Tools

Effects of Fluorophore Concentration on Fluorescence Spectra: Inner Filter Effects

  1. Prepare 10-3 M, 10-4 M, 10-5 M, and 10-6 M solutions of anthracene in spectroscopic grade cyclohexane. Note that very dilute solutions are not prepared directly. Lower concentration solutions are prepared via dilution of higher concentration solutions.
  2. To take a particular solution, first click on the appropriate concentration on the concentration selection bar and then on the volumetric flask. For measurement, start with the most dilute solution first (here, 10-6 M) and proceed to next higher concentration and so on.
  3. Carry out absorption and emission measurements as follows.
  4. Take a quartz cuvette (path length, 1 cm x1 cm) by clicking on it for spectrophotometric measurement. Quartz cuvettes for spectrophotometric measurements are transparent only on two opposite sides, unlike the all-side transparent quartz cuvettes used for fluorescence measurements.
  5. Click on the 5 mL capacity pipette to collect 3 mL of the experimental solution which will be transferred into the quartz cuvette. In real operation, one has to set the volume to 3 mL in the pipette and an appropriate tip should be attached prior to dipping it in the solution.
  6. Click on the pipette to draw the solution into it.
  7. Click on the pipette to take it out of the volumetric flask.
  8. Click on the pipette again to transfer the solution into the cuvette.
  9. To start the absorption spectral scan, click on the pop-up Start Absorption Measurement .
  10. Turn on the spectrophotometer clicking on the power button. In real operation, it takes approx. 30 min for initialization of the instrument.
  11. Open the lid of the sample chamber of the spectrophotometer by clicking on the lid for placing the sample in the cell-holder.
  12. Click on the cuvette to place it in the sample holder. One has to use pure solvent as the sample blank or reference in this measurement. Here a double beam spectrophotometer is shown.
  13. Close the chamber lid by clicking on it.
  14. Open the measurement set-up screen by clicking on the absorption measurement icon on the computer monitor.
  15. On the screen, enter the wavelength range: Start: 400 nm End: 260 nm. In real operation, the wavelength range of incident light for the sample is chosen and the wavelength scan is run via the accompanied computer software. One can run the scan in absorbance (A) or transmittance (%T) mode.
  16. Click on the green 'Start' button on the measurement set-up screen to run the wavelength scan. Observe the wavelength scan.
  17. Click on 'Close' button when spectral scan is complete. In real operation, the scan data are stored in the computer. The instrument stores data and therefore asks for the Sample File name. One enters a file name to save the data.
  18. To take the cuvette out of the sample chamber, first click on the sample chamber lid to open it and then on the cuvette.
  19. Close the sample chamber lid by clicking on it.
  20. Click on the pop-up: Start Fluorescence Measurement .
  21. Turn on the spectrofluorimeter by clicking on the power button. In real operation, it takes approx. 30 min for initialization of the instrument.
  22. Click on the spectrophotometric quartz cuvette to transfer its content into an all-side-transparent quartz cuvette of path length 1 cm x1 cm for the fluorescence measurement.
  23. For placing the sample in the instrument, open the lid of the sample chamber of the spectrofluorimeter by clicking on the lid.
  24. To place the cuvette in the sample holder of the spectrofluorimeter, click on the cuvette.
  25. Close the lid of the sample chamber by clicking on the lid.
  26. Open the instrument set-up screen by clicking on the fluorescence icon on the computer monitor.
  27. Select the Emission Scan Mode on the screen.
  28. On the screen, enter the Excitation wavelength: 360 nm, Emission Start Wavelength: 370 nm and Emission End wavelength: 460 nm. One chooses the Excitation Slit(nm) and Emission Slit(nm) values (here 5 nm/5 nm) and the scan speed value (here medium ) also.
  29. To run the wavelength scan for emission spectrum, click on the 'OK' button on the set-up screen. One has to be sure that the solvent blank does not fluoresce in the wavelength range of interest.
  30. Click on 'Close' button when spectral scan is complete. In real operation, the scan data are stored in the computer. The instrument stores data and therefore asks for the Sample Filename. One enters a file name to save the data.
  31. To take out the cuvette out of the sample chamber, first click on the sample chamber lid to open it and then on the cuvette.
  32. Close the lid of the sample chamber by clicking on the lid.
  33. Click on 'Reset' button to start over the measurements.
  34. Select the next higher concentration solution (10-5 M) for measurement by clicking on the concentration selection bar and carry out the Emission scan. If one uses the same cuvette for all the measurements, the cuvette should be rinsed a few times with the experimental solution to be analyzed prior to filling it up with the solution.
  35. Repeat the Emission scan measurements for two other solutions (10-4 M and 10-3 M).
  36. Collect all data by clicking on the Data tab.
  37. Find out the absorption-emission overlapping region for 10-6 M anthracene. Determine the Stokes Shift.
  38. Discuss how the maximum emission wavelengths and the intensities of the shortest wavelength emission bands of anthracene change with increasing concentration of anthracene.