Practicum Spectroscopy Fall 2010 Fluorescence spectra of 1-Pyrenebutyric acid, concentration dependence and dynamic quenching phenomena LUM Jorge Ferreiro, study degree Chemistry, 5 th semester, fjorge@student.ethz.ch Alex Lauber, study degree Chemistry, 5 th semester, allauber@student.ethz.ch Assistant: Dr. Xuewen Chen Abstract: Excitation and emission spectra of PBA were investigated. The concentration dependence of fluorescene was studied showing a maximum intesity at a concentration of 0.039 ± 0.04 mm. 2D-spectroscopy proved the independence of λ em on λ ex. Rayleigh and Raman scattering were observed, showing the shift wavelength ν shift = 3800 ± 800 cm 1, which corresponds to the as. st. vibration of water molecules. The dynamic quenching effect of O 2 on PBA was studied in two different matrices (silicone, NaOH-solution). The dynamic quenching constant K D = 34000 ± 2100 M 1 was calucalted with k Q = 2.41 10 13 ± 0.16 10 13 M 1 s 1. Zürich, the 16.11.2010 J. Ferreiro A. Lauber
1 Introduction Luminescene describes the transition of a system from an excited state Φ n to the ground state Φ 0 by emission of light. The two main types of luminescence are fluorescence and phosphorescence. Fluorescence describes the electronic transition S 1 S 0, phosphorescence the transition T 1 S 0. Since the transition in phosporescene is forbidden (total spin conservation), the intensity is much weaker. The electronic states S n are split into different vibronic states S nν. In order to Fig.1 Jablonski-diagram of electronic states in an fictive molecule. By internal conversion the molecule loses the excessive energy in form of heat without electromagentic radiation. Through a non-radiative spinreversal process, the electronic structure changes from the lowest singlet state into a triplet state. After excitation the electron undergoes vibrational relaxation in the excited state before a photon is emitted and the ground state is reset (Kasha s rule). [2] reach the vibration level ν of excited states, a photon of higher energy must be absorbed. Normally in condensed phase vibronic and electronic relaxation occurs much faster than emission and so the transition in fluorescence occurs from S 10 S 0ν with the highest transition from S 10 S 00 (Kasha s rule). After absorption the dipolmoment of the fluorophore changes and only shortly before emission the solvent realignes to lowest energy arrengement, resulting in a bathochromic shifting of the fluoresceneline compared to the absorptionline, known as Stokes shift. The concentration of the fluorophore in a solution has an impact on the intensity of fluorescence. The intensity increases with the amount of molecules in the solution but on the other hand the penetration depth of the beam decreases and only a small amount of molecules will be excitated. The process is described by Lambert-Beer s law: I ex (z) = I ex (0) 10 ɛ Mc M z (1) with I ex (0) and I ex (z) indicating excitation intensities, ɛ M the extinction coefficient, c M the concentration and z the cell width. Highly diluted samples appear to have two main scattering effects: Rayleigh and Raman scattering. The effects are explained by collision of a photon with energy hν 0 and a molecule. While in an 2
elastic Rayleigh-collision the energy of the photon remains constant, in an inelastic Raman-collision it s shifted. The wavenumber-difference does not depend on the irradiated energy. ν rm = ν 0 ν shift (2) ν rm indicates the Raman wavenumber, ν 0 the initial wavenumber. By adding a certain substance to the fluorophore, fluorescnene can be supressed. This process is called quenching. In dynamic quenching the excited fluorophore M is deactivated by diffusion controlled collision with the quencher Q. To determine the dynamic quenching constant K D the rate between the quantum yield without quencher Φ 0 F and the fluoroescene quantum yield Φ F is given by: Φ 0 F Φ F = 1 + τ F,0 k Q c Q = 1 + K D c Q (3) k Q describes the rate constant, c Q the concentration of the quencher and K D the quenching constant, which can be determined with linear regression from equation (4). 3
