User:Eleni N. Kalivas/Notebook/CHEM-571/2013/10/01

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Objective

Protocol

the following protocol was taken from Matt Hartings

Three sets of measurements will be performed today.

  1. UV-Vis Absorbance of reactants, catalysts, and products
    1. horseradish peroxidase (Use stock solution)
    2. luminol (use stock solution)
    3. 3-aminophthalic acid (product of reaction between luminol and H2O2 catalyzed by HRP. In order to take this measurement, react (in 1:1 ratio or with slight excess H2O2) luminol with H2O2 in presence of HRP. Allow the reaction to proceed for 5 minutes; take the spectrum)
  2. Chemiluminescence of luminol oxidation reaction initiated by stopped flow mixer
    1. Add HRP/Luminol stock solution to stopped flow mixer
    2. Add H2O2 stock solution to stopped flow mixer
    3. equilibrate mixer tubes with sample.
    4. Initiate Mixing
    5. Measure light produced as result of reaction, integrated over a specific time range
    6. Integrate area under the curve
  3. Kinetics of luminol oxidized by changes in absorption spectrum, reaction carried out in stopped flow mixer
    1. Add HRP/Luminol stock solution to stopped flow mixer
    2. Add H2O2 stock solution to stopped flow mixer
    3. equilibrate mixer tubes with sample.
    4. Initiate Mixing
    5. Using luminol and 3-aminophthalic acid spectra as endpoints, determine the kinetics of 3-aminophthalic acid synthesis.