HPLC for Pharmaceutical Scientists 2007 (Part 5)

High-performance liquid chromatography (HPLC) is a separation tool par excellence for the analysis of compounds of wide polarity. Since its inception approximately four decades ago, HPLC has revolutionized numerous disciplines of science and technology. Among the various modes of HPLC, reversed-phase and normal-phase chromatography (NPC) are employed most commonly in separation. Normal-phase chromatography was the first liquid chromatography mode, discovered by M. S. Tswett in 1903, and it is well established as evidenced by a plethora of books and articles that have been published in recent years. In this chapter we describe a simplified overview of the theory...
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HPLC for Pharmaceutical Scientists 2007 (Part 5)5NORMAL-PHASE HPLCYong Liu and Anant Vailaya5.1 INTRODUCTIONHigh-performance liquid chromatography (HPLC) is a separation tool parexcellence for the analysis of compounds of wide polarity. Since its inceptionapproximately four decades ago, HPLC has revolutionized numerous disci-plines of science and technology. Among the various modes of HPLC,reversed-phase and normal-phase chromatography (NPC) are employed mostcommonly in separation. Normal-phase chromatography was the first liquidchromatography mode, discovered by M. S. Tswett in 1903, and it is well estab-lished as evidenced by a plethora of books and articles that have been pub-lished in recent years. In this chapter we describe a simplified overview of thetheory and practice of normal-phase chromatography.5.2 THEORY OF RETENTION INNORMAL-PHASE CHROMATOGRAPHYUnlike the more popular reversed-phase chromatographic mode, normal-phase chromatography employs polar stationary phases, and retention is mod-ulated mainly with nonpolar eluents. The stationary phase is either (a) aninorganic adsorbent like silica or alumina or (b) a polar bonded phase con-taining cyano, diol, or amino functional groups on a silica support. The mobilephase is usually a nonaqueous mixture of organic solvents. As the polarity ofthe mobile phase decreases, retention in normal-phase chromatographyHPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBruttoCopyright © 2007 by John Wiley & Sons, Inc. 241242 NORMAL-PHASE HPLCFigure 5-1. Hypothetical representation of the adsorption mechanism of retention innormal-phase chromatography. S denotes sample molecule, E denotes molecule ofstrong polar solvent, and X and Y are polar functional groups of the stationary phase.Prior to retention, the surface of stationary phase is covered with a monolayer ofsolvent molecules E. Retention in normal-phase chromatography is driven by theadsorption of S molecules upon the displacement of E molecules. The solvent mole-cules that cover the surface of the adsorbent may or may not interact with the adsorp-tion sites, depending on the properties of the solvent. (Reprinted from reference 1, withpermission.)increases. Figure 5-1 illustrates the mechanism of retention in NPC [1]. Reten-tion is governed by the extent to which the analyte molecules displace theadsorbed solvent molecules on the surface of the stationary phase. This reten-tion model based on adsorption was first proposed by Snyder [2–5] to describeretention on silica and alumina adsorbents and later extended to explain reten-tion on polar bonded phases, such as diol-, cyano-, and amino-bonded silica.Snyder assumed a homogeneous surface so that adsorption energies for soluteand solvent molecules are constant. The stoichiometry of solute–solvent com-petition can be given by Sm + nEa ↔ Sa + nEm (5-1)m and a refer to solute (S) and solvent (E) molecules in the mobile andadsorbed phases, respectively. n is the coefficient that takes into account dif-ferent adsorption cross sections for solute and solvents; that is, adsorption ofa solute molecule displaces n solvent molecules in the adsorbed monolayer.For a binary mobile-phase system consisting of a weak nonpolar solvent anda strong polar solvent, adsorption of the weak solvent can be ignored. There-fore, solute retention can be expressed by AS ln(k2 ) = ln(k1 ) − ln( N E ) (5-2) AETHEORY OF RETENTION IN NORMAL-PHASE CHROMATOGRAPHY 243Here, AS is the solute cross-sectional area, AE is the molecular area of thestrong solvent, NE is the mole fraction of the strong solvent in the mobilephase, k2 is retention factor of the solute in the binary mobile-phase mixture,and k1 is the retention factor in the strong solvent alone. Yet another adsorption-based retention model similar to that of Snyder wasproposed by Soczewinski [6] to describe the retention in NPC. It assumes thatretention in NPC is the product of competitive adsorption between solute andsolvent molecules for active sites on the stationary phase surface. The sta-tionary-phase surface consists of a layer of solute and/or solvent molecules,but, unlike the former, the latter model assumes an energetically heteroge-neous surface where adsorption occurs entirely at the high-energy active sites,leading to discrete, one-to-one complexes of the form Sm + qEa -A* ↔ S - A* + qEm (5-3)A* is an active surface site and q refers to the number ...