TLC separation and extraction of phospholipids

The thin layer chromatography plate (for analytical TLC: Merck Silica gel 60, non-fluorescent, 0.25 mm thick and for preparative TLC: Merck Silica gel 60, non-fluorescent, 2 mm thick, glass plate) was equilibrated in acetone before use. The lipid extract was dissolved in 50 µL of CHCl₃ and applied onto the TLC plate before the development in a mixture of dichloromethane (DCM), MeOH and water (65:28:4) until the solvent front was 2–4 cm from the top. The plate was dried for 2 min under a hood to evaporate excess solvent. Next, the lipids were either stained in case of reference spots of PE to correlate migration or extracted from the TLC plate in case of purification of PE-alkyne. Lipids containing amine groups were stained with ninhydrin (Ninhydrin spray Sirchie, USA), spots of phosphatidylethanolamine (PE) appear as pink/purple. For the extraction of PE¹⁴, the areas of the silica gel containing the PE was scraped with a scalpel blade and the silica gel was transferred to a centrifuge tube. Each sample was eluted from the silica gel by resuspending the powder in eluting solvent through gently tapping the tube. The first and second elution were performed with the developing solvent DCM: MeOH: water (65:28:4) using a volume of 6 and 4 mL respectively, by subsequent vortexing and sonication. After centrifugation, the supernatant was collected. The third elution was made with 4 mL of MeOH, vortexing, sonication, centrifugation and collection of the supernatant. The fourth elution was obtained with 4 mL of MeOH: acetic acid: water (94:1:5). All supernatants were gathered, filtered on a Pasteur pipette with a cotton plug to eliminate silica particles, evaporated in a speedvac and stored at −20 °C.