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- 1. 3High-Performance LiquidChromatographyThomas H. Stout*University of Cincinnati, Cincinnati, OhioJohn G. DorseyFlorida State University, Tallahassee, FloridaI. INTRODUCTIONChromatography is a general term applied to a wide variety of separation tech-niques based on the sample partitioning between a moving phase, which can bea gas, liquid, or supercritical uid, and a stationary phase, which may be eithera liquid or a solid. The discovery of chromatography is generally credited toTswett (1), who in 1906 described his work on using a chalk column to separatepigments in green leaves. The term chromatography was coined by Tswett todescribe the colored zones that moved down the column. The technique lan-guished for years, with only periodic spurts of development following innova-tions such as partition and paper chromatography in the 1940s, gas and thin-layerchromatography in the 1950s, and various gel or size-exclusion methods in theearly 1960s (2). Then in January 1969, the Journal of Gas Chromatography of-cially changed its name and became the Journal of Chromatographic Science.This change reected the renewed interest in the technique of liquid chromatogra-phy (LC) and ofcially signaled the beginning of the era of modern LC. Thisrenewed interest in the oldest of chromatographic techniques was brought aboutboth because of the successes and because of the failures of gas chromatography(GC). On the one hand, GC provided a rm theoretical background on which* Current afliation: Eurand America, Inc., Vandalia, Ohio.Copyright 2002 by Marcel Dekker. All Rights Reserved.
2. modern LC could build. However, this renewed interest in LC was being driven because of the inability of GC to handle thermally unstable or nonvolatile com- pounds. It has been estimated that fewer than 20% of organic compounds have sufcient volatility and thermal stability to traverse a GC column successfully. Admirers of LC also liked the selective interaction of its two chromatographic phases, and easy sample recovery because of its nondestructive detection methods and room-temperature operation.In the following years the technique was known by many acronyms. It was at various times designated high-pressure liquid chromatography, high-speed liquid chromatography, high-performance liquid chromatography, high-priced liquid chromatography by cynical research directors, and nally modern liquid chromatography. The modiers were all used to signify the difference in the technique brought about by small-particle-diameter stationary phases and the re- sulting high pressure needed to drive the mobile phase through the densely packed columns. These differences resulted in much faster separations with higher reso- lution than traditional column chromatography. Through the decade of the 1970s the technique grew with astounding speed, and for nine consecutive years from the middle 1970s to the early 1980s the technique led all other analytical instru- ments in growth rate in an annual survey conducted by Industrial Research and Development magazine.A discussion of the history of liquid chromatography is beyond the scope of this volume; an excellent treatment has been published by Ettre (3). However, some perspective on the beginnings of modern liquid chromatography is useful for an understanding of the technique as now practiced. The rst comprehensive publication on the technique was a book edited by Kirkland (4), which grew from a short course offered by the Chromatography Forum of the Delaware Valley. This then laid the foundation for the denitive book, now in its second edition, Introduction to Modern Liquid Chromatography (2).During the development of modern liquid chromatography, advances have been driven by both instrumentation and chemistry. The technique as now prac- ticed produces elegant separations that have been developed through chemical manipulation of the stationary and mobile phases, which are then effected and detected by modern instrumentation. This chapter is therefore divided into three broad sections: the rst, on the fundamentals of chromatography, provides the necessary foundation for the second, on instrumentation, and the third, on separa- tion modes. II. FUNDAMENTALS OF CHROMATOGRAPHY Chromatography is a separation method which employs two phases, one station- ary and one mobile. As a mixture of analytes being carried through the systemCopyright 2002 by Marcel Dekker. All Rights Reserved. 