Anal. (Chem. 1982, 54, 323 9-363 R
Column Liquid Chromatography Ronald E. Majors” Varian Associates, Walnut Creek Instrument Division, 2700 Mitchell Drive, Walnut Creek, California 94598
Howard G. Barth‘ Hercules .Inc., Research Center, Wilmington, Delaware 19899
Charles H. Lochmuller Duke Unharsity, P. M. Gross Chemical Laboratory, Durham, North Carolina 27706
INTRODUCTION This review covers the fundamental developments in the field of liquid chromatography (LC) during the period of 1980-1981. Earlier significant articles which were published in foreign journals, the patent literature, and other sources which were not available a t the time of the previous review ( l a ) are also included. References to oral presentations, conferences,and other unpublished works have been excluded. This review does not attempt to be a comprehensive coverage of all LC literature or merely a recital of LC developments. The authors have attempted to be critical in their selection of those references which do the most in extending the fundamental development in theory, methodology, and instrumeintation. Applications were selected which provided a better umderstanding of the fundamentals of LC, gave some insight into future trends, or represented a significant advance in using ZC to solve previously insoluble problems. The emphasis of this review will be on high-performance liquid chromatography (HPLC) since most of the current research (anddevelopment in the LC technique has been directed toward this “modern” approach. However, any significant development in HPLC is bound to carry over to all forms of liquid column chromatography, be it high, medium, or low pressure work. For this reason, the term LC will be used thrcughout the text. For any particular citation which pertains only to work involving small particles packed into columns which require high pressure to provide flow, the term HPLC will be used. Assignment of literature references to a given section was made on the basis of emphasis of the particular citation. The L,ockheed computer-based library files, Preston Technical Abstracts (LC), CA Selects (HPLC, GPC), Liquid Chromatography Abstracts (Chromatography Discussion Group, United Kingdom), and the primary chromatographic literature were used to locate the references cited in this work. The most recent market study by Centcom (2a) indicated that LC (consisting of HPLC, column chromatography, amino acid analysis, and ion chromatography) is by far the single largest 1981 analytical instrument market ($513 million) and will contiinue to grow at an average rate of 20% per year through 1984. The HPLC market will grow the fastest (26%/ye0ar)and be the dominant market of all analytical ) also instruments by 1984. Other market studies ( 3 a - 6 ~have indicated that HPLC will be a growth leader throughout the 1980s. The increase in literature citations has echoed these market surveys. Compared to previous Anal. Chem. biennial reviews, the period of 1980-1981 represented the years of increased applications of LC to solve real problems. About 6000 publications occurred in LC during this period and about 65% were devoted to applications of the technique. Of the more fundamental papers, although columns and methodology ‘This author’s contribution was prepared in part while he was on the faculty of the Department of Chemistry, Northeastern University, Boston, MA. 0003-2700/82/0354-323R$06.00/0
continue to receive widespread attention, the search for ithe ultimate detectors is still on-going since about a third of these papers me devoted to investigation of new detection principlles or extension of old ones, for example by post- and precoluimn derivatization using fluorescence or UV detection. Compared to gas chromatography, the growth in HPLC could have been expected. Approximately 70% of all known compounds are nonvolatile and as more research and applications are conducted in the life sciences and water pollution, and on polymeric materials, and inorganic compounds, LC will continue to be a dominant technique.
BOOKS AND REVIEWS With the rapid growth in HPLC, review articles and monographs abound. The second edition of the popular book “Introduction to Modern Liquid Chromatography” by Snyder and Kirkland (23b) was published in late 1979. Although lengthy, 863 pages, this book is a must for every practiciing liquid chromatographer as well as those considering using the technique. Several general books on basic HPLC have itppeared (see References, Books); those new ones from instrument companies may have a slight bias but represent gctod price/value. Several new series of advanced monographs have appeared on the scene. These volumes are generally multiauthored, written by noted chromatographers in their areas of expertise, and usually address specialized topics which only receive cursory coverage in the basic texts. For example, the tvvovolume series “High-Performance Liquid Chromatography: Advances and Perspectives”, edited by Horvath, covers topics such as historical overview of LC, practical operation of bonded phase columns, secondary chemical equilibria, and gradient elution in volume 1(lob),while volume 2 ( l l b )covlers column optimization, normal phase columns, and a comprehensive treatment of reversed-phase chromatography. More specialized chromatographically oriented texts such as that on porous silica by Unger (24b),polymers by Ca:ces and Delamare (2b),and nucleic acids by Wehr (2%) provide in-depth coverage on topics of limited general interest. The text on porous silica (24b) represents a very useful monograph for those desiring a knowledge of the backbone material used for the preparation of most current LC column packings. The maintenance and troubleshooting of HPLC systems are always a topic of practical interest and the text by Runser (19b)helps in this area. Of particular usefulness are the chapters on column care and maintenance and on detectors. The latest volumes in the “Advances in Chromatography” series have been published (8b,9b) and much of their contents are devoted to LC. The LC topics covered in volume 18 (13b) are long chain fatty acids and their derivatives, ion pair chromatography on normal and reversed-phased systems (a particularly good review through 1979), current state of art in analysis of free nucleotides, nucleosides, and bases in biological fluids, and resolution of racemates by ligand exchange. Volume 19 (9b) is solely devoted to LC topics such as applications in nuclear medicine, polymer characterization by 0 1982 American Chemical Society
