SAPONIFIABLE LIPIDS °^ 



Stance, cuticle waxes may rise to the surface as a film if fruits, leaves, etc. , are merely 

 immersed for a few minutes in boiling water. Such a preparation would contain no phospho- 

 lipids and probably only traces of triglycerides. 



Procedures have not been given for preparation of the rarer types of lipids which 

 have been mentioned. Since at the present state of our knowledge each one is a special 

 case, the original papers on them should be consulted. Although suberin and cutin are 

 hardly rare in the plant kingdom, they are practically defined by the arbitrary and in- 

 volved methods by which they have been prepared, and small variations in experimental 

 details might yield quite different products. For suberin see the paper of Zetsche and 

 Rosenthal (31); for cutin the paper of Legg and Wheeler (32). 



CHARACTERIZA TION 



Many common methods used for characterization of saponifiable lipids are well 

 described in elementary texts of organic chemistry and biochemistry. They include such 

 determinations as iodine number, saponification value, etc. , and depend for their inter- 

 pretation on a knowledge of what type of lipid is being dealt with. A recent journal tissue 

 is devoted to reviews of methods of lipid analysis (33). Pertinent references are also 

 listed periodically in The Journal of Lipid Research. 



Several color tests are available to indicate what types of lipids may be present in 

 a mixture. Phospholipids are indicated by color reactions for phosphorus such as may 

 be given by adding ammonium molybdate solution and concentrated sulfuric acid to an 

 ethereal solution. Glycolipids are indicated by positive tests for carbohydrate such as 

 the Molisch reaction. For identification of other components the simplest procedure is 

 probably saponification of the lipid mixture followed by separation of the non-saponifiable 

 material. The latter may be tested for the presence of sterols, long-chain alcohols, hy- 

 drocarbons, etc. by methods described in the appropriate chapters. The presence of soap 

 in the aqueous layer is obvious on shaking and indicates the presence of long chain fatty 

 acids. After acidification of the saponification mixture and removal of the fatty acids, 

 glycerol may be detected by evaporating to a small volume. If triglycerides or other 

 glycerol derivatives were present in the original lipid, the odor of acrolein will be apparent 

 on heating this concentrated solution strongly with sodium bisulfate. Sphingosine is re- 

 leased from sphingolipids only by a combination of basic and acid hydrolysis, but acidic 

 hydrolysis alone produces a free amino group which may be detected by its color reaction 

 with ninhydrin. The other types of nitrogenous compounds released on hydrolysis of 

 phospholipids may be detected by methods described in Chapter 14. Ester waxes may be 

 distinguished from triglycerides by the fact that after saponification long-chain alcohols 

 appear in the benzene -soluble fraction and may be identified by methods described in 

 Chapter 6. If the individual fatty acids are separated and identified, the appearance of 

 acids containing more than 30 carbon atoms is also evidence for the presence of ester 

 waxes. 



Characterization by means of paper chromatography has not been of much value 

 when applied to the triglycerides as such. However, Cormier et al. (34) have shown 

 that triglycerides as a group can be separated from some other types of lipids on paper 

 impregnated with silicic acid using as a solvent ether -petroleum ether-heptane 4:25:25. 

 Identification was made by dipping in an alcoholic solution of Sudan black, which is tightly 

 bound to lipids, and washing off background dye with alcohol at 50° C. Phospholipids have 

 been separated and characterized also using paper impregnated with silicic acid and a 

 solvent consisting of diisobutyl ketone-acetic acid-water 8:5:1 (26). Phosphatides are 

 detected by spraying with the dye Rhodamine 6G and observing in ultra-violet light while 

 the paper is still wet. Quantitative measurement is possible by eluting the spots and de- 

 termining phosphorus in them. A more sensitive detection reagent which shows up all 

 types of lipids is a dilute solution of protoporphyrin in hydrochloric acid (35). After wash- 



