Technology Report pubs.acs.org/jchemeduc
FluSpec: A Simulated Experiment in Fluorescence Spectroscopy Stephen W. Bigger,*,† Andrew S. Bigger,‡ and Kenneth P. Ghiggino§ †
College of Engineering and Science, Victoria University, PO Box 14428, Melbourne 8001, Australia Lonely Planet, PO Box 1, Footscray 3011, Australia § School of Chemistry, The University of Melbourne, Parkville 3010, Australia ‡
S Supporting Information *
ABSTRACT: The FluSpec educational software package is a fully contained tutorial on the technique of fluorescence spectroscopy as well as a simulator on which experiments can be performed. The procedure for each of the experiments is also contained within the package along with example analyses of results that are obtained using the software.
KEYWORDS: Second-Year Undergraduate, Upper-Division Undergraduate, Physical Chemistry, Analytical Chemistry, Computer-Based Learning, Inquiry-Based/Discovery Learning, Fluorescence Spectroscopy and Lab Book. It is supplied with a Supporting Information file that includes all theoretical and experimental material that can be printed from within the software. The Theory section presents a scrollable window containing discussion of the elementary theory pertaining to the study of fluorescence spectroscopy. It provides a systematic overview of topics such as electronic transitions,4,5 singlet and triplet states,6−8 vibrational states and the “zero-point” vibrational level,9,10 the Franck−Condon principle,11−16 Jablonski diagrams,17 the main photophysical processes,18 Kasha’s rule,19 the Stokes shift,17 the “mirror image” rule,17,20 spectrofluorimeters21−23 and the various functional elements of these, fluorescence quantum yield,24,25 analytical fluorimetry,21,22 bimolecular quenching, and the Stern−Volmer equation11,17,21 as well as diffusion controlled quenching.17,23 Among the Theory text are links to information plates that provide further details and Web references on selected aspects. The software enables the user to obtain a printed version of the theory notes as well as any of the information plates. The Experiment section presents a scrollable window containing a series of experiments that can be performed using the simulator contained within the Flu Spec section of the software. The first experiment is an exploratory one where the user is familiarized with the controls of the simulator. This is followed by experiments that illustrate the concepts that are introduced in the Theory section. Step-by-step instructions are provided in the window for each exercise. The software enables the user to obtain a hard copy of the experimental notes if required. Nonetheless, the user can easily switch between the
teady-state fluorescence measurements1,2 are now commonplace in many laboratories and quantitative fluorimetry stands as a most sensitive and selective technique in modern chemical analysis. In order to fully appreciate the applicability of these and many of the other spectroscopic techniques in general, it is important for students to have a sound understanding of the principles of light absorption and emission by molecules.
S
■
SIMULATION SOFTWARE The FluSpec desktop software package incorporates a tutorial on the basics of light absorption and emission by molecules as well as steady-state fluorescence spectroscopy. It also has a fluorimeter simulator that can be used to perform experiments. The software is most suitable for schools that do not have a fluorimeter and instructors who are looking for another option to introduce their students to either basic or more advanced fluorescence techniques and their applications. The educational package provides an ideal grounding in the basics of fluorimetry that are needed before students embark on more advanced applications of fluorescence techniques such as fluorescence anisotropy that has also been treated recently in an analogous educational software package.3 It is anticipated that the more advanced exercises onboard the FluSpec software will be suitable for upper-level undergraduate students in courses such as physical chemistry. Depending on the extent to which the user wishes to explore the options available in FluSpec, it is estimated that the exercises can take between about 30 min (brief exploration) to about 90 min to complete (more complete exploration with multiple spectra run, etc.). The software incorporates four sections that are accessed by tabs appearing on the user interface: Theory, Experiment, Flu Spec, © 2014 American Chemical Society and Division of Chemical Education, Inc.