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Table I. Speciality Reviews Life Science/Pharmaceutical amino acids, PTH 6d antibiotics 5d antimicrobials 84d bile acids 15d, 52d bile pigments 23d biochemistry/ biomedical 10d, 39d, 56d cardiac glycosides 72d carbohydrates 46d, 65d, 71d clinical chemistry 22d, 26d, 37d, 38d, 61d, 63d, 83d (nuclear) toxicology 67d, 76d drug metabolism 43d drugs (urine/serum) 13d, 47d estrogens 68d hemoglobin, including variants 70d, 82d natural products 3d, 20d neuropeptides 7d neurosciences 8d, 48d, 53d nucleic acid constituents 9d, 27d peptides 36d, 58d, 69d, 73d peptide antibiotics 34d pesticide metabolism 28d proteins 24d, 49d, 59d proteins and peptides 4d, 30d, 56d, 60d, 62d, 75d steroid hormones 16d steroid (cholesterol biosynthesis) 29d steroid, adrenocortico74d vitamin A metabolites 45d Miscellaneous automation 2d forensic drugs 40d inorganic compounds 64d organometallic/coordination 81d chemistry optical isomers 77d trace enrichment 21d Industrial explosives surface active agents coal products hydrocarbons
35d 78d, 79d
Energy, Petroleum 12d 33d
Environmental, Industrial Hygiene carbamate pesticides 19d occupational health chemistry 54d pesticides 25d, 44d pesticide residues 32d, 50d Food bitter acids (hops/beer) chocolate, confectionary industry foods lipids
14d 31 d 18d, 41d, 42d, 55d, 57d, 66d, 80d lld
gel permeation chromatography, 2-4D herbicide products, aldosterine and metabolites, and more fundamental contributions on hydrophobic interactions and gradient elution theory. The later chapter by Jandera and ChuraEek explores the theoretical basis of gradient elution for both continuous and stepwise gradients and discusses retention effects on solvent strength changes for adsorption, bonded phase, ion pair, and ion exchange systems. Some practical guidelines on optimization of gradients, instrumental errors, factors affecting solvent demixing, and selection of mobile phases are presented. The “b” references give a summary of books published since the last review. 324R
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For those interested in historical overviews, the evolution of liquid chromatography has been written by Ettre (46). The author covers the era from Tswett’s original papers up to the beginnings of HPLC, a period of about 75 years. The nomenclature of liquid chromatography has joined the confusion present in gas chromatography and there is an equivalent dichotomy of terms even with the definition of HPLC itself (e.g., high-pressure LC, high-performance LC, “high-priced” LC!). Following an earlier GC article on the same subject, Ettre’s paper (5b) on the nomenclature of LC should help alleviate some of the disorder, provided those publishing papers in LC take heed. For those who prefer their LC by sight and sound, audiovisual cassette courses with well done colorful slides are available from SAVANT (15b-18b). Topics covered are basic LC, instrumentation of LC, column selection, and quantitation. The courses were reviewed by Keller (14b) who found that the cassettes LC-102 and LC-103 on instruments and columns, respectively, were exceptionally well done. A number of general review articles on basic LC or the recent developments in HPLC have been published in a variety of languages. These are covered in General Reviews. With the recent widespread applications of HPLC, a number of specialty applications reviews on the use of HPLC have been published. Although applications papers are not the thrust of this current manuscript, an alphabetical listing of these specialty reviews is given in Table I as a convenient, time-saving device for interested readers.
GENERALTHEORY Advances in the development of theoretical basis for chromatographic phenomena continue to appear. This section deals with work that is of significance to liquid chromatography in general. Work which has specific relation to a given mode of separation (e.g., reversed phase) is contained in the appropriate section for that technique. Huber has provided a review of the practical implications of “theory” in HPLC (17e). One of the continuing concerns is the development of a sound model for separation speed in chromatography. While the empirical optimization of a given separation can be achieved through the application of a variety of strategies, it is rare for any one of those techniques to provide real insight into the limiting factors which lead to adequate resolution in a minimum time. Guiochon (14e) has provided a comparison of the theoretical limits of separation speed in gas and liquid chromatography in a recent paper. High-speed, high-resolution liquid chromatography is currently an area of growing commercial significance and continued developments in this area are to be expected. A review of gradient elution in liquid chromatography was presented by Snyder (35e). The development of a more realistic, more soundly physicochemical basis