IV. ESTIMATION 215 



by other sterols occurs not only in activation by irradiation, but in the less 

 widely practiced activation in the vapor phase by a high-frequency elec- 

 trical discharge.^' 



IV. Estimation 



CHARLES E. BILLS 



Vitamin D is the only one of the better-known vitamins for which 

 physical or chemical methods of estimation have not largely replaced bi- 

 ological methods. The reason for this is plain: vitamin D is so potent that 

 even in the richer sources its concentration is usually minute. Conversely, 

 the substances which interfere with physical or chemical determination of 

 it are likely to be present in relatively overwhelming amounts. In biological 

 assays, however, rats or chicks show measurable responses to vitamin D 

 when it is present in their diet in amounts as little as 1 part in several 

 hundred million. Interference with the biological response occurs only when 

 comparatively gross ciuantities of materials such as phosphates are en- 

 countered. 



With the publication of Gyorgy's "Vitamin Methods,"^ the art of esti- 

 mating vitamins is presented in two volumes. Therefore, the techniques will 

 be presented here only in outline, in order to guide the reader to the 

 more detailed sources of information. 



A. PHYSICAL METHODS 



In very concentrated forms, vitamin D can be estimated by purely 

 physical means. Thus Pirlot- recommends infrared spectrophotometry for 

 determining calciferol in the irradiation products of ergosterol. For calciferol 

 itself, or concentrated oily solutions thereof, Penau and Hagemann^ em- 

 ployed ultraviolet spectrophotometry. The estimation, based on the ex- 

 tinction at X 2(55 mju, is straightforward, provided that the absorption curve 

 is normal. If there is displacement of the maximum, accentuated asym- 

 metry, or other abnormality, the presence of impurities or degradation 

 products is indicated. Concentrated oily solutions are diluted with choloro- 

 form and absolute alcohol to make a 0.0015% solution of calciferol. The 

 extinction is determined, and correction is made for the oil, if known. If the 

 oil is unknown, the unsaponifiable fraction is separated, and allowance is 

 made for saponification loss, which usually amounts to about 7.5 %. 



•2 C. E. Bills, F. G. McDonald, L. N BeMiller, G. E. Steel, and M. Nussmeier, 



/. Biol. Chem. 93, 775 (1931). 

 1 P. Gyorgy, Vitamin Methods. Academic Press, New York, 1950-1951. 

 ^G. Pirlot", Anal. Chin. Acta 2, 744 (1948) [C.A . 43, 7997 (1949)]. 

 3H. P^nau and G. Hagemann, Helv. Chim. Acta 29, 1366 (1946). 



