Good day,

I am currently stumbled upon a problem in my TPH analysis. Prior to this, I have zero experience in carrying such analysis and any guidance or advice is greatly appreciated.

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Below is my experimental procedure for TPH:

1. Extract is diluted to 1 mL of methanol.

2. Extract solution is added with 1 mL Folin reagent (10 fold dilution)

3. Mixture solution is added with 2 mL of 7.5% sodium carbonate solution - 5 mins after the addition of Folin solution.

4. Gallic acid calibration curve is prepared by diluting 10 mg GA in 80% methanol soln to get 0.1mg/mL concentration.

5. GA solution is further diluted to 0.06, 0.04 and 0.02 mg/mL concentration. All GA stock solutions are added with 1mL Folin and 2 mL sodium carbonate solution (similar to steps 2 and 3)

6. All extract and calibration solutions are subjected to wavelength 725 nm.

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For my results,

My extract obtained abs. value around 2.2.

For my calibration solutions, the values are roughly as follows:

Concentration Abs. value Colour

0.1 mg/mL 2.7 Black

0.06 mg/mL 3.05 Black

0.04 mg/mL 3.6 Bluish-black

0.02 mg/mL 2.01 Light bluish-black

From my understanding, abs. value decreases when GA concentration decreases. Only the most diluted samples showed promising result.

I believe the inconsistency may be due to the very dark coloured (black) calibration solutions at higher concentration, therefore the light could not penetrated fully into the solution during the analysis.

May I know how should I proceed from here? Should I dilute my GA solutions further?

Please let me know if I am doing something wrong along the way.

Thank you.