V. ESTIMATION 255 



with their true ascorbic acid couteut and cannot he accounted for by the 

 interfering substances ah-eady mentioned. The interference in most of these 

 instances can be attributed either to reductones (which can be produced 

 by heating sugar solutions with alkah in the absence of air) or possibly to 

 substances resembling reductic acid (produced by heating galacturonic acid, 

 pectin, or xylose with mineral acid). 



To differentiate ascorbic acid from these interfering substances, Lugg 

 made use of the fact that the vitamin reacts rapidly with formaldehyde at 

 pH 3.5 but slowly at pH 1.5."^ Other indophenol-reducing substances, 

 such as sulfite and cysteine, condense rapidly with the formaldehyde at 

 both pH 3.5 and pH 1.5, whereas reductones, thiourea, and ferrous salts 

 were found not readily to do so at either pH. Titration before and after 

 condensation with formaldehyde at pH 3.5 and pH 1.5 was therefore recom- 

 mended. 



Mapson, however, found that reductones and reductone-like substances 

 in caramelized, fermented foods and stored dehydrated vegetables did, in 

 fact, combine fairly rapidly at pH 3.5 but only slowly at pH 2.0 in the 

 presence of 8 % formaldehyde, and he was therefore able to suggest a 

 suitable modification to Lugg's method."^- '^o By a slight adjustment, di- 

 hydroxymaleic acid and the reducing substances present in malt and beet 

 molasses could also be differentiated from ascorbic acid. Snow and Zilva,^^^ 

 on the other hand, concluded that, with alkali-treated glucose solution, no 

 greater accuracy could be obtained by titrating at pH 2.0 instead of at 

 3.5, and they suggested a procedure which more closely approached Lugg's 

 original method. Further modifications were also put forward by AVokes 

 el al}" No simple quantitative method has been described for the deter- 

 mination of, and hence allowance for, hydroxytetronic acid or isoascorbic 

 acid in the presence of ascorbic acid, but there is no evidence that either 

 occurs in natural products. It seems certain that further chromatographic 

 studies Avill furnish alternative methods to differentiate these substances so 

 closely related to ascorbic acid.'-^ 



L. J. Harris and colleagues have devised a method which makes use of a 

 continuous flow apparatus.^-- ^^' '-^'' It was determined that, under suitably 

 controlled conditions, the rate of reaction of interfering substances with the 



'" J. W. II. Lugg, Mature 150, 577 (1942); Australian ./. Exptl. Biol. Med. Sci. 20, 



273 (1942). 

 i2« L. W. Mapson, Nature 152, 13 (1943). 

 '21 G. A. Snow and S. S. Zilva, liiochem. J. 38, 458 (1944). 

 '" F. Wokes, J. G. Organ, and F. C. Jacoby, J. Soc. Chcm. Ind. {London) 62, 232 



(1943). 

 »" L. W. Mapson and S. M. Partridge, Nature 164, 479 (1949). 

 123" M. N. Bland, B. J. Constable, L. J. Harris, and R. E. Hughes, Proc. 12th Intern. 



Congr. Pure and Appl. Chem., New York (1951). 



