Understanding Fluorescence Measurements through a Guided

In the Laboratory

Understanding Fluorescence Measurements through a Guided-Inquiry and Discovery Experiment in Advanced Analytical Laboratory Grazyna Wilczek-Vera* and Eric Dunbar Salin Department of Chemistry, McGill University, Montreal, Quebec H3A 2K6 *[email protected]

In the current age of automated analytical measurements, it is important to adjust teaching methods to adapt to the new situation. In the “old days”, a bachelors-degree chemist could have spent most of his or her time at work simply feeding samples into an instrument. Now, this is accomplished by an autosampler. Bachelors-degree chemists are expected to operate many types of instruments, troubleshoot them, develop new methodologies, report on their results, work in teams, and consult on their laboratory's capabilities and limitations. Thus, it is essential to prepare students so they can face these challenges. This is particularly important as there is a tendency for many to treat computerized instruments as magic black-boxes that, after being fed an unknown sample, immediately provide an investigator with all the knowledge required with stunning speed, precision, accuracy, and detail. CSI-type TV shows encourage this illusion. The experiment presented here was developed several years ago as part of an advanced instrumental analysis course that, in addition to standard spectroscopic analytical techniques, covers analog to digital data acquisition (1), signal processing, and elements of programming. Our goal is to encourage students to “open the black-box”, learn how to perform chemical analyses in a real world and, through this route, to become active observers and thinkers. We use the “learn-as-you-go” and direct “handson” approach, which ensures an active role of a student in the process of visualization and discovery of concepts. We also expect students to learn the mental processes involved in troubleshooting. Theory The theory and some aspects of practical applications of fluorescence spectroscopy are widely covered in standard textbooks of analytical chemistry (2a). Numerous articles related to this subject appeared also in this Journal (2b). Nevertheless, with the Nobel Prize 2008 awarded “for the discovery and development of the green fluorescent protein, GFP” (3a), the topic of fluorescence is more relevant than ever (3b). Methodology Articles in chemical education research present four basic styles of laboratory instruction: expository, inquiry, discovery, and problem-based (4a). All of them have their strong and weak points (4a, 4b). In our laboratory, we use a mixed approach that constitutes a combination of the expository and discovery styles. We call it a “spiral” approach, as students have to revisit the same topic on different levels of complexity and comprehension. The experiment described here is an example of this methodology. Students come to the lab with some theoretical preparation 216

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having read the assigned material and answering a few wellfocused questions. A short prelab verifies their basic knowledge. The experiment is a “hands-on” experience during which students discover first how the instrument operates and then explore basic principles of fluorescence. Armed with this knowledge and understanding, finally they perform an analysis of an unknown sample. The topics of fluorescence and the basics of instrumentation are also covered during the course lectures. If a group of students hears these lectures before the experiment, it is easier for them to perform their tasks in the lab. If, on the other hand, the lectures were scheduled after the relevant laboratory period, the students will profit more from the lectures having a much deeper prior understanding of the topic covered. Practical and theoretical exams at the end of semester ensure that students integrated properly the material covered. The data concerning students' learning are included in the supporting information. Experimental Details The experiment can be comfortably completed during one 4-h laboratory period by a group of 2-4 students. It starts with the determination of a dark current and a visible light spectrum. Subsequently, students explore fluorescence of aqueous solutions at high and low concentrations. The full and diluted range calibration curves are recorded. The determination of concentration of an unknown follows. The result is confirmed through the analysis of a diluted sample. The study of a pH effect on fluorescence intensity completes the experiment. The fluorescence data are acquired by a USB2000 Ocean Optics spectrometer (coupled with OOIBase32 acquisition software) system connected by a 1 mm fiber optic to a cuvette holder. The USB2000 is a very small spectrometer with a linear photodiode array detector with 2048 elements and an operating range from 380 to 1050 nm in our configuration. The sample is excited by a halogen-tungsten white light source (LS-1) channeled by a 1 mm fiber optic from the source to the linear variable filter (LVF), which selects the excitation wavelength, and then to the cuvette holder. In a typical fluorescence measurements arrangement, the detector is positioned at 90° versus the excitation light. The whole system is connected to a laptop computer through a USB port. Ocean Optics systems (5a) are widely used in undergraduate laboratories (5b); however, any hardware and software system able to record emission spectra would be suitable (5c). The photodiode array system does, however, offer a considerable time advantage over scanning systems. Students are provided with fluorescein solutions of the following concentrations: 2000, 200, 20, 2, and 0.2 ppm.

