Over 25 years ago, Horvath and Melander, in their fundamental work [1], discussed the reason behind the explosive popularity of reversed-phase liquid chromatography (RPLC) for analytical separations. It was estimated that about 80–90% of all analytical separations were performed in RPLC mode, and the authors noted that “the variation of eluent composition alone extends both retention and selectivity in HPLC [high-performance liquid chromatography] over an extremely broad range.” They compared gas chromatography with HPLC, citing “in gas chromatography a plurality of stationary phases has found practical application whereas HPLC tends toward the use of very limited number of columns. | 4 REVERSED-PHASE HPLC Rosario LoBrutto and Yuri Kazakevich INTRODUCTION Over 25 years ago Horvath and Melander in their fundamental work 1 discussed the reason behind the explosive popularity of reversed-phase liquid chromatography RPLC for analytical separations. It was estimated that about 80-90 of all analytical separations were performed in RPLC mode and the authors noted that the variation of eluent composition alone extends both retention and selectivity in HPLC high-performance liquid chromatography over an extremely broad range. They compared gas chromatography with HPLC citing in gas chromatography a plurality of stationary phases has found practical application whereas HPLC tends toward the use of very limited number of columns and optimization of the separation by manipulating the composition of the mobile phase. To some extent the statement is true even today except that with introduction of capillary columns in GC today only a very limited number of stationary phases are used while in HPLC during the last 25 years of development thousands of different stationary phases have been introduced. Practically all reversed-phase separations are carried out on stationary phases with chemically modified hydrophobic surfaces. Minor variations in the surface chemistry and geometry can lead to noticeable differences in surface interactions and as a result to differences in chromatographic selectivity. Specific stationary-phase properties and their influence on the chromatographic retention selectivity and efficiency are discussed in detail in Chapter 3. HPLC for Pharmaceutical Scientists Edited by Yuri Kazakevich and Rosario LoBrutto Copyright 2007 by John Wiley Sons Inc. 139 140 REVERSED-PHASE HPLC Mobile phase eluent is by far the major tool for the control of analyte retention in RPLC. Variations of the eluent composition type of organic modifier pH and buffer concentration provide the chromatographer with a valuable set of variables for successful .
