Hi Alexandre,

Here is my protocol for the calibration curve with Gallic acid for the total phenolic concentration using Folin Ciocalteu.

Solutions :

- Folin Ciocalteu 2N

- Sodium Carbonate 20% w/v, warm up to 37°C before use

- Gallic acid stock solution in MeOH 2.5 g/L

Take the following volumens into a 10 mL assay tube:

     Stock Solution      MeOH          

1.   0.1 mL                    4.9 mL

2.   0.15 mL                  4.85 mL

3.   0.20 mL                  4.80 mL

4.   0.25 mL                  4.75 mL

5.   0.30 mL                  4.70 mL

6.    0.40 mL                 4.60 mL

7.     0.45 mL                4.55 mL

You will have 7 points for the curve (50-225 ug of gallic acid).

Then we use falcon tubes of 50 mL for the assay in triplicate each point

1. Add 1 mL of the sample/standar tube

2. Add 0.2 mL of Folin Ciocalteu

3. Add 20 mL of distilled water

4. Add 1 mL Sodium Carbonate

5. Complete to 25 mL with distilled water

6. Leave in the darkness for 60 min

7. Read at 725 nm

8. Build your calibration curve using the mass of gallic acid in each point (X axis) and the absorbance (Y axis).

9. Calculate the results of your sample interpolating the absorbance and getting the ug equivalents of gallic acid. Then adjust the value to the concentration that you prepared your sample. For example, we use 1 mg/mL normally, so in the 1 mL alliquot of Step 1, it will have 1 mg of Extract.

Hope this works for you, it took us a while to set the conditions, but since we follow this protocol we haven't had any troubles. Be aware of using fresh Folin Ciocalteu, sometimes that affects.

Best regards,

Article Antioxidant capacity, polyphenolic content and tandem HPLC–D...

Total phenolic content can be estimated by folin-ciocalteau method (Pitchumoni & Doraiswamy, 1998). Aliquot of the extract mixed with 2ml of sodium carbonate (2%) and after 2 min; 100µl of folin reagent (IN) added for colour development, which is read at750 nm after 30 min. Standard curve with gallic acid (0-100nM) is used to express results as gallic acid equivalent (GAE) / g material.

Thanks to both of you for your answer.

In fact i have the same protocol, but i just have a problem with the linearity of my standard curve with gallic acid, that is not linear between the high (50-100mg/L) and the low concentration (

Hi Alexandre,

I was wondering if you figured out GA's linearity. I have been working with GA (dissolved in water), and it is linear for concentrations

One does not expect a straight line fit in the wide range of 1 mg/L to 100 mg/L. A curve is expected in the said range. You may expect a linear fit of lower slope in the low range of 1 to 10 mg/L and a higher slope in the high range of 10 to 100 mg/L. You have to make the calibration curve in the range of your interest so that your absorption values are tightly covered by the absorption values used for calibration .

however, the slope changing in the range of 10 to 100 mg/L can not be explained by the above. Please check your dilution in case.

Is your x-axis (Gallic acid concentration axis) is plotted on a logarithmic scale or linear scale? Please check.