Headgroup and acyl composition determine lipid recovery from silica in preparative HPTLC workflows

Thin-layer chromatography (TLC) is a widely used, cost-effective and convenient method for separating moderately complex mixtures. TLC is well suited for small, apolar compounds such as lipids and offers several advantages over high-performance liquid chromatography (HPLC). In contrast to HPLC, which commonly employs reversed-phase materials, TLC of (phospho)lipids traditionally relies on normal-phase separation using unmodified silica gel. Although TLC is well established for the analysis of (phospho)lipids, in particular when coupled to mass spectrometry (MS), details of the interactions between lipids and the stationary phase are still not completely understood. In this study, we investigate the extent to which the headgroup, the chain length and the number of double bonds of a dedicated phospholipid influences its extractability from the developed TLC plate. The results indicate that lipid recovery from HPTLC silica is influenced mainly by headgroup charge, while fatty acyl chain length and the degree of unsaturation contribute to a lesser extent. The impact of these effects on quantitative data analysis is discussed.