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Vol. 88 No. 2 February 2011 pubs.acs.org/jchemeduc r 2010 American Chemical Society and Division of Chemical Education, Inc. 10.1021/ed1005129 Published on Web 11/19/2010

In the Laboratory

Figure 1. Comparison of the dark current signal acquired without and with the “correct for electrical dark” option with no black cloth covering the optical system.

The 100 mL volumetric flasks with these solutions are kept during the laboratory time in a dark box, which is stored outside lab hours in a refrigerator. Additionally, students are provided with 1 L of a pH 10 buffer and 200 mL of 1% HCl solution, which usually last for few laboratory periods.

Figure 2. Effect of the integration time and the numerical processing technique used on appearance of the acquired signal.

Hazards The fluorescein sodium salt is harmful if inhaled, swallowed, or absorbed through skin. Contact may cause irritation to the skin and eyes. Spills should be washed up with water. Its solutions can be discarded in a drain (6). Hydrochloric acid and sodium hydroxide are corrosive and cause burns to all body tissue. Borax may cause irritation to skin, eyes, and respiratory tract and may be harmful if swallowed or inhaled. Results and Discussion During the first part of the experiment, students explore how the instrument works using a hands-on, step-by-step approach. As the experiment progresses, students are asked questions, which are indicated in the manual by italic numbers in parentheses. Students should answer them as they go along. Numerical results need to be entered in a provided Excel template (see the supporting information). Qualitative observations should be included in written “Results” section. Students should also be able to discuss what their observations mean during an oral exam. To start, the students record a dark current with no light present in the system and learn what the instrument really does when asked to “correct for electrical dark” (Figure 1). It becomes evident that, although the corrected signal drops almost to the baseline, its apparent noise is not reduced. Some pixels, the socalled “hot pixels”, generate much higher signals than their neighbors producing what looks like noise. This effect is not random, but arises from the CCD manufacturing process. Additionally, after the removal of a piece of black cloth covering the optical system, students can clearly see the effect of scattered ambient light present in the laboratory. The next step is to explore the spectrum of visible light with help of a diffuser (a white 1 1 5 cm plastic block with a 2 cm high notch cut in it). The diffuser is inserted into the cell holder so that the light from the light source is reflected by 90° toward the fiber optic going to the detector. Students are asked to move the variable linear filter and record the color and the intensity of the observed light. They can also see the colors on the diffuser surface by looking down into the cell. The students can also observe some radiation in the IR region (Figure 2) caused by the

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Figure 3. Comparison of spectra of a blank and of 2000 ppm fluorescein solution.

inability of the LVF filter to block beyond 750 nm. This is also a good time to study the influence of the integration time and the data processing method on the intensity and the shape of observed peaks (Figure 2). At this time, students are ready to study the molecular fluorescence of aqueous solutions of fluorescein. The first step is to acquire the emission spectrum of a 0.2 ppm solution. Then, by maximizing the intensity of the emission peak by scanning the LVF, students find the optimal excitation wavelength. Both values compare well with literature (7). With the filter's guiding screws tightened (locked) at the excitation wavelength, students acquire the spectrum of a blank (Figure 3), the NaOH/borax buffer of pH 10. In the spectrum, they can clearly observe the Rayleigh scatter of the excitation light. Two Raman bands of water (8) at 531 and 585.5 nm are difficult to discern from the noise of the spectrum. The next step is to obtain a gross calibration curve acquired at the wavelength of the emission maximum of the 0.2 ppm fluorescein solution. The concentration range varies from 0.2 to 2000 ppm. During the acquisition process, students are invited to lift the cover of the cell and observe directly the light path of the excitation beam. The inner filter effect (2a) can be clearly seen starting from the concentration of 20 ppm, but it is spectacular at 2000 ppm (Figures 3 and 4) producing a negative blank corrected emission signal recorded at the standard detection wavelength. By overlaying all the emission spectra, students also discover the phenomenon of spectral shift (2a) (Figure 4).

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In the Laboratory

Figure 4. Fluorescence emission spectra of fluorescein sodium salt solutions in a pH 10 buffer.

Figure 7. Comparison of fluorescein emission intensity at pH = 10 and in a solution acidified with HCl.

The scope of the experiment can be also extended by using the collected data to study the behavior of the signal-to-noise ratio, which is an indicator used in optimization of instruments. Acknowledgment

Figure 5. Gross calibration curve of the fluorescein sodium salt solutions in pH 10 buffer.

