SEPARATION OF AMINO ACIDS AND PEPTIDES BY HIGH PERFORMANCE HYDROPHOBIC INTERACTION CHROMATOGRAPHY

Abstract

Chromatographic behaviors of amino acids and peptide derivatives were examined by high performance hydrophobic interaction chromatography(HIC) on two different packings,TSKgels Phenyl–5PW and Ether–5PW. Most amino acids hardly retained and eluted at an elution volume being the same as the column volume even with an initial buffer for hydrophobic interaction chromatography; aromatic amino acids showed weak retention. On the other hand, peptides retained in the column and their elution was roughly in the order of the molecular mass, although some of them showed extraordinary elution behaviors. Kunitz bovine pancreas trypsin inhibitor showed weak retention in spite of having larger molecular mass, which might be due to the steric conformation. Some peptides showed considerably stronger retention even under ordinary conditions, and the addition of organic modifier (20 % acetonitrile) in eluent was required for their elution. HIC using less hydrophobic packings examined was found to be adequate to separate peptides quantitatively, and this might be useful in removing peptides from contaminants of amino acids and proteins.