Separation of acylglycerols, FAME and FFA in biodiesel by size exclusion chromatography

Size-exclusion chromatography separates solutes according to their molecular sizes. Free fatty acids (FFA), fatty acid methyl esters (FAME) and monoacylglycerols (MG) of vegetable oils or animal fats have very close molecular sizes and they cannot be baseline-separated on a single Phenogel column (100 angstrom, 300 min x 7.8 mm ID, 5 mu m) by using tetrahydrofuran (THF) as the mobile phase. When toluene is used as the mobile phase, triacylglycerols (TG), diacylglycerols (DG), MG and FAME are well separated but there is no baseline resolution between DG and FAME. In addition, the elution order of MG and FAME is reversed. However, baseline separation of all the above lipid classes can be achieved by using toluene containing THF, acetone, dichloromethane, ethyl acetate or acetic acid as the solvent modifier. Acetic acid (0.25%) as the solvent modifier gives the best resolution and all the reference peaks are symmetrical. The detection limit of each class of lipids is 0.1 mu g. The correlation coefficient values (between 1 and 100 mu g) of all the lipid classes are better than 0.99. Thus, the determination of biodiesel products in the biodiesel reactor is very much simplified.