VII. KSTIMATIOX 449 



proiK-rtics difTcrc'd from tlios(> encountered in a crystalline siihst.ance ob- 

 tained from Strcplomyci's aurcofaciens, which was given the name vitamin 

 Bi2b.^ Later investigations^ ^ showed that subsequent attempts to prepare 

 vitamin Bi^i resulted in the isolation of a substance with the properties of 

 vitamin Bi2b rather than those described for \'itamin Bijh. Of much interest 

 was the observation that a band at 315 mn in the ultraviolet al)sorption 

 spectrum, which is a prominent characteristic of vitamin Bi2a,* tended to 

 disappear when the a(iueous solution stood at room temperature. Cooley 

 et al.^ have discussed the possibility that \'itamin Bi2b may be dehydrated 

 with a simultaneous redistribution of charge to give vitamin Bi2a. 



In addition to cyanide, hydroxyl, and nitrite, various other anions may 

 be coordinated in the cobalamin molecule to give a series of compounds 

 with differing absorption spectra. It may thus be seen that the measure- 

 ment of the vitamins of this group by means of their absorption spectra 

 is difficult to carrj' out with mixtures of the cobalamins. 



B. CHEMICAL 



Chemical methods for the determination of small (luaiitities of vitamin 

 Bi2 have been studied by Boxer and Rickards. Their lirst method^° was 

 based upon hydrolysis of the vitamin to yield 5 , 6-dimethylbenzimidazole 

 which was benzoylated to 4,5-dimethyldibenzoyl-o-phenylenediamine. This 

 compound was then treated with concentrated sulfuric acid to form 4,5- 

 dimethyl-o-phenylenediamine, which was determined colorimetrically or 

 fluorometrically. The procedure was as follows: vitamin B12 was hydrolyzed 

 with 6 iV HCl at 150° for 18 hours to liberate 5,()-dimethylbenzimidazole. 

 Treatment with benzoyl chloride produced 4,o-dimethyldibenzoyl-o-phen- 

 ylenediamine in the Schotten-Baumann reaction, following which the excess 

 benzoyl chloride was converted to sodium benzoate with alkali. The di- 

 amine was extracted with chloroform which was removed by evaporation, 

 and the residue was treated with concentrated H2SO4 at 150°. For the 

 colorimetric reaction, the sulfuric acid solution of diamine was partially 

 neutralized and treated with acetylacetone to produce 2 ,4,7 ,8-tetramethyl- 

 l ,5-benzyldiazepine, which has a purple color sensitive enough to determine 

 10 7 of original l^enzimidazole. A more sensitive procedure was the fluoro- 

 metric determination of o-phenylenediamine by condensation with alloxan 

 to form the fluorescent compound (»,7-dimethylalloxazine which was ex- 



7 J. V. Pierce, A. C. Page, Jr., K. L. R. Stokstad, and T. H. Juke-s, ./. Am. Chem 



Soc.n, 2953 (1949). 

 * E. A. Kaczka, R. G. Denkewaher, A. Holland, and K. Folkers, ./. Am. Chem. Soc. 



73, 335 (1951). 

 9 G. Cooley, B. Ellis, V. Petrow, G. H. Beaven, E. R. Holiday, and E. A. Johnson, 



J. Pharm. Pharmacol. 3, 607 (1951). 

 »» G. E. Boxer and J. C. Rickards, Arch. Biochem. 29, 75 (1950). 



