PKEPARATION OF THE CEREBROSIDES 481 



precipitate of cephalin which forms on the sides and bottom of the flask. 

 The precipitate is washed with absohite alcohol. This is added to the main 

 solution. The petroleum ether is removed by reducing the volume of the 

 solution under reduced pressure. The remaining liquid is brought to the 

 original volume with absolute alcohol. A hot saturated alcoholic ammonia- 

 cal lead acetate solution is added, and the precipitate is filtered after the 

 solution cools. A second precipitate was obtained when the process was 

 repeated. In the material from the 10 brains, 21 g. of the crude precipitate 

 separated on the first precipitation, and 4.5 g. on the second treatment. 



The lead precipitate (21 g.) is decomposed with hydrogen sulfide by di- 

 gesting a suspension in hot 96% ethanol (350 ml.) on a water bath for 90 

 minutes. After removal of the precipitate of lead sulfide, the filtrate is 

 refrigerated overnight. In the experiment described, a copious precipitate 

 of crude nervone was obtained. On concentration of the filtrate in vacuo, 

 an additional small crop of crystals separated. 



The phospholipid and other sugar-free impurities are removed by wash- 

 ing the precipitate with acetone acidified slightly with hydrochloric acid. 

 A further purification from the phosphorus-containing compounds involves 

 precipitation with cadmium acetate from a methanol solution. 



The ciiide nervone was found to be phosphorus-free and, on hydrolysis 

 Avith acid, it yielded galactose, sphingosine, and a mixture of saturated and 

 unsaturated acids. Nervone is believed to be mixed with another cerebro- 

 side. The iodine number is 50.7-51.8, compared w^ith a value for the pure 

 nervone of about 63. Cerebron and cerasine both have iodine numbers of 

 31. 



b. Purification of Nervone. Since it was assumed that the saturated 

 fatty acids which occurred in the crude nervone preparation were impuri- 

 ties present in cerebron or cerasine, the new cerebroside (nervone) was 

 necessarily in the fraction with the higher iodine number. The determina- 

 tijon of the iodine number was used by Klenk to follow the progress of the 

 purification. Several fractional crystallization procedures were emploj'ed. 



(a) First Method of Fractional Crystallization.^" The phosphorus-free 

 crude nervone obtained above was dissolved in 15 volumes of methanol 

 with heating. On cooling to 40 °C., a granular precipitate separated which 

 was filtered off (Fraction 1). When the filtrate was allowed to cool fur- 

 ther, a second crystallization started at 30°C. After cooling to 0°C., the 

 precipitate was filtered (Fraction 2). This was refractionated in the same 

 manner; most of the material appeared in the fraction precipitating at the 

 lower temperature (Fraction 2a). A further refractionation gave no fur- 

 ther purification. 



Iodine number 



Fraction 1 47.1,46.5 



Fraction 2 58.7 



