High-Performance Liquid Chromatography Past ... - ACS Publications

High-Performance Liquid Chromatography Past Developments, Present Status, and Future Trends

Phyllis R. Brown Department of Chemistry University of Rhode Island Kingston, RI 02881

High-performance liquid chromatography (HPLC) is about 20 years old. This REPORT will assess where we have been, where we are now, and where we are going with this separation technique. We will look at the 1970s, when LC equipment became commercially available, and the 1980s, when HPLC fell into routine use. We will also project trends for the 1990s. Progress in instrumentation rarely takes place in quantum leaps; it is an evolutionary process. Therefore, to explore the future of HPLC, we must look at this technique in the context of past developments and present achievements. The 1970s When liquid chromatographs first appeared on the market, they were known by pseudonyms such as "amino acid analyzer," "nucleic acid analyzer," or "urine analyzer." In the biochemical market, for which the instruments were developed, chromatographic instrumentation was not widely used, and most separations were done by using open-column, paper, or thin-layer chromatography (TLC). Open-column chromatography was time consuming and tedious, often requiring a large amount of sample; if the technique was not carried out in a cold room, the constituents of interest could degrade be0003-2700/90/0362-995A/$02.50/0 © 1990 American Chemical Society

fore the separation was completed. Fractions had to be collected and the components detected and quantified by another method such as UV spectroscopy. Other disadvantages included the use of large volumes of solvents (exposing the researcher to carcinogenic organic solvents for long periods of time), low sensitivity, and poor reproducibility and resolution. When paper chromatography and TLC were used, some of the detection problems were solved. Very small samples could be analyzed, and the reproducibility and resolution improved. The ability to assay many samples simultaneously, achieve faster separation times, and use smaller amounts of

REPORT solvent made these techniques more advantageous than open-column chromatography. However, quantitation was still inadequate and resolution of similar compounds was difficult. Gas chromatographs were available in the 1960s and 1970s, but because most biologically active compounds are nonvolatile, thermally labile, ionic, or of high molecular weight, derivatization was usually necessary. Unfortunately, in the derivatization step, errors could be introduced, and sometimes the quantitative results were questionable. Although on-line derivatization procedures were developed, GC was not used routinely in the ma-

jority of biochemical, pharmacological, or medical laboratories. To the analytical chemist, it was obvious that there was a tremendous need for an instrumental technique that could separate water-soluble, thermally labile, nonvolatile compounds with speed, precision, and high resolution. The development of HPLC was spurred by the discovery of DNA. A reliable technique was urgently needed for the separation and quantitation of subnanoliter quantities of nucleotides and nucleosides from the hydrolysates of DNA and RNA. The separation methods available at the time were inadequate. By 1969 a limited number of HPLC systems were commercially available. Columns were unreliable— retention times and characteristics were not reproducible, not only in columns from company to company, but in columns from the same company. Pump flow rates were inconsistent, and the question of whether it was better to have constant flow or constant pressure was debated. UV and fluorescence detectors were developed and widely used, but for many biologically active molecules such as triglycerides, saturated organic compounds, fatty acids, and carbohydrates, detection continued to be a problem. Moreover, there was no sensitive universal detector for HPLC as good as the GC flame ionization detector (FID). Most researchers in biologically oriented laboratories continued to resist using HPLC. However, it was finding its way into analytical as well as a

ANALYTICAL CHEMISTRY, VOL. 62, NO. 19, OCTOBER 1, 1990

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REPORT few biochemical, pharmaceutical, and medical laboratories, and even clinical laboratories were using it for specific assays such as monitoring the level of theophylline in the serum of asthma tics (1). The development of microparticle chemically bonded packings was a ma jor breakthrough in making HPLC a valuable technique for the life sciences (2,3), and the introduction ofreversedphase packings made it a more univer sal tool (4, 5). High-resolution separa tions that could not be readily accom plished by ion-exchange, adsorption, or normal-phase partition chromatogra phy were achieved by using reversedphase liquid chromatography (RPLC) (e.g., the separation of nucleosides and their bases in one analysis, as shown in Figure 1 [6]). Publications on the mechanism of RPLC appeared, and re

ports on the use of HPLC and RPLC were published not only in analytical and chromatography journals, but also in biochemical, pharmaceutical, and medical journals. By the late 1970s, HPLC was an accepted technique in any laboratory requiring good separa tions (7, 8). The 1980s

