In the Classroom edited by
JCE DigiDemos: Tested Demonstrations
Ed Vitz Kutztown University Kutztown, PA 19530
Classroom Demonstrations of Concepts in Molecular Fluorescence submitted by:
Jonathan P. Blitz,* Daniel J. Sheeran, and Thomas L. Becker Department of Chemistry, Eastern Illinois University, Charleston, IL 61920; *
[email protected] checked by:
Neil D. Danielson Department of Chemistry & Biochemistry, Miami University, Oxford, OH 45056
The chemistry department at Eastern Illinois University has recently completed a multiyear upgrade of its general chemistry instrumentation, replacing aging Spectronic 20 spectrophotometers with fiber-optic probe multichannel spectrophotometers. In this process additional applications for the new spectrophotometers in the context of classroom demonstrations have been developed. This contribution describes the demonstration of principles of fluorescence spectroscopy using these instruments. Numerous laboratory experiments demonstrating the principles of fluorescence have appeared both in this Journal (1–10) and in books (11–13). Fluorescence lecture demonstrations have also been described (14–20). In all of these cases light emission is observed visually in the classroom. Portable multichannel spectrophotometers equipped with a fiber-optic probe make it possible to conveniently capture light in a lecture setting. Since many classrooms are now equipped with digital projectors, live spectra are easily viewed by students in any size classroom (21). The combination of visual observation of fluorescence phenomena with live spectral acquisition provides for a powerful lecture demonstration. The instrumental configuration for the acquisition of fluorescence spectra, correlation of spectral emission with color, and various principles of molecular fluorescence are readily demonstrated.
hanced emission of rhodamine, eosin, and fluorescein samples. In all cases, except for intensity, the fluorescence emission spectra were identical regardless of whether the long or short wavelength UV source was used. The source was positioned above the sample since the glass container may filter UV output causing significantly reduced fluorescence intensity. The fiber-optic probe was positioned against the side of the beaker near the top of the solution where fluorescence is most intense. Spectra were acquired using an Ocean Optics Inc. (Dunedin, FL) CCD-array spectrophotometer (model USB2000) with an approximate wavelength range of 200–800 nm. This spectrophotometer is equipped with a standard fiber-optic probe (1 m, 300 µm i.d.) that was used for the collection of emission spectra. Ocean Optics software (OOI base 32) was used to acquire and process all data. The scope mode was used for continuous viewing of spectra as acquired. Emission spectra were typically obtained with the acquisition of 16 scans and an integration time of 250 ms. Absorbance spectra, using the same spectrophotometer, were acquired with an 8 ms integration time by averaging 12 scans with 7 point boxcar averaging using a quartz cuvette and a distilled water reference.
Experimental Quinine sulfate, fluorescein (disodium salt), eosin yellow, and rhodamine B were obtained from Eastman Kodak. In all cases 0.18–0.20% (m兾v) solutions were prepared for obtaining fluorescence emission spectra. All solutions except quinine were prepared in a 50:50 methanol兾water mixture. The quinine solution was prepared in an aqueous 0.05 M H2SO4 solution. Fluorescence emission and absorbance spectra of quinine in Canada Dry tonic water were also obtained. The absorbance spectrum of quinine in tonic water was obtained after a 1:1 dilution with distilled water.1 The experimental arrangement to collect the fluorescence spectrum of quinine from a commercial sample of tonic water is shown in Figure 1. Excitation was accomplished with a handheld UV source (Mineralight model UVG-54, shortwave only, or Spectroline model ENF-240C with both short and long wavelength UV output). The long wavelength UV source (output shown in Figure 3) resulted in moderately enhanced fluorescence emission of quinine and significantly en758
Journal of Chemical Education
•
Figure 1. Acquisition of a quinine fluorescence emission spectrum in commercially available tonic water. The blue fluorescence of quinine is visually detected while the fluorescence emission spectrum is simultaneously displayed.
