User:Michael S. Bible/Notebook/CHEM-671/690 Lab Notebook/2015/09/09: Difference between revisions
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The Figure above shows the Fluorescence spectra of various concentrations of lysozyme. | The Figure above shows the Fluorescence spectra of various concentrations of lysozyme. | ||
[[User:Matt Hartings|Matt Hartings]] What is your molar absorptivity for lysozyme in M<sup>-1</sup> cm<sup>-1</sup>? | |||
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ObjectiveThe purpose of today's work is to create a UV-Vis and Fluorescence calibration curve for Lysozyme.
ProtocolA 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.
CalculationsConcentration 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.
DataUV-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.
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. Matt Hartings What is your molar absorptivity for lysozyme in M-1 cm-1? | |
