When calculating the mg GAE/g of the sample, you need to consider the volume in which you dissolved your extract, i.e., the initial volume in which you dissolved your sample (10 mg of extract in 10 mL of methanol). As for the final volume used for the analysis, you must take into account the dilution factor.

In your case, if you took 0.5 mL of the initial solution (10 mg in 10 mL) and diluted it to 5 mL with Folin-Ciocalteu reagent and sodium carbonate, the dilution factor is 10 (since 0.5 mL was brought to 5 mL).

To correctly calculate the concentration in mg GAE/g of the sample, you need to:

So the formula becomes:

C = (mg/mL from the calibration curve) × (dilution factor) × (extraction volume in mL) / (sample mass in mg)

Make sure the units are consistent: mg/mL from the calibration curve * mL (extraction volume) / g (sample mass) = mg/g.

Finally, I would like to remind you that you are determining total polyphenols, not phenols. If anything is unclear, feel free to ask!

Thank you, dear Francesca, for the clarification.

However, most of the available literature does not include the dilution factor. Could you please provide a link or reference to an article that uses the dilution factor?

Regarding the formula in bold, where "sample mass" is given in milligrams, I assume it should be in grams.

Dear Francesca,

I am using the following procedure. Could you please have a look of it. I didn't find the DF in this procedure

Dear Iftikhar,

I have reviewed the protocol, and it aligns with the procedure I have used. However, from my experience, I can tell you that, depending on the matrix, some adjustments may be necessary. In particular, the dilutions can differ, which is why I take into account the specific dilutions made. For example, if I take 1 mL of extract and dilute it to a final volume of 10 mL, don't you think this should be considered in the calculations?

In the work I conducted on beans (Article NMR Metabolomics and Chemometrics of Commercial Varieties of...

Dear Francesca,

Your insights are valuable, and I appreciate your attention to detail. You're absolutely right that the dilution factor should be considered in the calculations, as it directly impacts the concentration of the analyte in the final solution. Adjustments may indeed be necessary depending on the sample matrix, and it's good practice to document these modifications clearly.

I would like to review this article as it clearly presents all the calculations. However, the paper does not account for the dilution factor (DF). In this study, the extract (5 mg) was initially dissolved in 100 mL of water. Then, 1 mL of this solution was taken and mixed with 5 mL of water, 0.5 mL of Folin-Ciocalteu reagent (FCR), and 1.5 mL of sodium carbonate, making a final volume of 8 mL. Later, the extract volume used was reported as 10 mL instead of 8 mL.

Do you consider 8 mL or 10 mL as the dilution factor (DF)? Additionally, 100 mL represents the total extract volume (V), while 5 mg (0.005 g) is the sample mass (m). In this article, the author used 10 mL as V rather than as the DF, while 100 mL was not included in the calculations.

I would appreciate your thoughts on this protocol.

Dear Iftikhar,

the calculation becomes more complex. Basically, I need to consider that I started with 200g of sample, and so I need to trace the amount of substance present in the original sample in mg/g, taking into account the dilutions made during the process. Here are the steps to follow:

You dissolved 5 mg of the residue in 100 mL of water. Therefore, the concentration in the initial solution is:

Initial solution concentration = 5mg/100mL= 0.05 mg/mL

You took 1 mL of this solution and diluted it to 8 mL. The total dilution is 8 times. The concentration in the final solution will be:

Final solution concentration = 0.05 mg/mL x 1/8 = 0.00625 mg/mL

Now, we need to trace the amount of substance present in the original sample, expressed in mg/g. The relationship between the final concentration and the original sample is given by:

Concentration in the original sample = Final concentration (ppm) x Final volume (L) / sample mass (g)

This is the concentration of the final diluted solution

In your specific caseIn this case:

• The mass of the sample is 200 g (this is the amount of sample you initially extracted).
• The final volume of the analyzed solution is 8 mL (which is 0.008 L).

The formula is:

Concentration in the original sample (mg/g) = Final concentration (ppm) x Final volume (L) / Sample mass (g)

Substituting the values:

Final concentration (ppm): calibration curve

Final volume (L): 0.008 L (since you diluted to 8 mL, which is 0.008 L).

Sample mass (g): 200 g (this is the amount of the original sample).

