Complete high-performance liquid chromatographic separation of phenylthiohydantoin- and 4-N,N-dimethylaminoazobenzene 4′-thiohydantoin-amino acids on an ultrasphere ods column with the same buffer system

Abstract

A high-performance liquid chromatographic method is described for the separation of twenty phenylthiohydantoin (PTH)-amino acids and eighteen 4-N,N-di-methylaminoazobenzene 4′-thiohydantoin (DABTH)-amino acids. These amino acid derivatives, commonly generated after Edman degradation by the phenylisothio-cyanate (PITC) or 4-N,N-dimethylaminoazobenzene 4′-isothiocyanate (DABITC)—PITC coupling method, can be separated by high-performance liquid chromatography on an Altex Ultrasphere octadecylsilane (ODS) column. PTH-degradation products of threonine and scrine were unambigously distinguished with a dual detection system at 254 and 320 nm. The separation was accomplished by use of a gradient program consisting of 10 m M sodium phosphate buffer, pH 6.6, 0.5% n- butanol and methanol. It is demonstrated that only the methanol concentration in the gradient system must be altered depending on whether a PTH- or a DABTH- separation is required.