418 VITAMIN Bi2 



streptomycin, aureomycin, and terramycin, after the removal of the 

 antibiotic. 



2. Cultures of microorganisms which produce B12 as the only valuable 

 product, e.g., B. megatherium. 



3. Activated sludge from sewage disposal. 



At the time of this writing (September, 1953) source 1 provides almost 

 the whole output of B12; small amounts of concentrates are produced from 

 source 2, whereas the use of activated sludge is still in a pilot plant stage. 



Concentration of the Starting Material: 1 to 2 7 of B12 per milliliter of broth 

 (or gram per cubic meter) is considered worth while for industrial extrac- 

 tion. The highest activity for B12 production reported in the literature is 

 8 7 per milliliter ;2 however, this figure was obtained by microbiological 

 assay, and no microbiological method is entirely specific for B12. 



A. ISOLATION 

 1. From the Mother Liquors of Antibiotics 



It is self-evident that a raw material which is a by-product and which 

 yields a very valuable end product is always welcome. The producers of 

 antibiotics with vitamin B12 as a by-product are in a favorable position. 



The isolation of an active compound from a broth with a dilution as high 

 as 1:1,000,000 is quite a technical achievement. It normally starts from 

 the filtered broth after removal of the antibiotic and the vitamin B12 

 from the liquid phase by adsorption with charcoal,^' ^ calcium mont- 

 morillonite (fuller's earth), sodium montmorillonite (bentonite,)^ or car- 

 boxy lie ion exchange resins (Amberlite),^ followed by elution with such 

 diversified solvents as aqueous pyridine or a-picoline,^' ■* aqueous solutions 

 of sodium cyanide or thiocyanate,* mixtures of acetone, water, and dilute 

 hydrochloric acid,^ and others. For further purification, countercurrent 

 distribution between two liquid phases (e.g., o-cresol,^ amylphenol,^ or 

 benzyl alcohol-water),^ extraction from aqueous solution with a solid solvent 



2 D. Perlman, W. E. Brown, and S. B. Lee, Ind. Eng. Chem. 44, 2000 (1952). 

 3F. A. Kuehl and L. Chaiet, (to Merck & Co., Inc.), U. S. Pat. 2,505,053 (April 

 25, 1950). 



4 E. L. Rickes and T. R. Wood, (to Merck & Co., Inc.), U. S. Pat. 2,563,794 (Aug. 7, 

 1951). 



5 S. Kutosh, G. B. Hughey, and R. Malcolmson, (to Merck & Co., Inc.), U. S. Pat. 

 2,626,888 (Jan. 27, 1953). 



6 W. Shive, (to Research Corp.), U. S. Pat. 2,628,186 (Feb. 10, 1953). 



■' N. G. Brink and T. R. Wood, (to Merck & Co., Inc.), U. S. Pat. 2,609,325 (Sept. 2, 

 1952). 



8 F. R. Koniuszy, N. G. Brink, and K. Folkers, (to Merck & Co., Inc.), U. S. Pat. 

 2,594,314 (April 29, 1952). 



9 N. G. Brink and F. J. Wolf, (to Merck & Co., Inc.), U. S. Pat. 2,607,717 (Aug. 19, 

 1952). 