During the past decade, rapid advances were made in instrumentation and HPLC equipment was refined, auto mated, and computerized. Columns and instrumentation became both smaller for analytical purposes and larger for preparative purposes. For an alytical and trace analyses, microbore and small-bore columns and "fast" HPLC became available. Micro columns, also called capillary columns, have a typical diameter of a fraction of

Figure 1. Separation of 0.1-0.5 nmol of 28 nucleosides, bases, nucleotides, aromat ic amino acids, and metabolites (40 μΐ of a 10~ 5 mol/L solution of each standard). Column is C18 on 10-μηη silica. Gradient is 0-60% Β in 87 min. Mobile phase: (A) 0.02 M KH2P04 at pH 5.6; (B) 60% methanol. Flow rate is 1.5 mL/min. Peaks: (1) cytosine, (2) orotidine, (3) uracil, (4) tyro sine, (5) cytidine, (6) hypoxanthine, (7) uridine, (8) 5-aminoamidazole carboxamide riboside, (9) 7-methylinosine, (10) 7-methyl xanthosine, (11) 7-methyl guanosine, (12) ^-nicotinamide adenine dinucleotide (NAD), (13) inosine, (14) guanosine, (15) 2'-deoxyinosine, (16) deoxythymidine, (17) 1-methylinosine, (18) Λ/,-methylguanosine, (19) W2-methylguanosine, (20) kynurenic acid, (21) adenosine, (22) theobromine, (23) /V2-dimethylguanosine, (24) theophylline, (25) dyphylline, (26) 6-methyladenosine, (27) indoles-propi onic acid, (28) caffeine. (Adapted with permission from Reference 6.)

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a millimeter (9). There are three types of microcolumns: open-tubular, par tially packed, and tightly packed. To use microcolumns, all parameters and instrumentation must be minia turized. Among the advantages of mi crocolumn HPLC is the ability to work with very small sample volumes (nanoliters) and reduced flow rates and sol vent consumption. In addition, be cause of the small volumes of effluent from microcolumns, miniaturized elec trochemical or laser-based detectors or mass spectrometers can be interfaced with HPLC more readily. Thus, using HPLC/MS, quantitative data can be obtained simultaneously along with peak identification and structure de termination. It is also possible to use multidisciplinary techniques such as HPLC/GC/MS if very small effluent volumes are involved. Because cost benefits are obtained when small volumes of mobile phase are used, exotic and usually expensive solvents can be used to facilitate a diffi cult separation. However, researchers are still trying to solve some problems in capillary column HPLC: the lack of flow rate reproducibility, need for im proved gradient elution, and accurate introduction of minute sample vol umes. In addition, miniaturized HPLC instrumentation t h a t is compatible with capillary columns is still not com mercially available. To use small-bore columns that have internal diameters of 1.0-2.0 mm, the instruments on the market have to be modified to retain resolution (10). The major modifications required are de creased flow rate and detector flow cell volume. The dead volume of the system must be reduced by decreasing the in ternal diameter of all tubing used. In addition, if gradient elution is desired, a micro static solvent mixing chamber is preferable to a conventional mixing chamber. Among the advantages of small-bore columns are a significant re duction in the volume of both the sam ple and the eluent required and an in crease in the mass sensitivity without the loss of resolution (11). In fast HPLC, the internal diameter of the columns is the same as those used in conventional HPLC (~ 4.6 mm), but shorter columns are filled with particles of small diameter (~ 3 μτα) (12). Dong and Gant (13) reported that the use of 30 X 3 mm columns filled with 3-Mm packings resulted in decreased analysis time, solvent use, and sample requirements. Subsequent reports on short columns (14,15) indi cated that a 40-fold increase in sensi tivity could be obtained, along with higher reproducibility, shorter analysis time, and decreased solvent consump-

