478 V. CHEMISTRY OF PHOSPHATIDES AND CEREBROSIDES 



containing 20% chloroform. The precipitate separating on coohng still 

 contains small amounts of phosphorus-containing compounds. 



In order to remove the impurities, a zinc reagent — Zn(OH)2+NH3 (in 

 methanol) — is added to a methyl alcohol solution containing 10% of chloro- 

 form and the mixture is treated until the precipitate flocculates. The pre- 

 cipitate chiefly contains compounds rich in phosphorus. The precipitate 

 originating on cooling the residue is filtered off, and it is again dissolved in a 

 mixture of methyl alcohol and 10% chloroform. Phrenosine separates 

 from the cooled filtrate chiefly in the form of glistening platelets or in an 

 amorphous state. 



(2) Preparation of Phrenosine and Cerasine by the Rosenheim Method*^^'*^"^ 



a. Separation of Crude Cerebrosides. In order to prepare the crude 

 cerebroside mixture, 10 kg. of macerated brain is mixed with 10 liters of 

 acetone and is allowed to stand for 24 hours, with frequent stirring. The 

 water-containing acetone is removed by pouring through fine muslin, and 

 the brain is treated repeatedly with fresh acetone until all the cholesterol 

 has been removed. After this treatment is completed, the acetone is re- 

 moved from the residue, and the latter is dried in thin layers on a warm 

 plate. It is then exhaustively extracted with petroleum ether as long as 

 any material is being removed. The residue is again dried as above. It is 

 ground to a fine powder, which amounts to about 13% of the fresh brain 

 (1300 g.). Batches of 500 g. of this powdered residue are extracted with 

 1500 ml. of pyridine (b.p., 115°C.) by warming for about 20 minutes to 

 45-50°C. After cooling, the filtrate is concentrated to a small volume, and 

 the galactosides are precipitated by the addition of 3 or 4 volumes of ace- 

 tone and by cooling to 0°C. After the residue has been filtered through a 

 glass filter without pressure, the residue is suspended in acetone, filtered by 

 suction, and the acetone is removed from the precipitate by drying in 

 vacuo. 



Further elimination of ether-soluble impurities is effected by extraction 

 with ether in a Soxhlet apparatus. The crude cerebroside is a yellow pow- 

 der which contains only about 0.5% phosphorus. The yield is about 2% of 

 the fresh brain. 



b. Removal of Phosphorus-Containing Impurities. The phosphatides 

 and other similar contaminants are readily removed by recrystallizing 

 them twice from alcohol containing 67% chloroform. The purified cere- 

 broside mixture is a white powder containing only a trace of phosphorus 

 (0.08%). 



c. Separation of Phrenosine and Cerasine.^-- Phrenosine and cerasine 



«i O. Rosenheim, Biochem. J., 7, 604-610 (1913). 

 ^" O. Rosenheim, Biochem. J., 8, 110-120 (1914). 



