increased exposure of tr tophan groups to the lar solvent while the second shift ingcated exposure to the g d r o hobic environment. When residence times on reverseds hase materials exceeded 15 min, differences in UV second ierivative spectra also changed indicating that protein conformation was being altered ((28)as contact time with the packin increased. Circular dichroic spectra studies demonstrated that the nature of the solvent also influenced the conformational changea induced u n protein adsorption (G9, CIO). Propanol and acetonitric common mobile phase modifiers in protein chromatography, induced increased cyhelix structure in a-chymotrypsin and calcitonin when they were in contact with stationary phase. FT-IR clearly demonstrated that 2-propanol and methanol themselves induce conformational changes and that the changes are not analogous to the denaturation brought about by exposure of the proteins to alkaline pH ( G I I ) . If the kinetics of conformational change are in the time scale of the chromatography experiment, band broadening or multiplet peaks may result. Moreover, isolation of separated proteins for subsequent characterization could be precluded. Addition of salt to common HPLC mobile phases may increase the conformational stability of proteins during reversed-phase chromatography (G12). Stationary Phases. The influence of the chemical nature of alkyl ligands that are bound to the siliceous surface on protein chromatography continues to be investigated. Retention and resolution were insensitive to alkyl chain length and ligand density (GI3, GI41 but recovery was greater and denaturation less with low-ligand density supports. For most proteins, pore diameters of 300 A ave optimal recoveries (G13, G15). Very small porous particts with diameters as low as 1 pm packed in 1-cm columns appear to be uite useful for protein chromatography (G16), resumablylecause of the unproved mms transfer kinetics. 8umg the past several years another approach for improving chromatographic efficiency by the use of non orous microspheres (1-3 pm) was introduced. Stable anzefficient columns, about 30 mm long, can be slurr packed with siliceous (GI 7)and pol eric microspheridreversed-phase packings (GI8). Use o g e polymeric packings allows protein chroma aphy to be performed over a wider mobile phase pH ran 11)than is possible with the silica-based materials. Wten the temperature of mobile phase and all instrument components were carefully controlled and extracolumn effects minimized, mixtures of six proteins were resolved in 2 min with steep gradient elution profiles
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(G19). The disadvantage of the lower available surface of these packings is offset by the advantage of shorter protein residence times, which reduces the probability of denaturation. Increases in band spreading with concomitant decreases in resolution and reductions in retention times with increased sample size were greater with the non orous ackings than s i reduction with 5-pm particles commonly used in &PLC. h in efficiency was a function of surface inhomogeneity not surface coverage (G20). Optimization. Because of the importance of protein reversed-phase chromatography in clinical analysis,biochemical investigations, and biotechnology, much effort has gone into the development of methods for prediction of retention so that method development time can be reduced. A method was devised that predicted retention of peptides containing up to about 20 amino acids given the experimental conditions and the amino acid composition for any gradient slope, flow rate, and column length ((721). The contribution to retention of each amino acid was determined empirically. If an additional correction was added to account for chain length, good predictions were made for peptides containing up to 50 amino acids (G22). Such prediction tools have been studied using larger data sets. Deviations from predicted retention behavior were attributed to interactions of free basic groups on pe tides with stationary phase and mobile hase components &23), chromatographic kinetic factors ( 8 2 4 ) ,and peptide conformation effects (G25). A different approach was undertaken for proteins. Column parameters and mobile phase composition were systematically evaluated by computer simulation and by experiment for 33 reference proteins representing a range of molecular weights, basicities, and hydrophobicities to arrive at o timal conditions that would suffice for most proteins (C267. Seventy proteins from E. coli were resolved with the optimized parameters obtained by this evaluation without additional refinement. Applications. It is impossible to cite but an infinitesimal samplin of the applications of HPLC in protein chemistry. Selectefexamples are given to indicate the power of reversed-phase HPLC. Because of their crucial role in protein