Vol. 83 No. 5 May 2006
•
www.JCE.DivCHED.org
In the Classroom
The portable UV lamp should not be looked at or pointed towards the audience because of significant UV output. All of the fluorescent compounds have received either complete or provisional approval by the FDA, so there are no significant hazards associated with their use. Methanol is combustible and should be kept away from heat and sparks. Sulfuric acid is highly corrosive, can cause severe burns to the skin in its concentrated form, and should be handled with extreme caution. Results and Discussion An earlier report (14) describes a fluorescence lecture demonstration in which solutions of different fluorophores fluoresce in different regions of the visible spectrum. This effective visual demonstration can be enhanced by correlation of the colors of the various fluorophores with their emission spectra. In Figure 2, the emission spectra of quinine (blue, λmax = 470 nm), fluorescein (green–yellow, λmax = 525 nm), eosin yellow (yellow, λmax = 575 nm), and rhodamine B (orange, λmax = 615 nm) are shown. The wavelength of maximum emission correlates with the color of the luminescence. This demonstration can be implemented in the general chemistry curriculum when introducing electromagnetic radiation and atomic theory. We perform this in our sophomore-level quantitative analysis course in the context of solution colors. The spectra above are contrasted with solution colors and spectra resulting from light absorption. The same CCD-array spectrophotometer is used for absorption spectra, and an overhead projector demonstration of absorption and solution color is also used as described in Harris (22). This leads to the first detailed discussion of signal generation (emission versus absorption) and spectrophotometry in our curriculum.
Emission Intensity / (100 ct)
eosin yellow fluorescein
30
Emission Intensity / (100 ct)
35
35
rhodamine B
quinine 25 20 15 10 5 0 350
A more detailed description of the fluorescence process and instrumentation is given in our junior-level instrumental analysis course. This is accomplished by focusing on the behavior of quinine. A commercial sample of tonic water is used (Figure 1), however essentially identical results are obtained from a slightly acidic aqueous solution of quinine sulfate. Figure 3 shows the UV excitation source spectrum (long wavelength), as well as the emission and absorbance spectra of quinine in tonic water. For comparison the emission spectrum of pure quinine sulfate in 0.05 M H2SO4 is also shown. The excitation source spectrum clearly overlaps with quinine’s absorbance spectrum, which demonstrates the role of excitation in the fluorescence process. The need to isolate excitation source radiation, so it does not interfere with detected light from sample fluorescence emission, is readily demonstrated. When the source is orthogonal to the fiberoptic probe, minimal source radiation is detected. This demonstrates why the excitation and emission monochromators in most spectrofluorimeters are oriented at 90⬚ with respect to one another. It is also clear in Figure 3 that the excitation wavelengths are at higher energy or shorter wavelengths than fluorescence emission. This red shifting of emission spectra relative to excitation spectra is used to introduce the role of vibrational relaxation in fluorescence emission spectroscopy (23, 24). Additional concepts can also be incorporated into this demonstration. Fluorescence quenching is readily demonstrated by adding a few drops of concentrated base, which immediately eliminates the blue color resulting from quinine fluorescence emission. The adverse effect of the absorbing characteristics of glass for UV excitation is shown by covering the beaker with a watch glass. A significant reduction in fluorescence intensity is observed when short wavelength UV excitation is utilized. It should be noted however that the watch glass minimally filters long wavelength UV radiation, so little reduction in fluorescence intensity is observed with this excitation source.
400
450
500
550
600
650
700
750
30
0.9
A
0.8 25
0.7
D
0.6
20
0.5 15
0.4 0.3
10
0.2 5 0 300
0.1 350
400
450
500
550
600
0.0 650
Wavelength / nm
Wavelength / nm Figure 2. Fluorescence emission spectra of various fluorophores covering the visible range.
www.JCE.DivCHED.org
1.0
C
B
Absorbance
Hazards
•
Figure 3. Overlay of four spectra to demonstrate principles of fluorescence spectroscopy: (A) The emission spectrum of the long wavelength UV light source (correlated with left y axis); (B) the absorbance spectrum of quinine in tonic water (correlated with right y axis), and (C and D) fluorescence emission spectrum of quinine in tonic water and quinine sulfate in 0.05 M H2SO4 (correlated with left y axis).