3. by the mobile phase passes over and through the stationary phase, the individualcomponents of the mixture equilibrate or distribute between the two phases. Xm Xs The corresponding thermodynamic distribution coefcient K is dened asthe concentration of component (X) in the stationary phase divided by its concen-tration in the mobile phase. [X] s K (1) [X] m The use of a typical equilibrium constant K in chromatographic theory indi-cates that the system can be assumed to operate at equilibrium. As the analyte(X) proceeds through the system, it partitions between the two phases and isretained in proportion to its afnity for the stationary phase. At any given time,a particular analyte molecule is either in the mobile phase, moving at its velocity,or in the stationary phase and not moving at all. The individual properties ofeach analyte control its thermodynamic distribution and retention, and result indifferential migration of the components in the mixturethe basis of the chro-matographic separation. The effectiveness of the separation, however, is a func-tion of both thermodynamics and kinetics.A. Thermodynamic ConsiderationsIn order to understand the thermodynamic considerations of the separation pro-cess, we need to look at the basic equations which describe the retention of asolute and relate the parameters of retention time, retention volume, and capacityfactor to the thermodynamic solute distribution coefcient. Figure 1 shows a model chromatogram for the separation of two com-pounds. The time required for the elution of a retained compound is given by t r,the retention time, and is equal to the time the solute spends in the moving mobilephase plus the time spent in the stationary phase, not moving. If a solute is unre-tained and has no interaction with the stationary phase, it will elute at t o, thehold-up time or dead time, which is the same time required for the mobile-phasesolvent molecules to traverse the column. Another fundamental measure of retention is retention volume, V r. It issometimes preferable to record values of V r rather than t r, since t r varies withthe ow rate F, while V r is independent of F. If we wish to describe the volumeof mobile phase required to elute a retained compound, then the retention volumeis the product of the retention time and the mobile-phase ow rate. Vr tr F(2)Copyright 2002 by Marcel Dekker. All Rights Reserved. 4. Fig. 1 A model chromatogram for the separation of two compounds. V o is a measure of the total volume of space available to the mobile phase in the system and correlates to t o as the volume necessary to elute an unretained compound.Vo to F (3)The most commonly used retention parameter in high-performance liquid chromatography (HPLC) is the capacity factor, k. While the distribution coef- cient, K, describes the concentration ratios, the capacity factor, k , is the ratio of amounts of solute in each phase. moles of X in stationary phase[X ] s V s KV sk (4)moles of X in mobile phase[X] m V m Vm Equation (4) shows that the capacity factor is directly proportional to V s, and so k for each solute changes with the stationary-phase loading on the silica support. The capacity factor is conveniently measured from retention parameters, since k also describes the amount of time the solute spends in each phase. tr to Vr Vok (5)toVoRearrangement of Eq. (5) givesV r V o (1 k)(6)The retention volume can then be related to the capacity factor by substitut- ing Eq. (4) for k in Eq. (6), where V m V o.V r V o KV s(7)Copyright 2002 by Marcel Dekker. All Rights Reserved. 5. B. Kinetic ConsiderationsIt is obvious from Fig. 1 that the chromatographic separation of components ina mixture is dependent on two factors: the difference in retention times of twoadjacent peaks, or more precisely, the difference between peak maxima and thepeak widths. It was shown in the preceding discussion that the retention of asolute is a thermodynamic process controlled by the distribution coefcient andthe stationary-phase volume. The peak width, or band broadening, on the otherhand, is a function of the kinetics of the system.Early papers on chromatographic theory described the technique in termssimilar to distillation or extraction. The Nobel Prize-winning work by Martin andSynge (5) in 1941 introduced liquidliquid (or partition) chromatography andthe accompanying theory that became known as the plate theory. Although theplate theory was useful in the development of chromatography, it contained sev-eral poor assumptions. The theory assumed that equilibration between phaseswas instantaneous, and that longitudinal diffusion did not occur. Furthermore, itdid not consider dimensions of phases or ow rates. An alternative to the platetheory which came into prominence in the 1950s was the so-called rate theory.The paper which has had the greatest impact was published by the Dutch workersvan Deemter, Zuiderweg, and Klinkenberg (6). They described the chromato-graphic process in terms of kinetics and examined diffusion and mass transfer.The popular van Deemter plot resulted, and the rate theory has become the back-bone of chromatographic theory.Several processes, which will be discussed shortly, contribute to the overallwidth of the band, and the contribution from each process can be described interms of the variance (2 ) of the chromatographic peak, which is the square ofits standard deviation ().The most common measure of efciency of a chromatographic system isthe plate number, N. Because the co