450 VITAMIN Bi2 



traded with chloroform, transferred to aqueous alkali, and the fluorescence 

 measured in a Farrand fluorometer. Under these conditions it was possible 

 to determine the benzimidazole over a range of 10 to 100 my. Riboflavin 

 did not interfere with the determination. This method does not differentiate 

 vitamin B12 from other substances, including cobalamins and degradation 

 products, which contain 5,6-dimethylbenzimidazole. 



A more specific method for the determination of vitamin B12, outlined 

 subsequently by Boxer and Rickards, depended upon the determination of 

 cyanide which was liberated from vitamin B12 by photolysis with mono- 

 chromatic light. '"^ This procedure was reported to be sufficiently sensitive 

 to determine 1 part of cyanide in 10 parts of solution with a precision of 

 ±1.5%. Hydrogen cyanide was removed by aeration and determined by 

 developing a color reaction with chloramine-T phosphate reagent and 

 pyrazolone-pyridine reagent. The experimental procedure was as follows: 

 Stock solutions were prepared of: 



1. Sodium hydroxide, 0.1 A^. 



2. Sodium dihydrogen phosphate, 1.0 M. 



3. Ceric sulfate, 0.05 A'^, in 1 A'' sulfuric acid. 



4. Saturated solution of silver sulfate in 20% sulfuric acid. An excess of 

 solid silver sulfate should always be suspended in the solution. 



5. Aqueous solution of chloramine-T (recrystallized), 0.25%. This keeps 

 indefinitely in the refrigerator. 



6. Saturated aqueous solution of 3-methyl-l-phenyl-5-pyrazolone (re- 

 crystallized). A stock solution is kept with excess solid in a brown bottle 

 and is filtered as needed. This keeps indefinitely. 



7. Solution of bis(3-methyl-l-phenyl-5-pyrazolone) in pyridine (reagent 

 grade). This solution is prepared every second day and is kept in a freezing 

 compartment. The solution is discarded when visibly colored. 



Reagents Prepared from Stock Solution Prior to Color Development, (a) 

 Chloramine-T phosphate reagent. To 1 part of 0.25 % chloramine-T is added 

 3 parts of 1 M dihydrogen phosphate. Chloramine-T separates as a col- 

 loidal suspension. This reagent can be used as long as chloramine-T does 

 not separate in clumps (usually 15 to 30 minutes), (b) Pyrazolone-pyridine 

 reagent. Five parts of a saturated aqueous solution of methylphenylpyra- 

 zolone is mixed with 1 part of the solution of bismethylphenylpyrazolone 

 in pyridine. This reagent should be protected from the direct sunlight and 

 discarded whenever a pink color appears (60 to 90 minutes). 



Aeration Equipment. A simple aeration train was used throughout. The 

 reaction vessel used for volumes up to 25 ml. was a 25 X 200-mm. test 

 tube; for volumes up to 100 ml., a 38 X 200-mm. test tube; and for volumes 

 up to 500 ml., a 1-1. Florence flask. The reaction vessels were closed with 



11 G. E. Boxer and J. C. Rickards, Arch. Biochem. 30, 372, 392 (1951). 



