Team:Paris Bettencourt/Protocols/titration-acid-phytic

Titration of phytic acid using Megazyme© Kit
  • Preparation of reagent solutions (not supllied) :
    • Trichloroacetic acid (50% w/v) : 100mL
      • Add 50g of thrichloroacetic to 60mL of distilled water and dissolve. Make to volume (100mL) with distilled water. (Stable for > 6 moths at 4°C)
    • Hydrochloric acid (0.66M) : 1L
      • Add 54.5mL of hydrochloric acid to 945.5mL of distilled wtaer an mix. (Store at room temperature)
    • Sodium hydroxide (0.75M) : 200mL
      • Add 6g of sodium hydroxide pellets to 180mL of distilled water and dissolve. Make to volume (200mL) with distilled water. (Store at room temperature)
    • Phytic acid :
      • Pure phytic acid control sample may be required.
  • Sample extraction :
    • Weigh 1g of sample material.
    • Add 20mL of hydrochloric acid (0.66M).
    • Cover with foil and incubate for a minimum of 3 hours at room temperature.
    • Transfer 1mL of extract to a 1.5mL microcentrifuge tube.
    • Centrifuge 10 minutes at 13,000rpm.
    • Transfer 0.5mL of the resulting extract supernatant to a fresh 1.5mL microfuge tube.
    • Add 0.5mL of sodium hydroxide solution (0.75M) to neutralise.
  • Enzymatic dephosphorylation reaction :

    • <img src="ParisBettencourt_Tableau_1_dosage_acide_phytic.png" width="400px">

  • Colourimetric determination of phosphorous :

    • <img src="ParisBettencourtTab_PHYTIC_ACID_Colourimetric_determination.png" width="360px">

  • Preparation of phosphorous calibration curve :

    • <img src="ParisBettencourtTab_PHYTIC_ACID_Prep_calibration_curve.png" width="360px">

  • Calculation :
    • Phosphorous calibration curve :
      •      Determine the absorbance of each phosphorous standard. Substract the absorbance of STD0 from the absorbance of the others STD, therby obtaining ΔA(phosphorous).
     Calculate M as follows, for each standard :

      <img src="ParisBettencourt_Calcul_M_hytic_acid.png" width="400px"> 

           Use "Mean M" to calculate the phosphorous content of test samples.
    • Phosphorous / phytic acid content :
      •      Determine the absorbance of the both "Free Phosphorous" and "Total Phosphorous". Substract the absorbance of the "Free Phosphorous" sample from the absorbance of the "Total Phosphorous" sample. Obtaining ΔA(phosphorous).

      <img src="ParisBttencourt_Calcul_concentration_phytic_acid.png" width="400px"> Where :
      20 = original sample extract volume
      F = dilution factor
      10,000 = conversion from µg/g to g/100g
      1.0 = wigh of original sample material
      v = sample volume

      It follows for phosphorous :