Waters Symposium: High-Performance Liquid Chromatography
waters symposium: high-performance liquid chromatography
Modern Practice of Liquid Chromatography Before and after 1971 Lloyd R. Snyder LC Resources Inc., 26 Silverwood Ct., Orinda, CA 94563 This review deals with the birth of high-performance liquid chromatography (HPLC) and its subsequent growth into the mature technique of today. Let us begin by defining (or limiting) our subject with respect to classical liquid chromatography. First, HPLC is primarily a procedure for the rapid and precise analysis of mixtures of organic compounds. If we broaden this definition to include preparative-scale applications, we complicate the distinction between classical liquid chromatography (LC) and HPLC. Second, HPLC is a general technique that can be applied to any kind of sample. This feature allowed HPLC to challenge (and eventually surpass) gas chromatography (GC) as the preferred method for quantitative organic analysis. Third, HPLC is an automated procedure that can eliminate fraction collection or human intervention during the separation. Finally, HPLC implies that separation performance as measured by sample resolution, column efficiency, and/or run time has been “optimized” by the proper selection of experimental conditions (usually small particles and high-pressure operation). “Optimized” performance also implies a comprehensive and detailed understanding of the factors that affect separation. With this description of HPLC, it becomes possible to recognize important precursors to the method. It is also possible to state when and how HPLC became a reality, and what important developments occurred thereafter. Table 1 summarizes some of these events in the form of a time line that begins in 1941 with a publication by Martin and Synge (1). The title of the present review refers to an important event during the develop-
Table 1. Development of HPLC: A Time Line 1941
Martin and Synge (1 ): "the smallest HETP should be obtainable by using very small particles and high pressure difference across the length of the column"
1958
Amino acid analyzer
1964
Invention of GPC
1966–8 First reports of HPLC 1969
5th Intern. Symp. Adv. in Chrom., Las Vegas
1971
Modern Practice of LC (the book) First ACS short course (Modern LC)
1973
HPLC '73 (Interlaken)
1975
Biochemical separations (proteins)
1979
Chiral separations
1980
Systematic method development (computers)
ment of HPLC, namely the appearance of the first book that dealt with this subject: Modern Practice of Liquid Chromatography (1971), edited by Jack Kirkland (2). By the time this book appeared, commercial equipment and materials for HPLC had become available and the technique could now be applied to a wide range of “real” analytical problems (but by no means all samples). However, the practice of HPLC was still at an early stage. Numerous difficulties remained to be resolved before the prom-
The Annual James L. Waters Symposia at Pittcon The objectives of the annual James L. Waters Symposia at Pittcon are different from those of other symposia at either Pittcon or other conferences. Waters, founder of the well-known Waters Associates, Inc., and currently president of Waters Business Systems, Inc., arranged with the Society for Analytical Chemists of Pittsburgh (SACP) in 1989 to offer annual symposia at Pittcon to explore the origin, development, implementation, and commercialization of scientific instrumentation of established and major significance. The main goals were and still are to ensure that the early history of this cooperative process be preserved, to stress the importance of contributions of workers with diverse backgrounds, objectives, and perspectives, and to recognize some of the pioneers and leaders in the field. Important benefits of these symposia are creation of awareness of the way in which important new in-
struments and, through them, new fields, are created, and promotion of interchange among inventor, development engineer, entrepreneur, and marketing organization. The topics of the first six Waters Symposia, beginning in 1990, were gas chromatography, atomic absorption spectroscopy, infrared spectroscopy, nuclear magnetic resonance spectroscopy, mass spectrometry, and high-performance liquid chromatography. Publication of the papers presented at the Waters Symposia is a high priority of the SACP. So far, the papers of the first symposium have been published in LC.GC Magazine and those of the next four symposia in Analytical Chemistry. This is the sixth Symposium. The seventh Symposium, on the topic of ion selective electrodes, will be published in the February 1997 issue of the Journal. Administration of the Symposium, including selection of the topic and speakers, is handled by the SACP. J. F. Coetzee University of Pittsburgh Waters Symposium Coordinator
Vol. 74 No. 1 January 1997 • Journal of Chemical Education
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Waters Symposium: High-Performance Liquid Chromatography
ise of this technique could be widely realized, and systematic procedures for HPLC method development had yet to evolve. Equally important, only a few practitioners were adequately trained to be able to apply HPLC effectively. The scientific literature and short courses dealing with HPLC had so far reached only a limited audience. Prior to the appearance in 1966–1968 of the first published reports to describe HPLC (3–8), several developments required attention: equipment, columns, and an understanding of how separation could be controlled or a final HPLC method developed. This review will deal mainly with the role of theory and (especially) experience in the development of practical HPLC procedures. The discussion is divided into two parts, corresponding to the periods before and after the appearance of the book Modern Practice of Liquid Chromatography. These two intervals respectively encompass the emergence of a technique that could now be applied to practical problems, and its extension for convenient, routine application to any sample. Before 1971 Most of the people who contributed to the early development of HPLC have told their stories in the book 75 Years of Chromatography—A Historical Dialogue (9). I have used this material as well as my own recollections of this period to prepare the following account. The reader is also referred to Ettre’s history of liquid chromatography prior to the age of HPLC (10).
