Working with Lipids:- Adsorption Chromatography Separates Lipids of Different Polarity

Complex mixtures of tissue lipids can be fractionated by chromatographic procedures based on the different polarities of each class of lipid. In adsorption chromatography (Fig. 10–23b), an insoluble, polar material such as silica gel (a form of silicic acid, Si (OH)₄) is packed into a glass column, and the lipid mixture (in chloroform solution) is applied to the top of the column. (In high - performance liquid chromatography, the column is of smaller diameter and solvents are forced through the column under high pressure.) The polar lipids bind tightly to the polar silicic acid, but the neutral lipids pass directly through the column and emerge in the first chloroform wash. The polar lipids are then eluted, in order of increasing polarity, by washing the column with solvents of progressively higher polarity. Uncharged but polar lipids (cerebrosides, for example) are eluted with acetone, and very polar or charged lipids (such as glycerophospholipids) are eluted with methanol.

Thin-layer chromatography on silicic acid employs the same principle (Fig. 10–23c). A thin layer of silica gel is spread onto a glass plate, to which it adheres. A small sample of lipids dissolved in chloroform is applied near one edge of the plate, which is dipped in a shallow container of an organic solvent or solvent mixture—all of which is enclosed within a chamber saturated with the solvent vapor. As the solvent rises on the plate by capillary action, it carries lipids with it. The less polar lipids move farthest, as they have less tendency to bind to the silicic acid. The separated lipids can be detected by spraying the plate with a dye (rhodamine) that fluoresces when associated with lipids or by exposing the plate to iodine fumes. Iodine reacts reversibly with the double bonds in fatty acids, such that lipids containing unsaturated fatty acids develop a yellow or brown color. A number of other spray reagents are also useful in detecting specific lipids. For subsequent analysis, regions containing separated lipids can be scraped from the plate and the lipids recovered by extraction with an organic solvent.

FIGURE 10–23 Common procedures in the extraction, separation, and identification of cellular lipids. (a) Tissue is homogenized in a chloroform/methanol/water mixture, which on addition of water and removal of unextractable sediment by centrifugation yields two phases. Different types of extracted lipids in the chloroform phase may be separated by (b) adsorption chromatography on a column of silica gel, through which solvents of increasing polarity are passed, or (c) thin layer chromatography (TLC), in which lipids are carried up a silica gel coated plate by a rising solvent front, less polar lipids traveling farther than more polar or charged lipids. TLC with appropriate solvents can also be used to separate closely related lipid species; for example, the charged lipids phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol are easily separated by TLC. For the determination of fatty acid composition, a lipid fraction containing ester-linked fatty acids is trans esterified in a warm aqueous solution of NaOH and methanol (d), producing a mixture of fatty acyl methyl esters. These methyl esters are then separated on the basis of chain length and degree of saturation by (e) gas-liquid chromatography (GLC) or (f) high-performance liquid chromatography (HPLC). Precise determination of molecular mass by mass spectrometry allows unambiguous identification of individual lipids.

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مواضيع ذات صلة

The Composition and Architecture of Membranes:- All Biological Membranes Share Some Fundamental Properties

The Composition and Architecture of Membranes:- Each Type of Membrane Has Characteristic Lipids and Proteins

Working with Lipids:- Mass Spectrometry Reveals Complete Lipid Structure

Working with Lipids:- Gas-Liquid Chromatography Resolves Mixtures of Volatile Lipid Derivatives

Working with Lipids:- Lipid Extraction Requires Organic Solvents

Lipids as Signals, Cofactors, and Pigments: -Plants Use Phosphatidylinositols, Steroids, and Eicosanoidlike Compounds in Signaling

Lipids as Signals, Cofactors, and Pigments:- Steroid Hormones Carry Messages between Tissues

Lipids as Signals, Cofactors, and Pigments:- Phosphatidylinositols and Sphingosine Derivatives Act as Intracellular Signals

Structural Lipids in Membranes:- Sterols Have Four Fused Carbon Rings

Structural Lipids in Membranes:- Sphingolipids at Cell Surfaces Are Sites of Biological Recognition

Structural Lipids in Membranes:- Sphingolipids Are Derivatives of Sphingosine

Structural Lipids in Membranes:- Chloroplasts Contain Galactolipids and Sulfolipids