Separation and Determination of Wax Content Using 100-Phenogel Column

Rice bran wax (RBW) is a by product of rice bran oil refinery. Crude RBW from refineries in Thailand had only 20-40% of the wax ester. The major impurity was triglyceride (TG). Purification of RBW requires a rapid and reliable method of analysis. In this study, a modified size exclusion HPLC column (100- Phenogel) was reported. Degree swellings of the gel matrix were controlled by isooctane-toluene mobile phase ratio. With pure toluene as the mobile phase, the gel matrix is fully swollen. Wax and TG could not be separated. With 65:35 (v/v) of isooctane-toluene, wax and TG as well as other lipids were baseline separated. The resolution (Rs) between wax and TG was greater than 1.5. Acetic acid (0.1% or higher) in the mobile phase could suppress peak tailing and improved separation of the lipid containing active hydroxyl groups such as free fatty acid, diglyceride and monoglyceride without affecting retention times of the wax and the TG. Separation of lipids in crude RBW could be completed in a single run on the modified Phenogel column (100 ) with the total analysis time less than 15 min. The relationship between the amount of wax in the sample and the peak area was linear with the R (2) greater than 0.98.