Separation of 2 : 4 Dinitrophenyl Amino-acids on Buffered Silica Gel

Abstract

FOR some time past I have been making a study of the free amino groups of the peptides liberated by papain and sodium bisulphite acting on wool keratin¹, using Sanger's methods^2,3. At an early stage it was found that 'non-absorbent gels' which were of service in separating N-acetyl amino-acids were of little use in separating 2 : 4 dinitrophenylamino-acids, which all ran together as a single fast band using chloroform as solvent. Phillips and Stephen⁴ and Consden, Gordon, Martin and Synge⁵ have previously commented on the difficulty of preparing suitable gels with reproducible properties. Sanger² and Consden et al.⁵ considered that silica gels which permitted satisfactory separations 'absorbed' the dinitrophenyl amino-acids to some extent. I have observed that successful separations of these acids on silica gel can be achieved by using concentrated phosphate buffers instead of water as the stationary phase; by varying the pH of the buffer, the rate of movement of the band of a particular acid can be varied within wide limits, the higher the pH the slower being the rate.