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 methods2,3. 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.