44 THE VITAMINS 



and perchloric acid. With picrolonic acid and with gold chloride needle- 

 shaped crystals are formed. Pauly's diazo reaction is given with 

 sulfanilic acid and nitrous acid, followed by sodium hydroxide. Ele- 

 mentary analyses of the hydrochloride and gold chloride gave results 

 conforming closely to the empirical formulas CeHioONa.HCl and 

 CeHioONo.HCl.AuCls. From this work and the previous findings of 

 other investigators indicating that the antineuritic vitamin does not 

 react with nitrous acid and consequently contains neither primary nor 

 secondary amino groups, it is considered probable that the vitamin 

 contains either an imidazole or pyrimidine ring. 



In 1929, Jansen modified the above method by substitution of silico- 

 tungstic acid for phosphotungstic to increase the yield of purified 

 vitamin ; and cadmium chloride for platinic chloride for the sake of 

 economy. 



Kinnersley and Peters (1927) described further improvements in 

 their technique of preparing torulin (see above) which simplified the 

 procedure, doubled the yield of active material and increased the 

 activity to such an extent that only 0.5 to 1 mg. daily was required 

 in the curative tests. The principal change from the technique previ- 

 ously employed consisted in treating the filtrate from the neutral lead 

 acetate precipitate with cold saturated baryta before adding mercuric 

 sulfate. This served to flocculate the gums present in the autolysate and 

 thus facilitate subsequent filtrations. The optimum reaction for adsorp- 

 tion of the active material by norite was found to be at pH 7 for material 

 treated with mercuric sulfate. Starting with 14 pounds of bakers' yeast 

 the adsorption was found to be most complete when carried out in 

 two steps using 60 grams of norite in the first and 20 in the second 

 step. The active material was set free from each charcoal fraction by 

 cubic centimeters of tenth-normal hydrochloric acid, followed in some 

 two treatments on the hot water bath for an hour with about 200 

 cases by two extractions with about 150 cubic centimeters of 50 per 

 cent alcohol (by volume) containing 1 per cent strong hydrochloric 

 acid. The combined extracts, freed from the last traces of sulfuric 

 acid by dilute barium chloride, were concentrated in vacuo at a tem- 

 perature not exceeding 60° C. 



If it is considered desirable to avoid the use of mercuric sulfate, 

 as in the preparation of a concentrate to be used in long-continued 

 feeding tests, the bulk of the substances precipitable by this reagent 

 can be removed by adsorption on norite at pH 2.5 previous to the 

 adsorption of the active material at pH 7. The yield, however, is not 

 as high. 



