246 ASCORBIC ACID 



sample is important, and between 7:P^ and 4:P^ has been recommended. 

 Undoubtedly, however, the character of the sample must be considered. 

 A dehydrated and starchy vegetable, for instance, requires a much higher 

 ratio than a soft tissue sample. 



Although trichloroacetic acid was initially recommended for the ex- 

 traction of plant tissue,^^ this was subsequently replaced by metaphos- 

 phoric acid."' ^"^ ^^' ^^'^^ This acid has the advantage of giving a clearer 

 solution and also is a better stabilizer of ascorbic acid (especially in the 

 presence of cuprous ions) than trichloroacetic acid. The use of other 

 organic acids for this purpose has been investigated, and, of these, oxalic 

 acid has been found as reliable as and even superior to metaphosphoric.^^"^* 

 This acid, however, should be kept in the dark to avoid the formation of 

 peroxides which may destroy ascorbic acid in the presence of catalysts.^^ 

 If ferrous ions are present in the sample under test, the use of oxalic acid or 

 metaphosphoric acid is likely to catalyze the reaction of these ions -vvith 

 2 , 6-dichlorophenolindophenol unless suitable precautions are taken (see 

 p. 249). Extraction with 10% acetic acid containing 0.1 % oxalic acid, fol- 

 lowed by reduction of pH to 0.4 before titration with the dye, has been 

 suggested to avoid this difficulty,^^ and the use of sodium acetate-hydro- 

 chloric acid buffer of pH 0.65 has, for the same reason, been recommended 

 when extracting stored canned foods.^^ Mineral acids have also been advised 

 for samples where ferrous iron is likely to interfere, but some workers have 

 found higher results when extracting with hydrochloric acid or sulfuric 

 acid as compared with metaphosphoric acid. This finding, together with 

 an apparent increase in ascorbic acid content observed by some workers 

 when vegetables are cooked, has been attributed to the hydrolysis of pro- 

 tein bound or "combined" ascorbic acid.^^"^^'' Other workers have been 



22 H. J. Loeffler and J. D. Ponting, Ind. Eng. Chem. Anal. Ed. 14, 846 (1942). 



23 S. A. Goldblith and R. S. Harris, Anal. Chem. 20, 649 (1948). 



24 L. J. Harris and S. N. Ray, Biochem. J. 27, 303 (1933). 

 26 A. Fujita and D. Iwatake, Biochem. Z. 277, 293 (1935). 



26 C. M. Lyman, M. O. Schultze, and C. G. King, J. Biol. Chem. 118, 757 (1937). 



27 0. A. Bessey, /. Biol. Chem. 126, 771 (1938). 



28 W. L. Nelson and G. F. Somers, Ind. Eng. Chem. Anal. Ed. 17, 754 (1945). 



29 J. H. Roe, Science 80, 561 (1934); J. Biol. Chem. 116, 609 (1936). 



30 B. Willberg, Z. Untersuch. Lebensm. 76, 128 (1938). 



31 J. D. Ponting, Ind. Eng. Chem. Anal. Ed. 15, 389 (1943). 



32 W. S. Gillam, Ind. Eng. Chem. Anal. Ed. 17, 217 (1945). 



33 F. Wokes, Analyst 72, 63 (1947). 



34 L. P. Guild, E. E. Lockhart, and R. S. Harris, Science 107, 226 (1948). 



35 I. J. Babbar and B.Ahmud, Ann. Biochem. and Exptl. Med. {India) 9, 103 (1949). 



36 F. E. Huelin, Analyst 75, 391 (1950). 



37 F. E. Huelin and I. M. Stephens, Australian J. Exptl. Biol. Med. Sci. 25, 17 (1947). 



38 F. Brown and W. B. Adam, J. Sci. Food Agr. 1, 51 (1950). 



39 E. W. McHenry and M. L. Graham, Nature 135, 871 (1935). 



