Anal. Chem. 1986, 58, 2 11R-250 R (Q69) Shackleton, C. H. L.; Chai, W. In "Mass Spectrometry in the Health and Life Sciences"; Burlingame, A. L., Castagnoli, N., Jr., Eds.; Elsevier: Amsterdam, 1985; pp 491-505. (Q70) Jardine, I.; Scanlan, G. F.; Mattox, V. R.; Kumar, R. Biomed. Mass Spectrom. 1984, 1 1 , 4-9. (Q7l) Gaskell, S. J. Presented at the 33rd Annual Conference on Mass Spectrometry and Allied Topics, San Diego, CA, 1985; pp 140-141. (Q72) Moneti, G.; Agati, G.; Giovannlni, M. G.; Pazzagli, M.; Salerno, R.; Messeri, G.; Serio, M. Clin. Chem. 1985, 3 1 , 46-49. (Q73) Whitney, J. 0.;Ling, V.; Roitman, E.; Grunberger, D. I n "Advances in Mass Spectrometry, Vol. 10";Todd, J. F. J., Ed.; Wiley: Chichester, 1986, in press. (Q74) Bruno, I.; Minale, L.; Pizza, C.; Zollo, F.; Riccio, R.; Mellon, F. A. J . Chem. SOC.Perkin Trans. 11984, 1875-1883.
((275) Mlnale, L.; Pizza, C.; Plomitallo, A,; Rlccio, R.; Zollo, F.; Mellon, F. A. Gazzetta Chim. Italiana 1984, 114, 143-158. (Q76) Isaac, R. E.; Desmond, H. P.; Rees, H. H. Biochern. J . 1984, 217, 239-243. ((277) Pearlman, W. H.; LaMay, E. N.; Peng, L.-H.; Pearlman, M. R. J.; Hass, J. R. J . Bo/.Chem. 1985, 260, 5296-5301. (Q78) Raju, U.; Levitz, M.; Banerjee, S.; Bencsath, F. A,; Field, F. H. J . Clin. Endocrinol. Metab. 1985, 6 0 , 940-946. (Q79) Watson, D.; Taylor, G. W.; Murray, S. Biomed. Mass Spectrom. 1985, 12, 610-615. ((280) Smith, R. W.; Parker, C. E.; Farrow, P.; Rollins, K.; Gaskell, S. J. Presented at the 33rd Annual Conference on Mass Spectrometry and Ailied Topics, San Diego, CA, 1985; pp 504-505.
Column Liquid Chromatography Howard G . Barth* and William E. Barber' Hercules Incorporated, Research Center, Wilmington, Delaware 19894 Charles H. Lochmuller Department of Chemistry, Duke University, Durham, North Carolina 27705 Ronald E. Majors Varian Associates, Walnut Creek Instrument Division, 2700 Mitchell Drive, Walnut Creek, California 94598 Fred E. Regnier Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907
INTRODUCTION This review covers the fundamental developments in the field of column liquid chromatography (LC) during the period of 1984-1985. In an attempt to conserve space, we have excluded patents and theses and included only articles published in English, French, German, and Russian with some exceptions. As compared to our previous review (Anal. Chem. 1984,56,300R-349R), we have deleted all of the tables dealing with selected applications and have excluded sections on automation and data handling, ion chromatography, trace analysis and sample preparation, and novel applications. New sections that have been added are hydrophobic interaction chromatography,micellar mobile phases, fast LC, and indirect photometric direction. This review is not a comprehensive coverage of all LC literature. We have attempted to critically select only those references that reflect fundamental developments in LC theory, methodology, and instrumentation. Your suggestions and comments are most welcome and should be sent to the senior author (H.G.B.). Our main data base for this review was C A Selects (HPLC and GPC) from October 31,1983, to December 2,1985. This represented over 8000 abstracts of which 1200 were used. In addition, each author used other search routines to augment coverage from C A Selects.
There was a substantial increase in the number of liquid chromatography books published during this review period 'Present address: Hewlett-Packard, 2750 Monroe Blvd., Valley Forge, PA 19482. 0003-270Ql86l0358-21 1R$06.50/0
which are listed in the "Literature Cited" section. The more active publishing companies were CRC Press (CRC Handbook of Chromatography Series), Elsevier Scientific Publishers (Journal of Chromatography Library Series), Marcel Dekker (Chromatographic Science Series), and John Wiley & Sons (ChemicalAnalysis Series). The only text that appeared was "Contemporary Practice of Chromatography" by Poole and Schuette (40a). Books and symposia on size exclusion chromatography have been authored by Balke (2a),Belenkii and Vilenchik (3a), Janca (30a), and Provder (41a). Finally, Snyder and Dolm (46a) have produced an HPLC video course that consists of five 45-60 min video tapes aimed at providing a basic understanding of HPLC for the beginning chromatographer. Reviews related to specific areas of LC are given under the appropriate sections in this article.