REPORT tion. An example of a chromatogram of nucleosides and bases obtained with a 3.3-cm column is shown in Figure 2. The cost per analysis is significantly reduced, which is important in industry, quality control, or research applications when a large number of samples are to be analyzed. For the 30-mm columns, some instrument modification is required, but conventional instruments can be used with columns that are 50-100 mm long (16). For preparative separations, larger equipment, columns, and packing materials are used. The goals of preparative HPLC are different from those of analytical and trace analyses (17, 18). In preparative chromatography, the aim is to isolate or purify compounds; in analytical work, the goal is to obtain information about the sample. Therefore, the important parameters in analytical HPLC are resolution, sensitivity, and fast analysis time; for preparative HPLC, the focus is on the degree of solute purity that can be achieved and the amount of compound that can be produced per unit of time, known as throughput. HPLC is used not only to purify products of synthetic and naturally occurring reactions, but also to obtain intermediates of reactions, purify starting materials (19), and obtain enough of a purified compound for future testing or for structural determination (20). A preparative chromatogram of the 5'-monophosphate of AZT (3'-azido-3'deoxythymide), used as a starting material in the synthesis of analogues of AZT, is shown in Figure 3 (19). HPLC has also been scaled up for process work in industry (21). Because many of the processes are proprietary, fewer articles have been published on processscale separations than on preparative or analytical separations. HPLC has expanded to include ion chromatography, affinity, immunoaffinity, and chiral chromatography in addition to the more common modes of adsorption, ion-exchange, normalphase, reversed-phase, and size-exclusion chromatography. We have developed an abundance of packing materials—some that are highly specialized for specific applications and some that are all-purpose. Supports, besides the commonly used silica, include other oxides (22), carbon (23), polymeric resins (24), hydroxyapatite beads (25), and agarose (26). Packings come in a wide range of particle and pore sizes. There are specialty packings for affinity (27, 28) and immunoaffinity modes (29), chiral separations (30), macromolecules, and especially for biologically active macromolecules (31). Mixed-mode (32) and mixed-bed packings (33) have 998 A

been investigated as have been coupled columns (34) for compounds with multiple functional groups. Columns come in various lengths and internal diameters to solve any problem at hand. In addition to the commonly used stainless steel columns, glass-lined stainless steel and glass columns are now available as are plastic cartridges. The problem now is deciding which of the pleth-

ora of columns is the right one to buy, given that column prices are high and many academic laboratories have limited funds for equipment. The 1980s was a very exciting period in the area of applications. Few separation problems could not be solved with either HPLC alone or in combination with other techniques. The progress in the development of new packings for

Figure 2. Gradient separation of 11 nucleosides and bases on a 3.3-cm column packed with a Ci 8 packing. The linear gradient is 0.3-35% methanol in 0.02 M KH2P04, pH 5.6 in 4 min. Flow rate is 2 mL/min. The standard solution contains 60 pmol of each compound. The compounds are cytosine (Cyt), uracil (Ura), hypoxanthine (Hyp), xanthine (Xan), uridine (Urd), thymine (Thy), inosine (Ino), guanosine (Guo), adenine (Ade), thymidine (Thd), and adenosine (Ado).

Figure 3. Separation of 5'-monophosphate of 3'-azido-3'-deoxythymide and contaminants by preparative reversed-phase LC. Column isC 1 8 on 10-15 Mm silica, 20 X 7.3 cm. Mobile phase is 85:15 0.01 M ammonium acetate: methanol, pH 5.0. Flow rate is 150 mL/min. (Adapted with permission from Reference 19.)


REPORT biopolymer separations was initiated by Régnier and his group (35), who introduced rigid macroporous hydrophobic packings covalently bonded with a hydrophilic organic layer. These packings can separate high molecular weight multifunctional compounds, such as proteins, with high efficiency and recovery and without denaturing the proteins. In addition, with the appropriate mobile-phase conditions, these columns can be used in various modes such as size-exclusion, ion-exchange, hydrophobic interaction, metal chelate interaction, or affinity chromatography. For columns in which the mode of separation can be altered by changing the mobile phase to obtain the desired selectivity, Horvath and El Rassi (36) coined the term "polytyptic" or "polytyptic interaction" chromatography. This type of chromatographic behavior is also referred to as mixedmode chromatography. Although many columns can be used in the mixed mode, some packings are synthesized for specific purposes. Affinity chromatographic packings were developed to obtain high selectivity, especially for biological molecules. Since 1978, when Ohlson et al. (37) coined the term "high-performance affinity chromatography" (HPAC) and reported on the preparation of packings that could be used with HPLC instrumentation, many types of HPAC columns have been developed (38). Among them are

general-purpose affinity columns for separating groups of compounds with a specific functional group, such as borate columns for cis diols (39), and metal affinity and ligand columns (40) for specific biologically active moieties (ranging in size from nucleosides to nucleic acids and in biological function from enzymes to sugars). An exciting development is immunoaffinity columns in which the selectivity and specificity of immunological reactions are used. Figure 4 shows an immunoaffinity chromatogram of complement component C3 from human serum (41). In these columns, an immobilized antibody or antigen is used as the bioligand. Because an immunological reaction is the basis of separation, this method is very selective. Another fast-growing area is the development of columns specifically for the separation of chiral compounds. Shown in Figure 5 is the chiral separation of disopyramide in plasma (42). Since 1981, when Pirkle et al. (43) introduced chiral stationary phases, the number that are available has grown considerably. These packings can be used not only in analytical columns, but also in preparative work. There are several types of chiral stationary phases, such as those that use attractive interaction (44), inclusion (45), metal ligands (46), and protein complexes (47). The introduction of cyclodextrin bonded stationary phases has

