User:Michael S. Bible/Notebook/581/2014/10/08

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R6G Fluorescence: Calibration and Measurement

  1. Make stock concentrations (both groups can use the same solutions)
    • Initial stock: 2500μM, secondary stock: 500μM
    • 0.10μM
      • Made with 500μM solution
    • 0.50μM
      • Made with 500μM solution
    • 0.60μM
      • Made with 500μM solution
    • 0.75μM
      • Made with 500μM solution
    • 0.85μM
      • Made with 500μM solution
    • 1.0μM
      • Made with 500uM solution
    • 1.5uM
      • Made with 500μM solution
    • 2.0μM
      • Made with 2500μM solution
    • 0.75μM
      • Made with 500μM solution
    • 1.2μM
      • Made with 500μM solution

Thanks to Madeleine for making initial 2500μM solution of R6G.

  1. Take UV-Vis and Fluorescence spectra of these samples
    • Fluorimeter Settings:
      • 500nm excitation
      • 515-700nm scan range
      • 10.0nm slit width
      • 200nm/min scan speed
  2. Make a calibration curve based on UV-Vis.
    • Compare your data to some published values
  3. Make a calibration curve based on the fluorescence.
    • In order to do this, you'll need to measure the area under the fluorescence curve, not just the fluorescence peak height.


To calculate necessary amounts of stock solutions to add to enough water to have a total of 10 mL of calibration stock solutions:

  • C1*V1 = C2*V2
    • secondary stock: 500μM
      • (2500μM)*x = (500μM)*(10mL) → x = 2 mL 2500μM R6G
    • 0.1μM
      • (500μM)*x = (0.1μM)*(10mL) → x = 2 μL 500μM R6G
    • 0.5μM
      • (500μM)*x = (0.5μM)*(10mL) → x = 10 μL 500μM R6G
    • 0.6μM
      • (500μM)*x = (0.6μM)*(10mL) → x = 12 μL 500μM R6G
    • 0.75μM
      • (500μM)*x = (0.75μM)*(10mL) → x = 15 μL 500μM R6G
    • 0.85μM
      • (500μM)*x = (0.85μM)*(10mL) → x = 17 μL 500μM R6G
    • 1.0μM
      • (500μM)*x = (1.0μM)*(10mL) → x = 20 μL 500μM R6G
    • 1.2μM
      • (500μM)*x = (1.2μM)*(10mL) → x = 24 μL 500μM R6G
    • 1.5μM
      • (500μM)*x = (1.5μM)*(10mL) → x = 30 μL 500μM R6G
    • 2.0μM
      • (500μM)*x = (2.0μM)*(10mL) → x = 40 μL 500μM R6G


Using the data collected from the UV-Vis Spectrometer and the Fluorometer, the following calibration curves were created.

UV-Vis Calibration Spectra for R6G.png

Figure 1: The figure above shows the UV-Vis absorbance spectra for various concentrations of R6G. The spectra have been corrected by subtracting the spectrum values of water from each. The spectra have also had their respective absorbance values at 800 nm subtracted from every point in order to shift the baseline to zero across the board. For those spectra that had peaks shifted down below this baseline after the subtraction, the absorbance value of each of the graphs at 560 nm was subtracted from the whole spectrum instead so that the rise of the peak would occur at the baseline like the others.

UV-Vis Calibration Curve for R6G.png

Figure 2: It was noticed in figure 1 that the graph of 0.1 μM R6G had a peak at 0.003, which is extremely close to the detection limit of the instrument, so it was excluded from the calibration curve.

Fluorescence Calibration Spectra for R6G.png

Figure 3: The figure above shows the Fluoresence spectra for concentrations up to 1 μM of R6G. Concentrations above 1 μM gave values that were too high to show up on the fluorometer.

Fluorescence Calibration Curve for R6G Corrected.png

Figure 4: The figure above shows the calibration curve for fluorescence. Integration values were determined using Riemann sums with a base of .5 nm.


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