Identification of the Phenylthiohydantoin Derivatives of Amino Acids by High Pressure Liquid Chromatography, Using a Ternary, Isocratic Solvent System

Abstract

In recent years high pressure liquid chromatography has found increasing application in protein chemistry for the identification of the phenylthiohydantoin derivatives [PhNCS] of amino acids. For routine microsequencing none of the solvent systems so far published have proved ideal for this application. By using a ternary solvent system a rapid isocratic separation of the usual amino acid derivatives with the exception of the pair Gln > PhNCS/Ser > PhNCS. In practice discriminating between these 2 derivatives is seldom a problem because Gln > PhNCS is partially de-amidated during the conversion and is always accompanied by Glu > PhNCS. The dependence of the retention times from pH, buffer composition, buffer concentration and temperature will be discussed.