Phospholipids, separation of

Two-dimensional systems are occasionally used to separate phospholipids (Figure 12.5). After the separation of phospholipids using the chloroform methanol water (65 25 4) system, the plates can be dried and turned by 90°, followed by the second development in either n-butanoFacetic acid water (60 20 20) or chloroform acetone ... 

The separation of phospholipids by micellar electrokinetic capillary electrophoresis (MEKC) has been described (17-19). In this technique, solutes are separated based on their distribution between a mobile (usually aqueous) and a pseudostationary (micellar) phase. Szucs et al. found that the major soybean phospholipids were fully resolved in only 7 minutes using deox ycholic acid for micelle formation in combination with 30% n-propanol at 50°C (18). However, quantification of the separated compounds remains troublesome. This is due first of all to the fact that only UV detection can be used, thus making the response highly dependent on the degree of unsaturation of the phospholipids. Besides, the comparison of peak areas in MEKC is more complicated than in HPLC, because all compounds are moving with different velocities. 

JA Singleton, LF Stikeleather. High performance liquid chromatography analysis of peanut phospholipids. I. Injection system for simultaneous concentration and separation of phospholipids. J Am Oil Chem Soc 72 481-483, 1995. 

I have discussed normal phase separations on silica and hydrated silica columns in which polar compounds retain and nonpolars elute. Separations have also appeared in the literature using an acid phase silica column for the separation of phospholipids. 

Porter, N. A. and Weenan, H. (1981) High performance liquid chromatographic separation of phospholipids and phospholipid oxidation products, in Methods of Enzymology, Vol. 72, S. P. Colowick and N. O. K. Kaplan, editors, Academic Press, New York, pp. 34-40. 

GPL can be separated using either normal-phase (NP) LC or RPLC. NPLC allows the separation of phospholipids by class, i.e., first the neutral lipids, followed by GPEtn, GPtns, and GPA, while GPCho elute later. 

The following chapters in Advances in Lipid Methodology, Volume 3 (Cl) Chapter 3, Separation of phospholipid classes by high-performance liquid chromatography. W. W. Christie (C2) Chapter 6, Plant glycolipids Structure, isolation and analysis, E. Heinz. 

Lovem, J. The application of counter-current distribution to the separation of phospholipids. Biochem. J. (London) 1952, 51, 464-470. 

Two experimental methods have been described recently which should help the analysis of phospholipids. Details of a high-performance liquid chromatographic method for the rapid separation of phospholipids have been published, and hector-ite clay matrices have been used to stabilize phospholipid bilayers so that their vibrational spectra can be measured. The bilayers are incorporated into the clay to form ultra-thin, self-supporting films about 25 wm thick, and although some regions of the spectrum are masked by vibrations due to the clay, vibrations due to both the phosphoryl head-group and the acyl chain can be observed. 

One of the important methods of critical fiuid fractionation involves the countercurrent separation of phospholipids from a vegetable oil. A system to achieve this end is presented in Figure 7. Here, high pressure CO2 is fed into a pressure vessel packed with segmented gauze mesh packing (the refining... 

For the assay of PI kinase, A431 cell membrane and [y-32p]ATP were mixed in 20 mM HEPES buffer (pH 7.2). The reaction mixture was incubated for 20 minutes at 20"C and stopped by the addition of CHCI3, MeOH, and IN HCl (4 1 2). After vigorous vortexing, the lower phase was applied to a silica gel column for the separation of phospholipid and unreacted [y-32p]ATP (31). The phosphorylated lipid was eluted with CHCI3, MeOH, and 4N NH4OH (9 7 2) and the radioactivity was quantified by liquid scintillation counting. 

Lytle, C.A., Can, Y.D. and White, D.C. (2000) Electrospray ionization/mass spectrometry compatible reversed-phase separation of phospholipids piperidine as a post-column modifier for negative ion detection, youma/ of Microbiological Methods 41,22 7-234. 

Porter, N.A. and Weenen, H. 1981. High-performance hquid chromatography separations of phospholipids and phospholipid oxidation products, Meth. EnzymoL, 72, 34. 

Fig. 11.3.2. HPLC separation of phospholipid standards. Chromatographic conditions column, Micro-Pak SI-10 column (300 X 4 mm I.D.) mobile phase, acetonitiile-methanol-85% phosphoric acid (130 5 1.5, v/v/v) flow rate, 1 ml/min temperature, ambient detection, UV at 203 nm. Peaks LPC, lysophosphatidylchoUne (5 /ig) LPE, lysophosphatidylethanolamine (5 g) LPS, lysophosphatidylserine PA, phosphatidic acid PC, phosphatidylchoUne (0.5 /tg) PDME, phosphatidylmethy-lethanolamine PE, phosphatidylethanolamine (0.5 gg) PG, phosphatidylglycerol PI, phosphatidylinositol (2.5 gg) PMME, phosphatidylmonomethylethanolamine PS, phosphatidylserine (0.5 gg) SF, solvent front SPH, sphingomyelin (2.5 gg). Reproduced from Chen and Kou (1982), with permission.

Several other isocratic separations of phospholipids on silica have been reported using mobile phases comprising mixtures of hexane-propanol-water (Geurts van Kessel et al., 1977 Abood et al.,... 

