SOP: Colorimetric Determination of Total Chlorophyll
GIFSQ — Global Institute for Food Safety & Quality
Principle
Chlorophyll is extracted with acetone and its colour is measured at 660 nm against a calibration curve. The colorimeter is calibrated with a series of dilutions of a plant extract whose absolute chlorophyll concentration is determined spectrophotometrically at 660 nm and 642.5 nm (see SOP: Spectrophotometric Determination of Chlorophyll a and b), or with a calibration curve prepared from pure chlorophyll.
Apparatus required
- Mortar with a pointed spout, and purified quartz sand
- Volumetric flask, 250-ml
- Separating funnels
- Spectrophotometer and colorimeter
Reagents
a. 85% acetone.
b. Calcium or sodium carbonate.
c. Ethyl ether.
d. Fresh leaf (for the calibration standard).
Procedure
Sample
Weigh accurately 4 to 5 g of the sample; add a small quantity (0.1 to 0.2 g) of calcium or sodium carbonate
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Macerate in a mortar (having a pointed spout) with purified quartz sand for a short time
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Add 85% acetone a little at a time and continue grinding; decant the acetone extract
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Repeat the extraction with 85% acetone till the residue is colourless
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Filter the coloured acetone extract to a 250-ml volumetric flask; wash the filter paper and dilute to the mark with 85% acetone
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Dilute further if necessary, and measure the colour at 660 nm
Standard curve
Weigh accurately 4 to 5 g of fresh leaf; extract with 85% acetone and dilute the filtered extract to volume as for the sample — this is the stock solution
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Make a series of dilutions of the stock solution with 85% acetone and measure the colour at 660 nm
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Pipette 25 or 50 ml to a separating funnel and transfer the chlorophyll to ethyl ether; measure the colour at 660 and 642.5 nm in a spectrophotometer as in the spectrophotometric method
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Calculate the absolute chlorophyll content in the dilutions of the acetone extract
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Plot the absorbance of each dilution against concentration on graph paper
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From the curve, read the concentration of chlorophyll in the sample
Note: uncertainties are involved in using chlorophyll preparations as standards for the colorimeter; the use of a simple plant extract with a spectrophotometric standard for calibration is suggested in the source.
Calculation
Read the concentration of total chlorophyll in the sample extract from the calibration curve and calculate:
mg of total chlorophyll per 100 g = [Concentration read from the curve (mg/litre) × Volume made up (litre) × 100] ÷ Wt of sample (g)
Note: the source gives no calculation for this method; the formula above is the standard mass-balance form, not a printed source formula.
Where,
- Concentration read from the curve = total chlorophyll concentration in the extract (mg/litre)
- Volume made up = final volume of the acetone extract (litre)
- Wt of sample = weight of the sample taken (g)
Precautions
- For the calibration, transfer the chlorophyll to ethyl ether as in the spectrophotometric method and measure at 660 and 642.5 nm.
- Filter the acetone extract before making it to volume.
References
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.
- Petering, H.G., W. Wolman & R.P. Hibbard, Ind. Eng. Chem., Anal. Edn., 12, 148 (1940).
- Comar, C.L., E.J. Benne & E.K. Buteyn, Ind. Eng. Chem., Anal. Edn., 15, 524 (1943).