; ; Gallic acid determination 50 mg of sample tannin in 5 ml 2N H2SO4 are put into constricted test tubes and frozen. The tubes are vacuum-sealed and heated for 24 hours at 100 °C. The tubes are cooled, opened and the contents made up to 50 ml with water. Then 1.5 ml of freshly prepared 0.667% w/v
rhodanine in methanol and 1 ml of sample are mixed. After exactly 5 min 1 ml of 0.5 N KOH solution is added. After 2.5 min the mixture is diluted to 25 ml with distilled water and 5–10 min later the absorbance at 520 nm is measured. The measured absorbance obeys the relationship: A520= [0.13 × (mg of gallic acid) ] +0.03 Gallic acid is used as a standard and the data are based on experiments carried out in triplicate. ;Ellagic acid determination 10 mg of samples tannin in 1 ml 2N H2SO4 are put into constricted test tubes and frozen. The tubes are vacuum-waled and heated for 24 hours at 100 °C. Tubes were cooled, opened and the filtered content made up to 10 ml with
pyridine. Then 1.1 ml of pyridine and l ml of sample are mixed in a dry test tube. After adding 0.10 ml of concentrated HCl and mixing, the sample is brought to 30 °C. The sample is quickly mixed after 0.10 ml of 1% (w/v) NaNO2 in water and the absorbance 538 nm is immediately recorded. After a 36 min incubating period at 30 °C, the absorbance is again recorded. The difference between the initial absorbance and the absorbance at 36 min (D A538) is proportional to the ellagic acid concentration. The measured absorbance obeys the relationship: A538 = [0.03 × (mg of ellagic acid)] – 0.04 Ellagic acid is used as a standard and the data were based on experiments carried out in triplicate. ;Alkaline hydrolysis 4.8 g sample tannin in 9 ml water are refluxed in 4.2 ml 40% NaOH for 6h at pH 12 - 13. Neutralization to pH = 6.8 - 7 is performed with 62% H2SO4. ;HPLC determination == Uses ==