III. INDUSTRIAL PREPARATION 419 



(2,4,5-trichlorophenol),^'' and precipitation as copper or zinc cyanide- vita- 

 min Bi2 complex" may be used. Near the end chromatography on activated 

 alumina''- ^' and fractionated precipitation of the active substance in meth- 

 anolic or ethanolic solution with acetone^- ^^ is recommended. The final 

 crystallization takes place when an aqueous solution of the purified sub- 

 stance (50% purity) is treated with acetone (10 mg. + 1 ml. of water + 10 

 ml. of acetone); upon standing, vitamin B12 separates from the solution in 

 clusters of small red needles.* 



Each step in the complicated series of operations increases the potency 

 but includes a certain loss of active substance. A yield as high as 65 %, how- 

 ever, is claimed from the broth (1 : 1,000,000) to a concentrate of 5 % (U. S. 

 Pat. 2,582,589, example 2); from there on the losses are much higher: U. S. 

 Pat. 2,563,79-1 claims in example 6 only a yield of about 4 % in the form of 

 crystals (14.1 mg. from 364 mg.), starting from a concentrate with 0.4 7 

 per gram. 



The preparation of animal feed containing aureomycin and B12 is de- 

 scribed by Jukes^^ (Aurofac, Lederle, containing 1.8 to 5.0 g. of aureomycin 

 and up to 1.8 mg. of B12 per pound). A preparation of Pfizer, called TM 

 3 + 3, contains 3 g. of terramycin and 3 mg. of B12 per pound. 



2. Fermentation Processes Producing Only B12 



Fermentation processes producing B12 without any other highly valuable 

 product are economically justified only when they have definite advantages 

 over the fermentation producing antibiotics at the same time. Bacillus 

 megatherium as used by Lewis and coworkers^*' ^^ shows the unusual fea- 

 tures that propagation on a medium consisting of molasses, ammonia, and 

 inorganic salts is already completed within 6 hours (compared with the 

 usual three days), and that the vitamin-containing product is cjuantita- 

 tively recovered by centrifuging and drum drying, converting about 50 % 

 of the weight of sugar used to bacterial solids. The pale tan, palatable, non- 

 toxic, free-flowing, non-hygroscopic powder contains high nutritional value 

 in addition to 15 mg. of B12 per kilogram. 



The process is industrially used by one producer in the West (Pacific 

 Yeast Co., Wasco, California). '^ 



10 R. G. Denkewalter, G. B. Hughey, and S. Kutosh, (to Merck & Co., Inc.), U. S. 



Pat. 2,635,985 (April 21, 1953). 

 " A. J. Holland, (to Merck & Co., Inc.), U. S. Pat. 2,621,144 (Dec. 9, 1952). 

 12 H. H. Fricke, (to Al)l)ott Laboratories), U. S. Pat. 2,582,589 (Jan. 15, 1952). 

 " T. H. Jukes, (to American Cyanumid Co.), U. S. Pat. 2,619,420 (Nov. 25, 1952). 

 " J. A. Garibaldi, K. Ijichi, J. C. Lewis, and J. McGinnis, (to the United States), 



U. S. Pat. 2,576,932 (Dec. 4, 1951). 

 "J. A. Garibaldi, K. Ijichi, N. S. Snell and J. C. Lewis, Ind. Eng. Chem. 45, 838 



(1953). 

 i« Chem. Eng., 59, 244 (November, 1952). 



