tailieunhanh - 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. | 5 NORMAL-PHASE HPLC Yong Liu and Anant Vailaya INTRODUCTION 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 and practice of normal-phase chromatography. THEORY OF RETENTION IN NORMAL-PHASE CHROMATOGRAPHY Unlike the more popular reversed-phase chromatographic mode normalphase chromatography employs polar stationary phases and retention is modulated mainly with nonpolar eluents. The stationary phase is either a an inorganic adsorbent like silica or alumina or b a polar bonded phase containing cyano diol or amino functional groups on a silica support. The mobile phase is usually a nonaqueous mixture of organic solvents. As the polarity of the mobile phase decreases retention in normal-phase chromatography HPLC for Pharmaceutical Scientists Edited by Yuri Kazakevich and Rosario LoBrutto Copyright 2007 by John Wiley Sons Inc. 241 242 NORMAL-PHASE HPLC Figure 5-1. Hypothetical representation of the adsorption mechanism of retention in normal-phase chromatography. S denotes sample molecule E denotes molecule of strong 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 of solvent molecules E. Retention in normal-phase chromatography is driven by the adsorption of S molecules upon the displacement of E molecules. The solvent molecules that cover the surface .