ISOLATION 

 Folic Acid 



Folic acid was prepared from spinach leaves by H. K. Mitchell, 

 E. E. Snell and R. J. Williams/ and purified to such an extent that the 

 product was 137,000 times as active as a standard material (Wilson 

 liver fraction B). The spinach was extracted with water, acidified to 

 about pH 3 and the extract stirred with charcoal and filtered. The 

 adsorbate was eluted with hot 2-8 % ammonia and the eluate re- 

 adsorbed on charcoal. The second adsorbate was eluted with hot 

 aqueous aniline, and the adsorption and elution repeated once rnore. 

 The activity in the final eluate was precipitated first with lead acetate 

 and then with ammoniacal silver nitrate solution, the precipitates being 

 regenerated with ammonium sulphate and ammonium chloride respec- 

 tively. The solution from the silver regeneration was adsorbed on 

 fuller's earth and the adsorbate eluted with 5 % ammonia. Further 

 purification was effected by fractional elution from ammonia and by 

 precipitation of the acid from a concentrated solution of the 

 ammonium salt. 



Quantitative studies of the adsorption of folic acid on charcoal 

 were described by Frieden et al.^ They observed that folic acid was 

 more easily eluted from adsorbates made from crude preparations 

 than from adsorbates from relatively pure solutions. This difference 

 is attributed to the presence in the cruder solutions of interfering 

 substances that reduce the adsorption affinity of the charcoal for folic 

 acid. 



The L. casei Factors 



The method used by E. E. Snell and W. H. Peterson ^ for the 

 isolation of the norit eluate factor from " solubilised liver " was as 

 follows : The extract was diluted 20-fold with water, the pJi adjusted 

 to 3-0 with sulphuric acid and the liquor filtered and stirred with norit. 

 The adsorbate, after being washed with water, was stirred with 50 % 

 ethanol, filtered and then eluted three times with a mixture of pyridine- 

 ethanol water (1:2:1). In a modification of the method, elution was 

 carried out with 2 % aqueous ammonia. Further purification was 

 achieved by evaporating the eluate to dryness, dissolving the residue 

 in water and precipitating with picric acid. The picrate fraction, 

 which was insoluble in water and ether but soluble in alcohol, con- 

 tained the bulk of the activity. Considerable losses attended this 

 procedure. 



A somewhat similar method was used by E. L. R. Stokstad.* 

 " Solubilised liver " was adsorbed on norit and the adsorbate was 

 eluted with 0-5 N-ammonia in 70 % methanol. Final purification 

 was effected by fractionally precipitating the manganese salt of the 



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