In the Laboratory
An Easy and Inexpensive Flash Spectroscopy Experiment Mauro Maestri* Dipartimento di Chimica “G. Ciamician” dell’Università, 40126, Bologna, Italy Roberto Ballardini Istituto F.R.A.E.-C.N.R., 40129, Bologna, Italy Fernando Pina and Maria João Melo Centro de Quimica Fina e Biotecnologia e Departamento de Quimica, FCT-Universitade Nova de Lisboa, Lisboa, Portugal
Experimental Procedure The compound used in this experiment was the 4′,7-dihydroxyflavylium cation (DHF) as a perchlorate salt. It can be prepared easily following the literature methods (1). Water solutions of DHF (5 × 10{5 M) in acetate buffer (10{2 M) were used. To monitor the transient species we used a PerkinElmer Lambda6 spectrophotometer with a slightly modified sample compartment. The changes were as follows: (i) we opened a slit (5 mm wide and 20 mm high) on the external side of the sample holder in order to perform light excitation perpendicular to the analyzing beam; (ii) we shielded the whole sample compartment (except for the “new” slit described above) with black cardboard and black tape, to reduce as much as possible the flash light entering the exit slits. As a pulsed light source we used a commercially available Philips 38CT camera flash (like those commonly used by cameramen for flashlight photos), placed in close contact with the “new” slit opened in the sample holder. First we acquired an absorption spectrum of a thermally dark-equilibrated DHF solution at pH = 5 (Fig. 1). Then we started a time drive scan at 240 nm, setting the instrumental parameters as follows: response 0, interval time 0.1 s, 60 s total time. After 3 s the flash was triggered and the resulting spectral changes were monitored. Identical time drive scans were then performed at wavelengths up to 540 nm, changed in 20-nm steps. To increase the resolution in some important wavelength ranges, time drive scans were also run at 270, 390, and 450 nm. The absorbance changes vs. time at these wavelengths are reported in Figure 2. These illustrations clearly show at least two different decay processes: a faster one, completed in 10–15 s, and a slower one that continues for minutes. *Corresponding author.
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The second decay process is so slow that it was possible to scan directly, by means of the spectrophotometer, a differential absorption spectrum (for this system a transient spectrum) starting 10 s after the flash. This was done by exciting the solution outside the sample compartment (to increase the number of excited molecules), placing it in the sample holder as quickly as possible, and running a wavelength scan at 1440 nm/min (the faster scan speed allowed by the instrument) using a nonflashed DHF solution as a reference. To monitor the kinetics of the slow process, we ran additional scans over a longer time span at 270, 370, and 450 nm, using the following instrumental parameters: response 0 (or 1), interval time 3 s, 900 s total time. The absorbance changes at 450 nm in this time range are reported in Figure 3b. For these scans the solutions were again excited outside the sample compartment and immediately placed in the sample holder while the time drive was running. Note that all scans were performed on fresh solutions (flashed solutions were changed before restarting a time drive or a wavelength scan) placed in a spectrofluorometric cell. Results Time drive scans (run in the wavelength range 220– 540 nm) gave a series of absorbance traces like those reported in Figure 2. The difference between the initial absorbance (zero delay time) and the absorbance at different delay times from the flash (0.5, 1, 3, and 10 s) plotted vs. the wavelength of the corresponding traces gave the transient absorption spectra reported in Figure 4a (time-resolved spectra). The transient spectrum obtained in this way at 10 s delay is also reported in Figure 4b together with the differential spectrum instrumentally obtained (scanned) starting 10 s after the flash. The decay traces obtained at 270, 390, and 450 nm
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A
Flash spectroscopy is widely used in chemistry to monitor the formation and kinetics of transient species in the picosecond to millisecond time domain. Nevertheless, it has been neglected in university curricula owing to the high cost of a flash spectroscopy apparatus. While the cost of a flash spectroscopy system goes up rapidly with increasing time resolution, the principle of the technique is independent of time domain. With this in mind we developed an experiment suitable for introducing, in a chemical laboratory course, the concepts of transient decay kinetics and time-resolved spectra in an inexpensive and easy way. We suggest here a chemical system in which a polychromatic light flash forms transient species with lifetimes from subseconds to seconds. Their kinetics can be followed by means of a relatively simple computer-interfaced spectrophotometer capable of acquiring 10 or 5 points per second.
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Wavelength (nm) Figure 1. Absorption spectrum of an aqueous solution of 4′,7dihydroxyflavilium cation at pH = 5.
Journal of Chemical Education • Vol. 74 No. 11 November 1997
In the Laboratory (Figs. 2 and 3) were treated with the classical equation for first-order kinetics (2) and gave good straight lines.