synthesis, the chromatography of the ribosomal proteins has been studied and applied widely. Thirty proteins of ribosomes from a eubacteria were resolved on a reversed-phase column with an aqueous phosphoric acid/acetonitrile gradient. Correlation between retention times found experimentally and those predicted b computer simulation was better than 0.7% over all peaks (627). A frequent objective of ribosomal protein chromatography is the isolation of the proteins for further characterization and study. From this stand oint, two interesting conclusions are that 305 and 50s sugunits can be reconstituted successfully from roteins prepared by reversed-phase HPLC and that mogfied proteins can be separated from unmodified proteins (G28). This facilitates biochemical investigations of ribosome structure/function relationships. Histones, the basic proteins that are complexed with DNA to form the nucleosome, and histone variants of mature and immature erythrocytes were compared by a RPHPLC procedure that is simpler and more rapid than previous fractionation schemes (G29). Reversed-phase HPLC was used to isolate isoforms of human apolipoproteins (G30) that are important in heart disease. From retention data it was demonstrated that proteins of arterial plaques from different human anatomical sites varied in polarity and charge but had similar molecular weights (G31). Reversed-phase HPLC provided an independent comparative evaluation of subunits of organelle-specifictubulins, the major structural protein that forms microtubules (G32). The utilization of RP-HPLC of proteins has increased significantlyin the areas of agricultural and food science. For example, 13 new salt-soluble and seven previously identified proteins from barley were isolated with a single-step HPLC purification (6'33). In classic work, zeins, the storage protein fractions, from diverse varieties of maize were separated by RP-HPLC with aqueous TFA-acetonitrile gradient. Patterns of 20-25 peaks were obtained that were used to differentiate varieties and, with com uter analysis, to trace inheritance lines among hybrids (G34). ubsequent HPLC studies with a larger number of hybrids demonstrated that such data can be useful in the re istration, certification, and checking of hybrid pedigree ( 8 3 5 ) . Similar applications have been made with
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soybean cultivars ((736) and with wheat lines (G37), where chromatographic profiles were correlated with baking quality. The animal species could be identified from the HPLC profile of proteins extracted from meats parts and could be used to quantify meat species in meat blends (G38). The presence of soy protein at levels down to 2% in beef products was measured by RP-HPLC of the vinylpyridine derivatives (G39). Reversed-phase chromatography with electrochemical detection was emplo ed to study the modification of DNA by exogenous and endbgenous methylating agents. (The sensitivity of the method for the 7-methylguanidine adduct .was 10000-fold lower than that of optical methods (G40).) Mlxed mode columns were also introduced and evaluated for nucleic acid separations (G41). Formylated-transfer RNA was resolved from nonformylated RNA with mixed-mode HPLC, although this pair were not resolvable by reversed-phase chromatography alone (1242).Since on1 a small percentage of organic modifier was added to the mo ile phase, biological activlties of 98% were found for the recovered species tested.
i:
Hydrophobic Interaction Chromatography
Mechanisms. As stated in the previous section, interactions of proteins with a surface decrease with decreasing hydro hobicity of the surface. Since the supports for hydrophotic interaction chroma aph (HIC) are so prepared that the densities of nonpolar igan s are much lower than in reversed-phase supports, adsorption of proteins is reduced. In most cases, negative gradients of salt-containing buffers are used for chroma aphy. Organic solvents are not needed as mobile phase mo ifiers. The environment is presumed, therefore, to be a gentler one for proteins so that conformational integrity is more likely to be preserved. Studies with model proteins of relatively simple structure support predictions based on solvophobic theory that retention depends on the contact surface area of the protein and the surface tension raising effect of the mobile phase salt (G43). For a given rotein, plots of In k ’against salt molarity were linear at suflciently high salt concentrations. Derivations from the predicted behavior were found with magnesium chloride (G44) and for some proteins retention was a multimode1 function of salt molarity (G45). In the latter case, the observed retention correlated with a model based on the cooperative, sequential bindin of salt to roteins. When a surfactant (CHAPS) was use! to manipufke mobile hase surface tension, the linear contact