II. CHEMISTRY 173 



hydrocarbons and alcohols, (3) chromatographic adsorption on alumina gel, 

 aided at certain stages by an indicator dye, (4) removal of sterols by crys- 

 tallization from methanol and precipitation with digitonin, (5) esterifica- 

 tion to the 3,5-dinitrobenzoate, (6) chromatographic purification of the 

 crude esters, (7) recrystallization of esters, (8) saponification of purified 

 ester to free vitamin, and (9) crystallization of the free vitamin. 



The product thus obtained consisted of vitamin D3 with an admixture 

 of vitamin D2 to the extent of about 10%. Pre\'iously Brockmann had 

 made from tuna liver oiP^"* and halibut liver oiP^* preparations of vitamin 

 D which Avere crystallized as dinitrobenzoates but not as free vitamin. They 

 were regarded at the time as pure vitamin D3 esters, but in the light of the 

 later work they must be considered as somewhat less than pure. However, 

 in view of the known high efficacy ratio of halibut liver oil, it seems unlikely 

 that any ^dtamin D2 was present in this preparation. Zucker ei al}^^- ^^^ 

 obtained the vitamin D of tuna liver oil in the form of its 3 , 5-dinitroben- 

 zoate and allophanate, and that of cod liver oil as allophanate. Apparently 

 these products were about three-quarters pure. Haslewood and Drum- 

 mond^^^ also have w^orked wdth tuna liver oil, obtaining the allophanate of 

 the vitamin in semipure condition. 



In the isolation techniques the importance of the exclusion of air is 

 emphasized, even of air dissolved in solvents. Oxygen-free nitrogen and 

 carbon dioxide gases are used to replace air in apparatus and liquids. 

 Numerous modifications of the procedure of isolation described above are 

 possible. Alternative methods of removing vitamin A from vitamin D in- 

 clude the reacting of the vitamin A with maleic anhydride. ^^^ Presumably, 

 citraconic anhydride w^ould react similarly. ^^^ An alternative method of 

 separating vitamin D from hydrocarbon oils is to esterify the vitamin D 

 with phthalic anhydride, leaving the hydrocarbons behind.^^^ An alternative 

 method of purifying vitamin D by adsorption consists in the use of tri- 

 calcium phosphate instead of alumina gel.^"" 



3. Physics of Activation 



Since the absorption spectra of the several provitamins D are almost 

 identical in shape and position and not much different in height, it will 



19" E. J. H. Simons and T. F. Zucker, /. Am. Chem. Soc. 58, 2655 (1936). 



196 T. F. Zucker, E. J. Simons, H. C. Colman, and B. Demarest, Nahtnvissenschaften 



26, 11 (1938). 

 '" G. A. D. Haslewood and J. C. Drummond, J. Soc. Chem. Ind. (London) 55, 598 



(1936). 

 1^8 0. Dalmer, F. von Wcrder, and T. Moll, Hoppe-Seyler's Z. phijsiol. Chem. 224, 



86 (1934). 

 i«9 A. Windaus, O. Linsert, A. Luttringhaus, and G. Weidlich, Ann. 492, 226 (1932). 

 "« S. E. Miller, U. S. Pat. 2,179,560 (1939). 



