256 ASCORBIC ACID 



dye is different from and hence can be distinguished from ascorbic acid. 

 Good agreement was found between results obtained by the "continuous 

 flow" and formaldehyde methods when fresh, processed, and caramelized 

 foods were tested. Harris and Mapson^^ made an important contribution 

 to this field of study in obtaining confirmation of the validity of both these 

 methods by biological assays. 



(4) In Pigmented Solution. The presence of anthocyanins or other natural 

 coloring matters in tissue extracts may obscure the colorimetric end point 

 in the indophenol titration method. To overcome this difficulty and to 

 avoid the personal element in the visual titration of ascorbic acid, elec- 

 trometric titration has been used by Kirk and Tressler, working "with vege- 

 table and fruit extracts. i^'* A continuous drift in potential during titration 

 with the dye made the potentiometric method extremely difficult to oper- 

 ate, but L. J. Harris et al. obtained considerable improvement by using a 

 platinum and mercury electrode. ^^^ This was, however, unsuitable in the 

 presence of high concentrations of reducing substances other than ascorbic 

 acid and, in such instances, a bright platinum electrode was recommended. 



Various photoelectric colorimeters have been applied A\ith considerable 

 success to the estimation of ascorbic acid and have fulfilled the dual pur- 

 pose of correcting for the natural color and for spontaneous slow fading of 

 the dye in acid solution. Mindlin and Butler,^^^ working ^dth blood serum, 

 and Bessey,"'' with plant tissue, found that addition of excess 2 , 6-dichloro- 

 phenolindophenol with instantaneous measurement of the residual color 

 in a photocolorimeter avoided interference from the slower reducing non- 

 specific substances. 



Their work, based on the discussions of Rosen and Evelyn,^-" confirmed 

 the observations of Meunier^-* Avhich appeared about this time. Bessey also 

 showed that, with a photoelectric colorimeter, satisfactory results were 

 obtained, even in the presence of anthocyanins or in turbid solution, ^^■hen 

 buffers at pH 3.5 to 3.7 were used. This photoelectric method of assay, with 

 various modifications, has been applied to several colorimeters and to a 

 wide range of materials.'"- 22. 49, 129-132 



Oxidized 2 , 6-dichlorophenolindophenol can be quantitatively extracted 



124 M. M. Kirk and D. K. Tressler, Ind. Eng. Chem. Anal. Ed. 11, 322 (1939). 

 126 L. J. Harris, L. W. Mapson, and Y. L. Wang, Biochem. J. 36, 183 (1942). 

 126 n. L. Mindlin and A. M. Butler, J. Biol. Chem. 122, 673 (1937-1938). 

 1" C. Rosen and K. A. Evelyn, Proc. Roy. Soc. Can. 31, Ai)pendix B, cliii (1937). 



128 p. Meuiiier, Bull. soc. chim. hiol. 19, 877 (1937). 



129 K. A. Evelyn, II. T. Malloy, aiul C. Rosen, J. Biol. Chem. 126, 045 (1938). 



"0 M. A. Elliott, A. L. Sklar, and S. F. Acree, J. Research Natl. Bur. Standards 26, 



117 (1941). 

 "1 C. Carruthers, Ind. Eng. Chem. Anal. Ed. 14, 826 (1942). 

 i'^2 A. McN. Taylor, Biochem. J. 37, 54 (1943). 



