Enantiomeric analysis of triacylglycerols containing three different fatty acids

In natural fats and oils, fatty acids (FAs) are not randomly esterified to the different positions of triacylglycerols (TAGs), and the positional distribution of FAs affects nutritional, biochemical, and technological properties. This study aimed to contribute to the research area of lipid chirality by characterizing the chromatographic behavior and determining the elution order of TAGs with three different FAs.

Altogether, 49 enantiopure structured TAGs were synthesized in this study. Enantiomeric analysis of these samples, together with 16 commercial racemic TAGs, was carried out with two chiral columns and methanol as a mobile phase by using a sample recycling HPLC system with a UV detector.

Finally, 26 out of 33 mixtures were enantiomerically separated. Enantiomer pairs with two unsaturated FAs at the outer position were not separable with this method. However, in cases of two saturated FAs, the separation efficiency was dependent on the length of the carbon chain. The equivalent carbon number of the TAG was positively correlated with the retention time in all samples. The enantiomeric elution order was determined for each enantiomeric pair. The enantiomer with the unsaturated FA at the sn-1 position always eluted first. The difference in carbon chain lengths between FAs on different positions affects the separation efficiency.

This research provides the method and references for the analysis of elution rules and retention mechanisms for TAG containing three different fatty acids. To our knowledge, this is the first study where the chromatographic resolution of TAGs with three different FAs was studied systematically. The results are crucial for predicting the retention behavior of TAG isomers. This scientific knowledge is especially important when analyzing more complicated natural lipids and studying their importance for health and well-being.