II. CHEMISTRY 401 



mation of the molecular weight on the basis of x-ray crystallographic data 

 gave a range of 1360 to 15757 Vitamin B12 appears to be a polyacidic base, 

 as revealed by potentiometric titration in glacial acetic acid;'^ the basic 

 groups are not detected by titration in aqueous solution. In the titration of 

 vitamin Br. with perchloric acid in glacial acetic acid it was observed that 

 excess reagent caused precipitation of an amorphous orange-colored pre- 

 cipitate.-* Further in\'e8tigation proved it to be a simple salt of the vitamin 

 with 6 moles of perchloric acid. The vitamin B12 could be regenerated by 

 treatment with watei" and the liberated perchloric acid titrated with alkali. 

 The formation of this salt indicates the presence of six weakly basic groups 

 in the \'itamin. 



Estalilishment of the molecular formula for vitamin B12 by conventional 

 microanalyses is not readily feasible, because of the molecular size of the 

 compound. The first suggested formulation of vitamin B12 was C62H86-90N14 

 O13PC0 or C63H88-92N14O13PC0 for a sample dried in a weighing pig in vacuo 

 at 100° for 2 hours. ^^ Analyses on samples dried at room temperature gave 

 results indicating a fairly high degree of hydration supporting a formula 

 C63H97Xi402oPCo.^ IMore recently the perchloric acid salt of vitamin B12 has 

 been analyzed and formulated as C63H84Ni40i4PCo6HC104.^* 



Reports on the stability of vitamin B12 under a variety of conditions have 

 been published. As a generalization, it can be said that aqueous solutions 

 of vitamin Bio are most stable in the pH range of 4 to 7 at normal tempera- 

 tures.2*' 2^ However, exposure to sunlight brings about loss of microbiologi- 

 cal activity.^ Autoclaving solutions at 115° for 30 minutes causes slight but 

 significant decomposition.-^ Storage of aqueous solutions of vitamin B12 con- 

 taining 0.5 % phenol or 0.3 % cresol brought about no detectable decomposi- 

 tion as determined by spectrophotometric or microbiological tests.-* The 

 presence of small concentrations of acid or base brings about significant 

 loss of microbiological activity on storage.^ • -- Vitamin Bi2 in 0.015 N sodium 

 hydroxide solution (0.2 7 per milliliter) was microbiologically inactivated 

 at room temperature as follows: 20% (0.67 hour); 45% (6 hours); 90 % 

 (23 hours); 95% (95 hours). It was inactivated in 0.01 N hj^drochloric acid 

 solution (10 7 per millihter) as follo%vs: 18% (3 hours) 75% (23 hours), 

 89% (95 hours). The presence of oxidizing and reducing agents at the full 

 range of pH values is accompanied by loss of microbiological activity.^' -*• -^ 



Aeration of a solution of vitamin Bii brought about an unknown chemical 

 change which involved loss of the 280-m/i absorption maximum.-^ Chroma- 



" J. F. Alicino, J. Am. Chem. Soc. 73, 4051 (1951). 



^* F. Hartley, P. Stross, and R. E. Stuckey, /. Pharm. Pharmacol. 2, 648 (1950). 

 " C. A. Lang and B. F. Chow, Proc. Soc. Exptl. Biol. Med. 75, 39 (1950). 

 -6 H. G. Wijmenga, J. Lens, and A. Middelbeek, Chem. Weekblad 45, 342 (1949) 

 [C. A. 43,9193 (1949)]. 



