User:Melissa Novy/Notebook/CHEM-571/2012/09/19

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<!-- sibboleth --><div id="lncal1" style="border:0px;"><div style="display:none;" id="id">lncal1</div><div style="display:none;" id="dtext">09/25/2012</div><div style="display:none;" id="page">User:Melissa Novy/Notebook/CHEM-571/2012/09/19</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month"></div><div style="display:none;" id="year"></div><div style="display:none;" id="readonly">Y</div></div>

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Objectives

  • Complete horseradish peroxidase enzymatic activity assay from 2012/09/18.
  • Make 2.5 mM luminol (MW 177.16) stock solution for horseradish peroxidase chemiluminescence assay.
  • Using a fluorimeter, collect emission spectra at 425 nm for horseradish peroxidase chemiluminescence assay.

Luminol Stock Solution in H2O and Carbonate Buffer

  • Calculations:
    • 10 mL of 2.5 mM luminol stock solution requires the following:
  MW luminol: 177.16 g/mol
  Mass luminol:
        10 mL × (10-3 L/mL) × (2.5 × 10-3 M) × (10-3 mol/mmol) × 177.16 g/mol = 0.00443 g luminol
  Actual mass luminol: 0.0044 g
  Actual concentration: 
        0.0044 g × (1 mol/177.16 g) × (1/0.010 L) = 2.5 × 10-3 M in H2O
  • The concentration of the luminol stock solution was varied by serial dilution as follows:
    • 1.25 mM, 625 μM, 312.5 μM, and 156.25 μM.
    • 10 mL solutions of each concentration were made.
  • Notes:
    • Luminol did not dissolve completely in H2O, so Na2CO3 was added in a ratio of 4 g for every 500 mL of solution to aid its dissolution.
      • 0.08 g of Na2CO3 was added to the luminol solution.
    • After the luminol dissolved, 0.48 g NaHCO3 was added to adjust the pH of the solution to about 7.
      • 0.48 g of NaHCO3 was added to the luminol solution.

Notes

  • Please refer to the entry on 2012/09/25 for HRP-luminol fluorescence spectra and data analysis.