Baker and Williams's chromatographic fractionation technique, employing simultaneous temperature and solvent gradients along a column, has been successful in separating three diastereoisomer salt derivatives derived from optical isomers. Thed-andl-α-phenylethylamine diastereoisomers ofd-mandelic acid and thed-andl-tartrates ofl-brucine were separated by approximately 60%. A partial separation was achieved on thed-andl-histidine salts ofd-tartaric acid. An aqueous—alcohol—acetonitrile solvent system was used for the separations. Irregular diastereoisomer formation and dissociation of diastereoisomers were problems discussed in the development of the analytical method.