I am using fermented sorghum as my sample, I extracted the sample using MeoH. I facing problem my absorbance reading are not right, know that my samples are wet basis. I used catechin to do my standard curve.
To conduct a tannin content assay using the vanillin/HCl method, follow these steps:
1. Prepare the sample: Grind or crush the sample (e.g., plant material, extract, or beverage) to a fine powder. Ensure that the sample is representative and homogeneous.
2. Weigh the sample: Weigh accurately about 0.5-1.0 grams of the prepared sample using an analytical balance.
3. Prepare a standard solution: Dissolve 100 mg of tannic acid in 100 mL of distilled water to prepare a stock solution (1000 ppm). Dilute this stock solution further to obtain different concentrations (e.g., 50, 100, 150, and 200 ppm) for calibration purposes.
4. Prepare a blank: Take distilled water as a blank solution for calibration.
5. Prepare reagents:
- Vanillin reagent: Dissolve 1 g of vanillin in 100 mL of methanol.
- Hydrochloric acid (HCl) reagent: Dilute concentrated HCl with distilled water (1:3 ratio).
6. Calibration curve:
- Take different aliquots (e.g., 0.2 mL) of each standard solution into separate test tubes.
- Add 2 mL of vanillin reagent to each test tube.
- Mix well and incubate at room temperature for about 20 minutes.
- Measure the absorbance of each standard solution at a specific wavelength (usually around 500 nm) using a spectrophotometer.
- Plot a calibration curve by plotting the concentration on the x-axis and absorbance on the y-axis.
7. Sample analysis:
- Take an aliquot (e.g., 0.2 mL) of the prepared sample into a test tube.
- Add 2 mL of vanillin reagent to the test tube.
- Mix well and incubate at room temperature for about 20 minutes.
- Measure the absorbance of the sample at the same wavelength used for the calibration curve.
- Determine the tannin content of the sample using the calibration curve.
Note: It is important to perform all steps in a controlled environment, following good laboratory practices.
Fatemeh Gharahdaghigharahtappeh Thank you for your detailed answer. one more question does it affect if my samples are not dry? because my sample are batter so I took about 2 ml of sample and extracted it using methanol, after centrifuge it for 15min and filtered I used the extract directly the measure the tannin content.
When you extract by methanol. Methanol fraction contains other substance than tannins. If you want to estimate polyphenols(Tannins and others) then dissolve dried methanolic fraction in water and use folin ciocalteu reagent. I am attaching few article for your reference
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