SOP: Estimation of Lycopene
GIFSQ — Global Institute for Food Safety & Quality
Principle
The red colour of tomato is due to the pigment lycopene (C₄₀H₅₆). Its two absorption maxima lie at 473 nm and 503 nm; the molecular extinction coefficients for all-trans lycopene are 18.6 × 10⁴ at 473 nm and 17.2 × 10⁴ at 503 nm. A rapid method estimates lycopene from the absorption of the petroleum ether extract of the total carotenoids at 503 nm — the error is very small (2–5%), since carotene absorbs comparatively negligibly while lycopene absorbs strongly at 503 nm. In the more accurate method, the total absorbance is measured at 473 nm and, after chromatographic separation, a correction is applied for the absorbance due to carotene.
Apparatus required
- Chromatographic column and Buchner flask, as used for β-carotene
- Separating funnels
- Volumetric flasks, 50-ml and 100-ml
- Spectrophotometer with 1 cm cells
- Amber coloured bottle
Reagents
a. Acetone.
b. Petroleum ether.
c. Magnesium oxide.
d. Supercel.
e. Anhydrous sodium sulphate (Na₂SO₄).
f. Water, or water containing 5% Na₂SO₄.
g. Eluent: 3% acetone in petroleum ether (for Method II).
h. Pure lycopene (for the standard curve).
Procedure
Method I — rapid method
Weigh 5–10 g of the juice, puree or ketchup
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Extract repeatedly with acetone in a pestle and mortar or a blender until the residue is colourless
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Transfer the acetone extract to a separating funnel containing 10 to 15 ml of petroleum ether and mix gently
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Take up the carotenoid pigments into the petroleum ether by diluting the acetone (lower phase) with water or water containing 5% Na₂SO₄; transfer the lower phase to another separating funnel and the petroleum ether extract containing the carotenoid pigments to an amber coloured bottle
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Repeat the extraction of the acetone phase similarly with petroleum ether until it is colourless; discard the acetone phase
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To the petroleum ether extract add a small quantity of anhydrous Na₂SO₄, transfer to a 50 ml volumetric flask and dilute to the mark with petroleum ether
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Dilute an aliquot (5 ml if the test sample is tomato juice or ketchup, 2 ml if puree) to 50 ml with petroleum ether
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Measure the colour in a 1 cm cell at 503 nm in a spectrophotometer using petroleum ether as blank
Method II — chromatographic method
Prepare a chromatographic column of magnesium oxide and Supercel (1 : 3) as for β-carotene; place 1 cm of anhydrous Na₂SO₄ at the top of the column and wet the column with petroleum ether
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Pipette 5 or 10 ml of the petroleum ether extract (prepared as in Method I) onto the column and apply suction
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Wash the column continuously with the eluent (3% acetone in petroleum ether), adding successive portions of eluent when the preceding one is just barely visible above the Na₂SO₄ — continue elution until all the carotenoid pigments except lycopene (which remains adsorbed at the top) have moved off and the eluent is colourless
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Transfer the contents of the flask to a separating funnel and wash off the acetone with water
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Transfer the petroleum ether extract through a funnel containing anhydrous Na₂SO₄ on glass wool or cotton wad into a 50- or 100-ml volumetric flask; wash the anhydrous Na₂SO₄ with petroleum ether
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Measure the colour in a spectrophotometer at 473 nm using a 1 cm cell and petroleum ether as blank — call this reading A
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Pipette a similar aliquot to the one taken for chromatography (5 or 10 ml) and dilute to the same volume as that made after chromatography
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Measure the colour of the total carotenoid pigments at 473 nm — call this reading B
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From the absorbance at 473 nm for the total carotenoid pigments, deduct the absorbance due to carotene at 473 nm (B − A) — the value so obtained is the absorbance due to lycopene
Standard curve (for either method)
Weigh accurately 25 mg of lycopene, dissolve in petroleum ether, transfer to a 500-ml volumetric flask and dilute to the mark (1 ml = 50 μg)
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Dilute 25 ml of this solution to 250 ml (1 ml = 5 μg of lycopene)
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Pipette 10, 20, 30, 40, 50 and 60 ml of this solution into separate 100-ml volumetric flasks and dilute to the mark with petroleum ether so as to contain 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 μg of lycopene per ml
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Measure the colour at 473 nm or 503 nm depending on the method followed, and plot absorbance against concentration
Calculation
For Method I, the relationship is: an optical density (OD) of 1.0 = 3.1206 μg of lycopene per ml.
mg of lycopene per 100 g = [3.1206 × OD of the sample × Volume made up (ml) × Dilution × 100] ÷ [1.0 × Wt of sample (g) × 1000]
For Method II, using E¹%₁cm at 473 nm = 18.6 × 10⁴, the relationship is: an OD of 1.0 = 2.8874 μg of lycopene per ml.
mg of lycopene per 100 g = [2.8874 × OD of the sample × Dilution × 100] ÷ [1.0 × Wt of sample (g) × 1000]
Alternatively, read the concentration of lycopene in the sample extract from the standard curve and calculate:
mg of lycopene per 100 g = [μg of lycopene in the sample extract per ml × Dilution × 100] ÷ [Wt of sample (g) × 1000]
Where,
- OD of the sample = absorbance measured at 503 nm in a 1 cm cell (Method I); for Method II, the absorbance due to lycopene, i.e. reading B minus reading A at 473 nm
- Volume made up = volume to which the petroleum ether extract was made (ml)
- Dilution = dilution factor applied before colour measurement
- Wt of sample = weight of the sample taken (g)
Note: the molecular weight of lycopene is 536.85; 536.85 mg/ml gives an OD of 17.2 × 10⁴ at 503 nm, whence OD 1 = 3.1206 μg/ml — carried as printed in the source. The Method II formula is reconstructed from heavily garbled OCR in the source; the reconstruction is verified mathematically — with E¹%₁cm = 10E/M = (10 × 18.6 × 10⁴) ÷ 536.85, an OD of 1.0 corresponds to 2.8874 μg/ml.
To illustrate: the petroleum ether extract from 10 g of tomato juice was made up to 50 ml; 10 ml was diluted to 50 ml for colour measurement; the OD at 503 nm was 0.405 —
mg of lycopene per 100 g of juice = (3.1206 × 0.405 × 50 × 50 × 100) ÷ (1 × 10 × 10 × 1000) = 3.16
Precautions
- Collect the petroleum ether extract in an amber coloured bottle — the pigments are light-sensitive.
- In Method II, continue elution until all carotenoid pigments except lycopene have moved off and the eluent is colourless.
- When making Method I measurements, use petroleum ether as the blank and a 1 cm cell.
References
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.
- Beerh, O.P. and G.S. Bawa, Food Technol. (Chicago), 13, 414 (1959).
- Davis, W.B., Anal. Chem., 21, 1226 (1949).