Lipidos

Resolution and Analysis of Lipids
In exploring the role of lipids in a biological process, it is often useful to know which lipids are present, and in what proportions. Because lipids are insoluble in water, their extraction from tissues and subsequent fractionation require the use of organic solvents and some techniques not commonly used in the purification of water-soluble molecules such asproteins and carbohydrates. In general, complex mixtures of lipids are separated by differences in their polarity or solubility in nonpolar solvents. Lipids that contain ester- or amide-linked fatty acids can be hydrolyzed (saponified) by treatment with acid or alkali, to yield their component parts for analysis.
Lipid Extraction Requires Organic Solvents
Neutral lipids (triacylglycerols, waxes,pigments, etc. ) are readily extracted from tissues with ethyl ether, chloroform, or benzene, solvents in which lipid clustering driven by hydrophobic interactions does not occur. Membrane lipids are more effectively extracted by more polar organic solvents, such as ethanol or methanol, which reduce the hydrophobic interactions among lipid molecules but also weaken the hydrogen bonds andelectrostatic interactions that bind membrane lipids to membrane proteins. A commonly used extractant is a mixture of chloroform, methanol, and water, initially in proportions that are miscible, producing a single phase (1:2:0.8, v/v/v). After homogenizing tissue in this solvent to extract all lipids, more water is added to the resulting extract, and it separates into two phases, methanol/water (top phase)and chloroform (bottom phase). The lipids remain in the chloroform, and more polar molecules (proteins, sugars) partition into the polar phase of methanol/water (Fig. 9-21)
| Figure 9-21 Some common procedures used 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 ofunextractable 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 by a rising solvent front, less polar lipids traveling farther than more polaror charged lipids. TLC with appropriate solvents also can be used to separate individual lipid species from a single class; 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 esterlinked fatty acids is (d) transesterified in a warm aqueoussolution of NaOH and methanol, producing a mixture of fatty acyl methyl esters, which are then (e) separated on the basis of chain length and degree of saturation by gas-liquid chromatography. Precise determination of molecular mass, by mass spectroscopy (not shown), allows unambiguous identification of individual lipids. The lipid is ionized and volatilized by heat and the resulting molecular ion ispassed through an electromagnetic field, which deflects ions to a degree dependent on their size. By comparison with standard ions of known molecular mass, the mass of the unknown molecular ion is determined with such great accuracy that the structure of the lipid can be deduced.. |
Adsorption Chromatography Separates Lipids of Different Polarity
The complex mixture of tissue lipids can befractionated further by chromatographic procedures based on the different polarities of each class of lipid. In adsorption chromatography (Fig. 9-21), an insoluble, polar material such as silica gel (a form of silicic acid, Si(OH)4), is packed into a long, thin glass column, and the lipid mixture (in chloroform solution) is applied to the top of the column. The polar lipids bind tightly to the polar...