Resolution of phospholipid molecular species by 31P NMR

Phosphorus-31 NMR chemical shifts of phospholipids (PLs) solubilized in bile salts were studied with respect to variations in the structure of the acyl substituents. The presence of double bonds in the acyl chains of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidic acid (PA) induced small but consistent upfield shifts relative to the corresponding disaturated acyl PL signals. The magnitudes of the unsaturation-related upfield shifts were approximately twice as large in sodium cholate as in sodium deoxycholate. Chemical shift separations of PC, PE, and PG dipalmitoyl-dioleoyl species pairs increased slightly at lower temperatures. Resolution of the PC species pair was maximized and nearly independent of cholate-to-PL ratio at values greater than about 100 in 2% cholate. Only the PA species resolution varied significantly over the pH range 6.5 to 9.5, in the vicinity of its pK2. Shift differences for a homologous series of disaturated acyl PCs showed a logarithmic dependence on chain length. Spectra for a variety of PC standards were used to interpret the composite PC signals from egg yolk extract, soybean extract and whole human amniotic fluid. The 31P NMR analyses were consistent with published chromatographic studies. Interpretation of composite PL signals in the cholate system is simplified for mixtures having restricted acyl chain length and degree of unsaturation.