surface area relationsgp was conserved, but the relationship to surface tension was nonlinear (G46). To a first approximation, protein retention was explained by a competitive binding model. The low surface loading of nonpolar groups on HIC packinp does not preclude nonideal chromatography. &Lactoglobdm A, for example, self-aggregated under HIC conditions with HEPES-ammonium sulfate buffer to give multiple peaks (G47). Increased surface hydrophobicity of the stationary phase resulted in longer retention times, affected band shape, and favored conformationalalteration of a-lactalbumin (G48). Applications. As with other modes of HPLC, polymeric microspheres (G49)and nonporous beads (G50) were introduced for HIC applications. They have the strength and minimal tendency toward swelling or shrinking to be useful. Some example applications of HIC are given below. The major pancreatic secretory enzymes were separated with a four-step ammonium sulfate gradient. The conditions were sufficient1 mild that the identity of each enzyme could be determind by its activity. These same enzymes were reported to have been denatured by reversed-phase chromatography (G51).The enzyme that controls adipose tissue lipolysis was recovered from bovine omental tissue by HIC with a purity of 70%. Final purification to 95% was accomplished with a subsequent cation exchan e step (G52). Automated HIC was used to analyze blood glotin chains of normal subjects and of patients with hemoglobinopathies. Mutant chains were rapidly quantified (G53). Serum amyloid A and other highdensity apoli o roteins were isolated from serum free of lipid in one rapid E f C rocedure rather than several ultracentrifugation ste s a n f delipidation with organic solvents (G54). Studies of aykaline phosphatase with gradient hydrophobic interaction chromatography in combination with on-line low angle laser light scattering demonstrated that the enzyme undergoes rapid conformational interconversions (G55).
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Although experiments with such detectors are not trivial, requiring computerized base line subtraction techniques, complex instrumentation and considerable expertise for accurate interpretation useful information can be obtained. Cytochrome c oxidase was isolated for the first time from corn root mitochondria from HIC and a nonionic surfactant containing mobile phase. Ninety one percent recovery of total enzyme activity was achieved, along with a 12-fold increase in specific activity (G56). Separations of bio articles are illustrated by the following two examples. The Kydrophobic roperties of Prouidencia stuartii and other Gram negative gacteria were evaluated by HIC, which was found to a rapid and convenient means of screening strains for a property associated with bioadhesion ((357). HIC was an important step in the preparation of photosystem I particles from spinach leaves. Potassium ferricyanidewas added to the mobile phase to protect against dama e due to photoinactivation. Consequently, some previous y unknown pol eptides were preserved and the particles showed a very h i 3 activity in NADP photoreduction (G58).
f
Ion-Exchange Chromatography
Retention Mechanisms. Separation of proteins by ionexchange chromatography has been used widely by biochemists for some time is still preferred by many for protein isolation because the matrix and the salt, buffered and mobile phase resemble physiological conditions. Interactions between protein and column acking are primarily electrostatic in nature and involve Jsplacement of counterions from the acking sites of similar charge on the protein. Equilibrium Einding experiments with lysozyme, myoglobin, and bovine serum albumin were analyzed by a mass action model to determine the average number of binding sites and the mass action constant. Most of the ionized sites on the protein did not undergo ion exchange but were tied up by salt bridges. Scatchard plots of the data indicated that there was a competition between multiple binding forms of each protein studied. Even though the mass action and Langmuir models gave close fits to the data, they reflected an average interaction (G59). The three-dimensional structure of a biopolymer will determine those surface residues that are in a osition to interact with the packing surface so that multipre domains of the rotein may act cooperatively over considerable distances pG60). However, a change of a single amino acid residue in 275 amino acids influenced the retention of genetically engineered proteins. Both charged and uncharged residues had measurable effects on retention, the latter presumably because the interaction contact area microenvironment was altered (G61). Both electrostatic and hydrophobic interactions were demonstrated to be involved in retention and selectivity of 11proteins with a weak anion exchange column. Selectivity was