The first version of experiment was developed in the advanced instrumental analytical laboratory about five years ago. Many people contributed to its final shape. The authors would like to give special thanks to former teaching assistants Rebecca Lam, Josianne Lafleur, and David Duford and to undergraduate students Sayuri Friedland and Nicole Avakayan for making their reports available for this publication. The authors are grateful to the Ocean Optics Company for the Educational Grant that helped us to finance this project. Literature Cited

Figure 6. Determination of the concentration of an unknown using the linear range of the calibration curve from Figure 5. The limit of detection (LOD) and the limit of quantitation (LOQ) are also marked.

The resulting gross calibration curve (Figure 5) is highly nonlinear and it is clear that the same blank corrected fluorescence signal can correspond to two very different concentrations of an unknown sample. Thus, any determination of unknown concentration should include taking a signal of a diluted sample to confirm that its concentration lies in the linear region of the calibration curve (Figure 6). The final step of the experiment is to verify if the emission intensity of fluorescein solution is pH dependent (Figure 7). The effect is dramatic and can be explained by the extent of protonation of the fluorescein molecule (9). If time permits, one can also study the solvent effect, as described previously (10). 218


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1. Antler, M.; Salin, E.; Wilczek-Vera, G. J. Chem. Educ. 2005, 82, 425–427. 2. (a) Skoog, D. A.; Holler, F. J.; Crouch, S. R. Principles of Instrumental Analysis; Thomson/Brooks/Cole: Canada, 2007. Ingle, J. D. Jr.; Crouch, S. R. Spectrochemical Analysis; Prentice-Hall, Inc.: Englewood Cliffs, NJ, 1988. (b) Russo, S. F. J. Chem. Educ. 1969, 46, 374–377. Rivera-Figueroa, A. M.; Ramazan, K. A.; Finlayson-Pitts, B. J. J. Chem. Educ. 2004, 81, 242–245. Clarke, R. J.; Oprysa, A. J. Chem. Educ. 2004, 81, 705–707. Blitz, J. P.; Sheeran, D. J.; Becker, T. L.; Danielson, N. D. J. Chem. Educ. 2006, 83, 758–760. Cumberbatch, T; Hanley, Q. S. J. Chem. Educ. 2007, 84, 1319–1322. 3. (a) The Nobel Prizes Home Page. http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/press.html (accessed in Nov 2010). (b) Caldwell, B. A. J. Chem. Educ. 2009, 86, 71. 4. (a) Domin, D. S. J. Chem. Educ. 1999, 76, 543–547. (b) Allen, J. B.; Barker, L. N.; Ramsden, J. H. J. Chem. Educ. 1986, 63, 533–534. Chatterjee, S.; Williamson, V. M.; McCann, K.; Peck, M. L. J. Chem. Educ. 2009, 86, 1427–1432. 5. (a) The software and operating instructions for our equipment are available at http://www.oceanoptics.com/technical/softwaredownloads. asp, (accessed Nov 2010) and http://www.oceanoptics.com/technical/usb2000%20Operating%20Instructions.pdf, (accessed Nov 2010). (b) Bernazzani, P.; Paquin, F. J. Chem. Educ. 2001, 78, 796–797. Lorigan, G. A.; Patterson, B. M.; Sommer, A. J.; Danielson, N. D. J. Chem. Educ. 2002, 79, 1264–1266. Goode, S. R.; Metz, L. A. J. Chem. Educ. 2003, 80, 1455–1459. (c) Jones, B. T.; Smith, B. W.; Leong, M. B.; Mignardi, M. A.; Winefordner, J. D. J. Chem. Educ.


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9. Sjoback, R.; Nygren, J.; Kubista, M. Spectrochim. Acta, Part A 1995, 51, L7–L21. 10. Cook, A.; Le, A. The Effect of Solvent and pH on the Fluorescence Excitation and Emission Spectra of Solutions Containing Fluorescein. J. Phys. Chem. Lab 2006, 10, 44-49; http://www.uwlax. edu/faculty/loh/pdf_files/chm313_pdf/JPChemLab/ JPCL_F06_pdf_files/JPChemLab_F06_7_CL.pdf (accessed Nov 2010).

Supporting Information Available A PowerPoint presentation on the experiment, a detailed laboratory manual with instructions, Excel data submission and marking templates with example of students' results as well as some technical details. This material is available via the Internet at http://pubs.acs.org.


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Understanding Fluorescence Measurements through a Guided

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