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Fluorescence Study of the Kinetics of Energy Transfer Between Dyes 1. Introduction Coumarin 1 and Sodium fluorescein are two dyes which absorb and emit light in the visible region. By using a spectrophotometer we are recording an absorption spectrum and then determining the molar decade absorption coefficients, which will be used later to interpret and analyse the fluorescence spectra. For fluorescence spectra, there are two kinds, the excitation and the emission spectrum.

The excitation spectrum is obtained by measuring the intensity of the emission as the excitation wavelength is altered by scanning a monochromat or. The emission spectrum is obtained by measuring the emission intensity as a function of wavelength for excitation at a fixed wavelength. An absorption spectrum and an excitation spectrum are by their nature actually equivalent. The two dyes exhibit energy transfer properties.

The donor is coumarin and the acceptor the fluorescein. When the donor is excited, it is naturally decaying to the ground state. However in presence of an acceptor, this process is enhanced trough energy transfer. We will distinguish between collisional energy transfer and dipole-dipole transfer.

From here we will try to determine the quenching constant and the distance at which decay and energy transfer are equally probable, as well as prove that Stern-Volmer's law and F stern theory are obeyed. 2. Results 2. 1. Electronic Absorption Spectra We made up stock solutions for both salts and diluted them down to use in the UV/vis spectrometer. Coumarin 10 - 5 mol dm - 3 We obtained a spectrum with a maximum at 376. 5 nm at an absorbance of 1. 4573, using Beer-Lamberts law, we deduced the molar decade absorption coefficients.

Sodium fluorescein C = 2. 425 x 10 - 5 mol dm - 3 Here max was 500. 5 nm at an absorbance of 2. 0922 Summary of the results obtained. Dye max (nm) max (m 2 mol- 1) (m 2 mol- 1) (m 2 mol- 1) D (Coumarin) 376. 5 1. 82 x 1025 x 106 A (Fluorescein) 500. 5 5. 17 x 107 1. 41 x 106 8 x 106 2. 2. Fluorimetry a) Perylene standard /nm I Emission Spectrum 438 4. 063 EX = 434 nm 467 2. 620 Excitation Spectrum 410 2. 755 EX = 438 nm 437 4. 208 These are the values for the maxima in both spectrum, for the graphs, see attached sheet. b) Coumarin 1 C = 4 x 10 - 6 mol dm - 3 /nm I Emission Spectrum 374 1. 050 EX = 377 nm 443 4. 081 Excitation Spectrum 373 4. 156 EX = 443 nm 446 1. 026 b) Sodium fluorescein C = 1. 212 x 10 - 6 mol dm - 3 /nm I Emission Spectrum 516 7. 453 EX = 501 nm Excitation Spectrum 501 7. 516 EX = 516 nm On the graphs of the standard and the two dyes, we can nicely see that the excitation and emission spectra are mirror images of each other overlaid.

The excitation wavelength in one is the highest emitting one in the other. 2. 3. Energy Transfer Stern-Volmer equation[A] (1) with 0 / being the ratio of quantum yield and K being the Stern-Volmer quenching constant a) Experimental study To test the Stern-Volmer equation, as well as to prove the dipole-dipole transfer, we did fluorimetric measurements with mixtures of different concentrations of D and A. [D] [Arabia 0. 003 0 70. 238 1. 001188 0. 9883 0. 003 4. 042 E- 04 49. 052 1. 002212 1. 4288 0. 003 8. 084 E- 04 38. 766 1. 003237 1. 806 0. 003 1. 213 E- 03 31. 416 1. 004262 2. 2263 0. 003 1. 617 E- 03 25. 816 1. 005285 2. 7064 Table 1: Results from D-A mixtures experiments. with I being the intensity of light emitted m being a geometric correction factor and ratio the ratio of the quantum yields Those values were used to fit the model expression (1), using the following program NonLinear Regression. MODEL PROGRAM K = 1000000.

COMPUTE PRED = 1 +K conc. NLR ratio /OUTFILE = C: win 95 TEMPSPSSFNLR. TMP /PRED PRED /SAVE PRED RESID /CRITERIA SSCONVERGENCE 1 E- 8 PCON 1 E- 8. Non-linear Regression Iteration Residual SS K 1. 0089160488 1000000. 00 1. 1. 0031167306 1034392. 20 2. 0031167306 1034392. 20 2. 1. 0031167306 1034392. 20 Run stopped after 4 model evaluations and 2 derivative evaluations. Iterations have been stopped because the relative difference between successive parameter estimates is at most PCON = 1. 000 E- 08 Nonlinear Regression Summary Statistics Dependent Variable RATIO Source DF Sum of Squares Mean Square Regression 1 18. 55731 18. 55731 Residual 4 3. 116731 E- 03 7. 791826 E- 04 Uncorrected Total 5 18. 56043 (Corrected Total) 4 1. 79521 R squared = 1 Residual SS / Corrected SS = . 99826 Asymptotic 95 % Asymptotic Confidence Interval Parameter Estimate Std. Error Lower Upper K 1034392. 1957 12606. 399781 999391. 21871 1069393. 1726 The crosses are marking the values obtained and the line is the fit based on (1).

So the best fit value for K is 1. 034 &# 215; 106, with at confidence limit of +/- 3. 5 &# 215; 104. So K, the Stern-Volmer quenching constant is 1. 034 &# 215; 103 mol- 1 dm 3. Parent variance 2 ratio = 2. 43 &# 215; 10 - 4. The mean square residual s 2 ratio is 7. 79 &# 215; 10 - 4 Reduced chi-square 2 v = 3. 20. A value under 3 indicates a good fit, hence this shows that our data, is not really fitted onto the model.

b) Energy transfer rate constant For coumarin, = 0. 64 Its intrinsic fluorescence lifetime is 0 s = 1 / (1. 822 &# 215; 108) Therefore knows = 1 / (0 s) = 2. 846 s- 1 and ket = K knows = 2. 943 &# 215; 1011 mol- 1 dm 3 s- 1 This value as an error estimation of +/- 9. 961 x 109 mol- 1 dm 3 s- 1 And as ket >> knife, we can see that 98 % of the total energy transfer is due to dipole-dipole transfer. c) F rate energy transfer [A] 1 / 2 = 9. 67 x 10 - 4 mol dm - 3 Transformation of the raw data through SPSS gives us the following graph. Values obtained: JDA = 6. 897 &# 215; 1031 nm mo 14 nm (R 0) eff = 0. 584 nm based on equation (A 3 - 13) So we see that those are very clearly quite different, and that could show that it does not obey to Forsters theory. 3. Conclusion So we have shown that the quenching of Coumarin 1 by Sodium fluorescein is obeyed by Stern-Volmer kinetics, that there is mainly dipole-dipole transfer, but could not agree with Forsters theory.

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