User:Michael S. Bible/Notebook/CHEM-671/690 Lab Notebook/2015/09/09

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Objective

The purpose of today's work is to create a UV-Vis and Fluorescence calibration curve for Lysozyme.


Protocol

A stock solution of lysozyme was created using a 10 mL volumetric flask. The aim was to make a 50 μM stock solution but the actual concentration was 62 μM.

8.88 mg of lysozyme was added to a 10 mL volumetric flask

The following 5 solutions were made using a 10 mL volumetric flask and the volumes of water and other solutions listed below.

  • 15 μM
    • 2.419 mL of 62 μM stock solution
    • 7.581 mL of HPLC water.
  • 12.5 μM
    • 2.016 mL of 62 μM stock solution
    • 7.984 mL of HPLC water.
  • 6.25 μM
    • 5.0 mL of 12.5 μM lysozyme solution
    • 5.0 mL of HPLC water.
  • 3.13 μM
    • 5.0 mL of 6.25 μM lysozyme solution
    • 5.0 mL of HPLC water.
  • 1.56 μM
    • 5.0 mL of 3.13 μM lysozyme solution
    • 5.0 mL of HPLC water.

Calculations

Concentration of Lysozyme stock solution:

(0.00888 g)*[(1 mol lysozyme)/(14307 g lysozyme)] = 6.2*10-7 mols

(6.2*10-7 mols)/(0.010 L) = 62 μM


Standard solutions were made using the following formula C1V1 = C2V2

V2 = [C1V1]/C2

So, in order to make a 15 μM solution the following volume of stock solution was needed:

V62μM = (15 μM * 10 mL)/(62 μM) = 2.419 mL of stock solution

The method described above was used to determine the necessary volumes of previous serial solutions to use in each new dilution.


Data

UV-Vis and Fluorescence spectra were collected for each of the 5 solutions (excluding stock solution) above. The spectra were corrected using a HPLC water blank. A calibration curve for UV-Vis has been created using the absorbance at λ = 281nm.

The Figure above shows the UV-Vis spectra of various concentrations of lysozyme.

The Figure above shows the calibration curve for the absorbance at λ = 281 as a function of the concentration of lysozyme in solution. Using the equation of the line of best fit and Beer's Law, we see that A = εbc with b = 1 cm. This means that the slope of the line in the figure above is the Molar extinction coefficient. For lysozyme at λ = 281, ε = 3.57 × 104 M-1·L-1

The Figure above show the calibration curve for the Fluorescence intensity integration as a function of the concentration of lysozyme in solution.

The Figure above shows the Fluorescence spectra of various concentrations of lysozyme.



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