Polyphenol Analysis Lab Report

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ABSTRACT: Blackgram have the antioxidant properties because of the presence of the natural phenolic compounds which has beneficiary effects. In the present work, polyphenols are extracted using solid liquid extraction in a batch reactor at constant temperature. The polyphenol content is determined using the Folin- Ciocalteau method and expressed as Gallic Acid Equivalents (GAE). The effect of the solvents ethanol, methanol, acetone and water were studied on the parameters pH, extraction temperature and extraction time on Total Phenolic Content have been checked and it was optimized using a three variable, three-level Box-Behnken response surface methodology for extracting phenolic content. Validation of the model is based on the predicted…show more content…
The samples were withdrawn at different times and the total phenolic content (mg GAE/ g dry weight) was determined.

Effect of solvent concentration on extraction of phenolic compounds
Samples were extracted with the different solvent ranging from 25% (v/v) to 100% (v/v) by fixing extraction temperature at 180 min and at 300C respectively. The total phenolic content (mg GAE/ g dry weight) was determined.

Effect of extraction time on extraction of phenolic compounds
The solvent with the optimized concentration will be considered to study the effect of extraction time on the extract sample at 300C. At different time in the range of 5min to 300 min, the sample will be collected and its total phenolic content (mg GAE/ g dry weight) was determined.

Effect of stirrer speed on extraction of phenolic compounds
The solvent with the optimized concentration will be considered to study the effect of mixing on the sample at 300C. The stirring speed was maintained in the range from 200 rpm to 800 rpm. At different time in the range of 5min to 180 min, the sample was collected and its total phenolic content (mg GAE/ g dry weight) was determined.

Effect of pH on extraction of phenolic
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The significances of all terms in the polynomial were analyzed statistically by calculating the F-value at a probability (p) of 0.001, 0.01 or 0.05.

Determination of polyphenols using HPLC
The polyphenols present in the blackgram was determined using HPLC method with gradient elution. Phenolic acid and flavonoids of black gram extracts are analyzed at 215nm using UV detector using HPLC using C18 column. Acetonitrile and water are used as mobile phase in the ratio of 7:3 with a flow rate of 1ml/min under ambient temperature and 3500psi pressure. The detection is at 215nm. The phenolic acid and flavonoids present in the black gram are analyzed on the basis of retention time and it is compared with the standard chromatogram of gallic acid and rutine

Verification of model
Using the second order polynomial model of RSM, optimal conditions required for the extraction of total phenolic content from black gram was estimated. Under the certain optimal conditions, the model equation was verified for the predicted response values. The experimental and the predicted values of the TPC were checked to verify the validity of the model. Experimental runs were replicated under the optimized

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