Fractionation of soybean phospholipids by preparative high-performance liquid chromatography with sorbents of various particle size.
Normal-phase high-performance liquid chromatography was used on a preparative scale to seperate phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylcholine (PC) phospholipids from soybean. Separation was achieved using mixtures of three solvents, hexane, methanol and isopropanol. The optimized mobile phase compositions were experimentally determined while operating in a linear gradient mode using 15, 5-20, 25-40, and 40-63 microm preparative particles as well as 4 microm analytical particles. A gradient mobile phase was established on a commmercially available analytical Nova-Pak column such that hexane linearly decreased from 85 to 0 as isopropanol and methanol linearly increased in two gradient steps from 10 to 30 and 5 to 70 respectively. The total run time was 25 min at a flow-rate of 1.5 ml/min. A slight change in mobile phase composition was required to increase the resolution of phospholipids. The 15 microm particle size gave the best separation of the preparative particle sizes examined based on their resolutions between PE and PI and PI and PC. Finally, the retention factors of PE and PC were correlated in terms of mobile phase composition.
Method development for the separation of phospholipids by subcritical fluid chromatography
Abstract A packed-column subcritical fluid chromatographic separation of five phospholipids was achieved in less than 20 min by using a Luna octyl column (25 cm×4.6 mm I.D., 5 μm) employing evaporative light scattering detection. The chromatographic conditions used for the separation of (a) 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), (c) poly(ethylene glycolated)-1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPE–PEG), (d) 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) and (e) 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) were: 9% ethanol–methanol (50:50, v/v), containing 0.10% (v/v) trifluoroacetic acid for 2 min, increasing by 0.5%/min to 15% in 14 min, to 40% at 14.1 min, at a column temperature of 70°C, an outlet pressure of 135 bar and a mobile phase flow-rate of 2.0 ml/min. The separation was shown to be significantly influenced by the presence of a mobile phase acidic additive (e.g. trifluoroacetic acid) and its concentration, the overall modifier ramp rate and the column outlet pressure.
Improved determination of individual molecular species of phosphatidylcholine in biological samples by high-performance liquid chromatography with internal standards.
Phosphatidylcholine isolated from samples of bile, liver and plasma was converted into 1,2-diradylglycerobenzoate molecular species by hydrolysis with phospholipase C and reaction with benzoic anhydride. Up to seventeen molecular species were separated and determined by reversed-phase high-performance liquid chromatography with detection at 230 nm. The major improvement introduced here was the use of distearoylphosphatidylcholine as the internal standard, which corrected the results for incomplete hydrolysis and benzoylation. Other improvements concerned the clean-up of benzoyl derivatives and the chromatographic separation. The analytical results obtained were validated by comparison with the results of either lipid phosphorus or gas chromatographic determinations.