V. ESTIMATION 249 



natural from imitation fruit juices. In a later communication ''•' he reported 

 hioloj^ical tests which showed that this reduction of the dye to the leuco 

 form was a measure of the antiscorbutic activity of the lemon juice. Sub- 

 sequent observations by Tillmans and his associates'^" confirmed this 

 relationship, which was used by L. J. Harris and his coworkers for the 

 deA'elopment of a specific fiuantitative test for the estimation of ascorbic 

 acid.-^' '* Their more important modifications included the use of a pre- 

 liminary extraction process with trichloroacetic acid (later replaced by 

 metaphosphoric acid), titration hi relatively strong acid solution, and rapid 

 completion of titration (1 to 2 minutes), whereby the interfering action of 

 the majority of reducing substances other than ascorbic acid is inhibited. 

 Bessey and King'^ about this time put forward similar suggestions, the 

 main difference in procedure from the Harris method being that they pre- 

 ferred to titrate the dye against a fixed volume of sample extract and not 

 vice versa. This is possibly a point of personal preference, depending upon 

 whether discharge or appearance of the pink color is found to be the more 

 readily detected. 



It is generally accepted that the pH at which the titration is carried 

 out should lie between 1.0 and 3.5, although Bessey quotes a range of 2.0 

 to 3.0 as a necessary condition for specificity.'^" At pPI 1.0 some slight fading 

 of the dye occurs, but it has been show^n that interference by sulfhydryl 

 groups is eliminated at this point.''^ Because of the importance of the time 

 factor, a microtitration is usually preferred; 0.05 ml. of dye, ec|uivalent to 

 approximately 0.02 mg. of ascorbic acid, is titrated with the sample ex- 

 tract of such concentration that approximately 1 to 2 ml. is taken. In order 

 to maintain a relatively constant titer, the use of graded concentrations of 

 both sample extract and dye have been recommended for different ascorbic 

 acid levels.' The dye solution is usually prepared in a concentration of 

 0.025% to 0.100%,''^^ and the addition of small quantities of sodium 

 bicarbonate'^ or buffer at pH 6.8'^ has been suggested by some workers. 

 Storage in the dark and at a low temperature is advised, and renewal at 

 weekly intervals is usually recommended. '^*' '^ Daily standardization is 

 essential. This may be carried out by titration against pure ascorbic acid 

 in metaphosphoric acid'' or against ferrous salts^^ or by the reaction of the 



«^ J. Tillmans, Z. Untersuch. Lebensm. 60, 34 (1930). 



^'' J. Tillmans, P. Hirsch, and W. Hirsch, Z. Untersuch. Lebensm. 63, 1 (1932). 

 ^* J. Tillmans, P. Hirsch, and J. Jackisch, Z. Untersuch. Lebensm. 63, 241 (1932). 

 ^' J. Tillmans, P. Hirsch, and J. Jackisch, Z. Untersuch. Lebensm. 63, 276 (1932). 



68 T. W. Birch, L. J. Harris, and S. X. Ray, Biochem. J. 27, 590 (1933). 



69 O. A. Bessey and C. G. King, J. Biol. Chem. 103, 687 (1933). 

 ™ O. A. Bessey, /. Am. Med. Assoc. Ill, 1290 (1938). 



" C. G. King, Ind. Eng. Chem. Anal. Ed. 13, 225 (1941). 



" A. J. Lorenz and L. J. Arnold, Ind. Eng. Chem. Anal. Ed. 10, 687 (1938). 