Published: June 3, 2014 1081
dx.doi.org/10.1021/ed400748d | J. Chem. Educ. 2014, 91, 1081−1083
Journal of Chemical Education
Technology Report
Figure 1. User interface of the FluSpec spectrofluorimeter simulator.
plotting or further analysis. The data in the clipboard can also be captured and filed in the form of a tab-delimited ASCII text file that may later be read into a suitable spreadsheet program if the user desires. An image of the simulator screen along with its currently displayed spectra can also be printed if required. As the experimental data are captured and data files are created, a summary of the data files is compiled in the Lab Book section of the simulator. This allows the user to keep track of the files that are created during the course of running the various experiments. The Lab Book also contains access to model data obtained from each of the experiments that are displayed along with details of their corresponding analyses. The file summary data in the Lab Book as well as the processed model data screens can be printed if required. The FluSpec software is provided as a standalone application. Versions exist that run on standard Macintosh OSX or Windows environments. The software should be run in conjunction with a standard spreadsheet program for the plotting and labeling of the processed output data. This approach to handling the output data provides maximum flexibility with regard to its presentation and collation. The software comes with associated content, the reading of which prior to using the simulator is highly recommended in order for the student to gain maximum benefit from the educational package.
screen instructions and the experimental simulation without interruption to the continuity of the experiment. The Flu Spec section contains a graphic showing the functional elements of a standard laboratory fluorimeter along with a window that displays the spectra as these are recorded in accordance with the chosen settings of the simulator (see Figure 1). The simulator can be set to run the fluorescence emission or fluorescence excitation spectra of either Rhodamine B (RhB) or the fluorescence standard quinine bisulfate (QBS) under a variety of instrumental or experimental conditions. For example: (i) fluorescence excitation and emission spectra can be recorded and superimposed to illustrate the Stokes Shift17 and the “mirror image” rule,17,20 (ii) the RhB and QBS emission spectra can be used to calculate the fluorescence quantum yield24,25 of RhB, (iii) the concentration of RhB can be varied to illustrate the proportionality between the fluorescence intensity and the concentration of a fluorescent analyte at low concentrations,21,22 and (iv) different concentrations of a quencher (NaCl) can be added to the QBS solution to illustrate the Stern−Volmer equation.11,17,21 When spectra are being recorded, the simulator shows the status of the emission and excitation monochromators along with the light and signal paths within the instrument. The fluorescence “glow” of the sample is also displayed as well as its variation with excitation wavelength. For wavelengths in the visible region, the respective colors of the light beams are also shown and are varied as the wavelengths are scanned or changed. A scan guide lists recommended instrumental settings for running the spectra in order to assist students while they are becoming more familiar with the simulator. A cursor is available for identifying wavelengths of maximum emission and other spectral features. After a scan is completed all relevant information about the sample and the current instrumental settings are transferred to the Clipboard along with the wavelength-intensity data that constitute the recorded spectrum. This enables the data to be immediately transferred to a suitable spreadsheet program for
■
SYSTEMS It is anticipated the software will successfully run on a wide range of Macintosh and PC systems. The following systems are examples of ones that have been successfully trialed: Macintosh
• iMac 2.66 GHz Intel Core i5; Mac OS X 10.6.8 (Leopard); Fuji Xerox ApeosPort IV C5570 network printer. • Macbook 2.16 GHz Intel Core 2 Duo; Mac OSX 10.7.2 (Lion); HP Deskjet F4400 Series and Apple PDF printer. 1082
dx.doi.org/10.1021/ed400748d | J. Chem. Educ. 2014, 91, 1081−1083
Journal of Chemical Education
Technology Report
PC Systems
■
