Indeed, you're right. After all, the TPC equation (TPC = c x V/M) can be confusing at first, but let's demystify it so you can understand which values to use for your calculation.

C (concentration):

• You can obtain this value from the equation of your gallic acid standard curve (0.0085x - 0.123). Measure the absorbance of your sample at 765 nm and subtract the intercept (-0.123) from the absorbance value. Then, divide this result by the slope (0.0085x) to get the concentration (c) in mg/mL gallic acid equivalents (GAE).

V (volume):

• The appropriate volume (V) to use depends on the step you're considering in your TPC analysis: If using the concentration before adding Folin reagent: Use the initial volume of your extract before dilution (i.e., 30 μL in your example). If using the concentration after dilution: Use the total volume of the diluted extract solution after adding Folin reagent and other components (i.e., 240 μL in your example).

M (mass):

• Regardless of whether you use the concentrated or diluted extract solution, the mass (M) refers to the initial mass of the dried sample you used for the extraction process (i.e., 6 mg in your example).

Here's an example calculation for both scenarios:

Scenario 1: Using concentration before adding Folin reagent

• Concentration (c) from gallic acid standard curve (using your example values): Let's say your sample absorbance is 0.3. Then, c = (0.3 - (-0.123)) / 0.0085 = 0.494 mg/mL GAE.
• Volume (V) = Initial volume of your extract = 30 μL (0.03 mL)
• Mass (M) = Initial mass of dried sample = 6 mg

TPC = (0.494 mg/mL GAE) * (0.03 mL) / (6 mg) = 0.00247 mg GAE/mg extract

Scenario 2: Using concentration after dilution

• Follow steps 1 and 2 from scenario 1 to obtain the concentration (c).
• Volume (V) = Total volume of diluted extract solution = 240 μL (0.24 mL)
• Mass (M) = Initial mass of dried sample = 6 mg

TPC = (0.494 mg/mL GAE) * (0.24 mL) / (6 mg) = 0.01976 mg GAE/mg extract

Both scenarios will provide a TPC value, but they represent the phenolic content per unit volume of extract at different dilution steps. It's important to choose the volume (V) that aligns with the concentration (c) you determined.

Hope it helps

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