216 VITAMIN D GROUP 



B. CHEMICAL METHODS 



A drawback to direct spectrophotometric estimation is the fact that the 

 absorption maximum of vitamin D Hes at 265 mju, a region where ultra- 

 violet dirt is especially likely to show up. Accordingly, in the devising of 

 chemical methods of estimation, it has been sought to form strong and 

 stable colors in the visible range, less susceptible to irrelevant absorption, 

 and, of covu'se, as nearly as possible specific to the vitamin. 



The most widely used color reaction is that given by antimony trichloride 

 and first applied to pure vitamin Do by Askew et al} A deep yellow color 

 is developed, with the absorption maximum at 500 mju. Nield et al.^ in- 

 creased the sensitivity of the reaction to the point where they could measure 

 the color given by 0.2 y of calciferol, and they also increased the permanence 

 of the color. Milas et al.^ applied modifications of the reaction to the assay 

 of fish liver oils. In one procedure they made interference corrections for 

 sterols and vitamin A, and in their preferred method they first removed the 

 interfering substances by coupling them with maleic anhydride and then 

 measured the color developed at 500 mju. They were able to get fairly good 

 agreement with bioassays in the case of fish oils containing 10,000 units or 

 more of vitamin D per gram. 



Details of the antimony trichloride reaction were worked out by Vacher 

 et al.,'^ Shantz,^ Nielsen,^ and others. The important step of chromatographic 

 purification was introduced and improved successively by Ewing et al.,^^ 

 De Witt and Sullivan," Muller,^^ Ewing et al.,^^ and Fujita and Aoyama.^* 

 Under favorable conditions the pigment generated in this reaction from 

 vitamin D2 or D3 shows £'}^m. = 1800 at 500 m/x.^ This is an intense absorp- 

 tion, nearly four times as great as that of the vitamin itself at 265 mju, and 

 comparable with that of strongly absorbing dyes such as fuchsin. Hence it 

 seems unlikely that efforts to find a better color reaction will substantially 

 improve upon this one with regard to intensity of color, although there is 



4F. A. Askew, R. B. Bourdillon, H. M. Bruce, R. K. Callow, J. St. L. Philpot, 



and T. A. Webster, Proc. Roy. Soc. (London) B109, 488 (1932). 

 s C. H. Nield, W. C. Russell, and A. Zimmerli, /. Biol. Chem. 136, 73 (1940). 



6 N. A. Milas, R. Heggie, and J. A. Raynolds, Ind. Eng. Chem. Anal. Ed. 13, 227 

 (1941). 



7 M. Vacher, Y. Lortie, and H. Colson, Bull. soc. chim. biol. 26, 206 (1944). 

 8E. M. Shantz, Ind. Eng. Chem. Anal. Ed. 16, 179 (1944). 



9 P. B. Nielsen, Nutrition Abstracts & Revs. 19, 57 (1949). 



10 D. T. Ewing, G. V. Kingsley, R. A. Brown, and A. D. Emmett, Ind. Eng. Chem. 

 Anal. Ed. 15, 301 (1943). 



11 J. B. De Witt and M. X. Sullivan, Ind. Eng. Chem. Anal. Ed. 18, 117 (1946). 



12 P. B. MuUer, Helv. Chim. Acta 30, 1172 (1947). 



13 D. T. Ewing, M. J. Powell, R. A. Brown, and A. D. Emmett, Anal. Chem. 22, 317 

 (1948). 



" A. Fujita and M. Aoyama, /. Biochem. (Japan) 37, 113 (1950). 