I hope I was clear and above all helpful.

Thank You dear Francesca,

The calculation was quite complex but also interesting. The data provided in the article deviates significantly from your calculations.

The X value obtained from the calibration curve is 97.98 µg/mL (0.09798 mg/mL). Substituting this value into the given formula:

Concentration in the original sample (mg/g) = Final concentration (ppm) x Final volume (L) / Sample mass (g)

97.98 mcg/ml x 0.008 L / 200 g = 0.00391 (unit uncertain).

However if we expressed 0.008 L in ml it will be 8ml and the final results will be 97.98 mcg/ml x 8 ml/200g = 3.91 (unit uncertain)

If we convert 97.98 mcg to mg then it will be 0.09798 mg. Now putting these values in the formula will gives final value 0.09798 mg/ml x 0.008 L / 200 g = 3.91 (unit uncertain).

Even after ensuring unit consistency, the calculated value does not match the data presented in the article.

I suspect that the data in the article does not align with our calculations and requires further verification.

If you agree with my reasoning... the deductions on the article are immediate ;)

Thank You dear Francesca,

I appreciate the insightful discussion. Once I have analyzed the samples in our laboratory, I will calculate the total phenolic content and share the results with you for verification and feedback.

Dear Iftikhar,

thank you very much for your message. I will be happy to assist with any verification or feedback you may need.

I wish you a good evening

ChatGPT4 | Midjourney, [31-Mar-25 4:21 PM]

Okay, here's a detailed protocol for determining total phenols in a dried extract, along with considerations and troubleshooting tips. The Folin-Ciocalteu method is the most widely used, so I'll focus on that.

I. Principle of the Folin-Ciocalteu Method

The Folin-Ciocalteu assay is a colorimetric method based on the reduction of phosphotungstic and phosphomolybdic acids in the Folin-Ciocalteu reagent by phenolic compounds. The resulting blue-colored complex is then measured spectrophotometrically. The intensity of the blue color is proportional to the concentration of phenolic compounds in the sample. Gallic acid is commonly used as a standard.

II. Materials

• Dried Extract: Your sample, accurately weighed.

• Folin-Ciocalteu Reagent: Purchase commercially prepared reagent.

• Sodium Carbonate (Na2CO3): Anhydrous.

• Gallic Acid Standard: High purity.

• Solvent for Reconstitution: Choose a suitable solvent for your extract (e.g., methanol, ethanol, water, or a mixture thereof). The best solvent is the one that effectively dissolves your extract while minimizing interference with the assay.

• Distilled or Deionized Water:

• Volumetric Flasks:

• Pipettes and Micropipettes: Accurate and calibrated.

• Spectrophotometer: UV-Vis spectrophotometer with a suitable cuvette holder.

• Cuvettes: Use cuvettes appropriate for your spectrophotometer and the wavelength used (usually around 765 nm).

III. Procedure

1. Preparation of Standard Solutions (Gallic Acid)

• Stock Solution: Accurately weigh a known amount of gallic acid (e.g., 10 mg) and dissolve it in a known volume of solvent (e.g., 10 mL). This will give you a 1 mg/mL (1000 ppm) stock solution. Store in the dark and refrigerated.

• Working Standards: Prepare a series of working standard solutions by diluting the stock solution. A common range is 0, 20, 40, 60, 80, and 100 µg/mL. Prepare these fresh each day. For example:

* 0 µg/mL: Blank (solvent only)

* 20 µg/mL: Dilute 200 µL of the 1000 µg/mL stock to 10 mL with solvent.

* 40 µg/mL: Dilute 400 µL of the 1000 µg/mL stock to 10 mL with solvent.

* And so on...

2. Preparation of Sample Solution

• Accurately weigh a known amount of dried extract (e.g., 10 mg) and dissolve it in a known volume of appropriate solvent (e.g., 10 mL). This will give you a 1 mg/mL solution.

• If necessary, further dilute the sample solution to ensure that the absorbance falls within the range of the gallic acid standard curve. This is very important. Aim for a concentration where the absorbance will be roughly in the middle of your standard curve. Dilution is your friend! Too high a concentration will give you inaccurate results.

