508 THE TOCOPHEROLS 



ence by other reducing substances. At the dropping mercury electrode,^"* 

 the /3- and 7-tocopherols are oxidized at more positive potentials than is 

 the a, and the latter can be determined, polarigraphically, within 5 % in 

 the presence of oils or of traces of cholesterol.^^ The apparent oxidation- 

 reduction potential of a-tocopherol seems to be between the normal oxi- 

 dation potentials of mono- and dimethylhydroquinones/^ approximately 

 -f200 millivolts.^ The potentials of the four tocopherols (a to d) are re- 

 ported as +273, +343, +348, and +405 millivolts, respectively.^^ 



None of these methods distinguishes between all the different tocopherols, 

 and, since their biological activity is unequal, various chemical methods 

 were sought by which to confirm or replace the tedious bioassay. Chromat- 

 ographic separation of the pure tocopherols from mixtures of them is in- 

 complete.^^ In one of the differentiating chemical methods,^^ advantage was 

 taken of the fact that at 15° 13- and 7-tocopherols produce only about half 

 the color (Emmerie and Engel) which they would produce at 35°, whereas 

 temperature has no effect on color production by a-tocopherol. 



A differential method for 5-tocopherol was developed, ^'^' ^^ depending 

 on the fact that with the ferric chloride-dipyridyl reagent 5-tocopherol 

 gives a slow steady increase in color intensity (in 10 min.) after the initial 

 rapid oxidation is completed (23>^ min.). This probably indicates that 5- 

 tocopherol is oxidized beyond the p-quinone stage. The absorption spec- 

 trum of AgNOs-oxidized 5-tocopherol has a broad maximum, ^^ suggesting 

 a mixture of reaction products. With nitric acid oxidation the product has 

 an absorption maximum at 373 m/i which might be made the basis for 

 spectrophotometric differentiation from the other tocopherols whose maxi- 

 mum is at 460 to 480 m/x. 



An actual separation is partially secured by utilizing the fact that 13- 

 tocopherol does not couple with diazotized o-dianisidine.^^ A differential 

 method for the determination of mixtures of a-, 7-, and 5-tocopherols^^ 

 depends on the relative difference in color intensity of the coupled 7- and 

 5-tocopherols in alkaline solutions, measured at two wavelengths. 



All the tocopherols except the a react with nitrous acid to form nitroso 



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36 J. J. Beaver and H. Kaunitz, J. Biol. Chem. 152, 363 (1944). 



36 C. Golumbic and H. A. Mattill, /. Biol. Chem. 134, 535 (1940). 



37 W. Wacks, Biochem. Z. 319, 561 (1949). 



38 E. Emmerie, Ann. N. Y. Acad. Sci. 52, 309 (1949). 



39 E. L. Hove and Z. Hove, /. Biol. Chem. 156, 601 (1944). 

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« M. H. Stern and J. G. Baxter, Anal. Chem. 19, 902 (1947). 

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