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ln (A∞ – A) = ln (A∞ – A0 ) + k t
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A0 , A∞ , and A are the initial, final, and actual absorbance, respectively. The rate constant value obtained for the faster process, kf = 0.32 s {1, was found to be the same at the three monitored wavelengths. Identical values of the decay constant were also obtained for the slower process (ks = 1.8 × 10{3 s{1) regardless of the observed wavelengths. In Figure 3 are reported the decays (and linearizations) of the two processes monitored at 450 nm.
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time (s) Figure 2. Absorbance changes, at three wavelengths, of an aqueous solution of 4′,7-dihydroxyflavilium cation (pH = 5) upon flash excitation.
From the traces of Figures 2 and 3 it is clear that at least three transient species are formed after irradiation of the DHF solution at pH = 5. The first species is the singlet excited state of the reactant (A*), formed as a consequence of light absorption, that lives a few nanoseconds (Pina, F.; Maestri, M., unpublished results). Nothing can be said about what happens during the flash, but it is clear that at the end of the light flash a second species (B) is formed. B decays in about 10 s (kf = 0.32 s{1 ) to a third species (C), which in turn decays in minutes (ks = 1.7 × 10{3 s{1 ) to D. Since the absorption spectrum 30 min after the flash is almost identical to the initial spectrum as observed under steady-state irradiation (3), it is evident that the species we have labeled as D is the reactant A itself. Thus the light flash has the effect of rapidly changing the equilibrium concentrations of the system, and the species B and C are formed while the system is going back to the initial situation. This is shown in Scheme I: hv
kx
kf
ks
A → A* → B → C → A Scheme I
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0.12 y = m + k*x k = 0.32 s-1 R = 0.996
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ln((Af-Ai)/(Af-A))
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Time-resolved spectra of Figure 4a show that species B possesses, with respect to A, an extinction coefficient larger in the wavelength range 220–300 nm, lower between 300 and 400 nm and almost identical in the range 420–550 nm. Species C differs from B since its extinction coefficient is larger than that of A even in the range 420–550 nm. The explanation of the behavior of the 4′,7-dihydroxyflavylium salt upon flash irradiation could be found in the existing knowledge on this system. In moderately acidic solutions, synthetic flavylium salt undergoes structural transformations (4), like anthocyanins, as depicted in Scheme II:
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Figure 3. Absorbance changes (and kinetic linearization [see text]) at 450 nm of an aqueous solution of 4′,7-dihydroxyflavilium cation (pH = 5) upon flash excitation. a: Fast process. b: Slow process.
3
Scheme II
Vol. 74 No. 11 November 1997 • Journal of Chemical Education
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In the Laboratory The displacement of this equilibrium and the concentration of the various species are strongly dependent on pH. Recently we also proved (3) the existence of a photochromic process that leads to the formation of 4′,7-dihydroxyflavylium cation (4) upon irradiation of trans-chalcone (1); the reaction is (thermally) reversible in the dark. The kinetic schemes I and II could be correlated as follows. A corresponds to 1, B to 3, and C to 4. Thus flash irradiation of 1 leads first (within the flash) to the formation of 3, which decays in 10 s to 4. (Note that 4 is the steady-state irradiation product.) In a slower step (minutes), 4 decays back to 1, closing the cycle. The transient absorption spectrum of Figure 4b strongly supports this explanation, since it corresponds to the difference between the spectra of 1 and 4. A more detailed discussion can be found in reference 5.
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Conclusion
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The proposed system permits students of all levels to become familiar with the concepts of transient decay kinetics and time-resolved spectra by means of a simple and economic experiment.
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Acknowledgments
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This work was supported by JNICT Program STRDA/ CEN/438/92 (Portugal) and by CNR and MURST (Italy).
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wavelength (nm) Figure 4. Time-resolved spectra. a: Transient absorption spectra at different delay times, s = 0.5 s, u = 1 s; d = 3 s, j = 10 s. b: Comparison between the transient absorption spectrum at 10 s after the flash and a differential spectrum instrumentally scanned 10 s after the flash.
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Literature Cited 1. Michaelis, C.; Wizinger, R. Helv. Chim. Acta 1951, 34, 1761. 2. Frost A. A.; Pearson, R. G. Kinetics and Mechanism, 2nd ed.; Wiley: New York, 1961. 3. Figereido P.; Lima, J. C.; Santos, H.; Wigand, M. C.; Brouillard, R.; Macanita, A. L.; Pina, F. J. Am. Chem. Soc. 1994, 116, 1249. 4. Brouillard, R.; Dubois, J. E. J. Am. Chem. Soc. 1977, 99, 1359; Brouillard, R.; Delaporte, B.; J. Am. Chem. Soc. 1977, 99, 8461. 5. Pine, F.; Melo, M. J.; Bellardini, R.; Flamigini, L.; Maestri, M. New J. Chem. 1997, 21, in press.
Journal of Chemical Education • Vol. 74 No. 11 November 1997