Vol. 83 No. 5 May 2006
•
Journal of Chemical Education
759
In the Classroom
Conclusion Portable fiber-optic probe equipped multichannel spectrophotometers are becoming increasingly common and accessible in undergraduate education. Utilizing an instrument of this type, without any additional accessories, lecture demonstrations of various concepts in molecular fluorescence spectroscopy have been described. The demonstrations are easy to set up and project in a classroom equipped with a digital projector. Concepts include fluorescence spectrophotometer design geometry, the correlation of color with emission wavelength, excitation, and emission spectra. Note 1. Students in instrumental analysis quantitatively analyze quinine in tonic water from a variety of vendors by fluorescence. Typical concentrations range from 40–70 ppm, suggesting that the quinine concentration in the sample used to obtain the absorbance spectrum is in the range 20–35 ppm. Literature Cited 1. Duncan, R. L.; Kirkpatrick, J. W.; Neas, R. E. J. Chem. Educ. 1972, 49, 550–551. 2. Miguel, A. H.; Braun, R. D. J. Chem. Educ. 1974, 51, 682– 683. 3. O’Reilly, J. E. J. Chem. Educ. 1975, 52, 610–612. 4. Bower, N. W. J. Chem. Educ. 1982, 59, 975–977. 5. Sacksteder, L.; Ballew, R. M.; Brown, E. A.; Demas, J. N.; Nesselrodt, D.; DeGraff, B. A. J. Chem. Educ. 1990, 67, 1065– 1067. 6. Bigger, S. W.; Ghiggino, K. P.; Meilak, G. A.; Verity, B. J. Chem. Educ. 1992, 69, 675–677. 7. Peterson, J. J. Chem. Educ. 1996, 73, 262–263.
760
Journal of Chemical Education
•
8. Pardey, S.; Borders, T. L.; Hernandez, C. E.; Roy, L. E.; Reddy, G. D.; Martinez, G. L.; Jackson, A.; Brown, G.; Acree, W. E., Jr. J. Chem. Educ. 1999, 76, 85–87. 9. Delorenzi, N. J.; Aravjo, C.; Palazzolo, G.; Gatti, C. A. J. Chem. Educ. 1999, 76, 1265–1266. 10. Patterson, B. M.; Danielson, N. D.; Lorigan, G. A.; Sommer, A. J. J. Chem. Educ. 2003, 80, 1460–1463. 11. Sawyer, D. T.; Heineman, W. R.; Beebe, J. M. Chemistry Experiments for Instrumental Analysis; John Wiley & Sons, Inc.: New York, 1984; pp 266–285. 12. Christian, G. D. Analytical Chemistry, 5th ed.; John Wiley & Sons, Inc.: New York, 1994; pp 735. 13. Skoog, D. A.; West, D. M.; Holler, F. J. Fundamentals of Analytical Chemistry, 7th ed.; Saunders College Publishing: New York, 1996; pp 862–863. 14. Bozzelli, J. W. J. Chem. Educ. 1982, 59, 787–788. 15. Workman, M. O. J. Chem. Educ. 1971, 48, 303. 16. Roessler, N. J. Chem. Educ. 1979, 56, 675. 17. Chatellier, D. S.; White, H. B., III. J. Chem. Educ. 1988, 65, 814–815. 18. Marzacco, C. J. J. Chem. Educ. 1996, 73, 254–255. 19. Salter, C.; Range, K.; Salter, G. J. Chem. Educ. 1999, 76, 84. 20. Wagner, B. D.; MacDonald, P. J.; Wagner, M. J. Chem. Educ. 2000, 77, 178–180. 21. Knight, J. B.; Farnsworth, P. B. Spectrochim. Acta 1998, 53, 1889–1893. 22. Harris, D. A. Quantitative Chemical Analysis, 6th ed.; Freeman: New York, 2003; p 412. 23. Strobel, H. A.; Heineman, W. R. Chemical Instrumentation: A Systematic Approach, 3rd ed.; John Wiley & Sons: New York, 1989; pp 516–520. 24. Skoog, D. A.; Holler, F. J.; Nieman, T. A. Principles of Instrumental Analysis, 5th ed.; Saunders College Publishing: Philadelphia, 1998; pp 355–360.
Vol. 83 No. 5 May 2006
•
www.JCE.DivCHED.org