• Filter the diluted sample solution through a 0.45 µm syringe filter to remove any particulate matter that could interfere with the spectrophotometric measurement.

3. Color Development

• Label all tubes or cuvettes appropriately for the standards, sample(s), and blank.

• In separate test tubes or cuvettes, add the following:

* Standard/Sample: 0.5 mL of each gallic acid standard solution or diluted sample solution.

Folin-Ciocalteu Reagent: 2.5 mL of Folin-Ciocalteu reagent (diluted 1:10 with distilled water immediately before use). This is crucial – dilute fresh!*

* Mix thoroughly and let stand for 3-8 minutes. This allows the Folin-Ciocalteu reagent to react with the phenolic compounds.

* Sodium Carbonate Solution: Add 2.0 mL of 7.5% (w/v) sodium carbonate solution.

* Mix thoroughly again.

Incubate the tubes or cuvettes in the dark at room temperature for 2 hours (or 1-1.5 hours, depending on your specific protocol; consult literature for guidance). This incubation time is important for complete color development.*

4. Spectrophotometric Measurement

• After the incubation period, measure the absorbance of each solution at 7

ChatGPT4 | Midjourney, [31-Mar-25 4:21 PM]

65 nm against the blank (solvent + Folin-Ciocalteu reagent + sodium carbonate).

• Ensure the spectrophotometer is properly calibrated and zeroed before taking measurements.

• Readings should be taken relatively quickly after the incubation is complete, as the color intensity can change over time.

IV. Data Analysis

1. Standard Curve:

• Plot the absorbance values (y-axis) of the gallic acid standards against their corresponding concentrations (x-axis).

• Generate a standard curve by performing a linear regression analysis on the data points. Obtain the equation of the line (y = mx + b), where:

× y = absorbance

× m = slope of the curve

× x = concentration of gallic acid

× b = y-intercept

2. Calculate Total Phenolic Content (TPC):

• Use the equation of the standard curve to determine the concentration of phenolic compounds in your sample solution based on its absorbance value.

• Take into account the dilution factor used in the preparation of the sample solution.

• Express the total phenolic content as milligrams of gallic acid equivalents (GAE) per gram of dried extract (mg GAE/g DW).

• Formula:

ChatGPT4 | Midjourney, [31-Mar-25 4:21 PM]

```

TPC (mg GAE/g DW) = (C × V × DF) / W

```

ChatGPT4 | Midjourney, [31-Mar-25 4:21 PM]

Where:

× C = Concentration of gallic acid in the sample solution (µg/mL) determined from the standard curve

× V = Total volume of the sample extract (mL)

× DF = Dilution factor (if any)

× W = Weight of the dried extract used (mg)

V. Important Considerations and Troubleshooting

• Solvent Selection: This is critical. Choose a solvent that dissolves your extract well but doesn't interfere with the Folin-Ciocalteu reaction (e.g., by reacting with the reagent itself). If unsure, test the solvent alone as a "solvent control" to see if it produces any color. Water is generally a good starting point, but many extracts require a more polar organic solvent like methanol or ethanol.

• Folin-Ciocalteu Reagent: The reagent is sensitive to light and moisture. Store it in a cool, dark place. Discard if it appears dark or cloudy. Always prepare the 1:10 dilution fresh just before use.

• Sodium Carbonate Solution: Ensure the sodium carbonate is completely dissolved.

• Incubation Time: Follow the recommended incubation time carefully. Over- or under-incubation can affect the results.

• Sample Dilution: This is extremely important. If the absorbance values of your samples are outside the range of the standard curve (either too high or too low), dilute your samples accordingly and repeat the assay. High concentrations will give you inaccurate readings.

• Interferences: The Folin-Ciocalteu assay is not specific for phenolic compounds. Other reducing substances (e.g., sugars, ascorbic acid, certain amino acids) can also react with the reagent, leading to overestimation of the total phenolic content. If you suspect significant interference, consider using a more specific method like HPLC.

• Replicates: Perform the assay in triplicate or more to ensure reproducibility and obtain statistically valid results. Calculate the mean and standard deviation of your measurements.

• Standard Curve: Ensure your standard curve has a good linear fit (R² value close to 1). Use at least 5-6 data points for the standard curve.

