Difference between revisions of "Team:Paris Bettencourt/Protocols/titration-acid-phytic"

 
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       <li><b>Phosphorous calibration curve :</b></li>
 
       <li><b>Phosphorous calibration curve :</b></li>
 
       Determine the absorbance of each phosphorous standard. Substract the absorbance of STD0 from the absorbance of the others STD, therby obtaining ΔA(phosphorous).
 
       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 :  
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       Calculate M as follows, for each standard:  
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<img src="https://static.igem.org/mediawiki/2015/6/6b/ParisBettencourt_Calcul_M_hytic_acid.png" width="400px">
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\[
−
       Use "Mean M" to calculate the phosphorous content of test samples.
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\begin{align}
 +
M = \frac{P(\mu g)}{\Delta A (phosphorous)}
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\end{align}
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\]
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       Use "Mean M" to calculate the phosphorous content of test samples.<br>
 
       <li><b>Phosphorous / phytic acid content :</b></li>
 
       <li><b>Phosphorous / phytic acid content :</b></li>
 
       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).
 
       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="https://static.igem.org/mediawiki/2015/8/8f/ParisBttencourt_Calcul_concentration_phytic_acid.png" width="400px">
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\[
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Where :<br>
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\begin{align}
−
20 = original sample extract volume<br>
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C^{o} = \frac{mean(M)\times 20 \times F}{10000 \times 1.0 \times \nu} \times \Delta A(phosphorous)
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F = dilution factor<br>
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\end{align}
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10,000 = conversion from µg/g to g/100g<br>
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\]
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1.0 = wigh of original sample material<br>
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<br><i>Where :</i><br>
−
v = sample volume
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\(20\) = original sample extract volume<br>
 +
\(F\) = dilution factor<br>
 +
\(10000\) = conversion from µg/g to g/100g<br>
 +
\(1.0\) = wigh of original sample material<br>
 +
\(\nu\) = sample volume
  
−
<b><i>It follows for phosphorous :</i></b>
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<br><br>
 +
<b><i>It follows for phosphorous :</i></b><br>
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\[
 +
\begin{align}
 +
C^{o} ~= \frac{mean(M)\times 20 \times 55.6}{10000 \times 1.0 \times \nu} \times \Delta A(phosphorous)\\
 +
~= mean(M) \times 0.1112 \times \Delta A(phosphorous)
 +
\end{align}
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\]
 +
 
 +
<b><i>It follows for phytic acid :</i></b><br>
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\[
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\begin{align}
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C^{o} = \frac{phosphorus}{0.282}
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\end{align}
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\]
  
 
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Latest revision as of 16:40, 2 September 2015

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 :
  • Colourimetric determination of phosphorous :
  • Preparation of phosphorous calibration curve :
  • 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: \[ \begin{align} M = \frac{P(\mu g)}{\Delta A (phosphorous)} \end{align} \] 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). \[ \begin{align} C^{o} = \frac{mean(M)\times 20 \times F}{10000 \times 1.0 \times \nu} \times \Delta A(phosphorous) \end{align} \]
      Where :
      \(20\) = original sample extract volume
      \(F\) = dilution factor
      \(10000\) = conversion from µg/g to g/100g
      \(1.0\) = wigh of original sample material
      \(\nu\) = sample volume

      It follows for phosphorous :
      \[ \begin{align} C^{o} ~= \frac{mean(M)\times 20 \times 55.6}{10000 \times 1.0 \times \nu} \times \Delta A(phosphorous)\\ ~= mean(M) \times 0.1112 \times \Delta A(phosphorous) \end{align} \] It follows for phytic acid :
      \[ \begin{align} C^{o} = \frac{phosphorus}{0.282} \end{align} \]