Selective on-line enrichment and separation of peptides having aromatic amino acids at their C-termini by column-switching high-performance liquid chromatography using an anhydrochymotrypsin-immobilized precolumn

Abstract

A column switching high-performance liquid chromatographic system in which peptides retained on an anhydrochymotrypsin (AHC)-immobilized diol-silica precolumn were selectively transferred to and separated on a reversed-phase analytical column was developed. An investigation of the affinity characteristics of 40 peptides to the AHC precolumn showed that among eleven peptides having Tyr or Phe at their C-termini and more than five amino acid residues, ten were retained almost quantitatively on the precolumn, and two peptides having Trp at their C-termini were less retained. Two peptides having C-terminal PheNH 2 were also retained, but the peptide having C-terminal D-PheNH 2 was not retained. Among eighteen peptides having no aromatic amino acids at their C-termini, two were retained, one slightly and the other moderately. Calibration graphs for rat atrial natriuretic peptide constructed at various sample sizes were nearly identical, indicating that the peptide could be enriched by this system. The AHC precolumn showed no loss of analytical performance over 1 year, during which about 450 samples were analysed.