Method development for the separation of phospholipids by subcritical fluid chromatography

A packed-column subcritical fluid chromatographic separation of five phosph olipids was achieved in less than 20 min by using a Luna octyl column (25 c mx4.6 mm I.D., 5 mu m) employing evaporative light scattering detection. Th e chromatographic conditions used for the separation of (a) 1,2-dipalmitoyl -sn-glycero-3-phosphate (DPPA), 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC), (c) poly(ethylene glycolated)-l,2-dipalmitoyl-sn-glycero-3-phosphoc holine (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 aci d for 2 min, increasing by 0.5%/min to 15% in 14 min, to 40% at 14.1 min, a t a column temperature of 70 degrees C, an outlet pressure of 135 bar and a mobile phase flow-rate of 2.0 ml/min. The separation was shown to be signi ficantly influenced by the presence of a mobile phase acidic additive (e.g. trifluoroacetic acid) and its concentration, the overall modifier ramp rat e and the column outlet pressure. (C) 1998 Elsevier Science B.V. All rights reserved.