ASCORBIC ACID 301 



metaphosphoric acid has replaced other acids for extraction because 

 of the greater stability of the vitamin in this medium. Hence, in the 

 procedure to be given, metaphosphoric acid rather than the acetic 

 acid originally employed will be indicated. Ponting (1943) found 

 that oxalic acid may also be used. Ascorbic acid can be titrated with 

 the dye, and dehydroascorbic acid cannot be measured unless it is 

 reduced to ascorbic acid by an agent such as hydrogen sulfide. This 

 reduction is made preferably in a 3% metaphosphoric acid extract 

 buffered to pH 3.5 to 3.7 with citrate (Bessey, 1938) . In macro work 

 the hydrogen sulfide is passed through the solution for about 15 

 min., and after standing at room temperature for 2 hr., the sulfide 

 is removed by passing wet nitrogen through the liquid for 45-60 min. 

 An application of the procedure to micro work would require the ex- 

 perimental determination of the proper time interval for each of these 

 steps. For the colorimetric determination, see page 245. 



Glick Method for Ascorbic Acid 



SPECIAL REAGENTS 



Saturated Sodium 2,6-Dichlorophenolindophenol Solution (sodium 

 2,6-dichlorobenzenoneindophenol) . Shake a few mg. with 100 ml. 

 warm distilled water. Cool, filter, and store in a refrigerator. The 

 soln. may be used for about 10 days. [A stable soln. prepared in 

 dioxane was described by Stone ( 1940) : Distill the dioxane, dis- 

 carding the first and last 10%. Stir a weighed quantity of the dye 

 into the dioxane (about 0.4% equals 0.01 M) and add glacial 

 acetic acid to 1% of the volume. Stir well for about 15 min., and 

 filter through dry No. 42 Whatman filter paper. During titration 

 the dioxane soln. used should not exceed 10% of the total volume.] 



2-3% Metaphosphoric Acid. Store in a refrigerator and prepare 

 fresh every few days. 



Color Standard. Prepare a rose Bengal soln. ( about 1 part per mil- 

 lion) having a pink tinge when placed in a reaction tube and com- 

 pared with a similar vessel containing water. 



Standard Ascorbic Acid Solution. Prepare a fresh 0.1% soln. of 

 crystalline L-ascorbic acid in 2-3% metaphosphoric acid. 



PROCEDURE 



1. Standardize the dye soln. against freshly prepared ascorbic 

 acid soln. under the titration conditions employed for the unknown. 