for retention behavior and the effect of equilibrium dynamics on peak shape is an area of active interest. The use of nonequilibrium methods for the treatment of the chromatographic process, both thermodynamically and kinetically, is established but investigations continue (3e,le). The relation between isotherm linearity and sample capacity has been reexamined (7e). A review on the theoretical and experimental aspects of fractionation mechanisms in liquid chromatography on macromolecular stationary phases, either gels or bonded chains, has been published by Lecourtier and co-workers (22e). A variety of attempts to predict retention have been reviewed (15e). The Snyder eluant strength parameter has been correlated with Lewis acid-base strength through a modified Taft constant (20e). A review of quantitative structure-activity relationships and the role of chromatographic measurement was provided by Kaliszan (19e). Soczewinski (33e) has reviewed the question of quantitative, retention-eluent composition relations for liquid chromatography. Other papers of related interest are (31e,8e). The influence of flow, flow domain, mixing, and mass transfer kinetics on ultimate peak shape is important in that resolution can be degraded or limited by any of these factors. Golay and Golay and Atwood (lle,10e) have examined the influence of Poiseuille flow on peak dispersion. A mixingmodel approach for the estimation of extracolumn effects was presented by Lochmuller and Sumner (34e). Grushka and Mott and Halasz and Hoffmann (16e,12e) have examined the
COLUMN LIQUID CHROMATOGRAPHY Ronald E. Majws is Liquid Ch-tography Marketing Manager lor Varian Associates. Walnut Creek Instrument Divkion. Walnut Creek. CA. He recehred hk B.S.in Chemlsby at California State University. Fresno. in 1963 and his Ph.D. at Purdue Universib in 1968 under the direction of L. 0. Rogers. He hen pined the Cekmrse Research Corn pany in Summa. NJ. where he supe~ised the Separations Laboratory. Ronald returned to California in 1971 88 B Senior Research Chemist in the LC Research Group 01 VarlBn’s Aerogaph Divkion. I n his 11 years wim Varian. he har had several assignments in research. applications. and He is the Butno( 01 more than 50 publ&marketing in lhe U.S. and Eur-. lions in HFIC. Gc.and swface chamkby. He has served as Speck1 Edtw of the Jmmel of W w m m g r e P M c Scienn, for issues devoted to LC columns and Column technoloey. Dr. MBjor5 is a member of the American Chemical Society. AnafVticai Division. Califwnia Seaion. and the Chromatography DiScusrion Group (London). Howard 0. Earth is prerientiy senior research chemkt wnh the analvtlcal divishw of Hercules Research Center in Wllmington. DE. He rewived his B.A. (1989) and Ph.D. (1973) in anawical chemism hom N O W eastem Unlversity. Betwe plning Hercules. Inc.. in 1974. he was a postdoctoral fellow in clinical chemimy at Hahnemann Medical Colin Philadelphia. Howard spent the fail 01 1981 as assktant piolessor of chew isby at hk slma mater. HIS spmianies inch& characterimatian of water-soluble polymers. size exciusion chromatography. and HPLC. and he has published a number of papers in these areas. He has been essistant edhor and &tor of the Del-Chem Bulletin. the Delaware ACS ssct!m publication. and p r m m chairman and chairmanI of lhe Delaware ACS anatytlcsi topical group. Rmently he served as secretary of the Delaware ACS section. He is a member of lhe Divirion of Analytical Chemistry and lhe Division 01 Polymer Chemistry 01 the ACS. ASTM. AAAS. and the Delaware Chromatogaphy Forum.
Charles H. LochmBIIer is Rolesoor of Chemism and Chalrman of the Depaltment at Duke University. He rewived his B.S. in Chemism horn Manhattan College and lhe M.S. and Ph.D. hom Fwdham University. He was a pos1doc1or.a BMOCiat0 wim L. 0. Rogers at Purdue for 2 years prior to joining the Duke facuity in 1969. He teaches graduate courses in separation science and wM*ShOpS and training COU~SBS in gas and iiquld chromatography and has given numer0”s lnvned lectures of those subjects. He Is the m h w of more than 50 publications deallng wlih research findings in chromatqraphy. several chapters in bwks, and a number 01 review articles. Hk research interests are in fundamntai aDpects 01 liquid chromatography. bonded phase chemisby. resolution 01 optical isD mer% and detector design. He is a member 01 the American Chemical Scciety and an Anernate Carncllw 01 ns Anafytlcai Division. He SBNBS on the Editwiai Board 01 lhe J o m l Of Chromlographlc Science, an the A d v i ~ r y Board 01 lhe Handbaok of Tram Substances. and as a consultant to the USEPA in the area of Environmental Assessment programs. He 8150 serves as a Consultant to industry in the areas of instrument. column. detector design. and automation.
influence of mass transfer kinetics on solute profiles. Band spreading a t the end of columns is discussed in two papers by Ccq, Cretier, et al. (5e, 6 4 . Viscous dissipation in packed beds, a phenomena familiar to engineers hut only recently recognized as important in band-spreading phenomena, was examined by Horvath and Lin (23e). This phenomena-the evolution of heat in viscous flow-is a problem in the highspeed use of small-bore packed columns, especially as the gradient in temperature a c r m the bed leads to a distribution of capacity factors which results in peak deformation. A mathematical technique was described which can recognize multiple peaks that are not completely resolved by Chromatography ( B e ) . The method uses fast Fourier transfer (FFT) analysis and involves taking a chromatographic peak which is badly overlapped in the time domain and maps it
Table 11. LC ColumnIMode Usage chromatographic mode reversed phase (total) C18 C8 C2
phenyl other .-....
