(3) Fortuin, J. M. H., Anal. Chim. Acta 24, 175 (1961). (4) Hahn, F. L., 2. Anal. Chem. 163, 169 (1958). (5) Hartree, D. R., “Numerical Analysis,” pp. 86-9. Oxford University Press, London, 1958. (6) Hostetter, J. C., Roberts, H. J., J. Am. Chem. SOC.41, 1337 (1919). (7) Kolthoff, I. M., Laitinen, H. A., “pH
and Electrotitrations,” 2nd ed., p. 110, Wiley, New York, 1944. (8) Kolthoff, I. M., Sandell, E. B., “Textbook of Quantitative Inorganic Analysis,” p. 488, Macmillan, New York, 1952. (9) Korn, G. A., Korn, T. M., “Mathematical Handbook for Scientists and Engineers,” pp. 651-2, McGraw-Hill, New York, 1961.
(10) Lingane:, J. J., “Electroanalytical Chemistry, p. 70, Interscience, New York, 1958. JOHNSON F. YAN Department of Chemistry Kent State University
Ohio WORKsponsored by the National Institutes of Health, Project No. GM 08961-02.
Detection and Recovery of Biological Oxidation Products of Hydrocarbons by Gas Chromatography
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Figure 1. Chromatogram of a synthetic mixture of alcohols and fatty acids 0.25% Carbowax 2 0 M and 0.4% ;so-phthalic acid on 200-micron microbeads (acid-washed); 6-foot X ‘/cinch 0.d. column; He, 20 ml./min.; injector and detector at 2 4 0 ’ C.; programmed from 60’ to 180’ C. a t 4’/min. Instrument: F & M Scientific; Model 700, dual Hz flame ionization detector
SIR: Growth of certain microorganisms on long-chain alkanes and alkenes (GO to CM) leads to an accumulation of oxidation products in the culture medium. Thin-layer chromatographic (TLC) analyses of ether extracts of culture media invariably indicated the presence of long-chain monocarboxylic acids, usually longchain alcohols, and in some instances hydroxy and dicarboxylic acids. Monocarboxylic acids and alcohols were separated as classes by preparative TLC or column chromatography using silica gel. The two classes of compounds were further analyzed by gas liquid chromatography (GLC). In order to reduce the time required for analytical procedures and to minimize sample losses, fatty acids were determined as free acids with the column packing suggested by Nikelly who used acid-washed glass microbeads coated with a polar liquid phase and {so-phthalic acid as an additive to eliminate adsorption and dimerization of the fatty acids (3). This method proved expedient because glass microbeads are easy to coat and once coated the packing of the column is-readily achieved. Moreover, little if any column conditioning is needed before use. This column 1590
ANALYTICAL CHEMISTRY
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Figure 2. Preparative chromatogram of free fatty acids produced by Pseudomonas oeruginoso 3/a-inch a.d. X &foot aluminum column packed with 200-micron, acidwashed, glass microbeads coated with 0.35% Carbowax 2 0 M and 0.4% ;so-phthalic acid. Temperature 175’ C. Flow rate 150 cc. per minute. Wilkens Autoprep, Model A - 7 0 0 (1 ) Tetradecanoic acid (2) 13-Tetradecenoic acid
is also suitable for the analyses of longchain alcohols. Figure 1 shows a chromatogram of a programmed analysis of a known mixture of alcohols and monocarboxylic acids. Tetradecanoic acid and an unsaturated C14 acid, as well as several other saturated and unsaturated acid pairs, were identified from the culture fluid of Pseudomonas aeruginosa grown on tetradecene-1. Unsaturated acids were determined by peak disappearance after bromination ( 2 ) . Good separation of the saturated-unsaturated pairs prompted attempts to separate the pairs by preparative GLC using the same packing. The best results were obtained with a column 4 feet long packed with 200-micron, acidwashed, glass microbeads to which the amounts of Carbowax 20M and isophthalic acid added were 0.35% and 0.4% by weight, respectively. At best there was still some peak overlapping between saturated and unsaturated acid pairs (Figure 2). Therefore it was
more feasible to collect each pair by preparative GLC and then separate the pairs by column chromatography on Agl\’Os-impregnated silica gel (1). By these methods we have recovered a sufficient amount of the CI4pair to identify the components as tetradecanoic acid and 13-tetradecenoic acid. Analysis of the alcohol fraction is, a t the moment, preliminary. Thus far, long-chain saturated and some unsaturated alcohols have been tentatively identified by GLC on the free-fatty-acid column. LITERATURE CITED
(1) . . DeVries, B., J. Am. Oil Chemists’ SOC.40, 184 (1963). (2) . , James, A. T., Martin, A. J. P., Biochek. J. 63, i44 (1956). (3) Nikelly, J. G., ANAL. CHEM. 36, 2244 (1964). A. J. MARKOVETZ
M. J. KLUG
Department of Microbiology University of Iowa Iowa City, Iowa
