Thin-layer chromatography of endotoxins, their derivatives and contaminants

Thin-layer chromatographic (TLC) separation techniques were used to analyze the heterogeneity of various preparations which included smooth and rough endotoxins (ET), Lipid A precipitates and synthetic Lipid A samples and a novel cytotoxic bacterial lipid. Furthermore, carbohydrate-rich split products (PS) of ET were also separated on commercial silica-coated plates. Satisfactory results were obtained by two-dimensional TLC or by the combination of chromatography followed by high-voltage electrophoresis in the separation of PS of ET cleaved by mild acetic hydrolysis. Several spray reagents were found which were eminently suitable to detect carbohydrate containing compounds. Less specific but generally useful spray reagents were also developed which gave strong color reactions with lipids, proteinaceous and carbohydrate containing split products of the ET preparations. Improved chromatographic resolution has also revealed substantial heterogeneity in both rough and smooth ET samples. Three biological activities of the separated components could be determined. These were antigenicity detected by reactivity with monoclonal antibodies on the TLC plates, endotoxicity, determined by the Limulus amoebocyte lysate (LAL) test and direct cytotoxicity of P815 cells in vitro. Considerable amounts of non-endotoxic and non-antigenic contaminants could be detected in all preparations tested. Significant amounts of free Lipid A were also found in smooth ETs. Thus a new level of complexity is recognized by TLC within these preparations.