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    Experiment 7

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    Experiment 7

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    Experiment 7

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    Experiment 7

    Folin-Ciocalteu method: Determination of


    Total Phenolic Content (TPC)

    Introduction
    The Folin-Ciocalteu method, developed in 1927 by Otto Folin and Vintilă Ciocalteu, is a widely
    used technique for the quantification of total phenolic compounds in a variety of samples, ranging
    from plant extracts to food and beverages. The Folin-Ciocalteu method plays a critical role in
    assessing the nutritional quality and antioxidant capacity of different substances.

    The principle behind the Folin-Ciocalteu method is the oxidation of phenolic compounds by the
    Folin-Ciocalteu reagent, resulting in the formation of a blue-colored complex. The intensity of
    this color is directly proportional to the concentration of phenolic compounds present in the
    sample, allowing for a quantitative determination of their content. The method is versatile and can
    be adapted to various types of samples, making it a valuable tool for researchers in fields such as
    nutrition, pharmacology, and environmental science.
    Why Gallic Acid is used?

    Gallic acid is commonly used as a standard in the Folin-Ciocalteu method for its stability,
    simplicity, and representativeness of the phenolic compound class. It is also readily available and
    well-characterized.

    Why Sodium Carbonate is used?

    Sodium carbonate is used in the Folin-Ciocalteu method to:

     Neutralize the reaction mixture, optimizing pH for color development


     Stabilize the blue complex color, ensuring consistent measurements
     Enhance the reactivity of phenolic compounds with the Folin-Ciocalteu reagent
     Maintain consistent reaction conditions, leading to accurate and reproducible results

    In this laboratory experiment, we will explore the Folin-Ciocalteu method and its application in
    quantifying phenolic compounds. Our objective is to understand the underlying chemical
    reactions, establish the calibration curve, and analyze the phenolic content of a given sample.

    Materials and Equipment:

    1. Spectrophotometer
    2. Cuvettes
    3. Micropipettes
    4. Vortex mixer
    5. Test tubes
    6. Distilled water
    7. Water bath
    8. Folin-Ciocalteu reagent
    9. Sodium carbonate (Na2CO3) 7.5%
    10. Standard solution (e.g., gallic acid)
    11. Unknown sample (e.g., plant extract, food product)
    Procedure:

    Determination of Total Phenolic Content (TPC) Using the Folin-Ciocalteu Method

    First, prepare the following:

     Standard solution of gallic acid with a concentration of 1 mg/mL and subsequently perform
    serial dilution
     Folin-Ciocalteu reagent (ratio 1:9); 1 mL of Folin-Ciocalteu reagent for every 9 mL
    distilled water
     7.5% (w/v) Na2CO3 solution; 7.5 g in 100 mL distilled water.

    For the TPC determination:

    1. Take a volume of 100 µL of the gallic acid standard at each concentration.


    2. Add 500 µL of the diluted Folin-Ciocalteu reagent
    3. Vortex
    4. Add 400 µL of a 7.5% (w/v) Na2CO3 solution
    5. Vortex
    6. Place the tubes in the water bath at 60°C for 10 minutes
    7. Cool at 4°C for 10 minutes
    8. Use a UV–Vis spectrophotometer to measure the absorbance of the reaction mixture. Set
    the spectrophotometer to a wavelength of 750 nm.
    9. Create a calibration curve using gallic acid as the standard compound.
    10. Calculate the Total Phenolic Content (TPC) for any sample based on the calibration curve.
    Express the TPC as milligrams (mg) of gallic acid equivalent per gram of samples (mg
    GAE/g).

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