GENERALLCTHEORY This biennial review has seen no major breakthroughs in liquid chromatographic theory. However, there have been very significant insights developed as to the governing equations for band broadening and for retention as they relate to physical parameters that can be independently measured. Optimizationmethods continue to be applied which are based on either heavily modeled formalism or purely empirical functions. The latter suffer from all the typical problems of empirical approaches (SIMPLEX, steepest-ascent, surface modeling, etc.) in systems where there may be many local minima or maxima. The former become cumbersome outside of fairly simple systems as the number of parameters increases and the model grows more complex and more dependent on 0 1986 American Chemical Society
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COLUMN L I Q U I D CHROMATOGRAPHY
additional retention measurements to establish “parameters”. It may be that until some theoretical breakthrough occurs that empirical methods are the sole hope of the practitioner although it is obvious that some empirical approaches are better than others. For systems with less than ten components, the generation of resolution surfaces with the results of high-speed, high-efficiency separations is perhaps the most unequivocal ap roach a t this time. &he physicochemicalunderstanding of the processes leading to peak dispersion in liquid chromatography has been studied by Katz and co-workers (12b),who found that if extracolumn peak dispersion is minimized, the Van Deemter equation holds within experimental error over velocity ranges 0.02-1 cm/s for a wide range of particle size and solvent and solute type. Over 750 precise measurements of H and velocity were made which indicate that, for silica columns a t least, the relation holds. An interesting outcome of this work is the report by Katz and Scott ( I l b ) that solute molecular weight can be determined by measurement of the capacity factor and the bandwidth within 13% for 90% of the solutes studied providing the density of the solute lies between 0.85 and 1.25 g/mL. Knox et al. (13b)have examined the effect of the B and C terms of the Van Deemter equation and provided explicit equations for these terms involving processes inside and outside the particles of the column packing. Sout et al. (23b) studied the question of efficiency as a function of particle composition and geometry and found that the observed effect can be rationalized on the basis of a quantitative model which assumes two dominant effects: restricted diffusion of small molecules in narrow pores lined with long alkyl chains and surface diffusion along the pore wall surface. Martin and Guichon (16b) provided an interesting discussion of the statistical basis for the evaluation of complex chromatograms basing their approach on the analogy between depolymerization and separation. Seshadri and Deming (21b) used a simple chromatographic model to investigate the effect of sample interactions on elution behavior. Chromatography has always had one great limitation in practice, speed. Speed limits the sample throughput and the application of chromatography to process control. Grushka (8b) has examined the effect of high speed on peak capacity and introduced what appears to be a very useful parameter “peak capacity production” as a criterion for developing shorter analysis times. Erni (6b) provided a quasi-review of the “limits of speed” in HPLC. The growing importance of “affinity” separations has led to the rebirth of displacement chromatography. Displacement methods may be the most cost-effectiveprocess-scale approach in areas such as biochemical engineering. Displacement has the advantage of higher gram capacity and simplicity. Horvath (9b) has provided a useful review of the current theory in this area. Optimization remains a poorly defined term in chromatography (and perhaps in everyday chemometrics). For many the term means obtaining a chromatogram that solves their problem irrespective of time of analysis or quality of the chromatogram. In principle, an optimal chromatogram is one in which adequate resolution is obtained for all important peaks in an individual sample analysis time that is acceptable for the sample load in a given laboratory. A simple, pragmatic approach is given by Bounine and co-workers ( I b ) . There are a number of reviews on the subject (5b, l o b , 15b, 17b, 20b). One of the problems with response surface mapping and optimization is the choice of the response surface model. Utility functions for this purpose have been examined (25b) and, as is commonly observed, the major problem is the inability to define an unequivocal minimum. A demonstration of artificial intelligence, which is far from cybernetic, is contained in the paper by Lu and Lu (14b). Bounine and Guichon (2b) suggested an alternative approach. Glajch and co-workers (7b) presented an interesting example of mixed-statistical design applications to the optimization of phenylthiohydantoin amino acids. A given analytical problem may be solved in many ways to the satisfaction of the user. Purnell(18b) provided a review of “window analysis” as an approach to “optimization”. Some 18 different solutions to the problem of the separation of chlorocarbons were developed, if time were neglected. This reduces to one column and condition if time were included. Tomlinson et al. (24b) compared the “window diagram” me212R * ANALYTICAL CHEMISTRY, VOL. 58, NO. 5, APRIL 1986
thod to the solvent selectivity triangle method for optimizing solvent systems. Although the latter was useful for setting up a new solvent system, it had limitations when used for the separation of isomers. Isocratic methods have the advantage of simplicity and do not require a reequilibration delay between sample runs. Gradient methods offer numerous advantages in terms of individual analysis time, improved limits of detection due to narrower band shape, and increased degrees of freedom in retention control. The theoretical principles of gradient elution are an important area of current development. An interesting contrast in approach is afforded by the paper of Cohen et al. (3b) and that of Stadalius and co-workers (22b). A subsequent paper (19b) treated the question of solvent demixing caused by sorption of mobile phase components by the stationary phase and contains expressions that predict retention times to within 1% of the total gradient time. Many computer programs exist by which gradient conditions may be estimated. An interesting approach is that found in ref lob.
