For characterizing keto acids, paper chromatography of their DNPHa is most widely used irrespective of the disadvantages (1)) some of which are as follows: According to the methods reported before 1962, the DNPH formed by the usual method were extracted from water solution with an organic solvent such as ethyl acetate or ether. Then they were transferred to the water solution by shaking the organic solvent layer with sodium carbonate solution to separate them from the unreacted reagent (2). However, the recovery of DNPH of most keto acids in the latter extraction was incomplete, especially in the case of less polar keto acids, as has already been pointed out by the authors (3). A well-known inevitable disadvantage of the DNPH derivative is that most a-keto acids give a mixture of cis-and trans-DNPH
(1, 4) and that an isomerization occurs between both isomers depending upon conditions (4, 5). These properties of DNPH have caused some obstacles in the characterization of a-keto acids. In paper chromatography, DNPH were usually developed in a free form (6), or as the sodium (7) or ammonium salt (8). However, isomerization was found to occur when DNPH was developed in a free form (5). The DNPH, which was eluted from the single spot on the chromatogram thus obtained, often gave double spots upon rechromatography.
The solvent containing ammonia for the development of the ammonium salt of DNPH caused a decomposition of some