User:Alicia Rasines Mazo/Notebook/CHEM-581 Experimental Chemistry I/2014/10/01

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Task for October 1

  • To determine fluorophore concentration

Fluorescence

Procedure followed as directed by Dr. Hartings on Oct. 1<br.>

  1. Made stock concentrations of Rhodamine 6G (both us and Eleni's group used the same solutions)
    1. 0.10μM
    2. 0.50μM
    3. 0.75μM
    4. 1.0μM
    5. 1.2μM
    6. 1.5μM
    7. 2.0μM
    • Due to extremely low amounts of R6G required to make stock solutions, an initial stock solution of 2500 μM was prepared. This solution was further diluted to 500μM to make rest of solutions, except for 2.0 μM, which was prepared using 2500μM stock solution
      • (2500×10-6mol/L)*(0.010L)*(474.02g/mol)=0.01185 g (0.0120 g of R6G were measured out)
        • 2μM stock concentration: (2500μM)v1=(2.0μM)(5mL); v1=0.004mL
      • 500μM dilution: (2500μM)v1=(500μM)(5mL); v1=1mL of 2500μM R6G stock solution
        • 1.5μM stock solution: (500μM)v1=(1.5μM)(5mL); v1=0.0015mL of 500μM stock solution (Same calculations for rest of stock solutions)
        • 1.2μM stock solution: 0.012mL of 500μM stock solution were diluted to a total volume of 5mL with distilled water
        • 1.0μM stock solution: 0.010mL of 500μM stock solution were diluted to a total volume of 5mL with distilled water
        • 0.75μM stock solution: 0.0075mL of 500μM stock solution were diluted to a total volume of 5mL with distilled water
        • 0.50μM stock solution: 0.005mL of 500μM stock solution were diluted to a total volume of 5mL with distilled water
        • 0.1μM stock solution: 0.001mL of 500μM stock solution were diluted to a total volume of5mL with distilled water
  2. Recorded UV-Vis and Fluorescence spectra of these samples.
    • Excitation: 500nm
    • Excitation range: 515 to 700 nm
    • Slit width: 10 nm
    • Scan speed: 200 nm/min
  3. Make a calibration curve based on UV-Vis.
    1. Compare your data to some published values
  4. Make a calibration curve based on the fluorescence.
    1. In order to do this, you'll need to measure the area under the fluorescence curve, not just the fluorescence peak height.

Calibration curve of R6G using UV-Vis

R6G UVVIS calibration.png<br.>

MolarAbsorp R6G 521.png

Calibration curve of R6G based on fluorescence

Fluorescence R6G.png<br.>

Fluorescence R6G calibrationcurve.png