Objective
Protocol
the following protocol was taken from Matt Hartings
Three sets of measurements will be performed today.
- UV-Vis Absorbance of reactants, catalysts, and products
- horseradish peroxidase (Use stock solution)
- luminol (use stock solution)
- 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)
- Chemiluminescence of luminol oxidation reaction initiated by stopped flow mixer
- Add HRP/Luminol stock solution to stopped flow mixer
- Add H2O2 stock solution to stopped flow mixer
- equilibrate mixer tubes with sample.
- Initiate Mixing
- Measure light produced as result of reaction, integrated over a specific time range
- Integrate area under the curve
- Kinetics of luminol oxidized by changes in absorption spectrum, reaction carried out in stopped flow mixer
- Add HRP/Luminol stock solution to stopped flow mixer
- Add H2O2 stock solution to stopped flow mixer
- equilibrate mixer tubes with sample.
- Initiate Mixing
- Using luminol and 3-aminophthalic acid spectra as endpoints, determine the kinetics of 3-aminophthalic acid synthesis.
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