Plant Lab Protocols / Methodology for Pigments
Lycopene
Lycopene is responsible for the red color of tomato and the fleshy part of water melon. It is a carotene having the formula C40H56. Though it has no nutritional value, its contribution to the color of tomato has a great role in consumer acceptability.
Principle
The carotenoids in the sample are extracted in acetone and then taken up in petroleum ether. Lycopene has absorption maxima at 473nm and 503nm. One mole of lycopene when dissolved in one liter light petroleum (40-60°C) and measured in a spectrophotometer at 503nm in 1cm light path gives an absorbance of 17.2 x 104. Therefore, a concentration of 3.1206mg lycopene/mL gives unit absorbance.
Materials
» Acetone (AR grade)
» Petroleum Ether 40-60 (AR)
» Anhydrous Sodium Sulphate
» 5% Sodium Sulphate
Procedure
1.
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Take 3-4 tomato fruits (sample) and pulp it well to a smooth consistency in a waring blender.
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2.
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Weigh 5-10g of this pulp.
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3.
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Extract the pulp repeatedly with acetone using pestle and mortar or a waring blender until the residue is colorless.
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4.
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Pool the acetone extracts and transfer to a separating funnel containing about 20mL petroleum ether and mix gently.
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5.
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Add about 20mL of 5% sodium sulphate solution and shake the separating funnel gently. Volume of petroleum ether might be reduced during these processes because of its evaporation. So add 20mL more of petroleum ether to the separating funnel for clear separation of two layers. Most of the color will be noticed in the upper petroleum ether
layer.
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6.
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Separate the two phases and re-extract the lower aqueous phase with additional 20mL petroleum ether until the aqueous phase is colorless.
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7.
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Pool the petroleum ether extracts and wash once with a little distilled water.
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8.
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Pour the washed petroleum ether extract containing carotenoids into a brown bottle containing about 10g anhydrous sodium sulphate. Keep it aside for 30 min or longer.
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9.
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Decant the petroleum ether extract into a 100mL volumetric flask through a funnel containing cotton wool. Wash sodium sulphate slurry with petroleum ether until it is colorless and transfer the washings to the volumetric flask.
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10.
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Make up the volume and measure the absorbance in a spectrophotometer at 503nm using petroleum ether as blank.
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Calculation
Absorbance (1 unit) = 3.1206mg lycopene/mL
mg lycopene in 100g sample =
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31.206 X Absorbance
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Wt. of sample (g)
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References
1. Ranganna, S (1976) In: Manual of Analysis of Fruits and Vegetable Products McGraw Hill New Delhi p 77.
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