VIII. STANDAUDIZATIOX OF ACTIVITY 173 



Solubility. One gram of vitamin J^n di«solves in about 80 ml. of water. 

 It is soluble in alcohol, but is insoluble in acetone, in chloroform, and in 

 ether. 



Idvntification. A. Determine the absorbency of the solution prepared 

 for the assay in a 1-cm. quartz cell with a suitable spectrophotometer, 

 using water as the blank. Maxima within dzl mix are found at 278 and 

 361 niM and within ±4 myu at 548 m/x. The ratios of the absorbencies so 

 observed are as follows: 



— — is not less than 1.62 and not more than 1.88 



^278 



-T^^ is not less than 2.83 and not more than 3.45 



-(4 548 



B. Fuse about 1 mg. of vitamin B12 with about 50 mg. of potassium 

 bisulfate in a porcelain crucible. Cool, break up the mass with a glass rod, 

 add 3 ml. of water, and dissolve by boiling. Add 1 drop of phenolphthalein 

 T.S. and 10% sodium hydroxide solution, dropwise, until just pink. Add 

 500 mg. of sodium acetate, 0.5 ml. of diluted acetic acid, and 0.5 ml. of 

 nitroso-R-salt solution (1 in 500) : a red or orange-red color appears at once. 

 Add 0.5 ml. of hydrochloric acid, and boil for 1 minute; the red color 

 persists. 



Loss on drying. Weigh accurately, on a microbalance, about 5 mg. of 

 vitamin B12 in a suitable microdrying vessel. Heat in a suitable vacuum- 

 dryhig apparatus at 105° and at a pressure of not more than 5 mm. for 2 

 hours, cool, and weigh: it loses not more than 12% of its weight. 



Assay. Weigh accurately, on a microbalance, about 2 mg. of vitamin B12, 

 transfer to a 50-ml. volumetric flask with the aid of 15 or 20 ml. of water, 

 add water to make exactly 50 ml., and mix well. Determine the absorbency 

 of the solution in a 1 cm. quartz cell, at 361 m/x, with a suitable spectro- 

 photometer, using water as the blank. 



Calculate the per cent purity of vitamin B12 by the following formula: 



^361 ^ 1 ^ 100 



0.0207 wt. of sample (mg. per 10 ml.) ( 100-1 oss on drying) 



L. leichmannii 313 (7830) Tube Assay. As mentioned earlier, a method 

 utilizing L. leichmannii 313 (7830) has been adopted by the U.S.P.^ and 

 has been the subject of a collaborative study among several laboratories. 

 A special culturing procedure for the test organism renders the assay par- 

 ticularly sensitive. The culture medium consists of 0.75 g. of yeast extract, 

 0.75 g. of peptone, 1 g. of dextrose, 0.2 g. of potassium biphosphate, 10 

 ml. of jQltered tomato juice, and 100 mg. of Tween 80 in 100 ml. of water. 



^ U. S. Pharmacopeia, 14th revision, 3rd Supplement, p. 15, 1951. 