2 Experimental 2.1 General Measurements were performed on a aminco-bowman ab-2 spectrofluorimeter controlled by the software aminco-bowman ab-2. Weighing was performed onto a ab-mettler toledo. Evaluation was done with the program R. O 2 - concentration was measured with a greisinger gmh 3630 digital oxymeter. For all purposes a PBA parent solution (0.002 M, DMSO) was used. All used substances are listed in the appendix. Confidence intervals are given t-student distributed with 95 % confidence interval. 2.2 Fluorescence and excitation spectra Excitation and emission spectra of a PBA-solution (0.3 mm in 0.01 M NaOH, aq) were acquiered with fixed λ ex (327 nm, 341 nm) and fixed λ em (390 nm, 450 nm). 2.3 Concentration dependence of fluorescence signal Fluorescene spectra as a function of the PBA-concentration (0.3 mm in DMSO) were measured at constant λ ex = 341nm. The PM-voltage was kept at 500 V. All concentrations are listed in Labjournal. 2.4 2D-fluorescence spectrum A 2D fluorescence spectrum of PBA (0.03 mm in 0.01 M NaOH, aq) was measured with λ ex 290-400 nm and λ em 350-450 nm. 2.5 Rayleigh and Raman scattering Using three different λ ex (390 nm, 410 nm, 430 nm) the Rayleigh- and Raman scattering effects were recorded three times each, using 800 V PM-voltage for Rayleigh-scattering and 1200 V PM-voltage for Raman. 2.6 Quenching with O 2 in silicone matrix PBA in a silicone-matrix was measured under different atmospheres (Air, Ar, O 2 ). 4
2.7 Quenching with O 2 in solution PBA-solution (0.02 mm, NaOH, aq) was deoxygenated with Ar and fed with air such that the concentration of O 2 increased. Subsequently ten measurements were performed with different O 2 -concentrations. 5
3 Results 3.1 Fluorescence and excitation spectra 3 Absorption Fluorescence λ ex = 327 nm Fig.2 Emission spectra (λ ex = 327 nm (lines), 341 nm (dots)) and excitation spectra (λ em = 390 nm (lines), 420 nm (dots)): Absorption occurs at lower wavelength compared to emission. I AU 2 1 λ em = 390 nm λ ex = 341 nm λ em = 420 nm 0 250 300 350 400 450 3.2 Concentration dependence of fluorescence λ nm 10 Fig.3 Maximal fluorescence intensity plotted versus different PBA-concentrations: The maximum intensity is achieved at c = 0.039 ± 0.04 mm. In a very diluted region, not enough fluorophores are present while in high concentrated region the beam can t penetrate the surface layer and excites only few molecules. Imax AU 8 6 4 2 0 0 0.039 0.1 0.2 0.3 c PBS mm 6
I/AU 3.3 2D-fluorescence spectrum νex cm 1 34000 32000 30000 28000 26000 28000 26000 24000 ν em cm 1 0.0 0.5 1.0 1.5 2.0 2.5 Fig.4 (Above) Contour plot of 2D-fluorescence spectra: If the excitation wavelength is increased, the emission maximum stays at the same wavelength but the intensity of the fluorescence is altered. (Bottom) The 3D-plot highlights the intensity drop towards lower frequencies. 4 3 2 1 0-26000 -28000-30000 -32000-34000 nu[ex]/cm^-1-28000 -26000 nu[em]/cm^-1-24000 7
3.4 Rayleigh and Raman scattering Fig.5 Rayleigh- and Raman- 10 scattering recorded at different λ ex. The difference between both scattering effects is ν shift = 3800 ± 800 nm, which is independent of the ν ex and belongs to the as. st. vibration of water molecules. The intesities for Raman-scattering are always a much lower than those for Rayleigh-scattering since the cross section for elastic Rayleighscattering is much higher than for inelastic Raman-scattering. I AU 8 6 4 2 0 ν ex = 25600 cm 1 ν ex = 24400 cm 1 ν ex = 23300 cm 1 20000 22000 24000 26000 3.5 Quenching with O 2 in a silicone matrix ν cm 1 1.3 Fig.6 Quenching effect of O 2, Ar and air on fluorescene: For increasing O 2 concentration the fluorescene decreases because the high energetic states of the fluorophore are deactivated by diffusion-controlled collisions leading to alternative realxations and therefore lower intenstities (Ar: 1.3 AU, air: 0.8 AU,O 2 : 0.4 AU). I AU 1.0 0.7 0.4 0.1 Ar Atmosphere Air Atmosphere O 2 Atmosphere 350 375 400 425 450 λ nm 8
