Acetaminophen Project
This project's goal was to quantify the concentration of Acetaminophen left after a reaction with hydrogen peroxide, Acetaminophen, & horseradish peroxidase. To do this, I made standards with known concentrations of Acetaminophen. Then, we ran the reaction in three separate vials with varying amounts of peroxide with the constant amounts of Acetaminophen and horseradish peroxidase (HRP).
My research partner, Josie Welker, ran a similar experiment with HPA, the link to his page can be found here.
Acetaminophen Standards
In order for our samples to be tested, a 2 mM Acetaminophen pH 5 solution, Hydrogen peroxide stock solution, and horseradish peroxidase solution were made.
2 mM Acetaminophen Buffer Stock Solution
Put the 2 mM Acetaminophen standard into a jar, and add the pH 5 buffer tablet. Stir until dissolved.
Hydrogen Peroxide Stock Solution
Add 5 mL of water into a scintillation vial. Add 283 uL of 30% Hydrogen Peroxide to the water. The solution is now at about 0.413 M.
Horseradish Peroxidase Solution
Preparing The Final samples
Measure out 5 mL of Acetaminophen into four different scintillation vials. Add differing amounts of Hydrogen peroxide solution into each of the vials (3uL, 6uL, 12uL, & 24uL). Add 10uL horseradish peroxidase solution to each of the vials.
Instrumentation
After preparing our solutions, we tested them on the HPLC to see where the peaks were at different concentrations of hydrogen peroxide.
The following link provides insight into how the HPLC works and how a user can correctly use the instrument.
What the HPLC Data Tells us
The Acetaminophen samples were ran at a wavelength of 263 nm because that is the lambda max for acetaminophen. Running the samples at this wavelength shows us how much absorption occurs at that wavelength.
Data Analysis
The data from the HPLC was exported as an arw file in order to be analyzed. This file was then loaded into Igor, which we used to make chromatograms. Chromatograms allowed us to gain a better understanding of the how each concentration of Acetaminophen absorbs at 263 nm for our standards, and how each sample absorbs at 263 nm. We used the information from the chromatograms to make standard curves in excel. The slopes of the standard curves gave us an equation we could use to find the concentration of a sample using the absorbance.
Gepasi
We used the software "Gepasi" to analyze the kinetics of the reaction between horseradish peroxidase (HRP) and hydrogen peroxide. This program simulates how the compounds will react with each other. The following link describes how to use "Gepasi":
Copasi
A newer, updated version of Gepasi is Copasi. This software is newer and will be more efficient in getting data to the user. A link on how to navigate and use Copasi is listed below.