SOP: Colorimetric Estimation of Tannins
GIFSQ — Global Institute for Food Safety & Quality
Principle
The colorimetric estimation of tannins is based on the measurement of the blue colour formed by the reduction of phosphotungstomolybdic acid (the Folin–Denis reagent) by tannin-like compounds in alkaline solution. A standard curve is prepared from tannic acid, and the tannin content of the sample aliquot is read from the curve and calculated as tannic acid.
Apparatus required
- 100-ml volumetric flasks
- 500-ml volumetric flask
- Spectrophotometer (760 nm)
- Nessler tubes (alternative comparison)
Reagents
a. Folin–Denis reagent: to 750 ml of water, add 100 g of sodium tungstate (Na2WO4·2H2O), 20 g of phosphomolybdic acid and 50 ml of 85% phosphoric acid (H3PO4). Reflux the mixture for 2 hr, cool to 25°C and dilute to 1000 ml with water.
b. Saturated sodium carbonate solution: to 100 ml of water, add 35 g of anhydrous sodium carbonate, dissolve at 70–80°C and cool overnight. Decant the clear liquid before use.
c. Tannic acid standard solution: dissolve 100 mg of tannic acid in 1 litre of water. Prepare a fresh solution for each determination (1 ml = 0.1 mg of tannic acid).
Procedure
Preparation of the standard curve
Pipette 0 to 10 ml aliquots of the standard tannic acid solution into 100-ml volumetric flasks containing 75 ml of water
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Add 5 ml of Folin–Denis reagent and 10 ml of sodium carbonate solution into each volumetric flask and make up to 100 ml with water
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Mix well and measure the colour after 30 min at 760 nm against an experimental blank adjusted to 0 absorbency
Preparation of the sample
If the sample is solid, boil 5 g of it for 30 min with 400 ml of water, cool, transfer to a 500-ml volumetric flask and dilute to the mark; shake well and filter
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If the sample is liquid, make use of the clear filtrate
Determination
Use an aliquot of the filtrate containing not more than 0.1 mg of tannic acid
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Proceed as in the standard curve and obtain the mg of tannic acid from the standard curve
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Alternatively, samples treated as above may be compared in Nessler tubes against freshly prepared tannic acid standards treated in the same manner
Calculation
Tannins as tannic acid % = (mg of tannic acid × Dilution × 100) ÷ (ml of sample taken for colour development × weight of sample taken × 1000)
Where,
- mg of tannic acid = milligrams of tannic acid in the measured aliquot, read from the standard curve
- Dilution = overall dilution factor of the sample
- ml of sample taken for colour development = millilitres of the sample aliquot used for colour development
- weight of sample taken = weight of the original sample (g)
Note: the printed layout of the denominator is partly obscured in the extracted source; the components above are carried as read.
References
Official Methods of Analysis, Association of Official Analytical Chemists, Washington, D.C., 11th edn., p. 154 (1970).
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.