Separation of amino acids on highly crosslinked polyacrylamide gel and some observations on the mechanism of adsorption

Abstract

Mixtures of amino acids were fractionated on a 1.2 cm × 140 cm column of BioGel P2 polyacrylamide gel, with 0.5 M acetic acid as eluant. Useful separations were achieved and these were highly reproducible when cytochrome c and ascorbic acid were employed as internal markers. Non-aromatic amino acids were eluted in a general order that was their reverse of their p K a values, but withinthe basic group this relationship did not appear to be valid. Aromatic amino acids, ascorbic acid and dehydroascorbic acid were retarded in a general order that appeared to depend on their electron polarisability. Hydrogen bonding did not appear to play a major role in interactions with the gel. The method is ideally suitable for the detection and isolation of species that fluoresce or absorb in the ultraviolet, since the eluting medium, unlike the conventional volatile buffers used to obviate desalting procedures following ion-exchange chromatography, is transparent in this region. The method can readily be scaled up to gram quantities. It is suggested as a method of choice for the preparative scale preliminary fractionation of protein hydrolysates, or other complex amino acid mixtures which may contain novel components.