Effect of Mobile Phase Composition on the Separation of Neutral Lipids, γ-Oryzanol and its Saponified Compounds on a 100-Å Phenogel Column

Abstract

. Monitoring of bioactive compounds in rice bran oil is generally required more than one approach. This study attempted to simultaneously analyze the bioactive compounds (γ-oryzanol (OZ), free sterols (both phytosterols and triterpene alcohols, FS), ferulic acid (FA), free fatty acid (FFA) and acylglycerols (triglycerides (TG), diglycerides (DG), monoglycerides (MG)) in RBO-based mixture using a size-exclusion HPLC column (100-Å Phenogel) with a modified mobile phase. These compounds cannot be separated on a 100-Å Phenogel column using 0.1% acetic acid in ethyl acetate as a mobile phase. To improve their separation, isooctane was added to the mobile phase to decrease the degree of swelling of the column gel matrix and reduced the elution strength of the mobile phase. The retentions of TG, FS and DG were less affected by the isooctane content. Whereas, it had a moderate effect on the elution of FFA and MG and strong effect on OZ and FA. The separation of these compounds was obviously improved with increasing the isooctane content up to 70%. However, peaks of the polar compounds were broadened, especially for the most polar, FA. The separation on the size-exclusion column did not follow the expected elution order (molecular sieving) and showed interactions between the polar groups of the compounds to the column gel matrix. Ethyl acetate/isooctane (50:50, v/v) with 0.1% acetic acid as a mobile phase shows a promising condition for simultaneous separation of neutral lipids, OZ and its saponified compounds with a total analysis time of 15 min.