82 SAPONIFIABLE LIPIDS 



to saponification and then identify the constituent acids and alcohols. This, of course, 

 gives no information as to how they were originally combined. Triglycerides are saponi- 

 fied by refluxing for 1-2 hours with about five times their weight of 6% potassium hydrox- 

 ide in 95% ethanol. The long chain ester waxes require more drastic conditions for com- 

 plete hydrolysis e. g. refluxing for 12 hours a solution of wax in benzene plus an equal 



volume of 10% KOH in alcohol. This difference in ease of hydrolysis makes it possible 

 to remove some triglycerides from an ester wax by saponification without too much loss 

 of the wax. The chief danger to be noted in saponification is that prolonged heating may 

 cause isomeric charges in the double bond positions of polyunsaturated acids. It is some- 

 times recommended that lecithin be hydrolyzed by standing with N potassium hydroxide 

 for 16 hours at 37° C. 



After hydrolysis long chain alcohols resulting from saponification of ester waxes 

 may be removed (along with any unsaponified material) by extraction with benzene, etc. 

 The remaining mixture is then acidified and fatty acids extracted with ether. 



Mixtures of long chain fatty acids either naturally occurring or derived by saponifi- 

 cation can be separated by various crystallization and distillation procedures. A common 

 method is to dissolve the fatty acids in acetone and cool to -60° to = -70° C. Polyunsatu- 

 rated acids remain in solution while others crystallize out. Redissolving the precipitate 

 and recrystallizing at -30° to -40° C. leaves most of the mono-unsaturated acids in solu- 

 tion. A final recrystallization from ether at -30° deposits most of the saturated acids. 

 The separations are not perfect, but by repetition and combination with other methods a 

 satisfactory fractionation can usually be achieved according to degree of unsaturation. 

 Another type of separation depends on the fact that lead salts of the saturated acids are 

 insoluble in ether or alcohol whereas those of the unsaturated acids are soluble. Further 

 fractionation may be achieved by distillation of methyl esters of the acids under reduced 

 pressure (0. 1-5.0 mm. of mercury). 



Chromatographic separation of the saponifiable lipids has not yet been widely ap- 

 plied although it is certain to become more prominent since it offers many advantages 

 over solvent separation methods. Partition chromatography is apparently more useful 

 than adsorption chromatography. Gas chromatography has also been successfully em- 

 ployed (25). Marinetti (26) has reviewed methods of phospholipid chromatography. Cole 

 and Brown (27) have described a procedure for fractionating plant waxes by chromatography 

 on alumina and silica gel. A general review of lipid chromatography on silicic acid has 

 been prepared by Wren (28). Silicic acid chromatography is particularly useful for anionic 

 lipids since less polar lipids move rapidly through the column. Thus phospholipids may 

 be readily separated from other lipids and then further subdivided among themselves. A 

 typical procedure would be to slurry the silica gel with chloroform-methanol 2: 1, add the 

 lipid solution in chloroform, and start development with pure chloroform. As non-polar 

 lipids move through the column, the concentration of methanol in the developing solvent 

 is gradually increased. Finally the most polar substances are eluted with pure methanol. 

 Carroll (29) has suggested that Florisil offers many advantages over silicic acid as an ad- 

 sorbent for lipid chromatography. Separations are similar but more convenient. 



The elution peaks can often be recognized by measuring absorption at 300 nijU. This 

 absorption is given by oxidation products of the unsaturated fatty acids when careful exclu- 

 sion of oxygen is not practiced. Another way of identifying lipid-containing eluate frac- 

 tions is to spot a small amount on a ferrotype plate. When the volatile solvent evaporates, 

 small amounts of lipid are easily visible (30). 



Although the above procedures have been written as though a given lipid mixture 

 might contain every possible constituent, the situation in nature is seldom quite so dis- 

 couraging. In many cases one lipid, or at least one type of lipid, will be predominant; 

 and the problem is only to remove small amounts of a few other constituents. For in- 