normal bonded phase (i.e., -CN,-NH,) liquid-solid chromatography ion exchanee cation anion exclusion organic aqueous
% column
usage
61 (48.5) (8.7)
11.0)
ii.ij 1.7)
6.8
14 12.2
The journals surveyed were: Clin. Chem. J. Chromatogr. Sci. Anal. Biochem. Anal. Chem. J.A.O.A.C. Chroma togmp hia J. Chromatogr. J. Chromatogr. (Biomed. Appln.) into the frequency domain. Several levels of peak complexity were investigated for s y n t h e t i d y generated LC data. Using a rapid scan multiwavelength diode array detector, the authors showed that by generating chromatograms a t several wavelengths, application of FFT analysis at each one permits determination of the maximum numbers of sample components. Additional work needs to be done before this procedure could be used to determine retention times of the individual uuresolved components. Optimization of liquid chromatographic separations is an area of continuing intemk One of the difficulties is in defming exactly what the term ‘optimization” should signify. Highest resolution (all wnes resolved at least to base line) is not often the optimum for time of analysis. Over the past few years, a variety of techniques have been applied to the problem, including SIMPLEX, multilevel factor analysis and the use of response surface methodology. h u h has presented a review of optimization strategies in chromatography and other areas (214. Deming, who has contributed widely to the application of various optimization strategies to analytical problems, and co-workers have published recently on the use of multifactor methods for the optimization of reversed-phase separations (29e,30e). In the simplest cases, it is possible to make certain assumptions about the elution behavior of solutes with changes in mobile phase composition and to achieve an acceptable separation with a minimum of mathematical manipulation (2e,24e). As Guiochon points out (4e, 13e),one can take the approach of ‘absolute” or ‘relative” optimization. In the former, one designs a column uniquely suited for a particular problem and a particular mobile phase. The latter approach, which is perhaps more practical, in general, is to determine the optimum mobile phase composition to be used with a given column for a given separation problem. The “absolute” approach is familiar to gas chromatographic problem solvers. The design of a uniquely suited column is less easily achieved for HPLC. A “relative” optimization scheme for isocratic analysis is discussed hy Schoenmakers et al. ( 3 2 4 . Basically, there are two approaches in isocratic methods development: (1) to run a systematic series of isocratic compositions and -zero in” on the optimum composition by “trial and error“, or (2) to run a gradient elution profile and from the solute elution pattern select the approximate isocratic composition, followed by “tine tuning”. This latter approach ia favored by a graphical scheme worked out by Schoenmakers et al. (32e) who also give experimental guidelines on how to accomplish this optimization in the reversed-phase mode. They also discuss how the change in organic modifier permits selectivity changes hy “secondary solvent effects”. Molnar (26e)presented a similar approach for an aqueous phosphate buffer-acetonitrile mobile phase system. The isocratic composition where a test solute (npropyl benzoate) would elute agreed with in 2% absolute of the isoeratic composition calculated from gradient elution data. ANALYTICAL CHEMISTRY. VOL. 54. NO. 5. APRIL 1982
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Nurok and Richards present an interestin aid to the selection of solvent systems for thin-layer and &quid chromatography based on the assumption that log k’ is directly proportional to the log of the mole fraction of the “strong” solvent (27e). Readers interested in those findings should also consult the work of Perry ( B e ) . An interactive mixture-design technique applied to reversed-phase separations has been put forth by Kirkland and co-workers (9e) which may provide substantial aid to those venturing into this area. Their general scheme combines the Snyder solvent-selectivity-triangle concept with a mixed design statistical technique to optimize solvent strength and selectivity. With “overlapping resolution mapping”, the approach can achieve optimum resolution of all components in a mixture (they used nine substituted naphthalenes) or, alternatively, a single pair of several different pairs of compounds within the mixture. Transferable, optimized separation methods are still in the future due primarily to the variability of column selectivity and capacity, and, therefore, the user wishing to apply a particular method may need to readjust conditionssomewhat. Such readjustments are best carried out with established optimization methods.
COLUMNS Tremendous advances have been made in the areas of LC columns and column technology in the last decade. In HPLC, columns packed with microparticles with average particle diameters of 10 pm and under are rather commonplace and even columns packed with particles down to 3 pm are commercially available. Recent work in LC column technology has evolved around optimizing column reproducibility, especially of the chemically bonded phases, the development of columns optimized for specific applications (e.g., protein and carbohydrate columns), and developing experimentalways to extend column lifetime (e.g., “saturator-”, guard-, and precolumns). Research work on reducing column diameters to microbore (0.5-1 mm i.d.) and capillary (8.5) whereas silica-based packings cannot. A number of interesting newer packing materials were patented: porous carbon (17f),activated carbon prepared by carbonizing thermosetting resins (34f), steric exclusion packings for biological macromolecules consisting of silica gel with cellulose or modified cellulose (35f),and polyimine skin coated on a silica gel backbone (31f). Classification/Characterization.Although air centrifugal classifiers are commonly used to obtain narrow particle size distributions of inorganic packing materials, the hydrodynamic elutriation technique is used to classify resins and other organic packings. Larsen and Schou (19f) described a simple elutriation apparatus for the size fractionation of LC packings. They demonstrated the fractionation of Merck thin layer grade silica gel into narrow distributions of d, f 10%. The particle size range covered was 14.5-29.5 pm. Once classified, the next step is to characterize the packing in terms of average particle size and distribution. There are many procedures to measure particle size and particle size distribution-among the more popular are microscopy, use of Coulter Counter, sedimentation, and column permeability. Unger and Gimpel (360 compared the results of photosedimentation and microscopy for silica microbeads of graduated pore size and found that there was satisfactory agreement except when the silica contained large macropores of 400 nm. For computing particle size distribution, the effective diameter of the solvent-filled porous particles had to be inserted into the Stokes equation while the reproducibility of diameter, estimated by photosedimentation, was largely dependent on the accuracy of the specific pore volume determination. Ohmacht and Halasz (230 pointed out that the particle diameters quoted by manufacturers of chromatographic packings are often different than the particle diameters obtained by using a Coulter Counter or by measurement by chromatographic permeability. Column Packing and Preparation Techniques