(9) Laidler, K. J. The World of Physical Chemistry; Oxford University Press: Oxford, U. K., 2001; p 324. (10) Einstein, A.; Stern, O. Einige Argumente für die Annahme einer molekularen Agitation beim absoluten Nullpunkt. Annalen der Physik 1913, 40 (3), 551−560. (11) Porter, G. B. Introduction to Inorganic Photochemistry: Principles and Methods. J. Chem. Educ. 1983, 60 (10), 785−790. (12) Atkins, P. W. Physical Chemistry, 5th ed.; Oxford University Press: Oxford, U. K., 1994; pp 592−594. (13) Franck, J.; Dymond, E. G. Elementary Processes of Photochemical Reactions. Trans. Faraday Soc. 1926, 21, 536−542. (14) Condon, E. A Theory of Intensity Distribution in Band Systems. Phys. Rev. 1926, 28, 1182−1201. (15) Condon, E. Nuclear Motions Associated with Electron Transitions in Diatomic Molecules. Phys. Rev. 1928, 32, 858−872. (16) Schwartz, S. E. The Franck−Condon Principle and the Duration of Electronic Transitions. J. Chem. Educ. 1973, 50 (9), 608−610. (17) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006; Vol. 1, pp 1−60. (18) Jaffe, H. H.; Miller, A. L. The Fates of Electronic Excitation Energy. J. Chem. Educ. 1966, 43 (9), 469−473. (19) Kasha, M. Characterization of Electronic Transitions in Complex Molecules. Discuss. Faraday Soc. 1950, 9, 14−19. (20) Byron, C. M.; Werner, T. C. Experiments in Synchronous Fluorescence Spectroscopy for the Undergraduate Instrumental Chemistry Course. J. Chem. Educ. 1991, 68, 433−436. (21) Harris, D. C. Quantitative Chemical Analysis, 6th ed.; W. H. Freeman: New York, 2003; p 424. (22) Skoog, D. A.; West, D. M.; Holler, F. J.; Crouch, S. R. Fundamentals of Analytical Chemistry, 8th ed.; Brooks-Cole: Independence, KY, 2004; pp 825−835. (23) Bigger, S. W.; Craig, R. A.; Ghiggino, K. P.; Scheirs, J. Fluorescence Anisotropy Measurements in Undergraduate Teaching. J. Chem. Educ. 1993, 70, A234−239. (24) Valeur, B.; Berberan-Santos, M. Molecular Fluorescence: Principles and Applications, 2nd ed.; Wiley-VCH: Weinheim, 2012; p 64. (25) Brouwer, A. M. Standards for Photoluminescence Quantum Yield Measurements in Solution. Pure Appl. Chem. 2011, 83, 2213−2228.
• Macbook 2.1.6 GHz Intel Core 2 Duo; Windows 7 Professional; HP Deskjet F4400 Series and Cute PDF Printer. • Windows XP SP2 Virtual Machine running on Macbook 2.16 GHz Intel Core 2 Duo; HP Deskjet F4400 Series and Cute PDF Printer. • Lenovo ESA4C406−86, Intel Core 2 Duo CPU, E8400 @ 3.00 GHz, 1.97 GHz, 3.25 GB of RAM, Microsoft Windows XP Professional Version 2002 Service Pack 3; Xerox Phaser 6360 Printer, Model P6360DT.
AVAILABILITY FluSpec is available on the Web, distributed through Github under the GPL license at http://stephenbigger.github.io/ FluSpec. The Github page also accepts bug reports and feature requests, and updates will be posted at this location. The core of FluSpec is written in LiveCode.
■
ASSOCIATED CONTENT
S Supporting Information *
FluSpec educational software package; all printable documentation contained within the simulator; theory notes; instructions for experiments; questions and exercises pertaining to the experiments; diagrams and information plates; typical results; derivations of equations. This material is available via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors are grateful to Dr. Efrat Eilam, College of Education, Victoria University, for her expert advice on the pedagogical approach used in this work and her assistance in proofing the Windows version of the software.
■
REFERENCES
(1) Bigger, S. W.; Ghiggino, K. P.; Meilak, G. A.; Verity, B. Illustration of the Principles of Fluorimetry. An Apparatus and Experiments Specially Designed for the Teaching Laboratory. J. Chem. Educ. 1992, 69, 675−677. (2) Bigger, S. W.; Watkins, P. J.; Verity, B. A Fluorometric Approach to Studying the Effects of Ionic Strength on Reaction Rates: An Undergraduate Steady-State Fluorescence Laboratory Experiment. J. Chem. Educ. 2003, 80, 1191−1193. (3) Bigger, S. W.; Bigger, A. S. FluAnisot: A Simulated Experiment in Fluorescence Anisotropy Measurements. J. Chem. Educ. 2013, 90, 386− 387. (4) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of Organic Compounds, 5th ed.; Wiley: New York, 1991; pp 289−294. (5) Crouch, S.; Skoog, D. A. Principles of Instrumental Analysis; Thomson Brooks/Cole: Australia, 2007; pp 335−398. (6) Griffiths, D. J. Introduction to Quantum Mechanics; Prentice-Hall: New York, 1995; p 165. (7) McQuarrie, D. A.; Simon, J. D. Physical Chemistry, A Molecular Approach; University Science Books: Sausalito, CA, 1997; pp 296−355. (8) Turro, N. J. The Triplet State. J. Chem. Educ. 1969, 46 (1), 2−6. 1083
dx.doi.org/10.1021/ed400748d | J. Chem. Educ. 2014, 91, 1081−1083
