VITAMIN B {B,) 67 



proved active in doses equivalent to 2 grams wheat embryo, con- 

 taining 1.3 milligrams organic solids of which 17.8 per cent was 

 nitrogen. Subsequent fractionation with 50 per cent alcohol, and 

 acidified absolute alcohol yielded a product active in doses con- 

 taining 0.6 milligram organic matter, and corresponding to Z.7 grams 

 embryo. 



The filtrates from the alcoholic fractionation were evaporated to 

 dryness in vacuo at 40° to 50° C, taken up in water, and adjusted to 

 pH 6.5, with sodium hydroxide, whereupon an inactive precipitate 

 could be removed. Treatment of the filtrate with an absolute alcohol 

 solution of picrolonic acid produced a semi -crystalline yellow precipi- 

 tate, which when freed from the acid by hydrochloric acid and ether, 

 was inactive in doses corresponding to 12 grams embryo, while the 

 supernatant fluid was active in doses corresponding to 4.5 to 6 grams 

 embryo and carrying 0.4 to 0.5 milligram organic matter. Nearly 11 

 per cent of the activity of the original extract of wheat germ was 

 present in this solution. 



Attempts to concentrate further the active material of this prepara- 

 tion were unsuccessful. These included: (a) a second precipitation with 

 phosphotungstic acid followed by fractional crystallization from hot 

 water and from mixtures of acetone and water; (b) adsorption on silica 

 gel at pH 3, 4 or 5 followed by subsequent extraction at pH 9.5 (Levene 

 and van der Hoeven, 1925, 1925a; Levene, 1928) ; and (c) fractiona- 

 tion with platinic chloride. 



The second method followed by Guha and Drummond in fraction- 

 ating their concentrate made from wheat embryo approximated that of 

 Jansen and Donath (previously described). Upon decomposing the 

 aqueous suspension of the platinum precipitate with hydrogen sulfide, a 

 colorless solution was obtained which was evaporated to dryness over 

 soda lime. The residue was found to be active in doses equivalent to 

 4.6 grams embryo and containing 0.015 milligram total solids. "About 

 11 per cent of the activity of the original extract was thus retained 

 in this material. The product was colourless and for the greater part 

 microcrystalline but obviously impure." The average day-dose in cura- 

 tive tests on pigeons was 0.0051 milligram. 



Upon extracting the active preparation from the platinum compound 

 with absolute alcohol and treating the filtrate with an alcoholic solution 

 of gold chloride a slight turbidity resulted which cleared up on warm- 

 ing. When the preparation was slowly evaporated in a desiccator over 

 calcium chloride a small amount of microcrystalline deposit was 

 formed, which did not appear homogeneous and did not show the 