It is now generally accepted that optimum high-performance LC columns (approximate reduced plate heights of two particle diameters) packed with microparticulates can only be obtained by the use of slurry packing techniques. For rigid packings, such as silica gel with and without chemically bonded phases, high pressures, typically 6000-12 000 psi, are used to force the particles into the column. For semirigid microparticulatepackings, such as polystyrene-divinylbenzene beads, more moderate pressures, typically 1000-3000 psi, are used in order to avoid undue compression. Besides the actual pressure used, the only differences in the slurry packing procedures are the solvents used to prepare the slurry or force it into the columns and whether one packs downward (most commonly used) or upward (15f, 16f). For chemically bonded phase packings, Kuwata et al. ( I S f ) used upward slurry packing with MeOH-2-propanol-cyclohexanol but varied the packing pressure depending on column length, followed by heat treatment of the packed column. For 3-pm octadecylsilane reversed-phase packings, Yamauchi and Kumanotani (44f)used a 1:l mixture of hexanol-1-methylene chloride as
COLUMN L I Q U I D CHROMATOGRAPHY
a slurry medium with good results. For porous carbon packings, Unger et al. (37f)found that a high viscosity slurry technique worked best. For those liquid chromatographers desiring to pack their own columns, the packing manufacturer should be consulted for their recommended optimum packing procedure. Some precautions should be noted in conjunction with high-pressure packing. Vonk and Bal (41f)reported the deformation of a stainless steel column of larger internal diameter when 5 and 10 pm particles are packed. Guiochon ( l o / , l l f ) , on the other hand, reports that some care should be exercised when working with organic solvents under high pressure after a colleague developed an infection after accidentally forcing heptane into his skin while unpacking a column. For larger internal diameter columns (i.e., 6-50 mm) and large particle size silica gel packings in various distribution increments within the diameter range of 40 to 200 km such as might be used for preparative columns, Gazda et al. (9f) found that a symmetrical particle size distribution of f 80 pm had little effect on column efficiency for d = 110 pm when a dry-packing impact method was employe$, This behavior significantly deviates from anal ical columns where efticiency losses of at least 50% are note when such wide distributions are used. The practical implications are that these larger preparative columns can use inexpensive wide distribution silica gel provided they are correctly packed. In their excellent article on preparative chromatography, Verzele and Geeraert (38f)discuss packing procedures for various diameters and lengths of preparative columns. Additional studies on dynamically coating or bonding prepacked columns have been reported. By adding a long chain quaternary amine to an aqueous buffered mobile phase, Hansen ( l 2 f ) demonstrated that an in situ reversed-phase packing could be created. Retention and selectivity could be controlled by varying the nature or concentration of the quaternary amine and also by varying the ionic strength, buffer pH, or concentration of or anic modifier in the mobile phase. Olieman et al. (24f)found that they could regenerate octadecylsilane reversed-phase columns by in situ silylation. As one might expect, higher silylation temperatures gave better coverage; however, prior to use the column must be flushed with a series of solvents in order to remove excess silylating reagent.
2
Column Hardware
Although stainless steel columns are most widely used in HPLC, glass columns have been the mainstay of column chromatographers for years. Therefore, work has proceeded on attempts to develop glass columns which could withstand the high pressures encountered in the packing and use of high-performance columns. Hara (13f)has patented a unique approach where the glass column ends are flanged and sealed by a conical poly(tetrafluoroethy1ene) end fitting. Vozka and co-workers (42f)report on heavy-wall glass columns which can withstand pressures of up to 60 MPa. Their glass columns are also flanged and the actual high pressure seal is made with a PTFE disk. The seal is contained so that when the end fittings are tightened, the PTFE is not deformed. Although such columns are in use in the authors’ laboratory, it is not known if these columns are available outside of Czechoslovakia. Another alternative is the use of glass-lined tubing which was studied by von Arx (40f). He found that the columns offered good corrosion resistance and could be packed reproducibly. Henderson et al. (14f)found that in the separation of divalent metal dithizonates, the glass-lined columns were also beneficial. Rather than glass, Volkov et al. (39f)claimed to achieve an inert surface by the application of a PTFE coating on the internal surface of a tube. In both of these cases of lining of the column interior walls, one must be aware that the use of stainless steel (SS) terminators and SS porous frits with these types of tubes may contribute more of a stainless steel surface than the bare column tube itself. In fact, Shih and Carr (32f)found that the silanization of stainless steel frits was necessary where performing trace analysis of metals. A concept that does away with end fittings is the radially compressed cartridges available from Waters Associates (8f). These columns, constructed of a flexible polyethylene tube (10 cm length x 8 mm i.d.1, fit into a special holder. This holder contains a hydraulic fluid which, when compressed around the column, is said to reduce wall effects by causing
the plastic wall to conform to the packed bed, thereby eliminating channels. Because the columns are 8 mm i.d., flow rates of 4-6 mL/min are used to give expected analysis times (If). Column Performance Evaluation
