Quantitative determination of Tri-, Di-,monooleins and free oleic acid by the thin layer chromatography-flame...

The separation conditions for hydrolysates of triglycerides by lipase and their quantitative determination are discussed for a thin layer chromatography-flame ionization detector system utilizing internal standards. The complete separation of glyceride hydrolysis mixtures (triolein 1,3-diolein, 1,2-diolein, 1-monoolein and oleic acid) was achieved on a 3% boric acid-impregnated Chromarod S-II by development with benzene/chloroform/acetic acid (70∶30∶2, v/v/v) (mobile phase A) or hexane/ ether/acetic acid (70∶30∶1, v/v/v) (mobile phase B). Mobile phase B had an advantage over mobile phase A in terms of free space to add internal standards for simultaneous quantitation and was employed.p-Hydroxybenzoic acid andp-carboethoxy benzyl alcohol, which appeared between 1,2-diolein and 1-moloolein, were adopted as the internal standards. The calibration curves relating internal standards to each glyceride were all approximated by the equations Y=aX(b) giving high correlations. The method was applied to hydrolysis of triolein by pancreatic lipase.