Quantitative estimation of vitamin C (2,6-dichlorophenol indophenol titration)
Ascorbic acid turns the dye 2,6-dichlorophenol indophenol colourless; the first faint pink that lasts marks the end point. The dye is standardised with pure ascorbic acid.
Aim and principle
Aim: To estimate the vitamin C (ascorbic acid) content of a fruit (guava) by titration with 2,6-dichlorophenol indophenol. Principle: Ascorbic acid is a strong reducing agent. It reduces the dye 2,6-dichlorophenol indophenol (DCPIP), which is blue in neutral solution and pink in acid, to its colourless reduced (leuco) form, and is itself oxidised to dehydroascorbic acid. One molecule of ascorbic acid reduces one molecule of dye. While ascorbic acid remains in the flask, each drop of dye from the burette loses its colour. When all of it has been oxidised, the next drop stays oxidised and shows as a faint pink in the acid medium: this is the end point. The dye is first standardised against a known amount of pure ascorbic acid to find its dye factor. The sample is extracted in 4% oxalic acid or 3% metaphosphoric acid, which keeps the medium acid, slows oxidation by air and stops the enzyme ascorbic acid oxidase. Note: DCPIP measures only reduced ascorbic acid; the 2,4-dinitrophenylhydrazine (DNPH) method measures total vitamin C, including dehydroascorbic acid.
Requirements
| Requirement | What it does |
|---|---|
| 4% oxalic acid (or 3% metaphosphoric acid) | Extracts vitamin C and keeps it stable; gives the acid medium for a pink end point |
| Standard ascorbic acid: stock 1 mg/mL (100 mg in 100 mL 4% oxalic acid); working 0.1 mg/mL (10 mL stock made to 100 mL) | A known amount of vitamin C to standardise the dye |
| DCPIP dye: typically about 50 mg dye (52 mg in some manuals) with 42 mg sodium bicarbonate, made up to 200 mL with distilled water | Titrant that vitamin C reduces; bicarbonate helps it dissolve and stay stable |
| Fresh fruit (guava, amla, orange) or juice (lemon) | Sample |
| Apparatus | Mortar and pestle or mixer, 100 mL volumetric flask, filter paper or centrifuge, burette, 5 mL and 10 mL pipettes, conical flasks |
Procedure
Steps 1-2 standardise the dye; steps 3-6 test the sample. Typical volumes are given. Follow the exact volumes in your lab manual.
- 1Pipette 5 mL working standard ascorbic acid (0.5 mg) into a conical flask and add 10 mL 4% oxalic acid.
- 2Titrate with the dye from the burette until a faint pink lasts about 15 seconds. Repeat for concordant values (V₁).
- 3Weigh 5 g guava, grind it quickly with a little 4% oxalic acid and make up to 100 mL with 4% oxalic acid.work fast, away from sunlight
- 4Filter or centrifuge; pipette 5 mL of the clear extract into a flask and add 10 mL 4% oxalic acid.
- 5Titrate with the dye to the same faint pink (V₂). Repeat for concordant values.
- 6Check a blank of 15 mL 4% oxalic acid alone; it should need only a drop of dye.subtract it if larger
Worked example 1: vitamin C in guava (per 100 g)
Standardisation: 5 mL working standard (0.1 mg/mL) needed 4.0 mL dye. Sample: 5 g guava was extracted and made up to 100 mL with 4% oxalic acid; 5 mL of the extract needed 4.2 mL dye. Find vitamin C in mg per 100 g.
- Ascorbic acid in the standard taken = 5 mL × 0.1 mg/mL = 0.5 mg.
- Dye factor = 0.5 mg ÷ 4.0 mL = 0.125 mg ascorbic acid per mL of dye.
- Ascorbic acid in 5 mL extract = 4.2 × 0.125 = 0.525 mg.
- In 100 mL extract (from 5 g guava) = 0.525 × 100 ÷ 5 = 10.5 mg.
- Per 100 g = 10.5 × 100 ÷ 5 = 210 mg.
- In one line: mg/100 g = (0.5 ÷ V₁) × V₂ × (100 ÷ 5) × (100 ÷ sample weight) = 0.125 × 4.2 × 20 × 20 = 210.
Guava contains about 210 mg vitamin C per 100 g.
Worked example 2: lemon juice (per 100 mL)
Using the same dye (factor 0.125 mg/mL), 10 mL lemon juice was made up to 100 mL with 4% oxalic acid. 10 mL of this needed 3.2 mL dye. Find vitamin C in mg per 100 mL of juice.
- Ascorbic acid in 10 mL of diluted juice = 3.2 × 0.125 = 0.4 mg.
- In 100 mL of diluted juice (= 10 mL lemon juice) = 0.4 × 100 ÷ 10 = 4.0 mg.
- Per 100 mL juice = 4.0 × 100 ÷ 10 = 40 mg.
The lemon juice contains about 40 mg vitamin C per 100 mL.
Precautions and result
- Wear gloves and goggles. Oxalic acid is poisonous: never pipette it by mouth; use a pipette filler.
- Work quickly, away from sunlight, heat and copper or iron utensils, which all destroy vitamin C and give low values.
- Standardise the dye on the day of use and keep it in a brown bottle in the refrigerator, as it is unstable.
- Take the first faint pink that lasts about 15 seconds; do not wait for a permanent colour, as slow-reacting substances fade it later.
- Strongly coloured extracts hide the end point, and other reducing substances (Fe²⁺, sulphite) also decolourise the dye and give high values.
- Result: The vitamin C content of the given sample (guava) is ___ mg per 100 g (or ___ mg per 100 mL for a juice).
Vitamin C is needed to make collagen; lack of it causes scurvy (bleeding gums, poor wound healing). It also greatly improves the absorption of non-haem iron from plant foods, so it helps prevent anaemia. The ICMR-NIN 2020 RDA is 80 mg/day for men and 65 mg/day for women. Because vitamin C is easily lost to heat, air, alkali and storage, this titration is used to compare fresh, stored and cooked foods, check juice labels, and show why amla and guava are such rich sources.
Short answer: “What is meant by the dye factor in vitamin C estimation?” It is the mg of ascorbic acid that reacts with 1 mL of the dye. It is found by titrating a known amount of standard ascorbic acid: dye factor = mg ascorbic acid taken ÷ mL dye used.
Short answer: “Why is the sample extracted in oxalic or metaphosphoric acid?” The acid keeps vitamin C stable by slowing air oxidation and stopping ascorbic acid oxidase, and it gives the acid medium in which the end point shows as pink.
Structured question: “Explain the estimation of vitamin C in a fruit by the 2,6-dichlorophenol indophenol method.” Principle: ascorbic acid reduces the dye to its colourless form and becomes dehydroascorbic acid; the first faint pink is the end point. Standardise the dye with 0.5 mg ascorbic acid to get the dye factor. Extract the fruit in 4% oxalic acid, titrate a 5 mL portion, and calculate: mg/100 g = dye factor × V₂ × (total extract ÷ portion) × (100 ÷ weight), e.g. 210 mg/100 g for guava.
"Blue in the bottle, pink in the acid, gone with vitamin C": DCPIP is blue in neutral solution, pink in acid, and colourless once reduced.