498 V. CHEMISTRY OF PHOSPHATIDES AND CEREBROSIDES 



(a) Separation of the Saturated Fatty Acids. The fatty acids remaining 

 in solution are extracted by repeated treatment with petroleum ether. 

 The methyl alcohol present, which is extracted by the petroleum ether, can 

 be removed by washing the petroleum ether solution with water. The 

 residue remaining after removal of the petroleum ether consists largely of 

 the methyl esters of unsaturated acids and small amounts of the methyl 

 esters of the saturated acids. The total amount recovered in the two frac- 

 tions is approximately theoretical. Two procedures may be employed 

 from this point for the separation of the saturated fatty acids: 



a'. Thierfelder Procedure :^^" The first procedure involves solution of 

 the solid methyl esters in ether, followed by treatment with an excess of 

 0.5 A^ alcoholic sodium hydroxide. The sodium salts which precipitate are 

 filtered; they are next suspended in ether, filtered again, and washed fur- 

 ther with ether. They are decomposed by shaking in mineral acid and 

 petroleum ether. The acids which are formed dissolve in the petroleum 

 ether. This solution is washed with water and dried with sodium sulfate; 

 the ether is then distilled off. The residue is cerebronic acid. 



The petroleum ether filtrate which contains the methyl ester of ligno- 

 ceric acid is shaken with dilute sulfuric acid and then with water. The 

 ester remaining after distillation of the ether is saponified with 30 volumes 

 of 0.5 A'' methanolic sodium hydroxide for 1 hour. The sodium salt of 

 lignoceric acid, which precipitates at 0°C., is filtered off and obtained as the 

 acid by the process used above for the preparation of cerebronic acid. 



b'. Thierfelder-Ivlenk Procedure:^ The second method for the separa- 

 tion of cerebronic and lignoceric acids also requires a saponification of the 

 methyl esters. The fatty acid mixture is dissolved in 70 parts of ethyl 

 alcohol. The solution is heated to boiling, and an excess of a hot saturated 

 solution of magnesium acetate in methyl alcohol is added. The precipitate, 

 which is the magnesium salt of cerebronic acid, is filtered from the solution 

 while hot; the salt is then suspended in diethyl ether and is changed to the 

 acid by the addition of a drop of concentrated sulfuric acid and by shaking 

 the mixture. After thorough washing, the ether solution is dried with 

 sodium sulfate and the cerebronic acid is obtained by evaporation of the 

 ether. 



The hot filtrate obtained after removal of the magnesium cerebronate is 

 cooled, and magnesium ligiiocerate crystallizes. This precipitate is fil- 

 tered off and the free acid is obtained from this product by a procedure 

 similar to that described above for cerebronic acid. 



(6) Separation of the Unsaturated Acids. The ester mixture which re- 

 mains in solution after the precipitation of the esters of the saturated acids 

 in the cold consists largely of methyl esters of the unsaturated fatty acids. 

 This is saponified with 35 volumes of 0.5 N methanolic sodium hydroxide 

 by refluxing for 1 hour. The mixture, after cooling, is diluted with 2 



