Lipids column chromatography

Lipoproteins (from human plasma). Individual human plasma lipid peaks were removed from plasma by ultracentrifugation, then separated and purified by agarose-column chromatography. Fractions were characterised immunologically, chemically, electrophoretically and by electron microscopy. [Rudel et al. Biochem J 13 89 1974.]... 

High performance liquid chromatography (HPLC) has been by far the most important method for separating chlorophylls. Open column chromatography and thin layer chromatography are still used for clean-up procedures to isolate and separate carotenoids and other lipids from chlorophylls and for preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography. The whole analysis should be as brief as possible, since each additional step is a potential source of epimers and allomers. 

Following their use in the behavioral trials, sub-sets of the experimental females (n = 8) and experimental newly-emerged males (n = 8) were immediately sacrificed with an overdose of brevital sodium and their skin lipids were extracted with hexane overnight (LeMaster and Mason 2002). After removal of the animals, the extracts were returned to Western Oregon University where the methyl ketones composing the female sexual attractiveness pheromone were isolated using column chromatography (described in LeMaster and Mason 2002). 

Heptachlor and heptachlor epoxide have been measured in samples of human milk using GC/ECD and GC/MS (Mussalo-Rauhamaa et al. 1988 Polishuk et al. 1977b Ritcey et al. 1972). Sample preparation steps for milk involve homogenization with chloroform/methanol, lipid extraction with petroleum ether, hexane or acetone-hexane, clean-up by column chromatography, and elution with acetonitrile, hexane, methylene chloride, or dichloromethane-petroleum ether. Precision, accuracy, and sensitivity were not reported for most of the studies however, one study reported a sensitivity in the low-ppb range (Ritcey et al. 1972). 

Homogenize with chloroform/ methanol lipid extract with petroleum ether or hexane clean-up by column chromatography elution with acetonitrile, hexane, and methylene chloride. 

Bamba, T., Fukusaki, E., Minakuchi, H., Nakazawa, Y, and Kobayashi, A., Separation of polyprenol and dolichol by monolithic silica capillary column chromatography. Journal of Lipid Research 46(10), 2295-2298, 2005. 

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. 

Prepare the lipid for chromatography by dissolving 50 to 75 mg of crude lipid in a minimum of hexane (5 to 10 mL). Open the stopcock and allow excess solvent to drain from the column until the level of solvent just reaches the top of the silica gel column. Very carefully add the solution of crude lipid to the top of the column. This should be done by using a Pasteur pipet and allowing drops of the solution to run down the inside of the glass column to the top of the silica gel bed. It is important not to disturb the top of the silica gel column. After addition of the lipid solution, begin to collect a fraction from the bottom of the column into a 25-mL Erlen meyer flask. Set the flow rate to about 2 drops per second. When the level of solution in the column reaches the top of the silica gel, turn off the stop-... 

The quality of FAME prepared by the methods described in this unit must be examined by GC analysis. Generally, impurities in the extracted lipid samples are not removed before methylation. If the GC results are not satisfactory due to sample contamination, additional steps may be necessary to clean the sample either before or after methylation. Commonly used techniques for purifying lipid samples are thin-layer chromatography (TLC), solid phase extraction (SPE), and column chromatography. 

Another point of interest was the time required to equilibrate the system after changes were made in solvent composition. While the ChromSpher Lipids column had a column volume of ca. 3 ml, an increase in ACN concentration was not noted until the introduction of 7-8 ml of solvent (determined with refractive index detector). The problem of ACN-silver ion interaction and subsequent ACN retention is not new and may be noted in all forms of chromatography employing silver ions in the stationary phase. In the isocratic system, the column was equilibrated with the appropriate solvent mix for at least 0.5 h before sample injection. Since ACN dissolves very slowly into hexane, the ACN-hexane solvent mix was thoroughly stirred for 5 min before use. To obtain reproducible retention times, thorough mixing of the ACN and hexane is essential. 

Fig. 30 Silver ion high-performance liquid chromatography (Ag-HPLC-FID) with flame ionization detector (FID) analysis of the triacylglycerols of chromatographed Crepis alpina seed oil. Ag-HPLC-FID conditions 0.5-mg sample 5-micron Chromspher Lipids column (Chrompack International, Middelburg, The Netherlands) (4.6 X 250 mm) mobile phase 0.5% acetonitrile in hexane (v/v) flow rate 1.0 ml/min FID. Chromatogram peak triacylglycerol fatty acid abbreviations S, saturated (palmitic and stearic) O, oleic L, linoleic and Cr, crepenynoic fatty acids.

Kaul and Lester (10) reported the preparation of six novel glycophosphoceramide fractions from the above crude concentrate from tobacco leaves. The crude concentrate was first resolved into two groups by column chromatography on diethyl aminoethyl-cellulose. The first group contained no acetyl residues, whereas the second group contained one N-acetyl per phosphorus. Three lipid fractions from each group were further resolved by chromatography on Porasil columns. The chemical composition and the percent of the total P in the crude concentrate of these lipid frac-... 

Isolation of the neutra-L glycosphingolipids. In a typical extraction procedure, 10purified human neutrophils yielded 100-150 mg of total glycosphingolipids. As shown in Table I, glycosphingolipids account for approximately 10% of the total cellular dry weight of the neutrophil. Separation of the total neutrophil lipids by DEAE-sephadex and silicic acid column chromatography yielded 70-100 mg of neutral glycosphingolipids from lO cells. 

A constant observation when the MRP were separated by various methods was that antioxidative effect was found in many different fractions. Both the dialysates and the retentates from dialysis were antioxidative to some extent. All the electrophoresis fractions exhibited some antioxidative effect. Attempts to separate the MRP by column chromatography on Sephadex G-50 have resulted in several fractions with some antioxidative effect, and so on. This indicates that several antioxidative products are formed by the Maillard reaction, possibly differing in molecular size and chemical structure, but perhaps with one single antioxidative functional group in common, such as a free radical function. However, it can not be excluded that the MRP contain a few entirely different antioxidants with different modes of action. Various mechanisms have also been suggested. Eichner (6) claimed MRP to inactivate the hydroperoxides formed by the lipid oxidation. There are also reports on the complex binding of metals by MRP (18, 19). 

---