PREPARATION OF THE CEREBROSIDES 479 



can be separated because of a differential solubility in acetone. 50 g. 

 of the purified cerebroside mixture is suspended in 3500 ml. of 90% ace- 

 tone and heated on a water bath at 56°C. The insoluble residue (about 

 15%) is filtered off and the filtrate is allowed to stand at 37°C. After 16 to 

 20 hours, the precipitate, Avhich adheres to the walls of the flask and which 

 contains the phrenosine fraction, is separated by filtration through a filter 

 heated to 37 °C. The filtrate is allowed to stand for at least 24 hours in the 

 refrigerator, after which time the cerasine is largely precipitated. This 

 cerebroside starts to precipitate when the solution temperature falls below 

 28°C. It is filtered by suction, washed with acetone, and dried in vacuo. 

 The phrenosine fraction amounts to about 50% of the total crude cerebro- 

 side, while the cerasine fraction is much smaller. 



d. Purification of Phrenosine. The crude phrenosine (32.5 g.) is pul- 

 verized, dissolved in 120 ml. of chloroform at 60°C., and 180 ml. of acetic 

 acid heated to 60°C. is added to it. The phrenosine precipitates after 

 standing overnight at 37°C. ; it is then filtered and washed with a mixture 

 of 3:2 acetic acid and chloroform at 37°C. The moist precipitate is 

 treated with 200 ml. of the same mixed solvents; it is then filtered and dried 

 in vacuo. About 6.6 g. of precipitate is obtained from the first 2 mother 

 liquors when the solution stands at room temperature; this precipitate is 

 worked up with the cerasine. The phrenosine preparation, which still con- 

 tains traces of cerasine, is purified by solution in 40 volumes of chloroform, 

 to which is added 60 volumes of warm acetone; the precipitate of phreno- 

 sine which originates when the solution stands at 37 °C. is filtered, and the 

 reprecipitation is repeated twice. The preparation is found to be cerasine- 

 free. The final purification employed involves crystallization from acetone 

 containing 50% pyridine and acetone containing 10% of water. 



e. Purification of Cerasine. For the purification of the cerasine, the 

 crude sample (10 g.) is dissolved in 40 ml. of chloroform at 50 °C., and 60 

 ml. of acetic acid heated to 60°C. is added to it. The solution remains 

 clear until the temperature drops to about 40 °C., after which the phreno- 

 sine present separates out. The solution is kept at 37 °C. for some time 

 and the precipitate is removed. On cooling of the filtrate to 26 °C., 

 separation of a gelatinous precipitate of cerasine occurs. This is filtered 

 and washed with a 3 : 2 acetic acid-chloroform mixture. The precipitate is 

 then suspended in acetone and is again filtered. A second purification is 

 carried out, using 50 ml. of the acetic acid-chloroform mixture, at which 

 time about 0.5 g. of phrenosine separates at 37 °C. WTien this is repeated 

 once more, no additional phrenosine precipitates from the mixture on cool- 

 ing to 37*'C., even if the solution stands for many hours. If the precipitate 

 still gives a qualitative test for phrenosine, the precipitate can be further 

 purified by dissolving it in 10 volumes of warm pyridine, and by adding the 

 same volume of acetone warmed to 45 °C. Any phrenosine which precipi- 