3.6 Quenching with O 2 in solution 1.1 1.0 0.9 ΦF Φ F 0 0.8 0.7 0.6 0 0.1 0.2 0.3 0.4 0.5 c O2 Fig.7 Stern-Volmer-plot: (Above) Since the O 2 -concentration in the solution increases fluorescence is supressed diffusion-controlled. (Bottom) From the slope of the linear fit results K D = 34000 ± 2100 M 1 with a fluorescence life time τ 0 F = 139ns according to [3]. The results are summarized in Tab. 2 in the appendix. mm 1.5 1.4 1.3 Φ F 0 Φ F 1.2 1.1 1.0 0.9 0 0.1 0.2 0.3 0.4 0.5 c O2 mm 9
4 Discussion 4.1 Fluorescene and excitationspectra As observed in Fig. 1, emission and excitation spectras are shifted. As mentioned in the introduction part this is due to the slower rearrengement of the solvent molecules compared to the fast alteration of the dipolemoment of the fluorophore which is induced by modification of the electronic structure after absorption/emission. So basically the results show the vibrational relaxation in the excited state, from S 1ν to S 10 before fluorescence occurs. 4.2 Concentration dependence of fluorescence In a very diluted region, not enough fluorophores are present while in high concentrated region the beam can t penetrate the surface layer and excites only few molecules. So the result is a decrasing fluorescence intensity with higher concentrations. 4.3 2D-fluorescence spectrum In 2D-fluorescence spectroscopy results show that the emission maximum remains at the same wavelength at different excitation values. This means that the emission spectrum is independent of excitation wavelengths and thus Kasha s rule is fulfilled. 4.4 Rayleigh and Raman scattering The Rayleigh scattering shows more intense signals than Raman scattering because the cross section for elastic scattering is much higher than for inelastic. The inelastic collision leads to higher wavelengths since a certain amount of the initial kinetic energy of the photon is lost during the collision time as heat. The shift between both scattering effects remains constant independent of λ ex. The observed Raman shift corresponds mainly to the as. st. vibration of water molecules [5]. So in fact, the Raman shift for PBA wasn t really observed. 10
4.5 Quenching with O 2 in a silicone matrix The quenching effect of O 2 in Fig. (6) an proofs the predictions given through theoratical studies that the intensitiy of the fluorescene decreases due to lower fluourescene rates. The intensites are decreased to 30 % in O 2 atmosphere. 4.6 Quenching with O 2 in solution The results aren t good because the decrease of fluorescene doesn t appear as predicted [3]. One reason is the experimental set up. To flush a solution with Ar one should work in a closed experimental set up to prevent air coming into the solution. Nevertheless one sees with increasing O 2 concentation in the solution the quantum yields decrease slightly showing up the quenching effect of oxygen. 5 Literature [1] E. Meister, Grundpraktikum Physikalische Chemie, 2. Auflage, vdf Hochschulverlag an der ETH, Zürich,2006. [2] www.physik.unibas.ch [3] W.M. Vaughan, G. Weber, Oxygen Quenching of Pyrenebutyric Acid Fluorescence in Water. A Dynamic Probe of the Microenvironment, Biochemistry 9 (1970) 464. [4] Fluka Katalog (CH) Riedel de Haen, Sigma-Aldrich, 2007/2008 [5] E. Pretsch; P. Bühlmann; C. Affolter; M. Badertscher, Spektroskopische Daten zur Strukturaufklärung organischer Verbindungen, 4. Auflage, Verlag Springer, Zürich und Minneapolis, 2007 11
6 Appendix 6.1 Used abreviations DMSO Dimethylsulfoxide; PBA 1-Pyrenebutyric acid; λ ex excitation wavelength; λ em emission wavelength; ν ex excitation wavenumber; ν em emission wavenumber; PM photomutlplier; as. st. asymmetric stretch 6.2 Tables Substance Formula M m gmol 1 R-phrases S-phrases Argon Ar 39.9 - - DMSO C 2 H 6 OS 78.12 36,38 26 Oxygen O 2 32.0 8 (2), 17 PBA C 20 H 16 O 2 288.34 36,37,38 26, 37,39 Sodium hydroxide NaOH 40.00 35 (1,2), 26, 37,39, 45 Tab. 1: Substances used in experiments: Chemical formula, Molar mass (M m ) and R- and S- phrases. [4] m O2 /mg c(o 2 )/mm I max /AU 0.184 0.029 3.71 0.396 0.062 3.76 0.622 0.097 3.68 0.824 0.129 3.44 0.996 0.156 3.40 1.192 0.186 3.25 1.466 0.229 3.16 1.662 0.260 3.11 1.734 0.271 2.91 2.914 0.456 2.80 Tab. 2: Measurement of dynamic quenching: The table shows the measured oxygen concentrations with corresponding intensities. 6.3 Program codes 6.4 Labjournal 12