Improved reproducibility and quantitative analysis of phospholipids by flame ionization detection

Abstract

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 R F 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 μg 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 desribed. Scanning times and R F values of various phospholipids are compared to determine the best separation of the major phospholipids found in 3T3-L1 and leukocyte membranes.