Ca. 1% of the glycerides of milk fat contain oxofatty acids. The isolation, fractionation, and characterization of oxofatty acids were accomplished using the following sequence of steps: (A) transmethylation, (B) conversion into 2,4‐dinitrophenylhydrazones, (C) adsorption of the 2,4‐dinitrophenylhydrazones on magnesium oxide to eliminate the colorless lipid, (D) fractionation of the 2,4‐dinitrophenylhydrazones into non‐oxofatty acid and oxofatty acid fractions on alumina, (E) separation of the oxofatty acid 2,4‐dinitrophenylhydrazones into saturated and unsaturated classes by argentation column chromatography, (F) separation of these classes by chain length using liquid‐liquid column and thin layer partition chromatography, (G) resolution of positional isomers by thin layer chromatography, (H) regeneration of the positional isomer 2,4‐dinitrophenylhydrazones, and (I) analysis of the parent oxofatty acids by gas liquid chromatographymass spectrometry. In this manner, 36 saturated and 11 unsaturated oxofatty acids were identified tentatively or positively. The saturated oxofatty acids ranged in chain length from C10–C24, predominantly C18 and C16, and generally contained an even number of carbon atoms. The unsaturated oxofatty acids ranged from C14–C18, with C18 predominating.
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