Lin, S. S., Fischl, A. S., Bi, X. H., and Parce, W., Separation of phospholipids in microfluidic chip device apphcation to high-throughput screening assays for lipid-modifying enzymes. Analytical Biochemistry 314, 97-107, 2003. 

In addition to the MRM scan, full-scan data can be obtained using the EMS function on the QTRAP to provide valuable information for evaluation of the bioanalysis method. For example, dosing vehicle polyethylene glycol (PEG) has been found to cause ion suppression in electrospray ionization, and chromatographic resolution of PEG from the analytes of interest is crucial for reliable performance of a quantitative bioanalytical assay. The EMS scan of the incurred samples could provide valuable information on the presence and the retention time of PEG, which cannot be obtained by MRM (King et al., 2003). Phospholipids have been shown to cause ion suppression, especially when a generic extraction method such as protein precipitation is used. The EMS function on QTRAP can be used to elucidate the phospholipid profile and to monitor the separation of phospholipids from the analyte and the internal standard (King et al., 2003). 

TLC is probably the most common method for the separation of polar lipids. One-dimensional TLC is commonly used for rapid group separation or for small-scale preparative purposes, whilst two-dimensional TLC gives improved resolution. Silica gel G plates developed in chloroform-methanol-water (25 10 1 by volume) give useful separations of phospholipids. Solvent mixtures containing acetic acid or ammonia, e.g., chloroform-methanol-28 % aqueous ammonia (65 35 5) or chloroform-acetone-methanol-acetic acid-water (10 4 2 2 1) are among the wide range of solvent mixtures that have been used for TLC separations of polar lipids. 

Among these chemicals are silver nitrate - added to allow separation of cisitrans isomers, particularly for lipid analyses potassium oxalate - added to allow discrimination of polyphosphoinositides magnesium acetate - added to help in the separation of phospholipids ammonimn sulfate - added so that the plate is self-charring (after heating) carbomer -added for the analysis of mannitol/sorbitol and sodium hydroxide - added to improve the separation of bases and organometallics. 

Bradova, V., Simd, F., Ledvinova, J. et al. (1990) Improved one-dimensional thin-layer chromatography for the separation of phospholipids in biological material. Journal of Chromatography, 533, 297-9. 

Wang, W.-Q. and Gustafson, A. (1992) One-dimensional thin-layer chromatographic separation of phospholipids and lysophospholipids from tissue lipid extracts. Journal of Chromatography, 581, 139-42. 

Phospholipid molecular species from rat brain were determined by Ma and Kim (1995). Several separations of phospholipids are described in which not only class separation but also some molecular species separation were obtained. For example, PI, PE and PE plasmalogen (PLe) overlapped, making straightforward identification somewhat difficult. Ma and Kim... 

Matsuda, K. Matsuda, S. Saito, M. Ito, Y. Separation of phospholipids and glycoUpids using analytical toroidal-coil countercurrent chromatography. I. Separation of human hrain lipids. J. Liq. Chromatogr. Relat. Technol. 2002, 25, 1255-1269. 

A few articles are concerned with the simultaneous separation of phospholipids and glycolipids." The poly(vinyl alcohol) column gave excellent results. However, complex mobile phases were needed. 

Figure 24 shows other possibilities for linking up these individual critical fluid-based options into tandem processes. Here the previously discussed option is shown initially as well as the supercritical fluid extraction and chromatographic separation of phospholipids which was noted in Section 3.2.3. Also, our previously-cited example of subcritical water synthesis of fatty acids from natural oil feedstocks is noted, the end product in this case is a mixture of fatty acids contained in an aqueous emulsion. These can be separated from water via a membrane process or counter currently into supercritical or liquid carbon dioxide. Further rectification of the fatty acid mixtures would also be amenable to fractionation via the thermal gradient fractionation column mentioned previously. 

Juaneda, P., and Rocquelin, G. (1985) Rapid and Convenient Separation of Phospholipids and Non Phosphorus Lipids from Rat Heart Using Silica Cartridges, Lipids 20, 40-41. 

Solvents. Mixtures of chloroform and methanol containing a little water have been the principal solvents used for separations of phospholipids [128,150,163,210]. They have been combined in two-dimensional procedures with basic [1,69, 111, 186] or acidic [1, 111, 127,177] solvents. The solvents customarily employed for TLC of phospholipids can be used also for separating sulpholipids [69,210]. Glycolipids have been chromatographed with chloroform-methanol-water [73, 150, 172, 198] but especially with aqueous propanol [106, 107, 163, 199]. 

Polar Animal Lipids. The experimental conditions quoted in the first articles [70, 210] on thin-layer chromatographic separation of phospholipids in animal and human organs, have proved satisfactory. They have needed modification, however, in a few special problems [67, 163, 188, 200]. The range of application of TLC has been appreciably extended recently by introduction of the two-dimensional technique [159a, 177, 186] (see Fig. 151). 

Phospholipids are eluted, ignited and colorimetrically determined as phosphate [1, 47, 53, 159, 189] photodensitometric analysis on the layer is also possible [91, 132, 161]. The experimental conditions given in Fig. 151 are recommended for the two-dimensional separation of phospholipids, sulpholipids and glycohpids, prior to their quantitative analysis. Methods are available for determining ganghosides [73, 196] and other sphingolipids [71, 72, 164]. 

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