manipulated by var ‘ng salt type and through the use of ethylene glycol as a m o b e phase modifier (G62). A threeparameter e uation was developed and found to correlate well with the U-iaped plots of log k’for proteins against log salt molality observed on a variety of ion exchangers (G63). For polypeptides, hydrophobic interactions with either a siliceous or polymeric exchanger matrix were so strong, in some cases, that mobile phase organic solvent additives were required just to elute the peptides (G64). Column Packings. Traditionally, ion-exchange separations of proteins were carried out with beds of soft els repared from derivatized carbohydrate matrices. f n tgeir modern form, macroporous agarose beads were rendered impermeable to proteins by shrinking and cross-linking them in organic solvents. The surfaces were modified to give a ternary amino group which possessed ion-exchangeproperties. Packed beds of such beads were compressed under pressure to give efficient columns with improved mass transfer characteristics (G65). Resolution of proteins was little affected by flow rate and mixtures of six proteins were separated in 2 min with a 6 X 0.6 cm column and a positive salt gradient. Two micrometer nonporous silica particles were coated with polyethylenimine and cross-linked with glyceryl diglycidyl ether to roduce an anion exchanger. Protein separations were achievefin 40 s with recoveries 197% (G66). Similar chemistries followed by exhaustive methylation were employed to prepare a polystyrene-based strong anion exchange packing that allowed protein separations to be made in a minute (G67). ANALYTICAL CHEMISTRY, VOL. 62, NO. 12, JUNE 15, 1990
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While the trend is toward the use of these pellicular packings, small particles of the inor anic porous anion exchan er, hydroxyapatite, continue t o t e evaluated (G68, G69). klution was with a sodium phosphate gradient and proteins tended to elute in order of increasing PI. Twenty-one proteins were separated in less than a half hour with no loss of activity or evidence of denaturation. A new approach, which utilized polymerization of ion-exchange matrix directly inside short columns, may reduce the cost of protein chromatography in the future. A mixture of five proteins was resolved in 10 min at moderate pressures. Resolution was relatively independent of flow rate (G70). Applications. Cation-exchange HPLC produced an excellent separation of y-crystallins, the structural proteins of eye lens, and their photosynthetically modified forms. With a single analysis of total extract, the proteins were quantified and isolated using sodium acetate gradients (G71). Complete diagnostic profiles of various alkaline phosphatase isoenzymes in normal and pathological sera were obtained within 20 min with weak anion-exchange column. Quantification utilized an online enzyme reaction followed by spectrophotometric monitoring of the nitrophenol formed (G72). The structural characterization of glycoproteins by HPLC is often difficult because of the microhetero eneity of the oligosaccharide groups. In a systematic stu y of several modes of chromatography, peak broadening and asymmetry during anion exchange was attributed to carbohydrate charge heterogeneity. The effects were smaller in reversed- hase and hydrophobic interaction chromatography (G73). T\e rapid purification of a restriction endonuclease from the bacterium, Sphaerotilis sp was achieved by a two-step process. Nucleic acids and nonrestriction enzymes were removed with an anion-exchange solid-phase extraction cartridge. The endonuclease was subsequently urified to a 330-fold increase in s ific activity by cation-excgange chromatography (G74). T E e different botulinum neurotoxins, classically distinguished only by specific antisera, were resolved by chromatography for the first time using either cation- or anion-exchange columns (G75). Their purification was accomplished by a combination of the two modes and with an automated system. Macroporous beads and nonporous compressed beads were explored as matrices for hi h-performance chromatofocusing. In chromatofocusing, t e pH gradient is formed within the column and proteins move along the column near the points in the moving gradient that are equal to their isoelectric points. Separations were carried out in shorter times with the nonporous beads and their protein capacity was not much lower (G76). Mini chromatofocusing columns were found also to have provided a rapid estimation of isoelectric of plant enzymes (G77). Oligonucleotides were se arated on both porous and nonporous anion exchangers. f o r up to 10 bases, resolution was accomplished with a linear salt gradient. Above 10, a hyperbolic gradient was optimum (G78). Recovery of oligonucleotides was found to decrease as temperature was raised above 25 O C (G79). DNA, containing