COLUMNS Column advances in the 1980s have focused on improvements in bonding procedures, especially in phase reproducibility and the development (and, unfortunately, a proliferation) of selective bonded phases for specific applications (e.g., proteins, chiral separations, ion chromatography). There has been more widespread interest in the miniaturization of columns, with decreased length and/or internal diameters, and further research studies on open tubular columns and related instrumentation. New “inert” column materials (e.g., glass, titanium, etc.) as well as new designs in column hardware continue to be developed. Two reviews (13c, 16c) on trends in column usage noted the following: (1)use of reversed-phase chromatography columns dominates all applications of HPLC (-60%), mainly on C18 phases but with C8, phenyl phases, and polymeric phases also in use; (2) the 25 cm X 4.6 mm column is still the most widely used in analytical work; (3) the most popular particle size has an average diameter of 5 pm; (4) spherical packings are preferred 3:l over irregularly shaped packings; (5) guard columns are now in widespread use; (6) column lifetimes for silica-based packings are generally less than 6 months and the average columns per year of users is 5; (7) commercial prepacked columns are used by 90% of the practicing liquid chromatographers. Other general reviews on columns include state-of-the-art coverage (21c),advantages of prepacked columns (I&),commercial columns for amino acids, peptides, and proteins (28c), and new commercial columns introduced sipce the last biennial review (14c, 15c, 17c). The great amount of interest generated by “fast” LC columns and microbore columns is covered elsewhere in this article. Column maintenance and troubleshooting are always of practical significance. Abbott (IC)discussed column pressure problems and their diagnosis and cure. Retention time changes, symptoms, and cure were also covered (23c). Wehr (29c) provided tips on the care of prepacked HPLC columns commonly used in protein and peptide separations. Help with the design of columns was provided by Katz and co-workers ( I l c ) . Their design protocol utilizing three data bases (performance criteria, instrument constraints, and elective variables) provides optimum column length and radius and optimum particle size for minimum analysis time. Similar to other studies, available pressure is the limiting parameter for optimum separation of complex mixtures, but the authors conclude that 4000 psi appears to be adequate for most general work. Of course, these studies imply that columns must be well packed by an optimum procedure. Column Packing Technlques
High-pressure slurry packing techniques are most often used for the microparticulates. A Russian group (1Oc) has studied the effect of pressure on the slurry packing of 14-pm silica gel. They concluded that a t a constant rate of packing the absolute pressure per se does not affect the quality of the packing. The important factor is the pressure drop per unit
C M U M N LIQUID CHROMATOGRAPHY
Wwnd a. 6n.rlh b Senlor R e w f c h C h e m bt wim lhe Analyacal DMskm 01 Hercules Research Csmer in Wrmingm. DE. He is g.oup hem ot Me gas chromatogaphy. #quidchwnatcqaphy. panicle sbe analysis. and polymer analysir section*. He received , c his B.A. (1989) and Ph.0. (1973) In m a w cai chemistry from Natheastem University. Betwe joining Hsrcuies Inc. in 1974. he was a postdoctoral fellow in ~ l k i c a chemiSi try at Hahnemann Medical Col!-3ge In philadeiphb. Howard Spent the fall of 1981 as Aslistant Relessor 01 chermrtry at his a l m mater. He has taught at the University 01 h Delaware and is a freqmnt lecturer at S M courses smn~oredbv the DBhware Vallev Chomatoaraohv Forum HIS rpecianie; include pol;mer characterizationlsiie exci&n chromatography. and hi&pertormance liquid chromatography, Ha he5 published over 30 pap " s in these areas and also has edited a bmk. '"ModemMethods 01 Particle Size Anaivsir". oubiished bv Wllev in 1964. Barth 1s on the lnrtrumentation Advisory Panel 6f Anat&; Che&ishy and has been recently appointed ASsccbte Editor 01 Me Jownal of Applied Po@Sckwlce . He has been ASsistant Ednor and Edna of the Dsl-Chem Bulietin. the Debware ACS section publication: has been FTwam Charman and Chekman of the Delaware ACS Analylical t o p h l group: served as Secretary of the DBlaware ACS ~ection: and is presently Chairman 01 the A d i t CcmmMee. Dr. Barih Is a member 01 me divisions 01 Analytical Chemistry. Polymer Chemistry. and Polymeric MaIBrlalE Science and Engineering of the ACS. ASTM. AAAS. and Me DBbware Valley Chromatography Forum
&arch Chemist wiih me Analytical mbion f 01 Hercules Research Center in Wilmingtar. L E He received hi5 B.A. in chemistry at lhe University of Ver-t in 1977. Betore attending graduate school he spent 6 months at American CyanamM in Stamtad. CT. He received his m.0. in analytical chemistry at me univerrny of M I W S O ~ ~ in 1983 under the direction of P. W. Carr. Bill joined Hercubs Inc. in 1963 where he was involved In HPLC memods development and CwTdlnaIing analytical ettons for new product deveC opment. He has several publications in the areas 01 chromatographic peak shape analysis. detector linearity. indirect UV detection. and paired-ion ChrD matography. Bill is a member of the Division of Analytical Chemistly of the ACS and the Delaware Chromatography Forum.
