Improved reproducibility and quantitative analysis of phospholipids by flame ionization detection.

Quantitative analysis of phospholipids by flame ionization was improved by careful application of samples with a Hamilton syringe and use of a sealed dual tank system. Chromarods developed more consistently with reproducible scanning times or RF values (coefficient of variation of 1%) and with sharper peaks if development was carried out in a sealed dual tank system. The Chromarods were placed in the inner tank, which was the standard ground glass topped tank furnished with the Iatroscan TH-10 system. This inner tank was placed inside a larger thin-layer chromatography tank which was sealed with silicone grease and the lid held in place with a lead brick. Both tanks were lined with absorbent paper and contained the same solvent system. Biological samples quantified with these procedures and measured in amounts between 1 and 30 micrograms had coefficients of variation between 0.2 and 6%. An efficient method of completely separating neutral lipids from phospholipids and allowing quantitative determination of cholesterol is described. Scanning times and RF values of various phospholipids are compared to determine the best separation of the major phospholipids found in 3T3-L1 and leukocyte membranes.