Ascorbic acid reduces the oxidation-reduction indicator dye, 2,6dichloroindophenol, to a colorless solution. At the final point, the excess unreduced dye is rose pink in acid solution. The vitamin is extracted, and titration is performed in the presence of HPO3-CH3COOH or HPO3CH3COOH-H2SO4 solution to maintain adequate acidity for the reaction and to prevent autooxidation of ascorbic acid at high pH.

a) Extraction solutions

(1) Metaphosphoric acid-acetic acid solution.

• Dissolve, with stirring, 15 g of freshly powdered HPO3 or HPO3 granules in 40 ml CH3COOH and 200 ml H2O;
• Dilute to approximately 500 ml, and filter quickly through ribbed paper in a bottle with glass stopper. (HPO3 slowly changes to H3PO4, but if stored in a refrigerator, the solution remains satisfactory for 7-10 days.)

(2) Metaphosphoric acid-acetic acid-sulfuric acid solution. 

• Proceed as in (1), except use 0.3N H2SO4 instead of H2O 

b) Standard solution of ascorbic acid.-1 mg / ml.

• Accurately weigh 50 mg of USP ascorbic acid reference standard that has been stored in a desiccator away from direct sunlight. 
• Transfer to a 50 ml volumetric flask. 
• Dilute to volume immediately before use with HPO3-CH3COOH solution, (a) (1).

c) Standard solution of Indophenol.

• Dissolve 50 mg of 2,6-dichloroindophenol. Salt of Na (Eastman Kodak Co. No. 3463), which has been stored in a desiccator on soda lime, in 50 ml of H2O to which 42 mg of NaHCO3 have been added; shake vigorously, and when the dye dissolves, dilute to 200 ml with H2O. 
• Filter through fluted paper in a glass bottle with a glass stopper. Keep the cap out of direct sunlight and store it in the refrigerator. (Decomposition products that make the endpoint indistinct in some batches of dry indophenol and also develop over time in stock solution) 
• Add 5.0 ml of extraction solution containing excess ascorbic acid to 15 ml of dye reagent If the reduced solution is practically colourless, discard it and prepare a new stock solution. If the dry dye is defective, get a new source.) 
• Transfer three aliquots of 2,0 ml of standard ascorbic acid solution to each of three 50 ml Erlenmeyers containing 5,0 ml of HPO3CH3COOH solution, (a) (1). 
• Titrate promptly with an indophenol solution of 50 ml burette until light, but light pink, persists ≥5 s. (Each titration should require approximately 15 ml of indophenol solution, and titrations should be monitored at 0.1 ml.) 
• Similarly, titrate 3 blanks composed of 7.0 ml of HPO3-CH3COOH solution, (a) (1), plus volume of H2O ca equal to the volume solution of indophenol used in direct titrations. After subtracting average white space (usually about 0.1 mL) from the standardization ratings, calculate and express the concingestion of indophenol solution in mg of ascorbic acid equivalent to 1.0 mL of reagent. 
• Standardize the indophenol solution daily with standard freshly prepared ascorbic acid solution. (d) Blue thymol pH indicator. -0,04%. 
• Dissolve 0,1 g of indicator by crushing in agate mortar with 10,75 ml of NaOH 0,02 N and dilute to 250 ml with H2O. 
• Transition range: 1.2 (red) -2.8 (yellow).

• Grind the representative sample or express the contents of the capsule and add approximately 25 ml of HPO3-CH3COOH solution, (a) (1). 
• Test the pH by placing the thymol blue pH indicator in one hand or using a dot plate. (pH > 1.2 indicates appreciable amounts of basic substances.) 
• For liquid preparations, dilute the representative sample twice with HPO3-CH3COOH solution, (a) (1), before performing the test with the indicator.

For dry materials that do not contain an appreciable amount of substances.

• Spray the sample by gentle grinding, add the HPO3-CH3COOH solution, (a) (1) and crush until the sample is in suspension. 
• Dilute with HPO3-CH3COOH solution, (a) (1), at the measured volume. 
• Designate this volume as V ml. (Use about 10 ml of extraction solution / g of dry sample. The final solution should contain 10-100 mg of ascorbic acid / 100 ml). B. 

For dry materials containing appreciable amounts of basic substances.

• Spray the sample by gentle grinding, add solution of HPO3-CH3COOH-H2SO4, (a) (2), to adjust the pH to about 1.2 and crush until the sample is in suspension. 
• Dilute with HPO3-CH3COOH solution, (a) (1), at the measured volume. 
• Designate this volume as V ml. (Use approx. 10 ml of extraction solution / g of dry sample. Final solution should contain 10-100 mg of ascorbic acid / 100 ml.) C.

For liquid materials.

• Take the sample quantity containing approximately 100 mg of ascorbic acid. If there are appreciable amounts of basic substances, adjust the pH to about 1.2 with HPO3 CH3COOH-H2SO4 solution, (a)(2). 
• Dilute with HPO3CH3COOH solution, (a)(1), to a measured volume containing 10-100 mg ascorbic acid / 100 ml. 
• Designate this volume as V ml. For fruit and vegetable juices. 
• Mix thoroughly stirring to ensure a uniform sample and filter through absorbent cotton or fast paper. 
• Prepare fresh juices by pressing the fruit well pulped and filtering. Squeeze citrus juice by commercial device and filter. 
• Add aliquots of ≥100 ml of prepared juice to equal volumes of HPO3-CH3COOH solution, (a) (1). 
• Designate the total volume as V mL. 
• Mix and filter through fast folded paper (Eaton-Dikeman No. 195.18.5 cm, or equivalent).

Titrate 3 sample aliquots containing approximately 2 mg of ascorbic acid and perform blank determinations for correction of titrations as in 967.21B (c), using adequate volumes of HPO3CH3COOH, (a) (1) and H2O solution. If 2 mg ascorbic acid is contained in test solution aliquot <7 mL, add HPO3–CH3COOH solution to giv7 mL for titration.

mg Ascorbic acid/g, tablet, mL, etc.
= (X – B) x (F/E) x (V/Y)

where X = average mL for test solution titration, B = average mL for test blank titration, F = mg ascorbic acid equivalent to 1.0 mL indophenol standard solution, E = number of g, tab lets, mL, etc. assayed, V = volume initial test solution, and Y = volume test solution titrated.

Products containing ferrous Fe, stannous Sn, and cuprous Cu give values in excess of their actual ascorbic acid content by this method. 

• Add 2 drops 0.05% aqueous solution of methylene blue to 10 mL freshly prepared mixture (1 + 1) of test solution and HPO3–CH3COOH reagent and mix. 
• Disappearance of methylene blue color in 5–10s indicates presence of interfering substances. 

• To another 10 mL test solution to which 10 mL HCl (1 + 3) has been added, add 5 drops 0.05% aqueous solution of indigo carmine and mix.
• Disappearance of color in 5–10 s indicates presence of stannous Sn or other interfering substance.]

Download ascorbic acid determination protocol.