452 VITAMIN Bi2 



Colorimetric Determination. The collection tube containing 0.005 to 0.4 

 7 of cyanide in 1 ml. of 0.1 A^ NaOH is thoroughly chilled in ice. Two- 

 tenths milliliter of chloramine-T-phosphate reagent is added, and the 

 contents are mixed by drawing them once or twice partially up the inlet 

 tube by suction. The tube is replaced in the ice bath, and, after al)out 2 

 minutes, 3 ml. of the pyrazolone-pyridine reagent is added and the contents 

 mixed again. The tubes are remo\'ed from the ice bath and left at the 

 desired temperature for full color development. The color, once fully de- 

 \'eloped, is stable for at least 2 hours. The color intensity is measured in a 

 Coleman Model 14 spectrophotometer at 620 m/z, using a PC-5 filter in 

 front of the photocell. Beer's law is obeyed for the range from 0.005 to 

 0.40 7 of cyanide with a standard deviation of ±1.5%. Precise readings 

 are obtained with the scjuare cuvettes of 13-mm. depth for the range of 

 0.02 to 0.40 7, and with the cylindrical cuvette of 50-mm. depth and 3.5- 

 ml. capacity for the range of 0.005 to 0.10 7 of cyanide. The optical density 

 of the blank for the whole procedure (including aeration) was identical 

 with that of distilled water within the limit of errors. 



The method was adapted to the determination of total cobalamins by 

 transforming these substances to cyanocobalamin by treatment with cy- 

 anide. This was accomplished by adding a three- to fivefold molar excess 

 of freshly prepared aqueous potassium cyanide and holding the solution 

 in the dark for 3 hours at pH 5 or below. The excess cyanide was then re- 

 moved by aeration in the dark, following which the sample was illuminated 

 and "bound" cyanide was split off from cyanocobalamin by photolysis. 

 The difference between the values for vitamin B12 before and after the 

 treatment with cyanide was due to the presence of cobalamins other than 

 cyanocobalamin which were capable of binding cyanide and releasing it on 

 illumination. Data were presented describing the assay of vitamin B12 in 

 liver concentrate and other natural materials, and the results were com- 

 pared with the microbiological procedures. 



C. BIOLOGICAL 



1. Assay w'ith Chicks 



Various modifications of all-vegetable diets are used in the asvsay of 

 vitamin B12 with chicks. The use of high levels of soybean meal in the diet, 

 as described by the Beltsville group,'* has gained wide acceptance. It is 

 necessary for satisfactory results to use chicks hatched from eggs laid by 

 hens which are on a diet low in vitamin B12 because the "carry-over" of 

 this vitamin is pronounced. Furthermore, the hens must be restrained from 

 obtaining appreciable amounts of vitamin B12 from the litter on the floor 

 of the hen house. 



13 M. Rubin and H. R. Bird, ./. Nutrition 34, 233 (1947). 



