Separation of phenylthiohydantoin-amino acids by high-pressure liquid chromatography

A method for the separation of all phenylthiohydantoin (PTH)-amino acids except PTH-arginine and PTH-histidine by high-pressure liquid chromatography on a silica column is described. Elution is performed with a concave solvent gradient from hexane-methanol-propanol (3980:9:11) to methanol-propanol (9:11). A complete run is achieved in 40 min with a pressure drop of 1000 p.s.i. over the 250 mm X 2.1 mm column. Eluted peaks of 2-5 nmole are easily detected by their ultraviolet absorption at 254 nm. This method is superior to existing gas-liquid and thin-layer chromatographic techniques since all PTH-amino acids except PTH-arginine and PTH-histidine may be both separated and quantitated in a single run of 40 min. The use of the technique in conjunction with an automated peptide sequence analyser is illustrated.