Phospholipids column chromatography

Phospholipids have usually been isolated from milk lipids by silicic acid column chromatography. This is a difficult separation because the phospholipids are only 1% of the total as compared to 98% TG. Gent-ner et al. (1981) have not only separated the phospholipids from the remainder by TLC but have also resolved the major types of phospholipids on one plate. 

Buffered Tetrahydrofuran. In 1973, Tettamanti et al. [19) described an improved procedure for the extraction, separation and purification of brain gangliosides. In this method, the brain tissue was subjected to homogenization and extraction with buffered [potassium phosphate buffer, pH 6.8) tetrahydrofuran. Following centrifugation, diethyl ether was added and the mixture separated into organic and aqueous phase. The gangliosides, recovered exclusively in the aqueous phase, were then freed of residual phospholipids and other minor contaminants [i.e. peptides)by column chromatography on silica gel.This procedure, as shown by the authors,was superior to the commonly used chloroform/methanol... 

As described earlier, the total cellular lipids can be recovered by use of a neutral organic solvent system, such as chloroform-methanol-water. Silicic acid column chromatography, thin-layer chromatography, and high-pressure liquid chromatography (HPLC) are well suited to isolation of the ethanolamine-rich phospholipids. Only column and thin-layer chromatographic purification will be discussed at this juncture. 

Some 40 years ago in the halcyon days of study of the then new technique of silicic acid column chromatography of lipids, it was apparent to several investigators that there were nitrogen-free phospholipids in these samples. Also, these unique, acidic phospholipids did not contain inositol. In the usual protocol, lipids were applied to a silicic acid column in a nonpolar solvent such as hexane (or petroleum ether, b.p. 30-60°C), and elution continued with increasing amounts of diethyl ether. These solvent mixtures would elute the... 

We used DEAE-cellulose gel column chromatography at subzero temperature in an ethylene glycol—water mixture to isolate the microsomal cytochrome P-450 in its stable form. The already known stabilizing effect of ethylene glycol and low temperature contribute to the stabilization of the enzyme. The preparation of rat liver microsomes and the assays were carried out as described earlier. The amount of phospholipid was measured by the method of Ames and Dubin (1960). 

Extracted lipids were then separated into neutral, glyco-, and phospholipid fractions by silica gel column chromatography. Neutral lipids were eluted with 10 ml of chloroform per g of silica gel. Glycollplds were eluted with 20 ml/g of acetone, followed by phospholipids with 20 ml/g of methanol. The neutral lipid fraction... 

The triethylsilyl ether 147 thus formed regiospecifically from the diol 150 [Rl=(BnO)2 (0), R2=Bn], which was optically resolved by a chiral column chromatography, was transformed to Ins(2,4,5)P3 and Ins(l,4,5)P3 (Schema 3-23). At this stage, temporary protection of OH-1 with the silyl group is not necessary, i.e. 150 can be directly phosphorylated by the phosphite-phosphonium approach as described in the section on phosphorylation (Scheme 2-6). H The diol 150 was used furthermore as a versatile synthetic intermediate for the synthesis of myo-inositol 1,2-cyclic-4,5-trisphosphate 152 (Scheme 3-23), 8 2-acyl analogues of Ins(1,4,5)P3, and inositol phospholipid. 

The separated lipid classes can then be collected as discrete fractions in solvent. This is particularly useful for either the direct detection and quantitation of isolated lipid fractions or for further derivatization and analysis of lipid components. For example, acylglycerols, wax esters, and phospholipid classes can be separated by column chromatography and each fraction reacted as in Section 9.3.2 to analyze the FA present in each lipid class. 

The general application of reverse-phase HPLC can be employed directly in the separation and quantitation of total lipid classes. However, the high degree of resolution possible from HPLC columns is more frequently employed for the separation and analysis of individual lipid components from complex mixtures or individual lipid classes previously separated by other chromatographic procedures (e.g., column chromatography). Examples include HPLC analysis of acylglycerols (Plattner, 1981 Kuksis, 1994) and phospholipids (Porter and Weenen, 1981). 

High-performance liquid chromatography (HPLC) is the newest of the chromatographic procedures to be used for the isolation and purification of fatty acids and lipids and already shows great potential. It has been developed mainly in the analytical mode for acids and esters, triacylglycerols and phospholipids. It is usually more efficient and quicker than conventional column chromatography. It has not been extensively employed in the preparative mode for fatty acids and their derivatives. 

Various types of column chromatography are also very useful. Silicic acid columns are often the method of choice. Simple solvent systems can be used to separate neutral lipids and phospholipids. Some examples are given in Spanner (1973) and Sweeley (1969). Alternatively, silicic acid columns can be used to fractionate the phospholipid fraction at the... 

Applications of countercurrent distribution to lipid purification were already reported in the 1950s. These included the isolation of PC, SPM, or cerebrosides from brain tissue, or the placenta. It was then mentioned that lipids easily emulsify, and this adversely affects the ability to separate them. Therefore these methods were only used for crude separation. The method also requires a long time for phase separation before each phase transfer, and this procedure needs to be repeated 500-3000 times. Otsuka and Yamakawa reported the application of droplet CCC to the purification of phospholipids and glycohpids. Because the stationary phase retention is much more stable in TC-CCC than with HS-CCC, it has become possible to select appropriate two-phase solvent systems. In this study, we showed the successful separation of human brain lipids by using TC-CCC. Additionally, if an isolated band can be observed on HPTLC, the lipid can be purified by using silica column chromatography after TC-CCC cmde separation. 

Phospholipids and glycolipids have been usually fractionated from total lipids by thin-layer chromatography (TLC) or column chromatography. Then, they were later analyzed by HPLC separately. 

Fractionation of phospholipids by column chromatography is more difficult than the separation of neutral lipids. By using chloroform methanol mixtures and increasing the concentration of methanol up to 100% some fractionation is possible, although each fraction is still contaminated by other phospholipids (Hirsch and Ahrens 1958, Zollner and Kirsch 1960). 

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