User:Alexander Cvitan/Notebook/Experimental Biological Chemistry Lab/2013/10/01

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  • Complete the procedure as shown by Dr.Hartings, HERE
  • Bullet points number 1 and 3 were completed form Dr.Hartings procedure today.


  • Stock solutions
    • HRP: .77uM
    • Luminol: 1.51mM
    • Buffer: 5.1mM
    • Note that the concentration of Hydrogen Peroxide needs to be determined using UV-Vis. This is because the solution itself is unstable and can quickly break down.


  • Using the UV-Vis analysis using Beers law, and the abs and molar absorptivity the concentration of the hydrogen peroxide stock solution was calculated to be .03mM. This was calculated using wavelength 240nm. As a result the abs was 1.219 and the molar absorptivity was 40000 (1/M*cm)<br.>
  • Using UV-Vis analysis the solutions were diluted until peaks were under an absorbance of 1. As a result the concentrations of the solutions used to complete Dr.Hartings procedure was a 20x dilution of hydrogen peroxide, a 20x dilution of luminol, and just the stock solution of HRP. <br.>
  • The graph below represents the UV-Vis spectra of the solutions used for analysis.<br.>

Oct 1st combined data.png

  • After completing Dr.Hartings procedure none of the peaks changed position after the reaction started. It is thought that the HRP solution was to concentrated and the reaction ended up occurring to fast to catch using the equipment we have.<br.>

Due to the nature of the concentrations used for our group, below is Trisha's groups data who used slightly different concentrations

  • Trisha's group used a diluted solution of luminol with a concentration of .126mM and used the stock solutions for her reaction.
  • The graph for Trish's data is represented on tomorrow data. <br.>