Qualitative tests for vitamin A (demonstration)
Vitamin A in chloroform gives a fleeting blue colour with antimony trichloride (Carr-Price). Work dry, work fast and work in dim light.
Aim and principle
Demonstration experiment (shown by the teacher; you write it up and answer the viva). Aim: To detect vitamin A in the given sample (cod liver oil or a vitamin A capsule) by colour reactions. Principle (Carr-Price reaction): Vitamin A (retinol and its esters) is fat-soluble, so it is dissolved in chloroform. Antimony trichloride is a strong Lewis acid (an electron-pair acceptor). It pulls the -OH group off retinol and leaves a positively charged ion with a long chain of conjugated double bonds (a carbocation). This ion absorbs orange-red light near 620 nm, so the solution looks intensely blue. The ion is unstable and breaks down, so the blue fades within a few seconds. Sulphuric acid test: concentrated H₂SO₄ also dehydrates vitamin A and gives a violet to purple colour that soon turns reddish brown. Alternative: SbCl₃ is corrosive and very sensitive to moisture, so some methods use trifluoroacetic acid (TFA, the Neeld-Pearson method) or trichloroacetic acid (TCA) in chloroform instead. They give a similar short-lived blue, also read at about 620 nm.
Requirements
| Item | What it does |
|---|---|
| Sample: cod liver oil or a vitamin A capsule | Source of vitamin A (mostly as retinyl esters) |
| Chloroform, dry and alcohol-free | Dissolves fat-soluble vitamin A; water or alcohol in it spoils the reagent |
| Carr-Price reagent (saturated SbCl₃ in chloroform) | Reacts with vitamin A to give the transient blue colour |
| Acetic anhydride (1-2 drops) | Removes traces of water so SbCl₃ does not turn cloudy |
| Concentrated sulphuric acid | Dehydrates vitamin A for the violet-to-brown colour test |
| Apparatus | Dry test tubes and stand, dry graduated pipettes with a pipette filler, droppers, amber bottles, fume hood; a colorimeter at about 620 nm if the colour is to be measured |
Procedure
Shown as a demonstration. Typical volumes are given; follow the exact volumes in your lab manual. Work in a fume hood, in dim light, with gloves and goggles.
- 1Dissolve 1-2 drops of cod liver oil (or the contents of one capsule) in about 5 mL dry chloroform.keep it in an amber bottle
- 2Carr-Price test: take 1 mL of this solution in a dry test tube and add 1-2 drops of acetic anhydride.
- 3★Add about 2 mL Carr-Price reagent quickly (burette or pipette filler), mix and watch at once.blue appears instantly and fades in seconds
- 4Blank: repeat step 3 with 1 mL plain chloroform in place of the sample.no blue expected
- 5Sulphuric acid test: to 1 mL of the sample solution add 1 drop of concentrated H₂SO₄ down the side of the tube and shake gently.
- 6Optional, to show light sensitivity: leave a portion of the solution in bright light for about an hour, then repeat step 3 beside a fresh portion.compare the strength of the blue
★ one-way, controls the pace
Observation and inference
| Test | Observation | Inference |
|---|---|---|
| Carr-Price: sample + acetic anhydride + SbCl₃ in chloroform | Intense blue colour at once, fading within a few seconds | Vitamin A present |
| Blank: chloroform + Carr-Price reagent | No blue colour | Reagent and solvent are free from interfering substances |
| Sulphuric acid test: sample in chloroform + 1 drop conc. H₂SO₄ | Violet to purple colour that soon turns reddish brown | Vitamin A present (less specific; carotenoids also react) |
| Wet tube or moist reagent (what goes wrong) | White turbidity instead of a clear blue | Moisture has turned SbCl₃ into antimony oxychloride; repeat with dry glassware |
| Light-exposed portion vs fresh portion, Carr-Price | Much fainter blue in the exposed portion | Vitamin A is destroyed by light and oxygen |
Why vitamin A is so easily destroyed
Retinol has five conjugated double bonds (alternate single and double bonds). These give it its activity and its colour reactions, but they are easily attacked by oxygen (oxidation) and changed by light, especially ultraviolet; heat and acids speed this up. Antioxidants such as vitamin E protect it. So vitamin A samples are kept in dark, closed bottles, solutions are made fresh, and the test is done quickly in dim light. Carotenoids such as β-carotene also give a blue colour with SbCl₃, so the test is not fully specific.
Precautions and result
- Use completely dry glassware and dry, alcohol-free chloroform; moisture turns SbCl₃ cloudy.
- Observe (or read) the colour immediately; the blue fades within seconds.
- Protect the sample from light, air and heat: amber bottles, dim light, fresh solutions.
- SbCl₃ and conc. H₂SO₄ are corrosive and chloroform is toxic: wear gloves and goggles, work in a fume hood, and never pipette by mouth.
- Result: The given sample gave a transient blue colour with Carr-Price reagent and a violet colour turning brown with conc. H₂SO₄, so it contains vitamin A.
Vitamin A deficiency causes night blindness, Bitot's spots and xerophthalmia, and is still a public-health concern in young Indian children. Colour reactions like Carr-Price were the basis for measuring vitamin A in fish liver oils, fortified foods and serum; a serum retinol below 20 µg/dL (0.70 µmol/L) indicates deficiency. Because vitamin A is destroyed by light and air, storage and cooking losses matter in diet planning and in fortified oil and milk.
Short answer: “What is the Carr-Price reaction?” Vitamin A dissolved in chloroform gives an intense but short-lived blue colour with antimony trichloride in chloroform (absorbing at about 620 nm). It is used to detect and estimate vitamin A.
Short answer: “Why is acetic anhydride added in the Carr-Price test?” It removes traces of water. Moisture turns SbCl₃ into insoluble antimony oxychloride, which makes the solution cloudy and spoils the blue colour.
Structured question: “Describe the qualitative tests for vitamin A.” Aim. Principle: SbCl₃, a Lewis acid, removes the -OH of retinol and forms an unstable blue carbocation (about 620 nm). Procedure in 3-4 lines. Table: Carr-Price, fleeting blue; blank, no colour; conc. H₂SO₄, violet turning reddish brown. Precautions: dry glassware, dim light, read at once. Result line.
D-D-Q for Carr-Price: Dry glassware, Dim light, Quick reading, because the blue fades in seconds.