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

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Figure 1. Corrected Absorbance Spectra of preliminary kinetics
Figure 1. Corrected Absorbance Spectra of preliminary kinetics
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[[Image:HRP diluteluminol h2o2 zem.png]]
[[Image:HRP diluteluminol h2o2 zem.png|500px]]
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*The H<sub>2</sub>O<sub>2</sub> stock solution appears to be too dilute to produce a spectrum.
*The H<sub>2</sub>O<sub>2</sub> stock solution appears to be too dilute to produce a spectrum.

Revision as of 19:02, 7 October 2013

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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)


Figure 1. Corrected Absorbance Spectra of preliminary kinetics

  • The H2O2 stock solution appears to be too dilute to produce a spectrum.
  • A 1/20 dilution of luminol was used in order to produce an absorbance peak with a value below 1.

Kinetics of luminol oxidized by HRP


General Protocol:

  1. Add the desired HRP/Luminol stock solution to the stop flow mixer.
  2. Add H2O2 stock solution to the stop flow mixer.
  3. Equilibrate mixer tubes with sample
    • Note: For the steps described above, no air bubbles can be present in the sample. Air bubbles will affect the kinetics data(oxygen will act as an oxidant and produce unreliable data)
  4. Initiate Mixing
  5. Take a absorbance spectrum of the sample every 30 seconds for 10 minutes in order to monitor the reaction
  6. Using the luminol and 3-aminophthalic acid spectra as endpoints, determine the kinetics of the 3-aminophthalic acid synthesis


Sample preparation of
Figure 2. Absorbance spectra of the oxidation of luminol by HRP. 50x
Figure 3. Absorption spectra of the production of 3-aminopthalate by the oxidaiton of luminol by HRP. 50x



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