Working with Lipids:- Lipid Extraction Requires Organic Solvents

Neutral lipids (triacylglycerols, waxes, pigments, and so forth) are readily extracted from tissues with ethyl ether, chloroform, or benzene, solvents that do not permit lipid clustering driven by hydrophobic interactions. Membrane lipids are more effectively extracted by more polar organic solvents, such as ethanol or methanol, which reduce the hydrophobic interactions among lipid molecules while also weakening the hydrogen bonds and electrostatic interactions that bind membrane lipids to membrane proteins. A commonly used extractant is a mixture of chloroform, methanol, and water, initially in volume proportions (1:2:0.8) that are miscible, producing a single phase. After tissue is homogenized in this solvent to extract all lipids, more water is added to the resulting extract and the mixture separates into two phases, methanol/water (top phase) and chloroform (bottom phase). The lipids remain in the chloroform layer, and more polar molecules such as proteins and sugars partition into the methanol/water layer.

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:- Adsorption Chromatography Separates Lipids of Different Polarity

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