Separation of Lipid Classes

The choice of methods for separation of lipid classes depends on the desired degree of separation. Simpler methods permit concentration or separation of only a few fractions, whereas newer methods based mainly on chromatographic procedures permit fractionation into all main lipid classes. The selection is further dependent on the amoimts of material available. 

Fractionation of lipid mixtures into phosphorus containing polar constituents and non-polar neutral fats including cholesterol is frequently required and can be achieved by several procedures. 

Since phospholipids are insoluble in acetone they may be precipitated from concentrated ethyl ether, petroleum ether or chloroform solutions by the addition of excess anhydrous acetone (Zuelzer 1899). Precipitation is completed by small amounts of magnesium sulfate (Nerding 1910) or calcium chloride (Katsura et al. 1933). The concentrations of these salts are critical since greater amounts lead to incomplete precipitation and may cause irreversible alterations of the lipids. 

Neutral fats and phospholipids can also be separated by dialysis (van Beers et al. 1958, Eberhagen and Betzing 1962). Phosphatides aggregate in non-polar solvents whereas neutral lipids do not. Therefore only cholesterol esters, glycerides, free fatty acids, and free cholesterol pass through a rubber membrane in petroleum ether solution. The duration of dialysis depends on the pore size of the membrane. Contamination by soluble rubber components should be prevented by prewashing the membranes. 

Separation of polar and non-polar lipids can also be achieved by column chromatography using heat-activated silica gel (Borgstrom 1952). A chloroform solution of lipids is applied to such a column and the neutral lipids are eluted with chloroform. Phosphohpids are obtained by elution with methanol and chloroform-methanol (1 1). 

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MN Vaghela, A Kilara. A rapid method for extraction of total lipids from whey protein concentrates and separation of lipid classes with solid phase extraction. J Am Oil Chem Soc 72 1117-1121, 1995. 

MA Kaluzny, LA Duncan, MV Merritt, DE Epps. Rapid separation of lipid classes in high yield and purity using bonded phase columns. J Lipid Res 26 135-140, 1985. 

WW Christie. Separation of lipid classes by high-performance liquid chromatography with the mass detector. J Chromatogr 361 396-399, 1986. 

W. W. Christie and R. Anne Urwin, Separation of lipid classes from plant tissues by HPLC on chemically bonded stationary phases,/. High Resolut. Chromatogr. 18 97 (1995). 

S. E. Ebeler and T. Shibamoto, Overview and recent developments in solid-phase extraction for separation of lipid classes, in Lipid Chromatographic Analysis (T. Shibamoto, ed.), Marcel Dekker, Inc., New York, 1994, pp. 1-49. 

High performance and low pressure liquid chromatography (adsorption) Separation of lipid classes, separation of lipids by molecular weight and degree of unsaturation Impractical for most preparative or large-scale processes... 

Separation of Lipid Classes. Lipid classes were separated according to Singleton and Stikeleather (4) on a silica column (25 X 0.46 cm, 5 pm, Luna, silica (2), Phenomenex, Torrance, CA) with a linear gradient of 2-propanol/hexane (4 3, vol/vol) to 2-propanol/hexane/water (4 3 0.75, by vol) in 20 min, then isocratically for 20 min. A prepacked silica saturator column (3 x 0.46 cm, 15-25 pm, Phenomenex) was installed between the pump and injector to saturate the mobile phase with silica before it reached the column. 

Fig. 1. The silica high-performance liquid chromatography (HPLC) radiochromatogram for the separation of lipid classes of total lipid extract (2 pL injected from the total of 100 pL methanol solution) from the castor microsomal incubation (60 min) with P" C]-oleic acid. (For HPLC conditions, see Experimental Procedures.) (1) AG (including FA, 2-5 min) (2) the unknown (3) PE (4) PC (5) ricinoleoyl-PC. FA, fatty acid AG, acylglycerol PE, phosphatidylethanolamine PC, phosphatidylcholine.

Indrasena, W. M., Parrish, C. C., Ackman, R. G. et al. (1990) Separation of lipid classes and carotenoids in Atlantic salmon feeds by thin layer chromatography with latroscan flame ionization detection. Bulletin of Aquaculture Association of Canada, 4, 36-40. 

Fig. 1. Radiochromatogram of the separation of lipid classes after the microsomal incubation of 2-[14C]oleoyl-PC.

Skipski, V. P., Smolowe, A. F., Sullivan, R. C., and Barclay, M. (1965). Separation of lipid classes by thin-layer chromatography. Biochim. Biophys. Acta 106 386-396. 

Freeman, C. P., and D. West Complete separation of lipid classes on a single thin-layer plate. J. Lipid Res. 7, 324 (1966). 

Figure 4.8 Schematic comparison of intrasource separation of lipid categories to the theoretical electrophoretic separation of lipid classes, (a) Schematically shows the selective ionization of different lipid categories under three different experimental conditions with or without adding a small amount of LiOH. (b) Schematically shows the imaginary chromatograms of lipid classes after electrophoretic analyses under corresponding experimental conditions. PC, TAG, FA, PE, and AL stand for phosphatidylcholine, triacylglyceride, nonesterified fatty acid, phosphatidylethanolamine, and anionic lipids, respectively. Christie and Han [lb]. Reproduced with permission of Elsevier.

Collectively, IM-MS clearly provides separation of lipid classes according to their charge properties, individual molecular species of a lipid class based on their molecular size (including chain length and unsaturation), and isobaric/isomeric species possessing different conformational structures [83]. This in situ drift time/collision cross section variation could be used as an additional variable to the other separation variables (e.g., intrasource separation, LC-MS elution, and optimal selection of MALDI matrix for ionization) described earlier as an aid to providing 3D analysis of complex lipid mixtures. 

Han, X., Yang, K. and Gross, R.W. (2008) Microfluidics-based electrospray ionization enhances intrasource separation of lipid classes and extends identification of individual molecular species through multi-dimensional mass spectrometry Development of an automated high throughput platform for shotgun lipidomics. Rapid Commun. Mass Spectrom. 22, 2115-2124. 

Lipid analysts were relatively slow to adapt HPLC to the separation of lipid classes, largely because of limitations in the availability of a suitable detector. In spite of this, some excellent separations have now been achieved [168], and the technique is rapidly supplanting TLC in many laboratories. In comparison to the latter, it offers superior resolution, easier quantification together with a degree of automation, cleaner fractions, and a more hygienic working environment. 

FIGURE 9.10 Separation of lipid class representatives. (1) PAR (paraffin, liquid), (2) WE (n-hexyldecyl palmitate), (3) CE (cholesteryl palmitate), (4) FAME (stearic acid methyl ester), (5) TAG (glycerol tripalmitate), (6) FOH (hexadecyl alcohol), (7) FEA (stearic add), (8) CHOL (cholesterol), (9) 1>DAG (glycerol-1,3-dipalmitate), (10) 1,2-DAG (glyc-erol-l,2-dipalmitate), (11) MAG (glycerol monopalmitate) and (12) FAA (erucylamide). For chromatographic conditions, see Section 9.2.5 concentrations 20—50 mg/1, except CHOL 100 mg/1. Source Reprinted from R. [41] with permission from Elsevier. 

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