Fatty acid profiling of lipid classes by silica rod TLC-thermally assisted hydrolysis and methylation-GC/MS

Advances towards the coupling of thin layer chromatography (TLC) Chromarods to gas chromatography/mass spectrometric (GC/MS) analysis were accomplished by introducing an intact TLC silica-coated rod (15 cm) into a vertical furnace pyrolyzer coupled to a GC/MS instrument. Using this technique, a fatty acid profile corresponding to the neutral and polar fraction of a lipid extract previously separated by TLC was obtained. The fatty acids were analyzed directly off the TLC rod, as their methyl derivatives were obtained following thermally assisted hydrolysis methylation (THM) in the presence of trimethylsulfonium hydroxide (TMSH). The results show that the THM reaction is successful on lipids that are adsorbed on the silica surface. Using lipid standards, differences in the fatty acid yield for the THM reaction of phospholipids (PL), triacylglycerides (TAG) and free fatty acids (FFA) were observed. Compared to conventional TMSH/lipid solution analysis, there was a significant decrease in the degree of isomerization for polyunsaturated fatty acids (PUFA) using the TMSH/silica-supported reaction, when a relatively high TMSH/lipid molar ratio (>2500) is used. The fatty acid profile for a marine (cold water) lipid sample using silica-supported THM compared well with results obtained by conventional lipid extraction followed by BF3-methanol esterification. This study's success of in situ analysis of intact chromatographic bands illustrates the strong potential for TLC-GC/MS.