ISOLATION 



Vitamin Be and Vitamin Be Conjugate 



Vitamin Be was isolated from beef liver by the following procedure.' 

 An extract was made with acidulated water and, after concentration, 

 alcohol was added to 50 % by volume and filtered. The precipitate 

 was washed with 50 % alcohol and the combined filtrate and washings 

 were concentrated to a syrup. This was re-dissolved in water, acidified 

 to pH I and stirred with fuller's earth. The adsorbate was eluted 

 with 0-2 N-ammonium hydroxide and the eluate concentrated, acidi- 

 fied to pK I and stirred with Superfiltrol. The adsorbate was eluted 

 with I % ammonia in 50 % alcohol and the eluate concentrated. 

 Various purification procedures were tried out at this point, e.g., norit 

 adsorption at pK 3 and elution with 10 % ammonia ; adsorption at pK 

 4-3 on Amberlite IR4 and elution with 10% ammonia ; and precipita- 

 tion with barium hydroxide, zinc sulphate or phosphotungstic acid. 

 The last method was the only one that gave a sufficient degree of purifi- 

 cation together with a sufficiently high recovery to be of value. 



The method used by Pfiffner et al.^ for the isolation of vitamin Bo 

 (pteroylglutamic acid) from hog liver w*as as follows : The autolysed V 

 tissue was extracted with hot water, the extract was concentrated 

 under reduced pressure and filtered through a column of Amberlite 

 IR4. The adsorbate was eluted with aqueous ammonia, and the 

 eluate concentrated and stirred with Superfiltrol. This adsorbate was 

 eluted with aqueous alcoholic ammonia, and the eluate concentrated 

 and stirred with norit. This third adsorbate was also eluted with 

 aqueous alcoholic ammonia and the adsorption on norit and elution 

 then repeated. The final concentrate was extracted first at p^ 5-6 

 with butanol and the extract discarded and then at _/)H 3. This second 

 extract, which contained the activity, was evaporated and the result- 

 ing solid was extracted four times with boiling 90 % methanol. The 

 combined extracts were treated with baryta, and the insoluble barium 

 salts were filtered off. The water-soluble barium salts were then pre- 

 cipitated by the addition of zinc acetate and the precipitate decom- 

 posed with ammonium oxalate. The zinc oxalate was filtered off and 

 the filtrate was acidified to ^H 2-8 when the crude vitamin separated 

 out. It was leached out with baryta and re-precipitated by acidifying 

 the solution to pYi 2-8. It was then crystallised from hot w^ater. 



One process was simplified by esterifying the crude zinc salt with 

 methanolic hydrogen chloride and precipitating the methyl ester by 

 adjusting the concentrated solution to pYi 3. 



Horse liver was found to be a much richer source of vitamin Be 

 than was hog liver and, using this as the starting material, the process 

 could be still further simplified by omitting the butanol extraction and 

 the precipitation of the barium salts from methanol. 



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