Normal Liquid Chromatography
Normal phase HPLC is well suited for the separation of the phospholipids. Various silica gel columns yield excellent separations of the major phospholipid classes which, in many instances, also provide a readily discernible separation of the minor components. Figure 01 shows the separation obtained with a silica gel column for the ethanolamine, choline, inositol and glycerol glycerophospholipids and sphingomyelin isolated from a subfraction of human high density lipoprotein preparation with online MS detection. In the positive ion mode only the choline-containing phospholipids are readily detected, although the ethanolamine glycerophospholipids can also be seen at low intensity. The acidic glycerol, inositol and serine phosphatides, along with any ethanolamine phospholipids, are best detected in the negative ion mode. The negative ion mode also registers the choline phospholipids as the chloride adducts. There is a complete baseline separation for all phospholipids without significant resolution of molecular species, except for SM, which is separated into long chain and short chain species.
Figure 01. Normal phase HPLC resolution of high density lipoprotein glycerophospholipids and sphingomyelins in (A) the positive and (B) negative ion mode as recorded by online electrospray mass spectrometry. Peak identification is as given in Figure 1; PAF, platelet-activating factor. HPLC conditions: column, 5 μm Spherisorb (250×4.6 mm i.d.); solvent, a linear gradient of 100% A (chloroform/methanol/30% ammonium hydroxide 80:19.5:0.5, by volume) to 100% B (chloroform/methanol/water/30% ammonium hydroxide 60:34:5.5:0.5, by volume) in 30 min. (Unpublished results of Kuksis A and Ravandi A, 1997.)
Normal phase HPLC with online MS can be used to assess the molecular species present in the individual phospholipid classes. It is possible to obtain single ion chromatograms retrieved from the total positive ion current spectra for the major molecular species of the choline and ethanolamine phosphatides. In this normal-phase system the newly identified glycated diradylglycerophosphoethanolamine migrates with the front of the phosphatidylcholine peak. The single ion chromatograms retrieved by the computer from the total negative ion current permit accurate quantification of the major molecular species of the acidic glycerophospholipids.
Normal phase HPLC can be used for the separation of the alkylacyl, alkenylacyl and diacyl subclasses of the ethanolamine glycerophospholipids as the trinitrophenyl derivatives. The diradyl subclasses of the choline glycerophospholipids cannot be separated by chromatography of the intact parent molecules. For this purpose, diradylglycerophosphocholines must be dephosphorylated and the resulting diradylglycerols converted into UV-absorbing or fluorescent derivatives (Table 2) prior to HPLC separation unless an ELSD system is used. The molecular species separation of the diradylglycerols is carried out by reversed-phase HPLC, as described for the diacylglycerols derived from triacylglycerols by Grignard degradation.