Figure 4. Immunoaffinity chromatogram of human serum. Sharp peak is the complement component C3; large peak is unreacted serum components. Column is streptavidin (immunoaffinity), 100 X 4.6 mm. Mobile phase is 0.1 M phosphate buffer, pH 7.4. Flow rate is 1 mL/min; temperature, 4°C; sample size, 100 μL. (Adapted with permission from Reference 41.)

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also met with success in the separation of enantiomers and other isomers (48). The separation of optical isomers is very important in biochemistry and in the pharmaceutical industry, where one isomer of a drug may be therapeutically active and the other may be inactive, inhibitory, or toxic. Although progress has been made in the development and improvement of detectors, the lack of a reliable, sensitive universal detector is still a limiting factor in the growth of HPLC. However, recently a laser-based light-scattering mass detector (LSD) appeared on the market (49) that has the potential to overcome some of the problems encountered with the refractive index (RI) detector. For example, the LSD can be used with gradients, is free from the effects of ambient temperature, and has improved detection limits compared with the RI detector. Although HPLC/MS instrumentation is available, it has not yet gained the acceptance of GC/MS because of the problems of interfacing the liquid chromatograph with a mass spectrometer. Excellent progress has been made in the area of specific detectors. Laserinduced fluorescence (LIF) has greatly increased the sensitivity of previous fluorescence detectors (50), and indirect fluorescence detection shows promise for nonfluorescent materials (51). Radioactivity detectors are now available commercially (52) and are important in studying drug metabolism and metabolic pathways of normal subjects as well as those with various diseases. Electrochemical detectors are more reliable than in the past, and good quantitative data can be obtained for electroactive compounds (53). Optimization of separation conditions has become computerized. Programs are available (either built into the instrument or as stand-alone programs) that take the trial and error out of optimization. Programs such as "Drylab" (LC Resources) or the Perkin-Elmer Nelson systems are available for isocratic and gradient elution (54). Two areas that are finally being acknowledged as part of the chromatographic process are sample preparation and peak identification. If sample preparation is faulty, the results of the most sensitive and accurate chromatographic analysis can be invalid. In addition, in real-world samples, peaks must be identified unequivocally, not simply by retention times. Matrix effects and interferences must be considered and dealt with appropriately. The most exciting advances in the past decade resulted from the interaction of HPLC with biotechnology (55).

REPORT Not only must products be purified, but proof of their purity must be demonstrated and validated. In addition, methods are needed to follow the metabolism of drugs developed by biotechnology and to assess their toxicity and efficacy. The major separation methods used today are membrane separations and chromatography. The most important chromatographic technique is HPLC because the majority of the compounds of interest are biopolymers, such as proteins and peptides that are nonvolatile and thermally labile. Dramatic progress has been made in the separation of biopolymers since the late 1970s, when the peaks in chromatograms of proteins were broad and resolution of similar structures was poor. Reproducibility was difficult to achieve, separation times were long, and the results were rarely quantitative. Now, not only can proteins be readily isolated, but so can nucleic acids, synthetic long-chain oligonucleotides, and polypeptides. Usually not just one but several instruments in molecular biology laboratories are considered to be indispensable in daily operations. HPLC in the ion-exchange, reversed-phase, gel filtration, and affinity modes is used to separate and purify nucleic acids, proteins, and biologically active peptides from biological matrices. In the synthesis of DNA and RNA, HPLC is used to analyze synthetic mixtures for the presence of unreacted substrates (the nucleotides) and for the oligonucleotides that are formed in the first steps of the reaction, and to determine the purity of the final product. HPLC is used routinely in the same way in the synthesis of proteins. HPLC analyses for amino acids, peptides, polypeptides, and proteins are becoming fast and efficient (56). An example is Horvath and El Rassi's separation of proteins on a mixed binary ion-exchange column, shown in Figure 6 (36). Even the separations of the enantiomers of amino acids are readily accomplished, and most of the analyses are automated and computerized. An important application of HPLC is the structure determination of proteins and nucleic acids. Here, too, the instrumentation is automated and the immense amount of data generated is handled by computers. Enzymes are separated and purified using RPLC, and their activities can be rapidly and precisely determined (57). Moreover, it is possible to determine the activities of two or more enzymes concomitantly (58). Biochemical separations are not limited to compounds of interest in biotechnology; they include all biologically active moieties such as steroids, 1002 A

