Dear Abdulelah,

I believe you obtained the equation y = 0.0398x - 0.0006 , R² = 0.9971 by plotting the absorbance of different concentrations of gallic acid dissolved in methanol. If that is the case, put the absorbance reading of your sample as y into the equation to obtain a value, lets call it z in mg GAE/ml. Using the equation below you can calculate total phenolic content of that sample in mg GAE/g.

C = z mg GAE/ml * vol. of chemical used in assay (ml) * 1/mass of sample or crude extract.

Hope this helps.

Hello .... to calculate the value of GAE/gm ......

 First ... you have to prepare galic acid standard curve by plotting the absorbence  vs the concetration of galic acid ... from the standard curve equation ..Y=aX+b... then you can make the test using the same procedure fot the different plant extract ...and mesure the absorbences .... the absorbence value you obtain is the y value ... from the equation you can calculate the x value which is the concentration of galic acid (mg/ml ).... multiply the x value by the volume of the plant extract (ml)... now you have the amount of galic acid in that extract (mg)..... devide the value by the amount of plant used for pllant extraction (gm) ... now you have the amount of galic acid equevelent per gram plant extract .....

Dear Shabina,

I agree with Salam but please bear in mind that you need to consider the dilution of your sample in calculations. In the procedure that I know you add 5 ml of diluted sample, 0,5 Folin-Ciocalteu reagent and 0,5 ml of 7% Na2CO3 solution. So for example if you read from standad curve that concentration of gallic acid is 0.4 mg/l and sample was diluted 10 times then you need to multiply your result by 10 and you obtain 4 mg. You need to check total volum of the extract that you obtained initially, lets say 100 ml. If you took 5 ml to prepare dilution you need to multiply your result further according to the equation 4 mg x 100 ml/5 ml = 80 mg. Then you need to check the quantity of the sample that you used to prepare your extract, for example1 g. Lets assume that it contains 0,1 g of dry matter. This means that 800 mg of polyphenols expressed as gallic acid is present in 0,1 g of dry matter so if you multiply this result by 10 you get the information that 800 mg polyphenols is present in 1 g of dry matter. I hope this helps.

Dear Sir,

I have two doubts: 

1) GAE = [Absorbance (765 nm) / 0.0009567] + 0.02091, R2 0.9995.

2) Rutin Equivalent (RE) = Absorbance (506 nm) / 0.0002428 − 0.008141, R2 0.9987.

What is this (+ or -)? I don't understand it

Please go through the link: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746370/

Kindly explain me

Regards,

Rohan

Total phenolics content can be estimated using Folin- Ciocalteu reagent as described by Singleton et al. (1999) using gallic acid as a standard.

Dear Rohan,

The euqtion you quoted describes how the trendline fits to the points that are marked on your figure. If x axis describes concentrations of your standard solutions of gallic acid (catechin or others) then y axis describes absorbance reading obtained for them.R2 is a regression coefficient which says how well the trendline fits-the closer to 1, the better.+/- are just parts of functions obtained from the figure. If you have further questions please do not hesitate to contact me.

Dear Lukasz,

Please give a simple example detailing standard gallic acid and extract or any compound. How extract is expressed in terms of GAE?

It will help for better understanding for me.

Thanks again

Dear Lukasz,

Please look the link: http://www.japsonline.com/admin/php/uploads/1329_pdf.pdf

Table No. 1

Aqueous, 4 mg/ml       3.5725 ± 0.2336 (GAE)

I dont understand it

Dear Rohan,

The number you gave is a mean concentration of polyphenols expressed as gallic acid plus/minus standard deviation. The reason why you express the concent of polyphenols as gallic acid is that there is a huge variety of polyphenols and for the preparation of standard curve you need to choose one that is well known and have a composition which is characteristic to this group of nutrients.You may also come across paper were researchers use catechin instead. I hope this helps.

TPC is determined using the Folin- Ciocalteu reagent assay adapted by (Singloten and Rossi, 1965). Gallic acid is used as standard phenol and results are calculated in terms of Gallic acid equivalents (mg GAE/g) of dry leaf matrix.

This is how i perfomed it.. might help you...

The reaction mixture was prepared by using 2mg of papaya extracts and 100µl of freshly prepared 0.5N FolinCiocalteu reagent. The prepared mixtures were allowed to stand in dark for 15 min and then 2.5ml of 7.5% sodium carbonate was added. The resultant mixture was allowed to stand for further 30 min in dark. The absorbance was taken at 765 nm using UV visible spectrophotometer.

