42 THE VITAMINS 



the methods of separating the antineuritic vitamin from soluble inactive 

 material in aqueous solution may, in general, be classified as those which 

 depend upon differential solubility of the constituents of the mixture 

 in various concentrations of alcohol or similar solvents, those based 

 on precipitation by suitable reagents, and those based on fractional ad- 

 sorption. He inclined to favor the last as permitting the most complete 

 separation. He found that about 28 per cent of the original vitamin 

 activity of brewers' yeast remained in the coagulated protein which 

 separates out upon heating a suspension of the yeast in water. Three 

 successive adsorptions of the aqueous yeast extract on fuller's earth 

 (30 grams per liter) resulted in preparations of "activated solids" 

 containing respectively 55 per cent, 5 per cent, and practically none 

 of the activity of the original yeast. About 88 per cent of the vitamin 

 originally in the yeast was thus accounted for. 



Later (1929a) Seidell described a method for converting the dry 

 non-hygroscopic antineuritic concentrate (prepared by essentially the 

 method described in 1926) into a still more active fraction. To an 

 aqueous solution of the concentrate made alkaline with sodium car- 

 bonate an excess of benzoyl chloride was added, and the mixture was 

 extracted with chloroform. The aqueous layer was then acidified and 

 repeatedly extracted with chloroform. About 90 per cent of the vitamin 

 activity and 25 per cent of the nitrogen remained in the aqueous solu- 

 tion. When this solution was poured into 10 to 12 volumes of acetone, 

 salts were precipitated accompanied by nitrogenous material which pro- 

 tects pigeons from loss in weight on a diet of polished rice in doses 

 containing 0.15 milligram of nitrogen. Judged on the basis of nitrogen 

 content, this final product represents a concentration of antineuritic 

 substance of 100 times that of dried brewers' yeast. 



Jansen and Donath (1926, 1926a, 1927), working in the same lab- 

 oratory in which Eijkman carried on his pioneer studies on the anti- 

 neuritic vitamin, prepared from rice polishings a crystalline substance 

 which they believe to be the hydrochloride of the antineuritic vitamin. 



The method consists essentially in the extraction of the rice polish- 

 ings with water acidified with sulfuric acid to an acidity of pH 4.5, 

 and containing 20 cubic centimeters of alcohol per liter; adsorption 

 on acid clay (a kind of fuller's earth, hydrated aluminum silicate) ; 

 extraction of the activated clay with barium hydroxide at pH 12 or 13, 

 followed by sulfuric acid ; fractionation of the acid extract with silver 

 nitrate and barium hydroxide to pH 4.5, 6.5, and 8, followed, after 

 decomposition with hydrochloric acid, by a second treatment of the 

 combined silver fractions which concentrates the vitamin in the second 



