In addition, the response to gallic acid has been shown to be equivalent to most other phenolics in wine on a mass basis. Since the assay measures all phenolics, the choice of gallic acid as standard is based on the availability of a stable and pure substance, and gallic acid is both, and it is less expensive than other options. This assay is responsive to any reducing substance, so if applied to other types of samples, large errors could be encountered. It has a relative mass response of 0.68, so a wine with 30 mg/L ascorbate must be corrected by subtracting 20.4 mg/L from the total phenol value. It is usually not an important factor except for white wines with medium to high sulfite levels (>50 mg/L) and low phenol levels (<250 mg/L). Sulfites also cause an interference but the magnitude is variable. Reducing sugars such as glucose and fructose cause minor interferences and must be corrected. Results are reported at Gallic Acid Equivalent, GAE, because the phenols in wine contain mostly other phenols, and only small amounts of gallic acid. Do not neglect to multiply the observed concentrations by any dilution factor of the original sample. Alternatively, they can be left at 40☌ for 30 min before reading the absorbance.įor white wines, add 20 µL as for the calibration solutions, but in the case of red wines, dilute the wines by 10 first, then add 20 µL (or skip the dilution and add 2 µL if you have precise micro pipettors).Ĭreate a calibration curve with the standards and determine the levels in the samples. Leave the solutions at 20☌ for 2 hr and determine the absorbance of each solution at 765 nm against the blank (the "0 mL" solution) and plot absorbance vs. Wait for between 30 sec and 8 min, and then add 300 µL of the sodium carbonate solution, and shake to mix. Left over gallic acid solutions can be poured down the drain.įrom each calibration solution, sample, or blank, pipet 20 µL into separate cuvettes, and to each add 1.58 mL water, and then add 100 µL of the Folin-Ciocalteu reagent, and mix well. These solutions will have phenol concentrations of 0, 50, 100, 150, 250, and 500 mg/L gallic acid, the effective range of the assay. To prepare a calibration curve, add 0, 1, 2, 3, 5, and 10 mL of the above phenol stock solution into 100 mL volumetric flasks, and then dilute to volume with water. After cooling, add a few crystals of sodium carbonate, and after 24 hr, filter and add water to 1 L. Dissolve 200 g of anhydrous sodium carbonate in 800 mL of water and bring to a boil. Can be opened daily, but to store, keep closed in a refrigerator up to two weeks. In a 100-mL volumetric flask, dissolve 0.500 g of dry gallic acid in10 mL of ethanol and dilute to volume with water. Solutions containing the FC reagent must be disposed of as hazardous waste. Singleton and Rossi AJEV 1965, 16: 144-158, describe the preparation of the reagent from sodium tungstate, sodium molybdate, lithium sulfate, bromine, and some acids. This reduces waste and disposal volume.įolin Ciocalteu Reagent. This is usually purchased as the 2N reagent available from Sigma (F9252) or from Fisher Scientific (ICN19518690), and presumably others. If you cannot reproducibly measure such small volumes, try to reduce the volumes to the smallest you can. Total Phenol Analysis: Automation and Comparison with Manual Methods. American Journal of Enology and Viticulture 1977, 28: 49-55 only the volumes have been reduced. It is based on the method reported in Slinkard, K. Good micro pipets must be used for reproducibility. It uses the minimum volume of reagents and almost eliminates wasted reagent. The procedure is also used for analysis of total phenol in tea. This method is used routinely in our lab to measure total phenol.
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