One ml of supernatant was taken from ethanol extract prepared for TSS analysis and evaporated to dryness in water bath. In each test tube 1 ml of deionised water and 0.5 ml of 1N FCR (1:1, FCR: Water) was added and kept for 3 min. After this 2 ml of 20% Na2CO3 was added and mixed thoroughly. Then the test tubes were incubated in boiling water for exactly one minute and cooled in ice water. The absorbance was read at 650 nm against a reagent blank.

A standard graph was prepared in similar ways using pyrocatachol ranging between 0, 5, 10, 15, 20 and 25 g concentrations.

The amount of phenol present in the sample was calculated as:

Phenol (mg/g) = Sample O.D. × Dilution Factor × Graph Factor

How do to receive dilution factor and graph factor ?

Can i ask you my analyse.

i extracted 2,5 g dry weight in 250 ml solution. then i used 0,5 ml extract and diluted it with dH2O, FCR and sodium carbonate to 12,5 ml total value. i read absorbance at 750 nm. i prepared gallic acid standard curve as mg/l and my exuation is y=0,0059x. so when i use the curve i calculated the value as 34 mg/l. can anyone help me to find the final concantration as mg/100 g dry weight? thanks!

Thank you all for your great help.......

Dear Lukasz,

Please give clarification to me that I have found a formula from literature. Total Phenolic Content was calculated from the following equation: T=C X V/M where, T= Total Phenolic Content (mg/g) of extract as GAE, C= Concentration of GA established from the calibration curve in (mg/ml), V= Volume of the extract solution in ml & M= weight of extract in g.

So I would like to ask you, how to establish conc. of GA from calibration curve. I have done gallic acid calibration curve where I got y=0.155x-0.003, R2=0.994. Concentration of GA is whether 0.155 or 0.003?

It will help for better understanding for me.

Thanks again

Dear Oskal,

Calculations for your results will be as follows:

You obtained a value of 34 mg per litre which means that in 12,5 ml of diluted sample you will find (34 x 12,5)/1000 = 0,425 mg of gallic acid. This is the quantity that you may find in 0,5 extract that you took before diluting it. Now you need to find out how much of phenolic compounds was in your extract: (250 x 0,425)/0,5 = 212.5 mg. Finally you want to express your results per 100 g of dry matter 212,5 x 100/2,5 = 8500 mg. Could you please give me the details about the range of concentrations of gallic acid that you used for the preparations of your standard curve? Also, could you please give me the absorbance value for your sample what was your blank? The result is surprisingly high so it might be a good idea to double check the procedure you followed. I hope this will help you.

Dear Rohan,

When you prepare calibration curve you need to use a series of solutions of know concentration of gallic acid. I usually prepare solutions which contain from 0,1 to 15 of gallic acid per litre. When you make a figure for correlation between absorbance (y axis) and concentration of gallic acid (x axis) you fit in the trend line. The value of R2 describes how that line fits to the points that you got on your chart. Value 0.994 means that you did a great job;D Now, the equation that you obtained describes linear correlation between absorbance and concentration of gallic acid where y is absorbance and x is concentration of gallic acid. This means that if you want to find out the concentration of phenolic compounds based on absorbance value you need to transform your equation: x=(y + 0.003)/0.155. The only thing to do now is to insert the absorbance reading (y) for your sample and this is how you get the concentration of phenolic compounds expressed as gallic acid. I hope that this explanation will be useful to you.

Dear Lukasz,

YES!! It helped me a lot! I got it finally and doubt has cleared.

Thanks a lot!!!!

I express gratitude you from bottom of heart! 

Warm regards

Rohan

Hi all, I made Gallic acid standard curve in ethanol as solvent. And I need to know the phenolic content of ethanol extract of a plant. So, should I directly check the values of absorbance after dissolving that ethanol extract in ethanol and put in the regression equation (R2=0.995) for knowing  the phenolic content of plant extract or should I first perform FCR reaction with the plant extract???

Hi All,

I am estimating the Total Phenolic Content in a herbal formulation (Tablet or Liquid Formulation), In this case what blank i should used (Placebo as such dissolved in water or Placebo treated with Folin–Ciocalteu and all other reagents). What should i do in case if there is a absorbance   in blank. Please help. I am taking pyrogallol as standard. 

