236 THE VITAMINS 



and the unsaponifiable matter extracted with ether. This was then treated 

 with sodium ethylate for the removal of any remaining saponifiable 

 matter, nitrogen being used to displace air during this process and pre- 

 vent loss of vitamin A by oxidation. Complete removal of fatty acid 

 esters of cholesterol as well as of glycerol is important and was always 

 assured before proceeding to the next step, which was the extraction of 

 the unsaponifiable substances by means of ether and their recovery, on 

 evaporation of the solvent, as "a semi-solid crystalline mass with a waxy 

 appearance and varying in color from pale yellow to deep orange accord- 

 ing to the color of the original oil." The crystalline appearance was 

 found to be due to cholesterol which constituted about one-half of the 

 material at this point ; but which was removed, mainly by freezing out 

 from solution in methyl alcohol, and completely by subsequent pre- 

 cipitation with digitonin, an atmosphere of nitrogen being maintained 

 throughout the process to prevent loss of vitamin A by oxidation. The 

 cholesterol digitonide was removed by filtration, the filtrate evaporated 

 at low temperature and pressure to remove the solvents, the residue 

 dissolved in ether, and the small excess of digitonin washed out by 

 repeatedly shaking the ethereal solution with water in an atmosphere 

 of nitrogen. "Finally the ethereal solution is separated and dried with 

 anhydrous sodium sulfate and the solvent removed at low pressure. The 

 product thus obtained is a deep reddish orange oil possessing the char- 

 acteristic smell of the original unsaponifiable fraction. If it is carefully 

 prepared it shows a vitamin A value proportionate to the cod-liver oil 

 from which it was prepared." 



In a typical case, 10 liters of light-colored cod-liver oil yielded 30 

 grams of orange-colored product as just described; and this, when dis- 

 tilled in a current of super-heated steam and nitrogen, was separated 

 from its resinous matter and yielded a distillate of 25 grams of a clear, 

 pale yellow oil. On distillation at 3 millimeters pressure this oil yielded 

 the following fractions : 



° C. Grams 



(a) 100° 0.20 



(b) 100°-180° 1.20 



(c) 180'-230° 8.29 



(d) 230°-240° 1.40 



(e) 240°-26O° 6.23 



(f ) 260°-300° 1.73 



Undistilled residue 3.50 



22.55 



Fraction (a) was considered to consist of impurities from the ether 

 previously used as solvent. Feeding experiments with fractions (b) to 