catecholamines, carbohydrates, phospholipids, and prostaglandins. Although HPLC is widely considered to be a technique mainly for biotechnological, biomedical, and biochemical research as well as for the pharmaceutical industry, these fields currently comprise only about 50% of HPLC users. This technique is also used in the energy, food, cosmetic, and environmental fields. In fact, any industry that requires good separations for synthetic substrates, intermediates, or products, as well as good quality control over the purity of goods produced, usually uses some form of a chromatographic separation, and HPLC is the most universally used technique. Compounds analyzed by HPLC (especially by size exclusion) include commercial

resins, polymers, plasticizers, petroleum residues, and lubrication oils (59). HPLC is used in environmental work to monitor air and water pollutants and is a valuable tool in government laboratories. The Department of Agriculture uses HPLC routinely in trace analyses of pesticides, herbicides, and fungicides in our food, soil, and water. HPLC is also used in inorganic chemistry. It is now possible to investigate inorganic ions present in solutions (e.g., sulfate, nitrite, and chloride) as well as to determine the speciation and amount of ions present in ecological samples (60). Future trends Unfortunately, I do not have a crystal ball to see the developments that will

Figure 5. Isolation of disopyramide enantiomers from plasma. Column is aglycoprotein, 180 mg/g silica, 100 X 3.0 mm. Mobile phase is 1.95 mM N,N-dimethyloctylamine and 4.3% 2-propanol in 0.02 M phosphate buffer, pH 6.2. (a) Blank plasma, (b) blank plasma spiked with racemic disopyramide, and (c) plasma sample obtained after administration of racemic disopyramide. (1) R-disopyramide, (2) S-disopyramide. (Adapted with permission from Reference 42.)


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REPORT take place in the future. However, based on past experience and current needs, it is possible to predict trends in separation techniques that will be like ly in the next decade. The most obvious need for HPLC instruments will be in biotechnology and the life sciences. Currently there are more than 800 biotechnology com panies that must demonstrate that their products are nontoxic; therefore, new drugs must be tested and retested to meet stringent government regula tions. New challenges in environmental monitoring and forensic medicine will require sophisticated and reliable sep aration techniques. The results ob tained must be validated and be able to stand up in court. The tremendous human genome project embarked upon by the govern ment will require the most sophisticat ed separation instrumentation possi ble. HPLC will play an important role not only on the analytical scale, but also on the preparative scale. Both fast and microbore column HPLC will be used for various phases of the work, especially in mapping the human ge nome. HPLC will also be a vital tool in AIDS research and diagnosis and in de termining biochemical markers for de tecting and monitoring the course of many inherited genetic defects and other diseases such as cancer.

In biotechnology, high-speed separa tions will be used for process monitor ing of fermentation and growth media. Microbore columns with sensitivities in the attomole range (10~18) will be used routinely, and single-molecule ma nipulation may be possible because extreme sensitivity (10~21) is within sight using either capillary HPLC or capillary electrophoresis (CE). Another potential use is the separa tion of cells and the rapid identifica tion of viruses and bacteria. HPLC will also continue to be the mainstay of the pharmaceutical industry, not only in analytical laboratories, but also for quality control of starting materials and final products; for on-line process monitoring; and, on the preparative and process scale, for the isolation and purification of drug products. The in creased sensitivity of capillary HPLC and the very rapid analyses that can be achieved with short columns will make this technique indispensable in bio chemistry, molecular biology, and the medical sciences. The great potential of HPLC-coupled techniques will be valuable in toxicology, pharmacology, and forensic medicine. Because greater emphasis will be placed on the stringent require ments of regulatory agencies, HPLC/ MS will become a necessity in many laboratories and will be used routinely