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C. W. Loch(yw b Rolesoa 01 Chembtry and Biochemical Englneerlng at Duke university. A Fellow 01 me Royal Society of Chemistry and me American Institute of Chemistry. he serves as a Member 01 Me Comminee 01 Revision 01 me Unned States Pharmocopeiai Convsntlon and is past Chairman 01 the DIYISIOII of Analytical Chemistry 01 Me American Chemical Sock ety. He SBNBS on the editaiai boards of several jovnsis devoted lo chemically mod!tied surfaces. chromatography. and c h e w mewics. He is the author of more than 75 artlCleS related to "Brious aspects 01 separation SCK)"CB.
Ronald E. M a w b LHe Sclence MalkeUng Manager l a Varian AI)(ME~~w. Walnut Creek Instrument Dtdaion. Walnut Creek. CA. He received his B.S. in chembtry at California State University, Fresno, In 1983 and his Ph.D. at Purdue University in 1966 under me dkeclbn 01 L. B. R-r. He then pined Me Ceianese Research Co. in S u m mil. W. where he ~ u p e ~ l ~ the e d%param s labaatay. Raoaid retumx( @ CBiitornb in 1971 as a Senw Research Chemist in tM LC Research OIoup of Varlen's AWCgraph Division. In his 15 years with VariBn. he ha5 had mveral assignments In research. aDDlicatiOnS. and marketing in the U.S. and Eirope. He is the author i t over 80 publications in HPLC. GC. and surface chemistry. He has served 80 Speclal Editor of the Journai of Chromsfogrephic S c W tor ISLWSdevoted to LC columns and Column technol41y and is currently edna tor a monthly feature, "Column Watch in LC Msgazle for which he is also ar e d i t d l board. Dr. M a w is a member of Me Amedcan Chemical Society. Analytical Division. California Section. Computers in Chemistry DivIsIon. and SBNBS on the Executive Comminee 01 me newly tamed Sutdivbion of Chromatography and Separations Chemistry. He is also a member of the Chromatographic Sociely. Dr. Majors is also the Chairman 01 upcoming HPLC '86. the loth International Symposium on Column Liquid Chromatography. to be held in Sa" Francisco in May.
Fred E. R@ is a Rotessm of Bbchem istry at Purdua University. He recieved his B.S. in chemlatry from Nebraska State Teachers College and the ph.D. from Dklahama State University. Afler postdoctorates in biochemistry and biology at the University of Chicago and Harvard. respectively. he joined the tacuity at Purdue in 1966. He
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ligud chroma14lraphy 01 biopolymers. He 15 a member of the American Chemical Sacieiy. the American Society of Biological Chemists. and Sigma Xi. Dr. Regnler serves on the editorial board of Anaiyiical Bi0Chem;stry. Jom.41 of Liquid Chromalography. and Liqdd Chromatography Magazine.
c o l u m n l e n g t h during t h e p a c k i n g . F o r reversed.phase col. u m n s t h a t have lost their efficienry, a repacking procedure was d e v e l o p e d t h a t c a n p r o v i d e a1 least 9590 oi t h e p r e v i o u s best r e s o l v i n g c a p a c i t y ( 3 ~ ) D. r y p a c k i n g is n o t u s u a l l y a d v o c a t e d for p a r t i c l e s
