uch of modern chemical technology is based on our ability to separate and analyze complex samples, especially those of interest to the biological and medical sciences. Classical column chromatography, invented by Tswett in the early 1900s, experienced a rapid increase in use after its reintroduction in 1930 by Kuhn and Lederer. The time line above charts some important variations on classical column chromatography. The chromatographic innovations shown were introduced at roughly decade intervals, most notably HPLC in the mid-1960s. At the time, each technique offered major improvements in convenience, speed, resolving power,
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detection, quantitation, and applicability to new sample types. GC, TLC, and HPLC are used today to a much greater extent than are either older or more recent procedures, such as supercritical fluid chromatography (SFC), CE, and capillary electrochromatography (CEC). MS is currently an alternative to chromatographic procedures for analyzing some multicomponent samples, and we are likely to see an increase in the use of LC/MS. The early days of HPLC have been reviewed (1–4) and titles of related papers from 1966 to 1982 have been organized according to subtopic (5). To trace the expanding capabilities of
Lloyd R. Snyder • LC Resources, Inc.
HPLC from its beginning to the present, readers can consult three popular reference books (6–8). Also informative are the biennial analytical reviews published by Analytical Chemistry in even-numbered years. GC gained popularity because of its superior resolution, better automation, and more accurate and convenient quantitation. However, GC required volatile samples, which ruled out ~75% of all samples. Early versions of HPLC were introduced in the period 1959–64 for the analysis of certain sample types: amino acids (Spackman, Stein, and Moore [1]) and synthetic polymers (Moore [9]). During the mid-1960s, Horvath, Huber, and Kirk-
land developed means for the similar separation of any sample (2) through improvements in equipment and columns. These and other efforts led to the development of what we now call HPLC (Figure 1). During this period, it should also be noted that the development of GPC by Waters Associates in the United States and Tosoh and Showa Denko in Japan created equipment and columns that would require little modification for HPLC. With the commercial introduction of general-purpose equipment a few years later, HPLC began an explosive growth that, by the 1990s, would raise it to first place in total analytical instrument sales—and in scientific impor-
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Autosampler
Column
Injector
Pump
°C
Detector
Data system Fraction collector
Solvent reservoir(s)
System controller
FIGURE 1. Equipment schematic of an HPLC. Heavy lines and bold text refer to components that were present in the first HPLC systems.
tance as well. HPLC’s present popularity is attributable to its convenient separation and analysis of almost any sample that can be dissolved and that it can solve various related problems. Applications are generally characterized by exceptional resolving power, speed, and detection limits of nanomoles per liter or less. For example, HPLC is a key technology in pharmaceutical R&D for purifying synthetic or natural products; characterizing metabolites; assaying active ingredients, impurities, and degradation products generated by accelerated aging; dissolution assays; pharmacodynamic and pharmacokinetic studies; and verifying system cleanliness during the manufacturing process. Recent competitors such as SFC, CE, and CEC have not yet made serious inroads into the HPLC market, despite their promise of better resolving power and more sensitive detection for some samples. Their future impact is as yet uncertain.
Faster separations In principle, it would have been possible to carry out some chromatographic separations before 1965 that were equivalent in resolution to those later achieved by HPLC. For many samples, however, it was not possible to achieve these separations within a reasonable time. Figure 2 illustrates the theorybased separation of a model mixture by representative columns and equipment available between 1955 and 1985. For each of the separations in Figure 2, the same small-molecule sample (
