In general, any chromatographic separation, in which the mobile phase is more polar than the stationary phase and polar solutes move with this mobile phase rather than remain with the less polar stationary phase, is called reversed-phase chromatography. More specifically, reversed-phase chromatography (particularly in HPLC) refers to those procedures in which the stationary phase consists of silica beads that are completely covered with covalently bound hydrophobic n-alkyl chains (eg, octyl, C8, or octadecyl, C18). In reversed-phase chromatography, the adsorption of a solute to the reversed-phase matrix is generally driven by hydrophobic interactions, and the solutes migrate in decreasing order of net charge, extent of ionization, and hydrogen-bonding capabilities.
Because of its excellent resolution, experimental ease, high recoveries and excellent reproducibility, reversed-phase HPLC now plays a critical role in the biochemical/biomedical studies (see reviews 1, section V of Ref. 2, pp. 273-421, 3 and 4). Detailed practice is beyond the scope of this volume, and interested readers are directed to the reviews cited previously and to a number of excellent monographs (5, 6).