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choice for obtaining structural infor mation or for positive identification of an analyte, as in court cases or drug testing in athletes. The good old workhorse of the field, the UV-vis detector, will continue to hold its place of honor for routine anal yses of compounds containing a UV chromophore. Its ruggedness, reliabil ity, ease of operation, and reproducibil ity make this detector desirable for lab oratories where the ultimate in sensi tivity is not necessary. In our laboratory, the UV-vis detectors pur chased in the early 1970s have been used by a large number of graduate stu dents and are still giving good results. Multiple detectors will also play a major role in HPLC. Two or more de tectors will be used either in series or via stream splitting. It can be helpful to have a universal detector determine the number of solutes present in the effluent and one or more very selective detectors identify the compounds. The primary constraint is that the solvent system must be compatible with all de tection systems. Computer optimization is here to stay even though some people are still reluctant to use these programs. As the younger chemists become more com puter literate, resistance will diminish and a tremendous amount of time will be saved by computer optimization. The use of robots will also increase exponentially in the next decade. Ro bots will be used for all routine tasks and will be especially necessary in sam ple preparation and handling of poten tially dangerous samples, such as phys iological fluids from AIDS patients, other viral and bacterial materials, contaminated environmental samples, and radioisotopes. Robotics will im prove laboratory safety and will help to fill the gap due to the diminishing pool of science graduates available for jobs in industry and académie. In addition, the use of robots can increase the number of samples reliably and efficiently processed per day, which is clearly an advantage in high-volume analytical laboratories (61). Control programs that use artificial intelligence will be the next step in robotics. In the future, multitasking instruments will be commonplace. For example, after preliminary sample preparation by specialized robots, several instruments will be interfaced so that all the steps from rigorous sample preparation to separation, peak identification, structure determination, and quantitation will be done automatically. Not only will the instrument have several columns and/or detectors, but it will be interfaced with CE, GC, or MS. This combination of instruments


and techniques (62) is often referred to as multidimensional chromatography. In protein analysis, peptide mapping may be included in the multitasking program as will the determination of the base sequence in nucleic acids in genome investigations. HPLC, alone or in combination with one or more techniques, will continue to be the separations technique that is the backbone of the analytical laboratory, especially in the pharmaceutical and biotechnology industries, because of the ease of sample preparation, automation, speed, and sensitivity. In the future, fast HPLC may be used instead of flow injection analysis to monitor real-time fermentation processes that are used in biotechnology. Although the dominant applications will be in the life sciences, HPLC will face new challenges in environmental testing and in forensic medicine. Better separation techniques are also needed in the chemical industry for the manufacture of adhesives, sealants, catalysts for petroleum processing, and materials used in the manufacture of microcircuits. Good preparative and process HPLC techniques are required to separate, isolate, and purify most consumer products such as food, drugs, vitamins, and cosmetics. Several separation techniques will be competitive with HPLC in the next decade: CE, SFC, countercurrent chromatography (CCC), and field-flow fractionation (FFF). However, I do not believe that these techniques will replace HPLC. Because of its ruggedness, versatility, and separating power—especially for water-soluble, nonvolatile, thermally labile compounds—HPLC will maintain its solid position. Although CE is a strong competitor in the separation of large biopolymers and biologically active molecules, more research is needed in injection systems, detectors, and peak identification before it can achieve its full potential. HPLC will be used for routine analysis for molecules with molecular weights in the range of 200 to several thousand. GC will continue to be the method of choice for thermally stable, volatile molecules that have a molecular weight below 200, whereas CE will be the method of choice for biopolymers. SFC will be the preferred preparative technique if more mobile phases are found in which analytes are soluble. A major advantage of this technique is the ease of removing the solvent from the product solution. CCC is also useful for preparative work and has been used recently in the purification of peptides and proteins (63). However, many researchers prefer HPLC because they are able to scale up their analytical sep-

a r a t i o n s easily. I n s t r u m e n t a t i o n for F F F currently is available, a n d in t h e n e x t decade will be used in laboratories for t h e s e p a r a t i o n of m a c r o m o l e c u l a r or particulate species (64). Although other separation tech niques are available, H P L C will n o t be replaced as t h e m a i n s t a y of t h e analyti cal laboratory, w h e r e all t y p e s of m e d i um-sized molecules m u s t be s e p a r a t e d . Instead, in t h e 1990s GC a n d H P L C will be joined in t h e l a b o r a t o r y b y C E and, for specific n e e d s or p r o b l e m s , by SFC, CCC, a n d F F F .

This REPORT is based on Phyllis Brown's 1989 Dal Nogare Award Address at the Pittsburgh Con ference, Atlanta, GA. References

(1) Franconi, L. C ; Hawk, G. L.; Sandmann, B. S.; Haney, W. G. Anal. Chem. 1976, 48, 372. (2) a. Kirkland, J. J. J. Chromatogr. Sci. 1971, 9, 206. b. Kirkland, J. J. J. Chroma togr. 1972,10, 593. (3) Majors, R. E. J. Chromatogr. Sci. 1977, 15, 334. (4) Howard, G. Α.; Martin, A.J.P. Biochem. J. 1951,49, 215. (5) Halasz, I.; Sebastian, I. Anglu. Chem. 1969,8, 453. (6) Hartwick, R. Α.; Assenza, S. P.; Brown, P. R. J. Chromatogr. 1979,186, 647.