Dear Shreya,

After you prepare your stnadard curve you need to dilute your ethanolic plant extract in ethanol anyway because the concentration of total polyphenols would be out of standard curve. Then you can carry out the whole procedure foryour diluted extract. I am not sure what you want to gain in your experiments but generally speaking methanol is better for the recovery of polyphenols. I hope that attached paper will help.

Kind regards,

Lukasz

I am having the same issue. I am trying to measure the total flavonoid content in a freeze dried plant material as catechin equivalents. I made the calibration curve with a range of concentrations of (0.1 - 5mM) of catechin and I got the concentration of my sample against the absorbance (1.05mM) in which I divided by 1000 and multiply by the molecular wt of catechin to get the g/ml. I am using the Sodium borohydride/chloranil based assay in which the final volume is 7ml. Do I need to take anything else into consideration besides the dilution factor?

Thank you

Dear Noor,

Could you please provide me more detail about the protocol that you used? I need all the information about the weight of your sample that you used for extraction, volumes of solvents, dilutions etc. I assume that the concentration of catechine for your standard curve is expressed in mM per litre, is that correct? It would also help if send me detailed calculations.

Dear Lukasz,

Thank you for responding. First I added 1mg of freeze dried arugula sample and dissolved it in 1 ml of THF:ethanol (1:1 , v/v) then I added 0.5 ml of sodium borohydride and 0.5ml of AlCl3, and another 0.5ml of sodium borohydride was added after shaking for 30 minutes. 2ml of cold acetic acid solution was added and 1ml of chloranil was added and heated with shaking for 1 hr then the volume was made to 4ml with methanol. Then, 1ml of vanillin was added followed by mixing, and 2ml of HCl was added and the final volume was 7ml. I took 1ml from this solution and measured the absorbance.

Yes the concentration of catechin for my standard curve was expressed in mM/L.

This is equation for the standard calibration curve (0.1, 1, 3, and 5 mM) which I prepared daily according to the method:

y=0.1566x+0.183, R2 =0.9868; I did correct for the blank in this equation.

One of the absorbances I got for my sample is 0.322 which gave me a concentration of 0.89mM. But when I calculated the amount of catechin eq./g of sample, I would get a large number.

Thank you

I determined the total phenolic content as GAE and total flavonoids as Rutin E is there any way to determine the tannin content from both calculations?

Dear Heba,

I wouldn't do this because extraction efficiency of polyphenols varies, depending on the solvent used. Please see the link for simple colorimetric method for tannin determination. I hope this will help you.

http://link.springer.com/article/10.1007%2FBF01196209#page-1

Dear Lukasz,

Thanks a lot for your help, I will download the paper and see, it might help.

Dear all

Could you help with calculating total phenolic contents of a plant extract sample?

Experiment:

Weight of sample: 20g

Weight after extraction: 7.83 g

The crude extracted 7.83 g were dissolved in 40 ml methanol>

Amount used in FC assay: 300µL of crude extract mixed with 600µL of FC reagent(10%) and 2.4 mL of sodium bicarbonate(700mM). Total volume was 3.3 mL

y = 0.0398x - 0.0006 , R² = 0.9971 , abs =2.2329

GAE equivalent value: 56.088 mg/ml

How can I calculate the total phenolic content to express as (mg GAE/g extract)?

Thank you in advance.

Dear Abdulelah,

I believe you obtained the equation y = 0.0398x - 0.0006 , R² = 0.9971 by plotting the absorbance of different concentrations of gallic acid dissolved in methanol. If that is the case, put the absorbance reading of your sample as y into the equation to obtain a value, lets call it z in mg GAE/ml. Using the equation below you can calculate total phenolic content of that sample in mg GAE/g.

C = z mg GAE/ml * vol. of chemical used in assay (ml) * 1/mass of sample or crude extract.

Hope this helps.

Hello/Hi brothers and sisters. I have a question on how to calculate my total phenolic in mg GAE/g. Below are details:

sample extract: 1gram in 10mL

equation:    y=0.0457x - 0.0533, R2 = 0.9819

Abs : 0.601

Calculated x from equation is : 14.317

How can I calculate x in the form of mg GAE/g ?

Can anyone please help me. 

