You need to construct a standard curve for gallic acid and catechin. Same as protein estimation, draw a perpendicular line from obtained OD. Thus obtained concentration can be expressed as GAE and CE.

You need to construct a standard curve for gallic acid and catechin. Same as protein estimation, draw a perpendicular line from obtained OD. Thus obtained concentration can be expressed as GAE and CE.

Get it done Spectrophotometrically /colorimetrically as suggested above. For Total Phenolics you can refer the method of Singh and Sanwal (1979) Ind. J.Expt.Biol.

find the attachment 

Folin Denis or Folin Ciocalteu is fine using gallic acid as standard. For flavonoids, the Aluminium chloride method using quercetin as standard gives a good estimation. Please see appended paper

Depending on the phenols that are in large quantity in your sample, you will construct a calibration curve of a phenolic acid for total phenolics, measured with Folin  Ciocalteu reagent, and a calibration curve of a flavonoid for total flavonoid content. I am not saying to use gallic acid and catechin or quercetin for calibration curve, because is not correct to make a curve of gallic acid if the matrix does not contain gallic acid. The acid could be caffeic, chlorogenic, etc., but is preferably to be in the matrix you want to measure.  For flavonoids also, is not correct to make a calibraton curbe of catechin if in the sample there is no catechin. You check first which flavonoids are in the sample and than you make the curve. Anyway phenolics will be expressed as "..phenolic acid equivalents", so the real content is not the one you obtained, because is expressed as "equivalents" of something. But these are the consacrated methods, and these we are using. 

HPLC is the method of choice, not colorimetric determinations, which are prone to interference (Folin Ciocalteu measures reducing power and responds to ascorbic acid for sure, probably to sugars, etc, see a.o. George et al., JAFC 53 (2005) 1370-1373 . I have yet to see a convincing publication for the specificity (and homogeneity of response of the various flavonoids) of the Al method for flavonoids. Even the pH differential method for anthocyanins is prone to interference by tannins. You must be aware that some journals reject articles that only use colorimetric determinations, specially Folin.

What if we perform regression analysis of the data obtained (plot of concentration versus OD) and prepare a best line of fit to obtain a regression equation as y=mx + c. and validate it using quality control samples as per ICH norms and finally use the same regression equation to obtain the content of either phenolics or flavonoids in any unknown sample?

Indeed HPLC techniques are ultimate methods. The colorimetric methods though not precise have been routinely used in a high number of publications for estimation purposes and have been correlated with a number of biological activities. They still remain valid. However,  work can be fully complemented with the  identification and quantification of major individual componds from phytophenolic subclasses (phenolic caids, flavanones, flavones, flavonols, catechins etc..) by HPCL techniques. This would then provide an overall picture.

Total phenol contents of extracts are determined by the Folin-ciocalteu method. An aliquot of 0.5 ml of extract (1 mg/ml) is mixed with 2.5ml Folin-Ciocalteu reagent (previously diluted with distilled water 1:10 v/v) and 2ml (75% w/v) of sodium carbonate (Na2CO3). The tubes were vortexed for 15 s and allowed to stand for 30min at 40 °C for colour development. Absorbance was then measured at 765nm using UV/VISspectrophotometer. Total phenolics content of different extracts are expressed as mg/g gallic acid or tannic acid equivalent (mg/g) using the equation from the calibration curve.

Total flavonoid contents of extract are determined by method based on the formation of a flavonoid-aluminium complex. 0.5ml of various solvent extracts (1mg/ml) is mixed with 0.5ml of aluminium chloride prepared in (2% in ethanol). The resultant mixture is incubated for 60min at room temperature for yellow colour development which indicates the presence of flavonoid. Absorbance is measured at 420nm using UV–VIS spectrophotometer. Total flavonoid content is calculated as quercetin equivalent (mg/g) using the equation based on the calibration curve.

Chemicals and reagents used:

1.      Folin-Ciocalteau reagent: Folin-Ciocalteu reagent was diluted (1:10) with distilled water and used.

2.      Sodium carbonate solution:  202.5 g of sodium carbonate was dissolved in 1 litre of distilled water and used.

Preparation of test solutions: 10 mg the selected plant extracts were dissolved separately in 10 ml of methanol to get 1 mg/ml solution. These solutions were diluted with methanol to obtain lower dilutions

Preparation of standard solution of Gallic acid: 100 mg of gallic acid monohydrate was dissolved in 100 ml of distilled water to get 1000 μg/ml solution. It was serially diluted with distilled water to obtain solutions ranging from 50 μg/ml to250 μg/ml

Procedure

400 ml of the extract (1 mg/ml to 0.5 mg/ml) was mixed with 2 ml of Folin-Ciocalteu reagent and 1.6 ml of sodium carbonate solution. After shaking, it was kept for 2hreaction time. The absorbance was measured at 750 nm (Shimadzu UV-160 A Spectrophotometer, Shimadzu Corporation, Japan). Using gallic acid monohydrate, standard curve was prepared and linearity was obtained in the range of 10 to 100 mg/ml. using the standard curve the total phenol content of extracts was obtained. The total phenol content was expressed as Gallic acid equivalent in mg/g or %w/w of the extracts.

Hi Suresh, may I know the article paper for me reference. Thank you in advance

Hi everyone, Can anyone tell me the wavelength range for flavonoid estimation using vanillin reagent.

510 for Flavonoids and 765nm using UV/VIS spectrophotometer for Total phenolics content.