High-pressure liquid chromatography of alpha-keto acid 2,4-dinitrophenylhydrazones

Various α-keto acids were separated as their 2,4-dinitrophenylhydrazine derivatives by ion-pair, reverse-phase, high-pressure liquid chromatography. Excellent baseline resolution was obtained for a seven-component homologous series of α-keto acid dinitrophenylhydrazones at increasing carbon-chain length. Branched-chain keto acids were also separated. Resolution of syn and anti isomers of the α-keto acid derivatives was possible. Pyruvate from biological material was located and identity confirmed by an enzymic peak shift technique. Monitoring at 366 nm permits low-level (nanogram) amounts of keto acids to be detected. Ion pair versus ion exchange is discussed with regard to the mechanism of chromatographic separation.