SAPONIFIABLE LIPIDS 8 1 



is inactivation of degradative enzymes with heat before disintegration of the plant tissue. 

 This precaution is especially necessary for the phospholipids. A second precaution which 

 must be taken when isolating highly unsaturated lipids is to exclude oxygen and strong 

 light. 



The total lipid may be extracted from tissue with mixtures of methanol-chloroform, 

 ethanol-chloroform, or ethanol-ether. Boiling solvents have often been used, but homo- 

 genization at room temperature offers less chance for degradation to occur. If only non- 

 polar and unbound lipids are desired, the alcohol may be omitted from the extracting 

 medium or solvents like benzene, petroleum ether, etc. employed. Lipid-protein or lipid- 

 carbohydrate complexes may be insoluble in non-polar solvents. In the presence of alco- 

 hols these complexes are broken, so that the lipid extracted gives no indication as to how 

 it may have been complexed in situ. On the other hand, proteo- or peptido- lipids are 

 known which are soluble in lipid solvents but release amino acids on hydrolysis (19). In 

 the presence of phospholipids many non-lipids are extracted into lipid solvents. They can 

 be effectively removed by mixing a 2: 1 chloroform-methanol extract with 0. 2 its volume 

 of water. After centrifuging or long standing to separate the layers, non-lipid material 

 is found in the upper, aqueous layer (20). Proteolipids form a fluffy layer at the interface 

 and can be separated from other constituents (21). 



By addition of acetone to a lipid extract prepared as described above phospholipids 

 (with small amounts of sterolins and some waxes) are largely precipitated. Triglycerides, 

 other esters, sterols, terpenes, etc. remain in solution. However, if much triglyceride 

 is present, it will carry some phospholipid with it into the acetone -soluble fraction. Frac- 

 tionation of the phospholipid precipitate will depend to some extent on what types of com- 

 ponents are present. If inositol lipids are desired, the other phospholipids can be re- 

 moved by prolonged extraction with cold, glacial acetic acid and partition between glacial 

 acetic acid and benzene. Inositol lipid goes into the benzene phase. After evaporation of 

 the benzene, sterolins (if present) may be removed by extraction with a 2: 1 ethanol-chloro- 

 form mixture leaving relatively pure inositol lipid (11). Another method for fractionation 

 of the phospholipids depends on precipitating lecithin as a cadmium salt from alcoholic 

 solution. The cadmium salt is subjected to further purification steps and finally the leci- 

 thin recovered by dissolving the salt in chloroform and shaking this solution with an equal 

 volume of 30% methanol. Pure lecithin dissolves in the chloroform layer, and the cadmium 

 chloride goes into the aqueous methanol. After removal of lecithin the remaining phospho- 

 lipids may be fractionated by dissolving in chloroform and adding ethanol stepwise. Ino- 

 sitol lipids precipitate first followed by phosphatidylserine and finally phosphatidyle- 

 thanolamine. These processes may be repeated or refined by using countercurrent dis- 

 tribution methods with hexane and methanol as solvents (22). 



Going back to the solution of lipids remaining after removal of phospholipids, sepa- 

 ration of these constituents in their original form is very difficult. Free fatty acids may 

 be removed by extraction with sodium carbonate solution, long-chain hydrocarbons and 

 alcohols as urea complexes (Chap. 6), and sterols as digitonides or tomatinides (Chap. 8). 

 The lower terpenes and other volatile compounds may be removed by distillation (or steam 

 distillation); higher terpenoid acids will be extracted with free fatty acids into sodium car- 

 bonate solution. The long-chain fatty acids may then be separated as urea complexes from 

 the higher terpenoid acids (23). Glycolipids are not precipitated by acetone but can be 

 separated from everything except triglycerides by partitioning the mixture of acetone- 

 soluble components between methanol and heptane. Glycolipids (including sterolins) and 

 triglycerides go into the methanol layer. Further separation depends on chromatography 

 (24). 



A mixture containing chiefly triglycerides and/or ester waxes may be separated into 

 its ester components only by tedious and unsatisfactory procedures. Of most general ap- 

 plication are fractional distillation at very low pressures or fractional crystallization 

 from acetone at very low temperatures. The more usual procedure is to submit the esters 