Figure 1. HPLC separations from the author’s laboratory between 1967 and 1969. (1967) First separation of hydrocarbon mixture using 32-ft column packed with 75-µm alumina ( N = 2300); pentane mobile phase at 2 mL/min; (1968) separation of aromatic hydrocarbons with a 4-ft column packed with 20-µm silica ( N = 900); pentane mobile phase; (1969) separation of hydrogenated quinoline sample with a 4-ft column packed with 64-µm alumina. Reprinted with permission from refs 6, 9, 20.
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1941–1967 Before 1966 many scientists were carrying out work in classical liquid chromatography that would strongly influence the later development of HPLC. Especially noteworthy (see Table 1) was the prediction by Martin and Synge in 1941 that small particles and high pressures would be required (1). My own research in the field of chromatography began in 1954 and was soon directed toward the application and understanding of separations by adsorption chromatography (11). These early studies dealt mainly with sample retention as a function of sample composition and experimental conditions, but by 1966 I realized that column efficiency and automated liquid chromatographic separation also deserved further examination. This led to the independent development of HPLC in my laboratory (6). Figure 1 illustrates the progress we were able to make during a two-year period. In 1967 we carried out our first HPLC separations (Fig. 1a). Our early experiments focused on “higher” plate numbers (N ≈ 2000) without regard to run time. By 1968 we were able to achieve separations of reasonably complex samples in run times of less than 5 min, using columns packed with 20-µm silica particles (Fig. 1b). In 1969 we were applying the technique to samples of real interest (Fig. 1c), while accepting the usual compromise between resolution and run time. Other workers were approaching HPLC from the field of GC, which in many ways served as a blueprint for the development of HPLC. Many of the problems in GC that were being solved during the 1960s also had application to HPLC. Notable examples were the treatment of extra-column band broadening by Sternberg (12) and the theoretical analysis by Giddings (13), which showed that small particles and high pressures would be advantageous in HPLC (confirming the earlier qualitative prediction of Martin and Synge [1]). In my opinion, the first precursor of HPLC was the amino acid analyzer (AAA) described by Spackman, Stein, and Moore in 1958 (14). This instrument enabled the automatic separation and analysis of protein hydrolysates. Because of the importance of this application, other workers were stimulated to examine a number of important questions dealing with what would later become HPLC. The work of Hamilton during this period (15) was especially noteworthy for its aid in better understanding column efficiency and selectivity. A related development in the early 1960s was the development of gel permeation chromatography (GPC) by Moore (16). A short time later, commercial GPC equipment was made available by Waters Associates that with minor modification would also permit HPLC separation. The reader might well ask, why not refer to these earlier AAA and GPC separations as HPLC? The main distinction, in my opinion, was that each of these earlier procedures was designed for a narrow range of applications, whereas HPLC is in principle applicable to any sample. Although these early instruments were also restricted in terms of allowable operating pressure and required rather long run times, this does not seem to represent an essential difference compared to later applications of HPLC. 1968–1971 These years saw the development and commercialization of the first general-purpose HPLC systems, a rapid exchange of information among dozens of research groups working in this field, and important improvements in the technique (17). During the period 1966– 1968 when I was first investigating and applying HPLC
Journal of Chemical Education • Vol. 74 No. 1 January 1997
Waters Symposium: High-Performance Liquid Chromatography
at the Union Oil Co., I gradually became aware of similar efforts in other laboratories. Especially memorable was an Eastern Analytical Symposium meeting in 1968, where I presented some of our results and met Jack Kirkland for the first time. This led to a long, pleasant, and fruitful collaboration (see below). Soon after (January 1969), the Fifth International Symposium on Advances in Chromatography was held in Las Vegas. For the first time, a critical mass of HPLC practitioners was assembled in one location. The resulting interchange of information and enthusiasm made this a truly exciting event. Many of the papers from this meeting were published in the 1969 volume of what was then called the Journal of Gas Chromatography. The next few years saw several meetings devoted in part or entirely to HPLC, culminating in a 3-day HPLC course organized in April 1970 by the Du Pont Instruments Product Division and the Chromatography Forum of the Delaware Valley. Several of the speakers at this short course in turn became contributors to Modern Practice of Liquid Chromatography (2). That book therefore provides an accurate assessment of the state of the art at the beginning of 1971. I will next review some of this art as it relates to an understanding of separation and HPLC method development. Prior to 1971, numerous examples of HPLC separation had been published. For the most part, these chro-
(a)
Figure 2. Early separations of “real” samples by HPLC. (a) Methomyl insecticide bulk chemical separated by liquid–liquid chromatography with UV detection (18); (b) benomyl fungicide in bovine milk separated by anion exchange chromatography with UV detection (19); (c) commercial insecticide residue (Folidol ® ) in lettuce analyzed by liquid–liquid chromatography with polarographic detection (20); (d) ribonucleoside standards and yeast hydrolysate separated by anion exchange chromatography with UV detection (21) .
matograms originated either from HPLC companies or university laboratories and seldom represented “real” samples. Often a mixture of related compounds was used as sample to demonstrate separation, but the HPLC analysis of the actual sample (i.e., its composition) was of little practical interest. Sometimes the sample compounds were selected just because they could be separated easily and quickly by HPLC! The separation of one such sample, the three isomers of nitroaniline, was often offered as an example of “fast” HPLC separation (