(7) Brown, P. R. High Pressure Liquid Chromatography: Biochemical and Bio medical Applications; Academic Press: New York, 1973. (8) Snyder, L. R.; Kirkland, J. J. Introduc tion to Modern Liquid Chromatography, 2nd éd.; Wiley Interscience: New York, 1979. (9) Novotny, M. Anal. Chem. 1988, 60, 500 A. (10) Simpson, R. C ; Brown, P. R. J. Chromatogr. 1987, 385, 41. (11) Simpson, R. C ; Brown, P. R. J. Chromatogr. 1987,400, 297. (12) DiCesare, J. L.; Dong, M. W.; Vandemark, F. L. Am. Lab. 1981, No. 13, 52. (13) Dong, M. W.; Gant, J. R. LC Mag. 1984, 2, 294. (14) Dwyer, M. E.; Brown, P. R. J. Liq. Chromatogr. 1987,10(8 & 9), 1769. (15) Simpson, R. C. In High Performance Liquid Chromatography; Brown, P. R.; Hartwick, R. Α., Eds.; Wiley Interscience: New York, 1989; Chapter 9. (16) Simpson, R. C ; Brown, P. R.; Schwartz, M. K. J. Chromatogr. Sci. 1985, 23, 89. (17) Guiochon, G.; Colin, H. Chromatogra phy Forum Sept.-Oct. 1986, 21. (18) Colin, H. In High Performance Liquid Chromatography; Brown, P. R.; Hartwick, R. Α., Eds.; Wiley Interscience: New York, 1989; Chapter 11. (19) Turcotte, J. C ; Pivarni, P. E.; Shirali, S. S.; Singh, H. K.; Sehgal, R. J.; MacBride, D.; Jang, N-L; Brown, P. R. J. Chromatogr. 1990,449, 55. (20) Beebe, J. M.; Brown, P. R.; Turcotte, J. G. J. Chromatogr. 1988, 459, 369. (21) Shea, W. M. In High Performance Liq

uid Chromatography; Brown, P. R.; Hartwick, R. Α., Eds.; Wiley Interscience: New York, 1989; Chapter 12. (22) Unger, Κ. Κ.; Trûdinger, U. In High Performance Liquid Chromatography; Brown, P. R.; Hartwick, R. Α., Eds.; Wiley Interscience: New York, 1989; Chapter 3. (23) Knox, J. H.; Kauer, B. In High Perfor mance Liquid Chromatography; Brown, P. R.; Hartwick, R. Α., Eds.; Wiley Interscience: New York, 1989; Chapter 4. (24) Pietrzyk, D. J. In High Performance Liquid Chromatography; Brown, P. R.; Hartwick, R. Α., Eds.; Wiley Interscience: New York, 1989; Chapter 5. (25) Sutton, P. W.; Kemp, J. D. Biochem. 1976,15, 3153. (26) Porath, J.; Olin, B.; Grandstrand, B. Arch. Biochem. Biophys. 1983,225, 543. (27) Jaulmes, Α.; Vidal-Madjar, C. In Ad vances in Chromatography; Giddings, J. C ; Grushka, E.; Brown, P. R., Eds.; Marcel Dekker: New York, 1989; Vol. 28, Chapter 1. (28) Carr, P. W.; Bergold, A. F.; Hangge, D. Α.; Muller, A. J. Chromatography Fo rum Sept.-Oct. 1986, 31. (29) Phillips, T. M. LC/GC 1985,3, 962. (30) Wainer, I. W. Chromatography Forum Nov.-Dec. 1986,55. (31) Haff, L. A. Chromatography M a r c h 1987, 25. (32) Nahum, Α.; Horvath, C. J. Chroma togr. 1981,203, 53. (33) Crowther, J. B.; Hartwick, R. A. Chromatographia 1983,16, 349. (34) Halfpenney, Α.; Brown, P. R. Chromatographia 1986,21(6), 317. (35) Chang, S. H ; Gooding, K. M.; Régnier, F. E. J. Chromatogr. 1976,125, 103.

