SEPARATION AND QUANTIFICATION OF PHOSPHOLIPIDS FROM MARINE-BACTERIA WITH THE LATROSCAN MARK-IV TLC-FID

Several procedures for latroscan TLC-FID analysis of phospholipids hav e been proposed in the literature but none has been found to provide s ufficient resolution to prevent peaks overlapping when the analysis pr oceeds from standards to natural samples. This was particularly eviden t in the separation of marine bacterial phospholipids characterized by the predominance of phosphatidylethanolamine (PE), phosphatidylglycer ides (PG), and diphosphatidylglycerides (DPG) over phosphatidylcholine (PC). In this study, a procedure has been developed which requires ne ither preliminary sample preparation nor impregnation of the Chromarod s. The first step entails 15 minute elution in the modified Innis and Clandinin system (chloroform - methanol - water - formic acid, 85 + 30 + 3 + 0.2, v/v), which moves the diphosphatidylglyceride peak away fr om the partially resolved remaining phospholipids, followed by a parti al scan for DPG. The second step is elution in a basic system (chlorof orm - methanol - 28 % ammonium hydroxide, 50 + 50 + 5, v/v) followed b y complete scanning of the Chromarods. With the latroscan Mark IV, hyd rogen and air flows of 160 and 2000 ml min-1 were found to give a sati sfactory response in the 0.25 to 3 mug l-1 concentration range for all the phospholipids. Maximum sensitivity was obtained for DPG, PEF, and PC. Under these analytical conditions, second order polynomial regres sion curves proved the best fits to the calibration curves obtained fo r the standards. Separation of lipid extracts from bacteria proved to be satisfactory. Results were confirmed by comparison of the quantific ation of bacterial lipid extracts both by classical two-dimensional TL C and by TLC-FID.