User:Michael S. Bible/Notebook/CHEM-671/690 Lab Notebook/2015/09/09
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The purpose of today's work is to create a UV-Vis and Fluorescence calibration curve for Lysozyme.
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.
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
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.
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.