Determination of Free Fatty Acids by Supercritical Fluid Chromatography on an ODS Silica-Gel Column

Abstract

Free fatty acids (FFAs) were determined by supercritical-fluid chromatography (SFC) on an inert ODS silica-gel column using carbon dioxide as a mobile phase without a modifier. The standard samples of FFAs with carbon numbers of 6 to 26 (even numbers) were completely separated using the pressure-programming mode. The retention behavior of FFAs was investigated in terms of the density of the mobile phase, and the FFAs were separated according to a reversed-phase mode in liquid-chromatographic terms. The composition of fatty acids of coconut oil was determined by the SFC, and was compared with gas chromatography.A good agreement was obtained. The chromatographic separation of FFAs obtained from fish oil as well as the enzymatic hydrolysis products of the fish oil were also conducted. KeywordsSupercritical-fluid chromatography, free fatty acid, ODS-silica gel columnFatty acids have generally been determined by gas chromatography (GC) or liquid chromatography (LC). In determining free fatty acids (FFAs) by GC, it is usually necessary to derivatize the FFAs into their esters.1 Recently, capillary GC has been applied to the determination of FFAs to give good results.2,3 In LC, poorer detection limits have been a drawback, because UV absorption and fluorescence detection are impossible for saturated FFAs.4 A fatty-acid column for reversedphase high-performance liquid chromatography (HPLC) has offered good results of direct fatty-acid analysis.5-' The separation profile of commonly encountered FFAs by this column after a group separation by TLC have been improved.8 A reversed-phase ion-pair HPLC method has been applied to the separation and determination of FFAs (C12 -C18) using a conductivity detector.9FFAs were analyzed by a capillary SFC using supercritical CO2 as a mobile phase, and the effects of the temperature and stationary-phase polarity on the efficiency and chromatographic peak shape were investigated.1o,u Capillary SFC, however, has some drawbacks, for example very small amounts of sample introduction and the lack of a capability for up-scaling to preparative separation. The separation and detection of FFAs in simple mixtures as well as those extracted from some oil samples by SFC/Fourier-transform infrared detection were carried out on packed columns using supercritical CO2 and Freon 23 as a mobile phase.12 The FFAs were separated on an ODS-silica column using a modifier control column containing formic acid installed before an injector and were detected by a flame ionization detector (FID).13 A packed-column SFC-FID has also been applied to the analysis of abused vegetable oils containing FFA components)4 FFAs and their esters were separated by SFC on an inert ODS silica-gel column using CO2 as a mobile phase without the addition of any modifier by our group.15 In this study, however, no FFA obtained from natural sources, such as vegetable oils, could be applied, since the peak shape of the individual FFA was not sufficiently sharp to provide a base-line separation, presumably because free carboxyl groups ...

Supercritical Fluid Chromatography and Extraction

“…. Conversely, Nomura et al studied fatty acid compositions with a C 18 -bonded column(175). A very inert column was required since the authors eluted free fatty acids with unmodified CO 2 .…”