The range of my gallic acid is : 20 - 100 ug/mL

 Dear Zaida,

From the data given, the calculated x is the GAE/mL. 

for you to calculate the TPC of the extract in mg GAE/g you should multiply the calculated x with dilution factor and in your own case the dilution factor is 10

Therefore, TPC = 14.317*10

                            = 143.17mg GAE/g if the the gallic acid concentrations are in mg. However, if the gallic acid concentrations are in ug then the calculation would as follows:

x =14.317ug/mL = 0.0143mg/mL

TPC = 0.0143*10 = 0.143mg GAE/g

Regards.

I think this link is useful, 

http://www.instanano.com/characterization/theoretical/concentration-calculation-from-uv-vis-absorbance/

I agree with Kabiru. The value of x you get from your gallic standard curve should be multiply by dilution factor(volume of extract/weight of pure extract).

 the calculation of total phenolic contents in a plant extract sample will be as follows

T =Cx V/M .

T is the total phenolic content in mg·g–1 of the extracts as

GAE, C is the concentration of gallic acid established

from the calibration curve in mg·ml–1, V is the volume of

the extract solution in ml and M is the weight of the extract

in g

Please find some help attached, I agree also with Lukasz Wojciech Wajda

Article Analysis of phenolic acids and anthocyanins of pasta-like pr...

Gallic acid monohydrate is  used for the calibration curve for phenolic content and a series with six dilutions should be   prepared to cover the range of 0.01mg/ml – 0.1 mg/ml. The total phenolic content of Gallic acid samples can be  determined spectrophotometrically using the Folin-Ciocalteu reagent (Povichit et al., 2010) and each sample should be  done in triplicate. Data is expressed as mean ± SD of three replicates. The calibration curve is  drawn with sample concentration of Gallic acid in   mg/mL vs. absorbance at 765 nm and get the equation as y = mx + c.Then the   Total phenolic content is  expressed as mg Gallic acid equivalent /g using the equation from the calibration curve  for Gallic acid by measuring the absorbance of  your sample prepared with known concentration following the same manner. Apply the sample absorbance to y of the equation and find the concentration of total phenolic content  in mg Gallic acid equivalent /g   of your sample. 

in Zaida Othman case....

how much sample she had taken will not be included or what? say suppose from sample extract she might have taken 0.05 ml or 0.5 ml or whatever it may be right for analysis of TPC....

let us consider that if she takes 0.05 ml from the sample extract then x will be 14.31 ug/0.05 ml right?

then convert it to 1ml then we will get 286.2 ug/ml,

therefore 0.2862 mg/ml , after this let us go back to sample extract, what she did there?, she made sample extract to 10 ml so 2.862 mg /10ml, this 10 ml corresponds to 1 g (because she put 1 g sample to 10 ml solvent) therefore for 1 g how much?..2.862 mg / 1 g...then the final ans for 100 g will be 286 mg/100g .....is it right or wrong??...if wrong, pl. mention where i made mistake.

data of zaida othaman is below

sample extract: 1gram in 10mL

equation:    y=0.0457x - 0.0533, R2 = 0.9819

Abs : 0.601

Calculated x from equation is : 14.317

How can I calculate x in the form of mg GAE/g ?

Please find the attached of these articles.

Hey guys, I wanna ask about my TPC (Total Phenolic Contain) calculation.

Here's what I've done

I've maserated 200g of fresh pomelo with 200 mL ethanol, then I've got my crude extract from rotary evaporation process.

I took 50 mg crude extract and diluted it with 5 mL of ethanol.

The next thing, I took 1 mL to test the TPC content.

Then I recieved my standard equation

Y= 0.001x + 0.061, with 0.082 absorbance. So I got x= 21 mg GAE/L.

Here's the question, have I done it right with this calculation 21 * 0.005L/ 0.05 g = 2.1 mg GAE/g extract?

And is it possible for me to convert it to mg GAE/ g fresh weight and mg GAE/ g dry weight (cause I've read a lot of literatures using those unit)

Thank you

follow the procidure:

1. Folin-Ciocalteu method

2. 200 mg will be extracted with 4 mL of acidified methanol (HCl/Methanol /Water, 1: 80: 10 V/V/V) at room temperature (25 0C) for 2 hrs

3. Aliquot of extract (200µL) will be added to 1.5 mL freshly diluted (10 fold) Folin-Ciocalteu reagent

4. mixture will be allowed to equilibrate for 5 min

5. then mixed with 1.5 mL of sodium carbonate solution (60g/L)

6. incubation at room temperature (25 0C) for 90 min

7. the absorbance of mixture will be read at 725 nm (6505 uv/vis spectrophotometer.

