II, CHEMISTRY 191 



and after saponification, it provides a means of determining the percentages 

 of free and esterified total sterols. Digitonin does not give a precipitate 

 with any form of vitamin D. The critical details of conducting an analysis 

 with digitonin are given by Penau and Hardy,-*^ Castille and Ruppol,^" 

 and Bilger ei al.-"^^ 



With the availability of modern apparatus, the spectrophotometric 

 method of determining ergosterol and other provitamins D is usually the 

 method of choice. The characteristic spectrum (Fig. 8) is shown by the 

 free pro\dtamins and also by their esters, provided that the acid radical 

 does not itself absorb in the critical region. ^^^' ^^^ If the unknown is free of 

 "ultraviolet dirt," it is only necessary to compare the optical density at 

 the 282 van maximum with that of pure provitamin D. To prove that the 

 unknown is not dirty, it is usually sufficient to measure the absorption at 

 the nearby minimum, 277 mju; the relation of this to the maximum should 

 be 0.8. If irrelevant absorption cannot be avoided, one may resort to the 

 procedure employed by Castille and RuppoP^'' in studies with yeast. This 

 consists in precipitating the total sterols with digitonin, dissolving the 

 washed and dried digitonides in absolute alcohol, and making the spectral 

 measurement on the resulting solution. In this case it is desirable to use 

 as the standard a preparation of pure ergosterol digitonide. 



The accepted formula of ergosterol is the work of many chemists. Tan- 

 ret^^* in 1889 recognized the single hydroxyl group and the molecule of 

 water of crystallization, and he presented analyses corresponding to the 

 formula C26H4oO-H20. In 1908 he^^^ re\ased the formula to C27H420-H20, 

 which remained accepted until 1932, when Windaus et aZ.,^^^-"^ by the 

 analysis of halogenated nitrobenzoic esters and other complex derivatives, 

 established the empirical formula as C28H440-H20. The structural formula 

 (Fig. 4) was established by Windaus ei aW^ in 1934. Confirmation of de- 

 tails followed in papers by Fernholz and Chakravorty"^ and Dimroth and 

 Trautmann."^ The complicated proofs of stmcture of ergosterol and related 

 sterols are reviewed by Rosenberg^* and Fieser and Fieser."^ 



6. Lumisterol 



C28H44O. Three double bonds. Melting point 118°. Principal absorption 

 maxima at 265 and 279 m/x. Molecular extinction coefficient at 279 m/x = 

 8500.280 [ajif = +192°, and [a]lli = +235° (in acetone). 



^''^ A. Windaus and A. Liittringhaus, Nachr. Ges. Wiss. Gottingen, Math, phijsik Kl., 



Fachgruppe III 4 (1932). 

 276 A. Windaus, H. H. Inhoffen, and S. von Reichel, Ann. 510, 248 (1934). 

 2" E. Fernholz and P. N. Chakravorty, Ber. 67, 2021 (1934). 

 "8 K. Dimroth and G. Trautmann, Ber. 69, 669 (1936). 

 *7' L. F. Fieser and M. Fieser, Natural Products Related to Phenanthrene, 3rd ed. 



Reinhold Publishing Corp., New York, 1949. 

 wo I. M. Heilbron, G. L. Moffet, and F. S. Spring, /. Chem. Soc. 1937, 411. 



