Reversed-Phase High-Performance Liquid Chromatography

Abstract

Reversed-phase high-performance liquid chromatography (RP-HPLC) involves the separation of molecules on the basis of hydrophobicity. The separation depends on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to the stationary phase, i.e., the sorbent. A schematic diagram showing the binding of a peptide or a protein to a reversed-phase surface is shown in Fig. 1. The solute mixture is initially applied to the sorbent in the presence of aqueous buffers, and the solutes are eluted by the addition of organic solvent to the mobile phase. Elution can proceed either by isocratic conditions where the concentration of organic solvent is constant, or by gradient elution whereby the amount of organic solvent is increased over a period of time. The solutes are, therefore, eluted in order of increasing molecular hydrophobicity. RP-HPLC is a very powerful technique for the analysis of peptides and proteins because of a number of factors that include: (1) the excellent resolution that can be achieved under a wide range of chromatographic conditions for very closely related molecules as well as structurally quite distinct molecules; (2) the experimental ease with which chromatographic selectivity can be manipulated through changes in mobile phase characteristics; (3) the generally high recoveries and, hence, high productivity; and (4) the excellent reproducibility of repetitive separations carried out over a long period of time, which is caused partly by the stability of the sorbent materials under a wide range of mobile phase conditions (1,2).