Separation of polar and neutral lipids from mammalian cell lines by high-performance thin-layer chromatography

The introduction of high-performance TLC (HPTLC) instrumentation that allows precise control of critical parameters has transformed the technique into an efficient and rapid tool for analyzing various metabolites, namely lipids. Although mass spectrometry (MS) has largely replaced lipid analysis techniques over recent decades due to its comprehensive lipidome profiling capabilities, it typically lacks the rapidity and simplicity of TLC. HPTLC remains advantageous due to its ease of use, simpler data interpretation, and compatibility with complementary techniques. In this study, we established a HPTLC protocol to fractionate both polar and non-polar lipids on a single normal phase plate. Twenty lipid standards were fractionated and the method was successfully applied to whole extracts from six mammalian cell lines. Standards and extracted lipids were applied with an automated sampler, and polar lipids were first fractionated in a 5-step automated gradient elution, followed by the fractionation of neutral lipids in a twin-trough chamber with three different elutions. Plates were automatically sprayed with a modified copper sulfate solution and charred to reveal lipids and obtain the respective chromatograms. LC-MS was used to identify ambiguous bands, thus ensuring the accuracy of lipid identification.