Once the column is packed, it is desirable to determine if the optimum results can be obtained from the column. Likewise, a way of comparing the relative performance of several different columns in order to select the best one is required, Furthermore, during use, a way of checking continued performance is useful. Important parameters in performance evaluation of a column are resolution (a combination of capacity factor, k’, selectivity, a, and efficiency, N), permeability (related to hack-pressure),and peak symmetry (%ding and/or fronting”) as was discussed by DeStefano (7f). Majors ( 2 l f ) , in his review on LC columns and packings, discusses some practical aspects of performance characteristics of prepacked columns. The useful BASIC program of Bristow and Knox (3f)for the standardization of test conditions for HPLC columns was converted to FORTRAN by Oliver and Sugden (25f). After a study of detector time constant (7) and its effect on peak height, peak skewness, capacity factor, theoretical plates, and peak symmetry correlation ratio, Low and Haddah (20f) made the recommendations for minimizing the effect of 7: (1)column efficiency should be quoted as 7 = 0 by extrapolation of a N vs. T, (2) for detectors which do not have a variable 7, column efficiency should be measured for a solute of k’ = 5-6; (3) peak symmetry correlation ratio, R,, should be determined a t 7 = 0 by extrapolation of linear R, vs. 7 plot, and (4) 7 should be l/l.oo of the peak width. It is doubtful that chromatographers wll take the time to make measurements recommended in (1) and (3) but (2) and (4) are plausible. Using the separation factors of three pairs of test compounds to evaluate 24 different lots from one manufacturer of a reversed phase material, Atwood and Goldstein (2f) concluded that lot-to-lot variations could be more easily spotted when the difficult-to-separate pair of polynuclear aromatic hydrocarbons anthracene/phenanthrene or a pair of steroids (androstenedione/testosterone)were used as test solutes. They also investigated eight other manufacturers’s reversed-phase materials and found these probes to be a suitable means to differentiate these columns while the biphenyl/naphthalene test pair gave similar separation factors. Their studies point out the importance of manufacturers’ maintaining rigid quality control standards on their lots of packings using meaningful test probes and if variations do occur to inform the user so that he may adjust his HPLC analytical method conditions accordingly. Column Maintenance and Troubleshooting
The microparticulate columns used in HPLC are expensive and somewhat fragile. Most are packed with small diameter silica or silica-based particles with chemically bonded phases with various functional groups of different degrees of reactivity. The packings are sometimes of irregular shapes, are operated a t relatively high back-pressure, and usually have small porosity frits at either end to contain the packing and prevent column contamination. With all these factors plus frequently used corrosive mobile phases, high temperatures, and use of samples which may not be sufficiently clean, it is no small wonder that column lifetime is of a major concern. Rabel has investigated ways and means of extending column lifetimes by the use of precolumns (27f),when injecting extracts of fat- and oil-based preparations (290, when using silica-based ion exchange packings (26f),as well as microparticulate columns in general (28f). The latter article is particularly informative, covering considerations of mobile phases, column storage, reconditioning, and sample preparation. In addition, Vunnam et al. (43f)have described a simple way to protect preparative chromatographic columns from running dry when solvent is fed by gravity. Anhydrous magnesium sulfate is added to the top of the column and its fine particle size and surface tension between solvent and salt are sufficient to prevent entrance of air into the system. When a reversed-phase column has obviously lost some of its bonded phase, Olieman et al. (24f)have described a way to regenerate the column by in situ silylation. If the inlet filter becomes plugged, Brown (4fi describes a reversible column which can be turned around and used in the opposite direction. ANALYTICAL CHEMISTRY, VOL. 54, NO. 5, APRIL 1982
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INSTRUMENTATION General
Conventional liquid chromatographic instrumentation is on par with gas chromatographic instrumentation in terms of flow rate precision, injection techniques and precision, column temperature control, and automation capability, but in general lags behind in detector sensitivity and selectivity. As will be seen in the Detector section, improvements are continuing to be made in conventional LC detectors. Besides several new books with chapters on the subject (covered in BOOKS section), a general review on HPLC instrumentation has been published by Henry and Sivorinovsky (8g). Also articles on new specific commercial instrumentation have been covered (5g, 7g, I7g, 23g, 26g). Pumps, Hydraulics, and Gradient Devices
Pum ing systems continue to improve with the main advancesI! eing made in better flow precision, in improved accuracy and precision in both programmed isocratic and gradient applications, and in the expandin use of multiple solvent capability, specifically ternary mo%ilephases. Most commercial pumping systems, especially those with gradient formation devices, have incorporated some degree of microprocessor control in their electronics, allowing for more sophisticated mobile phase composition changes as well as other time programmable features, Modern high-performance liquid chromatographic gradient pumping systems are of one of two types: (1)gradient formation on the high pressure side, generally havin multiple pumps, each of which pumps a single solvent; (27 gradient formation on the low pressure side where solvents are proportioned by valves, with one or more low pressure pumps, or by gravity feed. Billiet et al. (lg) has reported on comparative performance of seven commercial pumping systems as well as their own scheme where a single-pump solvent programmer used synchronized valve switching t o proportion pure solvents on the low pressure side. A different method was employed by Popovich et al. (18g) who created ternary mobile phases by using three low pressure metering pumps to feed mobile phases into a tee and flow sensor which was in turn connected to a high pressure pump that delivered the solvent mixture to the column. The flow sensor ensures that the total flow delivered by the metering pump matches the flow demand of the high pressure pump. In a single pump ternary device described by Klink (12g, I3g) two or three solvents are accurately proportioned during