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VITAMIN "C" DETERMINATION Use: The test is used to determine ascorbic acid concentration in single strength citrus juices

and reconstituted concentrates provided they do not contain ferrous, stannous, cuprous, sulfite or thiosulfate ions. Reagents: Methaphosphoric acid pellets HPO3, C.P. Sodium 2, 6-dichlorobenzenoneindophenol (Eastman No. 3463) Reagent grade. Store in dessicator. Ascorbic acid (l-Ascorbic Acid) (Eastman No. 4640) Keep cool, dry and store out of direct sunlight Acetic acid, glacial Equipment: Analytical balance 500 ml glass stoppered bottle, clear 500 ml glass stoppered bottle, amber 500 ml beaker 250 ml beaker 150 ml beaker 50 ml beaker 500 ml volumetric flask 100 ml volumetric flask 125 ml Erlenmeyer flask - 6

50 ml burette 50 ml graduated cylinder 10 ml pipettes - 6 5 ml pipettes - 6 Filter funnel Fluted filter paper Reagent Solutions:

A. Acid solution - Dissolve 10 gms. HPO3 pellets in warm distilled water (60EC) using successive portions until the acid completely dissolve, transferring portions to a 500 ml volumetric flask. Allow to cool to room temperature and add 40 ml glacial acetic acid and sufficient distilled water to make 500 ml. (HPO3 slowly changes to H3PO4, but this solution remains satisfactorily for 7-10 days when storied in refrigerator). B. Dye solution - 250 mg. of sodium 2,6-dicholrobenenoneindcphenol dissolve in approximately 500 mil warm (60EC) recently boiled distilled water. Cool to room temperature and transfer to brown bottle fitted with a tight stopper to protect from air and light. Store in refrigerator and filter before using. When dye solution fails to give a distinct endpoint with ascorbic acid plus acid solution, discard it. C. Standard Ascorbic Acid Solution - Weigh accurately a 50 ml beaker and without removing from the balance pan add exactly 100 mg pure ascorbic acid (mp-190-192EC). Add 10 ml acid mixture to beaker and dissolve the ascorbic acid. Transfer the solution to a 100 ml volumetric flask qualitatively, by rinsing the flask with successive portions of distilled water. Make up to volume. The solution is now exactly 1 mg of ascorbic acid per ml of solution. Make up fresh for each standardization. DO NOT STORE. D. Standardization of Dye Solution - Bring dye solution to room temperature. Transfer 10 ml aliquot of ascorbic acid solution to a 150 ml beaker and add 10 ml of the acid solution. Titrate rapidly the ascorbic

acid solution with dye solution from 50 ml burette until light but distinct rose-pink persists at least 5 seconds. E. Calculation for Dye TiterDye Titer (or Factor) = 10 (mg ascorbic acid used) = mg mls Dye Solution used ml



1. Take a 10 ml sample of orange juice and transfer to 125 ml Erlenmeyer flask. Add 10 ml acid solution. 2. Titrate immediately with Standard Dye Solution to first permanent pink endpoint. First endpoint may fade due to absorbed juice in pulp. 3. Calculation:
Dye Titer X titration X 100=Mg Ascorbic acid per 10 100 ml juice

RAPID METHOD OF VITAMIN "C" DETERMINATION IN CONCENTRATED ORANGE JUICE 1. 2. Take a 2 gm sample of concentrate and transfer to 125 ml Erlenmeyer flask. Add 10 ml acid solution. 3. Titrate immediately with Standard Dye Solution to first permanent pink endpoint. 4. Calculation:
100 Mg. Ascorbic acid per Dye Titer X titration X wt = 100 gms. concentrate

A fast and reliable method for standardization of Dye Solution is as follows: 1. Use 98 to 99% pure ascorbic acid

2. Dissolve 100 mg of ascorbic acid in 100 ml of distilled water. (Use a 200 ml vol. flask) 3. Fill burette with the dye solution that is to be standardized. 4. Titrate into sample containing a little acid mixture solution and 10 ml sample of the ascorbic acid solution (use 10 ml vol. pipette for ascorbic acid solution). 5. Results of Standardization: 10 ) number ml dye used equals Dye Titer (or Factor).

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