Ultraviolet detection of peptides by reversed-phase liquid chromatography using an in-line reactor containing copper metal

A general method has been developed for the selective ultraviolet detection of peptides using reversed-phase liquid chromatography. The method is based on the formation of copper(II)-peptide complexes by the reaction of peptides with copper(II) ions in an alkaline mobile phase through an in-line reactor containing copper metal. Amino acid, di-, tri- and tetra peptides used as model compounds (leucine, glycylleucine, leucylglycylglycine and alanylalanylalanylalanine) are separated on a column containing C18-bonded vinyl alcohol copolymer gel (Asahipak ODP-50, 5 μm, 150 mm × 6 mm i.d.) and an alkaline mobile phase (pH 11.0, 25 mM phosphate buffer). A detection limit of 200 pg (SN = 3) was obtained for alanylalanylalanylalanine.