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

< User:Melissa Novy‎ | Notebook‎ | CHEM-571‎ | 2012‎ | 09

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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 HRP assay report for all UV-vis and fluorescence spectra.
• Abigail E. Miller 18:03, 7 October 2012 (EDT):the report is not a substitute for your notebook. all data and results should be posted in your notebook. you then draw/drew from your notebook to write the report.