v. ESTIMATION- 257 



from at'id solution l)y xylene and other solvents in wiiicli \vater-solul)le 

 pigments are insoIul)le, and use can be made of this findinji; for \isual and 

 photometric assay of ascorbic acid in colored extracts of plant tissue and 

 blood and uiinc.^- -*•'"'• '^''^^ Care should i)e exercised in the choice of 

 xylene, since some batches have been found to introduce errors through the 

 presence of oxidizing substances.-* Rubin ct a/.'^^ described a method ^vhich 

 also determined dehydroascorbic acid, antl Robinson and Stotz''' suggested 

 the use of formaldehyde and hydrogen peroxide to allow for the presence of 

 reductones and other interfering substances. 



A further complication has recently been found with some colored ex- 

 tracts, since it has been shown that the biologically inactive anthocyanin 

 betanin in red beet also reacts with the dye in the indophenol-xylene ex- 

 traction method, even in the presence of formaldehyde.'-** It has also been 

 reported that, in some anthocyaimi-containing berries, a high percentage 

 of the total apparent ascorbic acid is not fixed by formaldehyde.'^^ This 

 fmdmg, wliich is not in accordance with the work of, for instance, Miller,*^ 

 is somewhat surprising in \iew of the good agreement shown between the 

 results from biological and chemical assaj^ on fresli fruits and \'egetables 

 (see p. 250), including some of the fresh berries cited and stored products 

 made from the fruit. ^' ^^ Further experimental data from biological tests 

 would undoubtedly be valuable for assessing the reliability of these various 

 methods wliich have been proposed for the estimation of ascorbic acid in 

 the presence of interfering substances. 



C. PHYSICAL METHODS 



The relative simpUcity of the chemical methods for estimation of ascorbic 

 acid has diverted interest from the possible apphcation of physical tech- 

 niques in this field. However, Kodicek and Wenig reported a successful 

 modification of the polarograpliic procedure for the estimation of the 

 v-itamin by making use of oxidation at a dropping mercury anode, instead 

 of the more usual reduction at a dropping mercury cathode.^'"' Investiga- 

 tions by other workers into the use of the polarograph in the assay of 

 ascorbic acid in natural products were encouraging.^'"'"*' Gillam subse- 



'" F. Bukatsch, Hoi)pe-Scyler's Z. physiol. Chem. 262, 20 (1939). 



•3< E. Stotz, J. Lab. Clin. Med. 26, 1542 (1940-1941). 



'" L. P. Pcpkowitz, J. Biol. Chem. 151, 405 (1943). 



'36 S. H. Rubin, F. W. Jahns, and J. C. Bauernfeind, Fruit Products ./. 24, 327 (1945). 



>" W. B. Robinson and E. Stotz, J. Biol. Chem. 160, 217 (1945). 



'38 G. F. Somers, W. C. Kelly, E. J. Thacker, and A. M. Redder, Science 110, 17 (1949). 



'39 G. F. Somers, W. C. Kelly, E. J. Thacker, and A. M. Redder, Food Research 



16,62 (1951). 

 '« E. Kodicek and K. Wenig, Nature 142, 35 (1938). 

 "1 K. Schwarz, Z. anal. Chem. 115, 161 (1938-1939). 