8. Acidified methanol will be used as a blank.

9. From the stock solution of standard gallic acid, a series of six standard solutions (0, 5, 10, 15, 20, 25, 30, 35ppm) will be prepared

10. total phenolic will be estimated from the calibration curve as gallic acid equivalent (GAE) in milligram per kilogram of sample.

11. HPLC analysis will be used for a simultaneous measurement of several phenolic compounds.

This material can be helpful:

For viny Trifera.

Your answer 2.1mg/g is absolutely right. y = 0.082-0.061/0.001 = 21mg/L * DF(in your case is 5) =105mg/L . For conversion in mg GAE /g it is = 105/50 =2.1.mgGAE/g. 50 comes from litter, if one ml contains 0.05g then liter contains 50g. You used 0.05g or 50 mg/ml so 1 liter comprise 50g.

The next part of your question is pertaining to conversion of FW into dry weight. So you should present it in dry weight.

For dry weight calculation first of all you should know the moisture content of sample, which can easily be determined from AOAC oven drying method. Once you calculated moisture content(eg. 21.6) you will get dry mass of sample also(100- Calculated Moisture content =dry weight).Now for calculation in terms of dry weight you go like

2.1mgGAE/21.6*100 = 9.722mgGAE/g dry weight

if you go ahead of this do 9.722*100 = 972.222mgGAE/100g

dry weight

I have same query like rohan,

How do to receive dilution factor and graph factor ?

Please reply.

I agree with the submissions above. After the cal. curve from GA, use the Equation to determine x with your sample absorbance as y, then input into TPC=x(vol of extract)/(mass of extract)

if I use %80 ethanol solvent for extraction, how can I calculate total phenolic content? I use evaporator for keep of methanol but water still in there what do I calculation?

Dear Merve,

You can use freeze drier to remove the water and get dried extract.

Dear Alkasim,

Thank you very much your answer. İt is helpful for My project.

Hello everyone,

I am trying to calculate the phenol amount using gallic acid as standard and i try to express the amount of phenols as mg GAE (Gallic Acid Equivalents) / gram sample. I did fount a lot information here on researchgate but need some specific help please!

So i made a stock by using 0.05 g gallic acid / 50 mL (95%) Methanol.

Then i made standards by diluting the stock.

Then i took from every standard 40 µL and added (360 µL demiwater, 800 µL F-C reagens, 800 µL (7%) NaCO3) and finally i measured the absorbance at 765 nm to produce a trendline!

I also did kinda the same with my samples:

BUT I solved 2.0 gram of sample in 10 mL of (95%) MeOH.

Then i took also 40 µL of this solution and added the same compounds with the same amount shown above and measured the absorbance.

The calculated concentration of sample was 0.28 mg/mL Gallic acid using the trendline.

So i want to know the concentration in mg GAE/g sample.

I think I make mistakes with the dilution factors but i am not sure. I calculated 1.37 mg GAE/g?

Thankyou very much u all!

I am not sure but did you check the amount of sugars in your samples. If your sample has free sugars that might be a problem.

Hi, so I did it all right following testes procedures. But the only problem was that I was having prolems by calculating the amount of phenols in mg GAE/g sample.

Wajdy Al-Awaida

Wajdy Al-Awaida

vol extract is the sample volume used in the dilution? or the total volume of sample prepared?

thank you already.

Dimitris P Makris

My doubt is that I made the dilution of 80, 60,40,20,10 µg / mL; to obtain the standard curve and for the tests with the sample (2mg), using the equation I obtain different concentration values, therefore different values of (YTP). What value do I adopt? or is it normal different values?

thank you already.

Dimitris P Makris ris P Mak

Thank you for your help! I used the equation of the line, found the C, then found the equivalent value of gallic acid for different concentrations due to dilution, my question is what value should I admit with the existence of several? in most articles they express a single value of equivalent, but not at what concentration of the dilution this value was obtained. So, do I get any value? my English is bad and I may not be understanding.

How do you calculate the antioxydant activities equivalent in trolox

in a plant extract sample?