Identification of the phenylthiohydantoin derivatives of amino acids by high pressure liquid chromatography, using a ternary, isocratic solvent system.

In recent years high-pressure liquid chromatography has found increasing application in protein chemistry for the identification of the phenylthiohydantoin derivatives 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 we have been able to achieve a rapid isocratic separation of the usual amino acid derivatives with the exception of the pair Gln greater than PhNCS/Ser greater than PhNCS. In practice discriminating between these two derivatives is seldom a problem because Gln greater than PhNCS is partially de-amidated during the conversion and is therefore always accompanied by Glu greater than PhNCS. The dependence of the retention times from pH, buffer composition, buffer concentration, and temperature will be discussed.