Principles of Lipid Extraction

The potential toxicity of the solvents to the users is another important factor for consideration. Therefore, it is always better to employ safer extraction conditions if possible. For example, the isopropanol-hexane (3 2 by volume) system was used in many cases due to their relatively low toxicity [21,22]. Unfortunately, this system is unable to recover some lipid classes such as gangliosides quantitatively. 

There are many cases in which alternative or modified procedures must be used. For example, butanol saturated with water appears to be the very useful solvent mixture to disrupt the inclusion complexes of lipids in starch and give the best extraction 

Lipid extracts obtained from biological samples, as aforementioned, tend to contain significant amounts of nonlipid contaminants, such as sugars, amino acids, urea, and salts. These must be removed before the lipids are analyzed. A common and classical approach is to use a simple washing procedure devised by Folch, Lees and Sloane Stanley [17], in which a chloroform-methanol (2 1, v/v) extract is shaken and equilibrated with one-fourth its volume of saline solution (i.e., 0.88% potassium chloride in water). The mixture partitions into two layers, of which the lower phase is comprised of chloroform-methanol-water in a proportion of 86 14 1 (by volume) and contains virtually all of the lipids, while the upper phase consists of the same solvents in the proportion of 3 48 47 (by volume), respectively, and contains much of the nonlipid contaminants. It is important that the proportion of chloroform, methanol, and water in the combined phases should be as close as possible to 8 4 3 (by volume), otherwise selective losses of lipids may occur. If a second wash of the lower phase is needed to remove any remaining contaminants, a mixture of similar composition to that of the upper phase should be used, i.e., methanol-saline solution (1 1, v/v). 

It should be recommended that in any extraction procedure, it is important that the weight of fresh tissue extracted is recorded, together with the weight of lipid obtained from it. For many purposes, it could be desirable to normalize the lipid content to the amount of dry materials or protein content in the tissue later. 

It should be emphasized that it is difficult to achieve a complete recovery of every lipid class by any known method of extraction. Any incomplete recovery will lead to an inaccurate measurement of the lipid content in a sample or an inconsistency in the results from different laboratories if the analytical methods are based on external calibration curves. To avoid this potential problem, internal standards may be 

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