HPA Project

Project Overview

For this project, the goal was to quantify the concentration of HPA remaining in solution after a reaction between hydrogen peroxide, horseradish peroxidase, and HPA is completed. To do this we made standards of known concentrations of HPA, made reaction vials of the hydrogen peroxide, enzyme (horseradish peroxidase), and HPA. We ran these through the HPLC to collect the data.

My research partner, Will Fox, ran a similar experiment with acetaminophen, the link to his page can be found here.

Making the Samples

This project required HPA standards to be made. The following link describes this process.

HPA Standards

Instrumentation

After making the standards, we tested the standards using the HPLC to see where the peaks were for the different concentrations of HPA.

The following link goes into more depth about working with the HPLC:

HPLC

What the HPLC Tells Us

We ran these samples through the HPLC at the wavelength of 273 nm, which is the lambda max for caffeine. Running the samples at this wavelength tells us how much the samples absorb at this wavelength.

Data Analysis

To analyze the HPLC data, the data was exported as an arw file. 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 HPA absorbs at 275 nm for our standards, and how each sample absorbs at 275 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.

Example standard curve data can be found here.

Equation

The following equation was used to find the concentration of caffeine in our samples. The equation is solved for x, which is concentration, then the absorbance is plugged in for y and solved.

y = 0.0041x + 0.0071

R² = 1

Data Summary