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REPORT (36) Horvath, C ; El Rassi, Z. Biochromatography Sept.-Oct. 1986, 49. (37) Ohlson, S.; Hansson, L.; Larsson, P. 0.; Mosbach, K. FEBS Lett. 1978, 93, 5. (38) Chaiken, I. M.; Fassina, G.; Caliceli, P. In High Performance Liquid Chromatog raphy; Brown, P. R.; Hartwick, R. Α., Eds.; Wiley Interscience: New York, 1989; Chapter 7. (39) Gehrke, C. W.; Kuo, K. C ; Zumwalt, R. W. J. Chromatogr. 1980,188, 129. (40) Lindner, W. F.; Hirschbock, J. J. Liq. Chromatogr. 1986, 9, 551. (41) Phillips, T. M. In Advances in Chro matography; Giddings, J. C ; Grushka, E.; Brown, P. R , Eds.; Marcel Dekker: New York, 1989; Vol. 29, Chapter 3. (42) Hermansson, J.; Schill, G. In High Per formance Liquid Chromatography; Brown, P. R.; Hartwick, R. Α., Eds.; Wiley Interscience: New York, 1989; Chapter 8. (43) Pirkle, W. H.; Finn, J. M.; Schreiner, J. L.; Hamper, B. C. J. Am. Chem. Soc. 1981,103,1964. (44) Oi, N.; Nagare, M ; Inda, Y.; Doi, T. J. Chromatogr. 1983, 265, 111. (45) Sybilska, D.; Zukowski, J.; Bojarski, J. J. Liq. Chromatogr. 1986, 9, 591. (46) Gil Av, E.; Tishbee, Α.; Hare, P. E. J. Am. Chem. Soc. 1980,102, 5115. (47) Blaschke, G. J. Liq. Chromatogr. 1986, 9,89. (48) Armstrong, D. W.; Li, W. Chromatog raphy March 1987, 43. (49) Stolyhwo, Α.; Colin, H ; Guiochon, G. Anal. Chem. 1985, 57, 1342. (50) Evans, C. E.; McGuffin, V. L. J. Liq. Chromatogr. 1988,11(9 & 10), 1907.

(51) Ruhr, G.; Yeung, E. S. Anal. Chem. 1988, 60, 2642. (52) Kessler, M. J. Am. Lab. June 1988, 86. (53) Horval, G.; Pungor, E. Chromatogra phy 1987, 2(2), 15. (54) Snyder, L. R.; Quarry, M. A. J. Liq. Chromatogr. 1987,10(8 & 9), 1789. (55) Borman, S. Anal. Chem. 1984, 56, 1548 A. (56) Engelhardt, H., Ed.; Practise of High Performance Liquid Chromatography; Springer-Verlag: Berlin, 1986. (57) Hartwick, R. Α.; Jeffries, Α.; Brown, P. R. J. Chromatogr. Sci. 1978,16, 427. (58) Halfpenny, A. P.; Brown, P. R. J. Chromatogr. 1985, 346, 275. (59) Yau, W. W.; Kirkland, J. J.; Blythe, D. D. In High Performance Liquid Chro matography; Brown, P. R.; Hartwick, R. Α., Eds.; Wiley Interscience: New York, 1989; Chapter 6. (60) J. Chromatogr. Sci. 1989, 29(8) (spe cial issue on ion chromatography). (61) Isenhour, T. L.; Eckert, S. E.; Mar shall, J. C. Anal. Chem. 1989, 61, 805 A. (62) Sagliano, Jr., N.; Raglione, T. V.; Hartwick, R. A. In High Performance Liquid Chromatography; Brown, P. R.; Hartwick, R. Α., Eds.; Wiley Interscience: New York, 1989; Chapter 16. (63) Ito, Y. In Advances in Chromatogra phy; Giddings, J. C ; Grushka, E.; Brown, P. R., Eds.; Marcel Dekker: New York, 1984; Vol. 24, Chapter 6. (64) Kesner, L. F.; Giddings, J. C. In High Performance Liquid Chromatography; Brown, P. R.; Hartwick, R. Α., Eds.; Wiley 'Interscience: New York, 1989; Chapter 15.

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Phyllis R. Brown is a professor of chemistry at the University of Rhode Island. She received her B.S. degree from George Washington University and her Ph.D. from Brown University. In 1979 and 1983 she was a visiting professor at Hebrew University in Is rael and was also a Fulbright Scholar there in 1987. Her current research in terests involve the optimization of chromatographic separations of nu cleic acid constituents and their ana logs on the analytical scale and the purification of new anti-AIDS drugs.

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