SIMULTANEOUS DETERMINATION OF KREBS CYCLE ACIDS BY GAS-LIQUID CHROMATOGRAPHY

Abstract

Carbowax-coated Teflon powder (2–3%, w/w, on Teflon 6, 90–100 mesh) columns (5 ft long, 1/8 in. diam.) were used with the Aerograph Hy-Fi gas chromatograph. Complete separations and symmetric peaks were obtained both isothermally (150 °C) and by temperature programming (100–160 °C) for the methyl esters of succinic, oxalacetic, malic, alpha-ketoglutaric, trans- and cis-aconitic, and citric acids. On account of enolization, the alpha-keto esters gave more than one peak, the proportions of which varied with the chromatographic conditions. The succinic and fumaric esters overlapped. The trans isomers were eluted ahead of the cis isomers.A linear relationship was obtained between the areas recorded and the quantities of the individual esters injected in the range of 0.01–10 μg. The absolute response varied with the oxygen content of each ester. Analysis of polycarboxylic acid mixtures from natural sources gave values comparable to those obtained by the more laborious conventional techniques.