Method development for the analysis of bioactive and minor compounds from vegetable oil by supercritical fluid chromatography (SFC)

Vegetable oils are complex mixtures composed of many compound families having varied masses and chemical structures. Supercritical fluid chromatography (SFC) with C18 stationary phase (C18-SP) is especially suited for the analyses of fatty acids, di- and triglycerides (the main compounds), due to the chemical properties of carbon dioxide (the mobile phase). However, the separation of minor compounds having interesting bioactive properties, for instance anthraquinones, from the lipids families is not achieved with the C18 bonded phases. To face this issue, the use of polar stationary phase (SP) was studied, with supercritical fluids. Because of the diversity of the type of polar stationary phases (silica, polyhydroxylated polymers, cross linked propane diol silica, amino, nitro, cyano and polyamide, triazole, polyvinyl alcohol and ethyl pyridine), an initial screening of 17 polar phases was achieved. The retention of compounds is reversed with this polar SP, meaning that anthraquinone retention becomes higher than the retention of lipid families, providing their separation and quantification. Depending on the chromatographic profiles of lipids families and anthraquinones, three clusters of polar SP were obtained. One column of each cluster was selected (XBridge BEH amide, Luna HILIC and Viridis 2-EP) to improve the separation method. The slope of the elution gradient (reaching two different final compositions of methanol, 20 and 30%) and the analytical temperature (15,25 and 30 °C) were studied. Finally, an ethyl-pyridine SP was retained, with a gradient of methanol/CO₂, from 2 to 30% of methanol in 19 minutes at 15 °C. In these analytical conditions, the retention of standard compounds was studied (Aloe emodin, emodin, rhein, juglone, alizarin, chrysophanol, physcion and plumbagin), allowing the identification and quantification of four anthraquinones.

Keywords: SFC, lipids, anthraquinones, vegetable oils, bioactive compounds.

No conflict of interest has been declared by the author(s).