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

Objectives

• Complete horseradish peroxidase enzymatic activity assay from 2012/09/18.
  • Please see entry on 2012/09/18 for absorbance over time graphs and data analysis.
• 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.