High-performance liquid chromatography on microcrystalline cellulose triacetate was examined for the enantiomeric separation of five racemic α-substituted α-methylbutyric acid derivatives. For four pairs the retention and the separation factor increased with increasing water content in methanol. The retention of the fifth derivative increased slowly, but separation did not occur. The concentration of the second-eluted enantiomer increased and the detection improved when a step gradient was used. Small amounts of the second-eluted enantiomer in the first could be determined.