ABSTRACT
Reversed-phase high-performance liquid chromatography has found wide acceptance in numerous peptide and protein laboratories around the world. Reversed-phase chromatography (RPC) columns provide rapid, high resolution separations together with generally excellent recoveries. This chapter explains how to maximize the resolving power of RPC columns. It discusses the concepts of ion-pairing and pH manipulation. By altering the nature and strength of the ion-pairing reagent and the pH of the solvent system, it is possible to exploit both the acidic and basic character of solutes in a systematic and predictable manner. Use of reagent grade chemicals leads to baseline problems and the appearance of spurious peaks. It is important to use high grade reagents to ensure optimal column performance. The chapter also discusses the preparation of solvents for RPC. The concept of ion-pairing in RPC is very useful in both developing and understanding the mechanism of action of solvent systems.