the pump fill stroke by high speed proportioning valves directly into the unique mechanical pump inlet valve. The proportioning valves were synchronized with the pump by an optical encoder and controlled by a microprocessor. Another ternary solvent programmer was described by Hathaway (7g). The solvent proportioning on the low pressure side of the pump is regulated by two three-way solenoid valves whose proportioning frequency is controlled by the pump flow rate through an electronic feedback circuit. Furthermore, the proportioning of the solenoid valve is synchronized with the pump intake stroke 80 that solvents are only proportioned during the intake stroke. The solvents are then mixed in a 1.5-mL dynamic mixer before being drawn into the pump. Two different investigations used a similar simple method in generating gradients. In using a well-known approach, Kaminski et al. ( I l g ) showed how gradients can be formed by varying the volume of a stirred mixing chamber on the low pressure side of the pump using a movable chamber piston while Simatupang ( 2 I g ) ,using a system of three-way valves, added the provision for automatidy regenerating the column prior to gradient formation. For such a system, retention time relative standard deviation averaged about 3% for 11 test compounds. A more general paper by Jandera et al. (9g) covers instrumental errors which can originate during gradient formation and influence chromatographic performance. Among those errors which were considered were solvent demixing, random or systematic deviation from the preset volume ratio of the two solvents caused by mechanical or electronic malfunctioning of the pump, the thermodynamic volume change connected with the mixing of a binary mobile phase, influence of solvent compressibility, and void spaces between mixer and 328R
ANALYTICAL CHEMISTRY, VOL. 54. NO. 5, APRIL 1982
column which cause discrepancies between the actual and expected gradient profiles. They performed tests on some of these potential errors on a commercially available two-pump liquid chromatograph. These results were compared to a two-plunger block gradient-generatingdevice with programmed stroke ratio that formed low pressure gradients the outputs of which were fed to a high pressure pump. An advantage of their gradient device is that it can correct for the volume contraction error resulting from the mixing of methanol and water. The possibility of using osmotic phenomena for an LC pump has been proposed by Shmidel et al. (20g). They showed that flow rates in the range of 10-40 KL/min could be generated with a semipermeable membrane of cellulose acetate separating two compartments of a rigid container. In one side was a saturated solution of magnesium sulfate; on the other side was distilled water. Osmotic pressure developed, driving an eluent (hexane) placed at the top of one compartment at a constant rate. An example of a separation was shown a t 3 mL/h which suggests that such a pump may be suited for micro LC columns. A theoretical design approach for two piston reciprocating pumps has been proposed by Luby et al. (14g). By use of analytical equations describing the shape of the cams which drive the pump piston, close to pulse-free operation could be achieved by providing a constant pressure with a minimum period required for pressure equilibration. A number of commercial pumps already use a similar concept. Recycling has found some use in HPLC, mainly in steric exclusion chromatography. In this technique, unseparated solutes are redirected from the detector outlet, back through the column by means of valves or through the chromatographic pump. A new concept in recycling was proposed by Minarik et al. (I6g). They developed a circulation valve which permitted eluent from the column outlet to be injected back into the column inlet without passing through a large dead volume. This valve was essentially an automatic two-way injection valve with a plurality of identical internal loops and was driven by a stepper motor. An example of a low pressure recycle experiment was shown, but no comments were made as to the valve’s suitability at high pressure operation typical of HPLC columns. Verillon et al. (24g) studied the possible use of a double chamber, single piston pump for recycling. Injection Devices
Typical HPLC injectors include septum, stop flow, loop valve (either variable or fLxed),and automatic sample injectors. The latter two are by far the most popular. A general review of injection techniques has been published by Simpson (22g). A comparison of various injection devices was carried out by Schmid (19g). He discussed the operation and design of various types of injectors commonly in use, the requirements for consideration of the “right” injector for a given application, and suggested a way to measure the band broadening contribution of an injector. After testing several commercial injectors, Schmid found a typical band broadening contribution uv,injwas about 15 mm3 for which he concluded may be too large for high-performance columns. Peak asymmetry contributions can also originate from the injector. In a similar study by Kelsey and Lascombe ( l o g ) ,they compared stopped flow injection using a syringe with valve injection with and without curtain-flow. They concluded for the particular injectors they evaluated that the stopped flow technique for syringe in’ection gave the highest efficiency with acceptable reproduci ility. For valve injection, the curtain-flow configuration was optimum. For a conventional valve injector, the use of PTFE insert in the column inlet could improve its performance. Using a single injector with open sampling loops, Coq et al. (3g) studied the input profiles and injection variance for the injection of large sample volumes. They found that the variance can be 30% to 80% higher than “plug” injection which assumes no contribution from the loop, Two methods were proposed to decrease the band broadening contribution: One method was to employ a larger than necessary loop and to make a rapid injection. The second method uses a sample loop packed with glass beads. Guidelines as to bead sizes and sample loop internal diameters were given in graphical form. Other sampling effects under mass overload conditions were investigated by Wall using a curtain flow injector employing a precolumn (25g). For both semipreparative and analytical separations by adsorption and reversed-phase chromatogra-
b
COLUMN LIQUID CHROMATOGRAPHY
Table 111. Survey of HPLC Detector Usage (1980-1981) % usage absorbance UV fixed UV filter spectrophotometric fluorescence refractive index electrochemical other
70.7 (28) (8.7)
(34) 15 5.5 4.4 4.4
Total Papers Surveyed: 356 Journals Surveyed: Am. Lab. (Fairfield, Conn.) J. Chromatogr.
