Improved separation of conjugated fatty acid methyl esters by silver ion-high-performance liquid chromatography

Operating from one to six silver ion-high-performance liquid chromatography (Ag+-HPLC) columns in series progressively improved the resolution of the methyl esters of conjugated linoleic acid (CLA) isomeric mixtures from natu ral and commercial products. in natural products, the 8 trans, 10 cis-octad ecadienoic (18:2) acid was resolved from the more abundant 7 trans, 9 cis-1 8:2, and the 10 trans, 12 cis-18:2 was separated from the major 9 cis, 11 t rans-18:2 peak. In addition, both ii trans, 13 cis-18:2 and ii cis, 13 tran s-18:2 isomers were found in natural products and were separated; the prese nce of the latter, 11 cis, 13 trans-18:2, was established in commercial CLA preparations. Three Ag+-HPLC columns in series appeared to be the best com promise to obtain satisfactory resolution of most CLA isomers found in natu ral products. A single Ag+-HPLC column in series with one of several normal -phase columns did not improve the resolution of CLA isomers as compared to that of the former alone. The 20:2 conjugated fatty acid isomers ii cis, 1 3 trans-20:2 and 12 trans, 14 cis-20:2, which were synthesized by alkali is omerization from ii cis, 14 cis-20:2, eluted in the same region of the Ag+- HPLC chromatogram just before the corresponding geometric CLA isomers. Ther efore, CLA isomers will require isolation based on chain length prior to Ag +-H PLC separation. The positions of conjugated double bonds in 20:2 and 18 :2 isomers were established by gas chromatography-electron ionization mass spectrometry as their 4,4-dimethyloxazoline derivatives. The double-bond ge ometry was determined by gas chromatography-direct deposition-Fourier trans form infrared spectroscopy and by the Ag+-HPLC relative elution order.