Fermentation Media by Gas-Liquid Chromatography

PACKETT, L. V. (Purdue University, Lafayette, Ind.), AND R. W. MCCUNE. Determination of steam-volatile organic acids in fermentation media by gas-liquid chromatography. Appl. Microbiol. 13:22-27. 1965.-Five gas chromatographic liquid phases (25% Carbowax 20M plus 4% H3PO4, 17.5% dioctyl sebacate plus 7.5% sebacic acid, 17.5% dioctyl sebacate plus 7.5% docosanoic acid, 5% Tween 80, and 20% LAC296 [poly(diethylene glycol adipate)] plus 2% H3PO4) were studied with respect to their utility in the separation and quantitation of steam-volatile organic acids commonly produced in fermentation. Optimal operating conditions and column stability for routine analysis were established. An Aerograph Hy-Fi gas chromatograph was used for all work, except the studies with Tween 80 in which an Aerograph A-90-C was employed. Chromatographic traces are presented of volatile fatty acid analyses with each of the liquid phases. Complete separation of all isomers of the fatty acids from C2 to C5 was accomplished by the Carbowax 20 M plus H3P04, dioctyl sebacate plus sebacic acid, and dioctyl sebacate plus docosanoic acid columns. The latter two liquid phases were extremely unstable and proved to be unsatisfactory for analysis of aqueous samples. A column of Carbowax 20 M + H3PO4 separated steam-volatile organic acids completely. The volatile fatty acid isomers were separated by 5% Tween 80 somewhat less completely, and the peak shapes were not as sharp and symmetrical as that desired for good quantitative work. LAC-296 (20%) plus 2% H3PO4 proved to be the most satisfactory of the liquid phases for routine analysis of deproteinated in vitro rumen fermentation media. The column has been used for routine analysis of rumen fermentation fluid and in vitro rumen incubation fluid. All the organic acids from C2 to C5 , except isobutyric, could be quantitated with this column. Stability of the column with the aqueous solutions was extremely good. The standard deviation of the analysis of each volatile acid component in a fermentation fluid was less than 0.5 molar per cent. The short-chain organic acids (C2 to C5) were shown to be extremely stable in aqueous solution for as long as 6 months after preparation for gas chromatographic analysis by protein precipitation with metaphosphoric acid-H2SO4 and refrigeration at 4 C in stoppered