Anal. Biochem. Chro matographia Clin. Chem. J. Am. Assoc. Anal. Chem.
J. Chromatogr. Sci. Chromatogr. Sci. (Bio.1 Biomedical Appl. Liq. Chromatogr. 2)
~
phy, his results suggest that in addition to reduced velocity and reduced plate height, a reduced sample load parameter should be considered when describing column performance. An interesting observation was that the reversed-phase packings showed little difference in their sample capacity compared to adsorbents in contrast to other reports. By slight modification of a simple loop valve, variable volume injections could be carried out by Minarik et al. (15g) without the need of partially filling a sample loop. With a four-port valve which has interconnected grooves leading to the valve inlets, one of the grooves serves as the sample loop and could be partially filled by turning the valve rotor against the valve body only part of a turn. A calibrated scale allows selectivity of microliter volume. Ferraris and Lauret (4g)have developed split-stream injectors for both analytical and preparative HPLC. A real advantage of this approach for preparative work was that by coupling the split stream injector with a spreader, the solute was allowed to be dispersed over the entire head of the column, thereby avoiding localized stationary phase overloading and subsequent band broadening. In a theoretical study of injection by Colin, Martin, and Guiochon (2g), the effect of the injection process on column efficiency was dependent on injection time, volume, technique of sampling, and, depending on experimental conditions, the particle size of the packing. A model was developed for the injection process which related the contribution to plate height to the length of the solute zone at the column inlet, the column length, and the concentration profile in the solute zone. Although various types of conventional injection devices were considered, few practical recommendations for new injector designs came out of this study. Clearly more work needs to be done in the improvement of injector design, especially for the short conventional microbore and capillary columns. Detectors General
It is generally agreed that detectors are the weakest link in the liquid chromatograph. A great deal of detector research and devielopment is currently in progress, mainly in academia and in instrument companies. The past 2 years has seen a rapid increase in the number of published papers dealing with some aspect of LC detection-the pursuit of new detection schemes, improvements in or extension of well-known detection principles. In order to reflect on current usage of LC detectors, we surveyed 356 randomly selected papers from applications of HPLC in 1980-1981 and categorized them according to detector types used. Table I11 shows that, by far, the optical detectors predominate, as expected, with absorbancedetectors the most widely used. The variable wavelength detectors are the most popular since undoubtedly the nature of the survey reflects a “research”bias. The fixed wavelength detectors are likewise popular since they have been available the longest and are suitable for use with a wide variety of aromatic com-
pounds. The indicated use of refractive index detectors, mainly used in the polymer industry in steric exclusion chromatography and in the food and beverage industry for sugars, is probably slightly low because of the journals surveyed and since those indicated industries do not generally publish their results. The most rapid growth in new detector applications is in the use of variable wavelength spectrophotometric detection and electrochemical detectors. While new detectors are being developed and commercialized, there has been a trend to get more information from currently available detectors, notably by the use of detector ratioing for peak confirmation or purity determinations, use of pre- and postcolumn derivatization (see section on these techniques), and other enhancement techniques (e.g., chemical amplification or use of laser sources). In addition to the coverage of detectors in the LC books cited earlier, several general reviews on HPLC detection have been published (4h-6h, 9h, l l h , 12h). The review by McKinley et al. (12h) is a short, concise, readily available general overview. Besides the obvious chromatographically important detector signal parameters of noise and drift, the dynamic range of a detector is a factor which is sometimes overlooked. Detector linearity is particularly important when working with samples of a wide concentration range, when doing quantitation with a data system, or when working with high detector background signals. Carr and associates (2h, I l h ) have published two papers dealing with the effect of detector nonlinearity on height, area, width, and moments of chromatographic peaks. The first paper (2h) develops a model relating chromatographic signal characteristics and calibration curve nonlinearity. By use of pure Gaussian peaks, calculations indicate that the peak center, symmetry, and odd higher moments are insensitive to detector nonlinearity while peak height is most sensitive. Peak area is less sensitive and peak width the least sensitive. Using an absorbance detector with a 2nm spectral slit width, the second paper ( l l h )demonstrates that such effects can be observed under reasonable LC conditions. Employing a shoulder at 212 nm for biphenyl for their measurements in order to maximize nonlinearity, the authors proceed to show that very nonlinear calibration curves can be obtained at relatively low (
