User:Moira M. Esson/Notebook/CHEM-571/2013/10/01

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Objectives

  1. Monitor the kinetics and yield of horseradish peroxidase catalyzed oxidation of luminol
    1. Measure UV-Vis absorbance of luminol, HRP, and Luminol+HRP+H2O2
    2. Monitor chemiluminscence of luminol oxidation using stop-flow techniques
    3. Measure kinetics of reaction using stop-flow techniques and absorbance


UV-vis

Stock solutions prepared before class:

  1. Luminol: 13.5mg luminol in 300μL DMSO added to 50mL 5mM Tris pH=8
  2. Buffer: 5mM Tris pH=8
  3. HRP: 1.7μM HRP in 50mL 5mM Tris pH=8
  4. H2O2: 117μL of 30%H2O2 in 50mL 5mM buffer.

Samples Run:

  1. 1/20 dilution of lumino1
  2. HRP stock solution
  3. Reaction of HRP+Luminol stock+H2O2 (prepared solution by taking 1/3mL of each sample and allowing to react for ~5min)


Sample notes:

  • Performed 1/20 dilution of 13.5mg luminol in 300μL DMSO added to 50mL 5mM Tris pH=8. (The luminol stock solution).
    • Make a 10mL stock solution of 1/20 dilute luminol stock solution.
  • Prepared stock solution of hydrogen peroxide was too dilute to measure (characteristic peak at 432 nm was not present)