Reversed-phase high performance liquid chromatography of phosphatidylcholine: a simple method for determining...

A convenient method for the separation of molecular species of phosphatidylcholine (PC) by reversed-phase high performance liquid chromatography (HPLC) is described. PC species from egg, bovine brain, and porcine liver were resolved into 11-13 separate peaks on a Nucleosil-5-C18 reversed-phase column with methanol-1 mM potassium phosphate buffer, pH 7.4, 9.5:0.5 (v/v) as the solvent. Detection was at 205 nm. PC species were primarily resolved due to specific hydrophobic interaction of their fatty acid side chains with the alkyl ligand of the stationary phase. The retention time and therefore the hydrophobic interaction of acyl chains of PC species increased logarithmically as the total number of carbon atoms in the chains increased in the homologous series. The retention times decreased nonlinearly as the number of double bonds in the fatty chains increased. Introduction of the first double bond in the side chain reduced the retention time to the greatest extent. From the chromatography data the reduction in the retention time was calculated to be equivalent to 1.8 carbon atoms compared to the fully saturated PC. Further introduction of 2, 3, 4, 5, and 6 double bonds in the same side chain reduced the retention time additionally, equivalent to 1.4, 1.2, 1.0, 1.0, and 1.0 less carbon atoms. Each molecular species of PC was assigned a "Hydrophobic Carbon Number" (HCN) based upon the total number of carbon atoms ad double bonds in the side chain. PC molecules with the same HCN had the same retention time. The average HCN was calculated for various PC preparations and found to be remarkably the same for all the three different naturally occurring sources considering a wide variety of different fatty acid compositions of each type. HCN provides a simple measure of relative hydrophobicity of each PC molecule.