66 THE VITAMINS 



sons of vitamin activity were made by finding the minimum amount of 

 supplementary food which would permit a weekly gain of 10 to 12 

 grams for 2 to 4 weeks. Longer experiments were avoided as the 

 authors believed that in too long an experiment the result might be 

 vitiated by some of the other factors necessary for growth. In a num- 

 ber of fractions the curative property was also investigated by sub- 

 cutaneous injection into rats exhibiting typical beriberi convulsions as 

 a result of prolonged deprivation of vitamin B (Bi). 



As starting material for their fractionations, wheat embryo was ex- 

 tracted twice with alcohol (50 per cent by volume) acidified with 

 hydrochloric acid, each extraction being conducted for 3 to 4 hours 

 between 60° to 70° C. The filtrate was concentrated in vacuo to a yellow 

 viscous mass which retained its potency (active in doses corresponding 

 to 0.5 gram embryo daily) for at least 10 months when stored in the 

 dark at 0° C. in well-closed vessels. 



This concentrate was subjected to two fractionation processes. In 

 the first, from a solution of the concentrate inert materials were re- 

 moved by precipitation with lead acetate ; to the filtered supernatant 

 liquid was added enough hydrochloric acid to make it just acid to Congo 

 red, and the resulting filtrate was freed from lead by hydrogen sulfide. 

 Upon subsequent removal of the hydrogen sulfide this solution was 

 active in rat experiments in doses corresponding to 0.5 gram to 0.75 

 gram embryo daily, and contained 2.3 per cent nitrogen in the organic 

 matter. 



The active material was found to be preferentially adsorbed under 

 their conditions on norite at pH 4 and 5. (Vitamin B from yeast ex- 

 tracts was found to be adsorbed by norite at pH 7, as stated by Kin- 

 nersley and Peters, 1928.) After two successive adsorptions at each of 

 these hydrogen-ion activities the active material was extracted from 

 the charcoal-vitamin complex by refluxing twice successively with 50 

 per cent alcohol containing 1 per cent hydrochloric acid, each extrac- 

 tion lasting 30 minutes at 60° C. The extracts corresponding to pH 4 

 and 5 were combined, and the acidity of the mixture brought to pH 5 

 by adding 2M sodium hydroxide, which process darkened the solution 

 and precipitated some inert material. The active material was then 

 precipitated by an excess of phosphotungstic acid in 5 per cent sulfuric 

 acid. After freeing the potent material by treatment with concentrated 

 hydrochloric acid and with amyl alcohol and ether, most of the active 

 material could be adsorbed on freshly precipitated silver oxide, from 

 which it could again be released by extraction with acidified alcohol. 

 After removal of the alcohol in vacuo the resulting red solution 



