38 THE VITAMINS 



both in solution and in the dried state, if, before drying, the silver 

 was removed by hydrochloric acid or hydrogen sulfide. A comparison 

 of the nitrogen content of the various fractions with their activity 

 showed that in the ammoniacal silver nitrate fraction a smaller amount 

 of nitrogen was required for protection than in the other fractions, 

 although this was almost four times as much as in an ammoniacal 

 silver nitrate fraction obtained after silver acetate precipitation. "Rea- 

 soning in this manner, it may be concluded that the extracts prepared 

 from activated solid are mixtures of vitamin with one or more other 

 nitrogenous bases. By silver fractionation the proportion of vitamin 

 may be increased to a certain extent, but a considerable degree of 

 purification can not be effected by the method." 



Abandoning the silver precipitation, Seidell (1924) next used picric 

 acid as a precipitant of the active material from the extract obtained 

 from activated solid as previously. The new method, an important pre- 

 liminary step of which was thought to be the removal of potassium 

 sulfate from the extract before the addition of the picric acid, was as 

 follows : 



The extract was dissolved in water and diluted to a definite volume. An aliquot 

 of this was evaporated to dryness, the residue ignited, and the resulting ash titrated 

 with tenth-normal sulfuric acid, with methyl orange as indicator. From the amount 

 of acid required the calculated amount of normal acid equivalent to the bases 

 present in the entire solution was added to the main solution and the mixture 

 distilled under reduced pressure almost to dryness. The residue was treated vvith 

 66 per cent ethyl alcohol and the insoluble potassium sulfate removed by filtration. 

 The protective dose of the alcoholic filtrate thus obtained was found to be 4 

 milligrams fed every other day. 



The picrate was prepared by adding to this alcoholic extract an amount of 

 picric acid dissolved in methyl alcohol, approximately equal in weight to the solids 

 present in the extract. The alcohol was gradually removed by evaporation under 

 reduced pressure, water being added at intervals to replace it. The precipitate 

 was finally separated by centrifugation and washed with small portions of water. 

 The crude picrate was purified by adding to 5 grams of the material 8 cubic 

 centimeters of 95 per cent acetone, stirring for 3 minutes, centrifuging, and decant- 

 ing the clear reddish yellow solution. The process was repeated successively with 

 4, 2. and finally 1 cubic centimeter of the acetone. Ten cubic centimeters of water 

 was added to the combined extracts and the mixture evaporated in a partially 

 evacuated desiccator containing sulfuric acid until the volume had been reduced 

 about one-third, when 2 cubic centimeters of water was added and the process 

 continued until the further addition of water caused no further precipitation. The 

 solid was then filtered on a paper filter resting on a small perforated porcelain 

 disc, and washed with a little water, and the entire process repeated. The twice- 

 crystallized picrate was reported to consist of irregularly-shaped transparent 

 crystalline flakes capable of protecting fully grown young pigeons in doses as low 

 as 2 milligrams daily. 



The portion of the crude picrate insoluble in 95 per cent acetone 

 proved to be soluble in dilute acetone, yielding on recrystallization pale 

 yellow rods or prismatic crystals possessing very little activity, although 



