Hello Milad, 

I got the following Absorbance for standard solutions (Gallic Acid solutions): 

g/L      Absorbance

0.3 --- 0.308

0.9 --- 0.879

1.5 --- 1.421

2.1 --- 1.950

3.0 --- 2.754

My doubt is about the Gallic Standard curve that I built. I used the results of the absorbance (axe Y) and the concentrations above (axe X) to build it. But due to the fact that I also diluted the initial concentration of gallic acid in the 50ml flask....I do not know which concentration of gallic acid I have to use to build the curve: the initial ones (0.3 g/L - 3g/L) or consider the dilution and use (0.003g/L - 0.03g/L)...?

What would you suggest?

Thanks!!

Pd: Correct, the C in my equation is TPC or the gallic acid equivalent. ;)

Follow the link 

Hello

As you have diluted your Gallic acid solutions, you should use the following concentrations 0.003g/l-0.03g/L

Thank you for your recommendations !!!! I As Wissam suggested ...it seems that I have to use the diluted concentration to build my curve. ;) Thanks!!

Hello, Wissam is right. when you measured absorbance make sure that the value are below 0.7 because  Scalbert, A., Monties, B., Janin, G., 1989 during the studies name: Tannins in wood: comparison of different estimation methods in Journal of Agricultural and Food Chemistry volume 35 and pages 1324–1329 have shown that after 0.7 in absorbance the line is not straight. when you have equation, follow the same procedure with your sample by replace gallic acid with your sample and have the absorbance of the sample. then replace the value of the sample absorbance in the equation Y = ax+ b (equation that you obtained by plotting absorbance of gallic acid against concentration) and have your value express in mg eq. gallic acid/g of sample. You can also this thesis (saha tchinda 2015,Caractérisation et valorisation des substances extractibles de cinq essences camerounaises majeures de l'industrie du bois : Ayous, Moabi, Movingui, Padouk et Tali)  were you have the construction at page 61 to 62

Thanks Jean, I have repeated the trial with a lower concentration of Gallic Acid for my standard. Now my values of absorbance are bellow 0.7. :) 

Well, I guess my response may be late but we can still learn something.

In constructing a calibration curve for gallic acid, I think you can firstly prepare a stock and then dilute this stock into different concentrations. For example you can dissolve 0.500 g of dry gallic acid in 10 ml ethanol and dilute with distilled water to 100ml.

From this stock solution of GA, you can now prepare a calibration curve by adding 0, 0.1, 0.2, 0.3, 0.5, 1.0 ml of the GA stock into a 10ml test tube and top with distilled water to the 10ml mark. They should contain 0, 50, 100, 150, 250 and 500 ml of GA.

Then you read their absorbance at 760 nm, and then plot concentration against absorbance to get your curve.

how to determine the suitable wavelength for the gallic acid? does it affect the absorbance value? why do we need to add in ethanol in preparing stock solution? is it necessary or we can just simply dilute the gallic acid with distilled water only?

Dear Dr. Courage Sedam Dzah in the details provided by you regarding dilution it seems concentration of GA in different test tubes will be 0, 50, 100, 150, 250 and 500 mg/l instead of ml.

The total phenolic content can be calculated as a natural compound (gallic acid) equivalent (GAE) by the following equation: T= C XV/M.

For making standard, do we need to add distilled water only as solvent?

or do we need to add some quantity of FCR and 20% Na2CO3 as well based on final volume say 10 ml for each standard concentration?

For sample analysis, before checking the absorbance, the FCR reagent, and NA2CO3, and distilled water is added along with sample extract so I am confused.

How do you make a standard curve from a catechin solution of 2000 microgram/ml in methanol.

As Dr Courage Sedem Dzah explained... I understand taking 1ml to make 100ml solution from 0.5g/100ml (which is 500mg/100ml) will give us 5mg/100ml solution. Am i right sir?