• Contamination: Use clean glassware and avoid contamination from other chemicals.

• Spectrophotometer Calibration: Regularly calibrate your spectrophotometer using appropriate standards.

• Reproducibility: If you get wildly different results between replicates, check your pipetting technique, the quality of your reagents, and the cleanliness of your glassware.

• Color Development Issues: If the color does not develop properly, check the age and quality of your Folin-Ciocalteu reagent and sodium carbonate solution. Ensure that the pH is within the appropriate range.

• Extract Stability: Phenolic compounds can degrade over time. Analyze extracts as soon as possible after preparation, or store them properly (e.g., in the dark at -20°C or lower).

VI. Example Calculation

Let's say you analyzed a sample extract and obtained an absorbance value of 0.600 at 765 nm. Your standard curve equation is y = 0.01x + 0.02, where y is absorbance and x is the concentration of gallic acid in µg/mL. The dilution factor for your sample was 10, and you dissolved 10 mg of dried extract in 10 mL of solvent.

1. Calculate the concentration of gallic acid in the sample solution:

0. 600 = 0.01x + 0.02

1. 580 = 0.01x

x = 58 µg/mL

2. Calculate the total phenolic content (TPC):

TPC (mg GAE/g DW) = (C × V × DF) / W

TPC (mg GAE/g DW) = (58 µg/mL × 10 mL × 10) / 10 mg

TPC (mg GAE/g DW) = 5800 µg / 10 mg

TPC (mg GAE/g DW) = 580 mg GAE/g DW

Therefore, the total phenolic content of your dried extract is 580 mg gallic acid equivalents per gram of dried extract.

By following this detailed protocol and addressing the potential troubleshooting points, you should be able to accurately determine the total phenolic content of your dried extract using the Folin-Ciocalteu method. Remember to adapt the specific parameters (e.g., incubation time, wavelength) based on the recommendations in the literature for your particular extract and research question.

Thank You dear Tsegaye,

You have described the procedure very briefly. Could you clarify how you diluted the dried extract? Additionally, why did you not consider the 5 mL reaction solution (0.5 mL sample + 2.5 mL FCR + 2 mL Na₂CO₃ = 5 mL) as part of the dilution factor? Overall, the dilution factor should be 10 × 5 = 50 rather than just 10 mL.

Dear Francesca,

I hope you're doing well.

I have completed the analysis of the total phenol content in the dried ethanol extract. The standard curve for gallic acid yielded the equation:

y = 0.0019x + 0.0013, with an R² value of 0.9994.

The absorbance of my sample is 0.127.

The concentration of the dried ethanol extract was 10 mg/mL, from which I prepared a 100 ppm working solution. I then mixed 1 mL of this 100 ppm solution with 5 mL of 10% Folin–Ciocalteu reagent and 4 mL of 7% sodium carbonate (Na₂CO₃), making the final volume up to 10 mL.

From the calibration curve, the X value I obtained is 0.066 mg/mL.

I have two questions:

Have I calculated the X value correctly?

Using the formula C = (c × V) / m, what would be the final result in mg of gallic acid equivalent per gram of extract?

Hi Iftikhar, I hope you are happy with it too. Regarding your question, are you sure that the concentration of your dry extract is 10mg/mL and you brought it to 100ppm? In that case it would mean having a dilution factor of 100, then add to that that when you did the analysis you diluted it another 10 times, so in total a dilution factor of 1000. I find that, based on your straight line, x is 0.066. This value must then be divided by the concentration of your dry extract which is 10mg/mL and by the dilution factor which is 1000. In the end it comes out as an exaggeratedly large value (6600 mg/g) in my opinion unless you actually have a sample very rich in polyphenols

Thank You Francesca,

I mistakenly stated the dried extract concentration as 10 mg/mL. However, according to the procedure, I actually used 10 mg in 10 mL. Could you please calculate the final concentration based on this correct value?"

In this case, the final dilution factor is 100 (10x10), so the result will be 661.6 mg GAE/g

Thank you Francesca.

I sincerely appreciate your valuable assistance and the time you took to support me. Your help made a meaningful difference, and I’m truly grateful for your guidance

Good luck with your search!

Dear Francesca,

Would you mind sharing your LinkedIn ID and email address with me, if possible?