Quantitative estimation of carotene (demonstration)
Carotenes are extracted with acetone, moved into petroleum ether, β-carotene is separated on a column and read at about 450 nm against a standard.
Aim and principle
Demonstration experiment (shown by the teacher; you write it up, do the calculation and answer the viva). Aim: To estimate the β-carotene content of a vegetable such as carrot or a green leafy vegetable. Principle: Carotenes are fat-soluble pigments. Acetone mixes with the water in fresh tissue and dissolves them. When water is added, the pigments move into a petroleum ether layer. On a column of an adsorbent (activated alumina, or magnesium oxide mixed with a filter aid), β-carotene, a pure hydrocarbon and the least polar pigment, is held weakly and washes out first as a yellow-orange band. The more polar xanthophylls and chlorophylls stay higher on the column. β-carotene absorbs blue light strongly, with a peak at about 450 nm (manuals use 436-452 nm). By Beer's law, absorbance is proportional to concentration, so the sample is compared with a standard β-carotene solution.
Requirements
| Item | What it does |
|---|---|
| Fresh sample (e.g. 5 g carrot or leafy vegetable) | Source of carotenes |
| Acetone | Extracts pigments from the wet tissue; grind until the residue is colourless |
| Petroleum ether (40-60 °C) | Takes up the carotenes as a non-polar layer; also the eluting solvent (a little acetone may be added) |
| Anhydrous sodium sulphate | Removes traces of water from the petroleum ether extract |
| Activated alumina, or magnesium oxide with a filter aid | Adsorbent in the column that separates β-carotene from other pigments |
| Standard β-carotene solution (e.g. 2 µg/mL in petroleum ether) | Reference for the calculation |
| Apparatus | Mortar and pestle, separating funnel, chromatography column, volumetric flasks, colorimeter or spectrophotometer at about 450 nm, fume hood, pipette filler |
Procedure
Shown as a demonstration. Typical quantities are given; follow the exact volumes in your lab manual. Work in dim light, in a fume hood, away from flames.
- 1Weigh 5 g of the fresh sample and grind it with about 10-15 mL acetone (a pinch of sand or anhydrous sodium sulphate helps).
- 2Pour off the acetone; grind again with fresh acetone until the residue is colourless. Pool the extracts.
- 3Transfer to a separating funnel with about 15-20 mL petroleum ether; add water and swirl gently.pigments move to the upper petroleum ether layer
- 4Discard the lower acetone-water layer; wash the petroleum ether layer with water and pass it through anhydrous sodium sulphate.
- 5Concentrate the extract to a few mL by gentle warming (below about 40 °C) or a stream of nitrogen.
- 6★Load it onto the column; elute with petroleum ether (with a little acetone if needed) and collect the first yellow-orange band.this is β-carotene
- 7Make the eluate up to a known volume (e.g. 50 mL) with petroleum ether.
- 8Read the absorbance at about 450 nm against a petroleum ether blank, together with the standard β-carotene.
★ one-way, controls the pace
Scheme at a glance
Xanthophylls and chlorophylls are more polar, so they stay higher on the column.
Worked example: β-carotene in carrot
5.0 g of fresh carrot was extracted and the β-carotene fraction was made up to 50 mL. Its absorbance at 450 nm was 0.45. A standard β-carotene solution of 2.0 µg/mL read 0.50. Find the β-carotene content per 100 g.
- Formula: β-carotene (µg/100 g) = (A test ÷ A standard) × concentration of standard (µg/mL) × total volume (mL) ÷ weight of sample (g) × 100.
- Concentration in the eluate = (0.45 ÷ 0.50) × 2.0 = 0.9 × 2.0 = 1.8 µg/mL.
- Total β-carotene in 50 mL = 1.8 × 50 = 90 µg.
- This came from 5.0 g of carrot, so per gram = 90 ÷ 5.0 = 18 µg/g.
- Per 100 g = 18 × 100 = 1800 µg.
- Check: 1800 × 5.0 ÷ 100 = 90 µg, and 90 ÷ 50 = 1.8 µg/mL.
1800 µg (1.8 mg) of β-carotene per 100 g of fresh carrot. If the eluate was diluted before reading, multiply by the dilution factor. Some manuals use a standard curve or the extinction coefficient of β-carotene instead of a single standard.
Expressing carotene as vitamin A
β-carotene is provitamin A: the body splits it into retinol, but not one-to-one, and absorption from food is incomplete. To express carotene as vitamin A activity, you divide by a conversion factor, and the factor depends on the system used. For example, the US retinol activity equivalent (RAE, Institute of Medicine 2001) counts 12 µg of food β-carotene as 1 µg of retinol, while the older retinol equivalent (RE, FAO/WHO) uses 6 µg. The ICMR-NIN 2020 Indian RDAs use 6 µg β-carotene = 1 µg retinol (12 µg for α-carotene and β-cryptoxanthin). So 1800 µg β-carotene is 300 µg retinol by the Indian factor but 150 µg RAE by the US factor. Always name the factor you use.
Precautions and result
- Work in dim light and quickly; carotenes are destroyed by light, air, heat and acid.
- Petroleum ether and acetone are highly flammable: no flames, warm only on a water bath below about 40 °C, work in a fume hood, wear gloves and goggles, and never pipette by mouth.
- Grind until the residue is colourless so no pigment is left behind.
- Do not let the column run dry or crack; collect only the first yellow-orange band.
- Read the absorbance soon after elution; dilute if the reading is too high for the instrument.
- Result: The β-carotene content of the given sample is ___ µg per 100 g (fresh weight).
β-carotene from green leafy vegetables, carrot, yellow-orange fruits and red palm oil is the main source of vitamin A in many Indian vegetarian diets. Estimating it supports food composition tables, diet surveys and programmes to prevent vitamin A deficiency (night blindness, xerophthalmia). It also shows how cooking, drying and storage reduce provitamin A, which guides advice such as short cooking with a lid and eating vegetables with a little fat for better absorption.
Short answer: “Why does β-carotene come off the column before xanthophylls?” β-carotene is a hydrocarbon with no polar groups, so the polar adsorbent holds it least and it is washed out first. Xanthophylls carry -OH groups and are held more strongly.
Short answer: “Why is the absorbance of β-carotene read at about 450 nm?” 450 nm is near its absorption maximum (it absorbs blue light most strongly), which gives the highest sensitivity.
Structured question: “Outline the estimation of carotene in a vegetable, with the calculation.” Principle: acetone extraction, partition into petroleum ether, column separation (β-carotene least polar, elutes first), Beer's law. Steps: grind, partition, dry, column, collect the band, read at about 450 nm with a standard. Formula: (A test ÷ A std) × C std × volume ÷ weight × 100. Result in µg/100 g. Two precautions.
A-P-C-450: Acetone pulls it out, Petroleum ether carries it, the Column sorts it, and it is read at 450 nm.