Lipid Analysis by Thin-Layer Chromatography—Detection, Staining and Derivatization

Thin-layer chromatography (TLC) remains a widely used, cost-effective and convenient method to separate small molecules, particularly in the field of natural products and (phospho)lipids. Despite advances in chromatographic methods such as high-performance liquid chromatography (HPLC), TLC retains several advantages, including simplicity and accessibility. However, a critical step is the visualization of the separated lipids on the TLC plate. Although the majority of the regularly used methods were established decades ago, there are still a number of potential pitfalls and widely unknown aspects. This review provides a concise overview about commonly used stationary phases and the solvent systems in TLC analysis of lipids. The main focus is on visualization techniques, spanning from non-specific, destructive (charring by semi-concentrated acids) to specific, non-destructive approaches (e.g., exposition to iodine to monitor unsaturated lipids). The advantages and disadvantages of the different methods will be critically discussed and frequently occurring problems highlighted. Furthermore, the combination of TLC with mass spectrometry (MS) detection will be introduced, covering both extraction-based electrospray ionization MS techniques as well as desorption techniques such as matrix-assisted laser desorption/ionization MS. MS detection, while generally more sensitive and offering molecular specificity, introduces higher technical and financial requirements compared to conventional staining. Nonetheless, the combination of TLC with MS holds significant potential for enhancing lipidomic workflows, particularly in complex biological samples.