482 V. CHEMISTRY OF PHOSPHATIDES AND CEREBROSIDES 



(6) Second Method of Purification.'^" The cmde nervone was taken up 

 by warming in 20 volumes of a 1:2 mixture by volume of chloroform and 

 methanol. The following fractions were obtained: 



Iodine number 



Fraction 3, by cooling at room temperature 55.8 



Fraction 4, by refrigeration 60 . 



Fraction 5, by recrystallization of the residue from the 



mother liquor from methanol 61.0 



The third fraction was considered to be the purest. 



(c) Purification as the Acetyl Compound. After acetylation by Thier- 

 f elder's method, ^^'^ fractionation of a methyl alcohol solution was employed. 

 One fraction (6) was obtained on cooling while the other fraction (7) was 

 obtained from the filtrate. After removal of the acetyl groups by boiling 

 for 30 minutes with 0.5 N methanol-sodium hydroxide, the two fractions 

 had the following iodine numbers : 



Fraction 6 52 . 1 



Fraction 7 62.0,62.6 



It is obvious that Fraction 7 is the purest preparation of nervone obtained. 

 The purer fractions of nervone comprised only about 10% of the original 

 crude nervone. Most of the nervone remained in the less pure fractions. 



4. Tests for Purity of the Cerebrosides 



Three tests are useful in establishing the purity of phrenosine and cera- 

 sine. 



(1) Proof of Absence of Phosphatides 



The phosphatides are the impurities most frequently present. A pure 

 preparation in fairly large amounts (0.3 g.) should give no phosphorus reac- 

 tion after incineration to an ash with sodium carbonate and potassium 

 nitrate with ammonium molybdate. 



(2) Crystallization Test 



When dissolved in a mixture of methyl alcohol and 10% chlorofoi-m by 

 treatment over a water bath, the clear solution changes to a mass of crystals 

 which resemble those of cholesterol. ^^^ 



(3) Selenite Plate Test 



To determine whether or not cerasine is mixed with phrenosine, the 

 Rosenheim test*^^ is most useful. This depends upon the variation in 

 properties of the two cerebrosides in polarized light. One dissolves 8-10 



«* H. Thierfcldcr, Z. plujsiol. Chem., SO, 248-250 (1914). 



