Flourescence
- 3. CONTENTS : History Introduction Types of fluorescence Quenching of fluorescence
- 4. The term fluorescence comes from the mineral fluorspar (calcium fluoride) when Sir George G. Stokes observed in 1852 that fluorspar would give off visible light (fluoresce) when exposed to electromagnetic radiation in the ultraviolet wavelength. History :
- 5. Introduction: Luminescence is the emission of light by a substance. It occurs when an electron returns to the electronic ground state from an excited state and loses its excess energy as a photon. It is of 3 types. Fluorescence spectroscopy. Phosphorescence spectroscopy. Chemiluminescence spectroscopy
- 6. Principle of fluorescence : The electronic states of most organic molecules can be divided into singlet states and triplet states : • Singlet ground state : All electrons in the molecule are spin-paired • Singlet excited state : Unpaired electrons of opposite spin present • Triplet state : Unpaired electrons of same spin present 6
- 8. Principle of fluorescence : Energy of emitted radiation is less than that of absorbed radiation because a part of energy is lost due to vibrational or collisional processes. Hence the emitted radiation has longer wavelength (less energy) than the absorbed radiation. Vibrational deactivation takes place through intermolecular collisions at a time scale of 10 -12 s (faster than that of fluorescence process) . 8
- 9. As electronic energy increases, the energy levels grow more closely spaced.It is more likely that there will be overlap between the high vibrational energy levels of S n-1 and low vibrational energy levels of S n.This overlap makes transition between states highly probable. Internal conversion is a transition occurring between states of the same multiplicity and it takes place at a time scale of 10 -¹² (faster than that of fluorescence process). The energy gap between S ₁ and S ₀ is significantly larger than that between other adjacent states → S ₁ lifetime is longer → radiative emission can compete effectively with non-radiative emission. Internal conversion : 9
- 10. Stokes shift Principle of fluorescence : The difference btw the max wavelength of the excitation light and the max wavelength of the emitted fluorescence lights is a constant – stokes shift 10
- 11. Mirror image rule : Vibrational levels in the excited states and ground states are similar. An absorption spectrum reflects the vibrational levels of the electronically excited state. An emission spectrum reflects the vibrational levels of the electronic ground state. Fluorescence emission spectrum is mirror image of absorption spectrum Principle of fluorescence : 11
- 12. Mirror-image rule typically applies when only S₀ → S₁ excitation takes place Deviations from the mirror-image rule are observed when S₀ → S₂ or transitions to even higher excited states also take place 12
- 13. Types of fluorescence : 1. Stoke’s fluorescence: The wavelength of emitted radiation is longer than the Absorbed radiation e.g . Conventional fluorimetric experiments. 2. Anti-stock’s fluorescence: The wavelength of emitted radiation is shorter than the Absorbed radiation e.g. Thermally assisted fluorescence. 3. Resonance fluorescence: When the wavelength of emitted radiation is equal to the Absorbed radiation e.g. Mercury vapour at 254 nm. 13 A) Based upon the wavelength of emitted radiation when compared to absorbed radiation : 3 1 2
- 14. 14 B) Based upon the phenomenon: 1) Prompt fluorescence: 2) Delayed fluorescence: Delayed Fluorescencere are delayed emissions whose spectra coincide exactly with the prompt fluorescence from the lowest singlet state, the only difference is in their life time. Types of fluorescence :
- 15. Two most important types of delayed fluorescence are: • E-type delayed fluorescence • P-type delayed fluorescence Delayed fluorescence : 15
- 18. Exception from kasha’s rule Azulene exhibiting S₂-fluorescence in violation of kasha’s rule : 18
- 19. Self Quenching/ Concentration Quenching: Fluorescence Concentration of fluorescing species Fluorescence Concentration of fluorescing species Calibration curve (Low con) calibration curve (High con) 20
- 20. Rigidity of structure The fluorescence intensity of 8-hydroxyquinoline is much less than that of the zinc complex. 21
- 21. • 22 Solvent effect on fluorescence spectra of a spirooxazine
- 22. 1. Matenova, M.; Horhoiu, V. L.; Dang, F. L.; Alster, J.; Burda, J.V.; Balaban, T.S, Phys. Chem. Chem. Phys. 2014, 31, 16755- 16764. 2. Beer, M.; Longuet-Higgins, H. C, J. Chem. Phys. 1955, 23, 1390– 1391. 3. Soroka, K.; Vithanage, R.S.; Phillips, D. A.; Walker, B.; Dasgupta, .K, Anal. Chem. 1987, 59, 629-636. 4. Química-Física, C.D, Phys. Chem. Comm. 2000, 5, 18-23. 5. Handbook of Fluorescence Spectroscopy and Imaging. M. Sauer, J. Hofkens, and J. Enderlein 6. Nishkiori, H.; Takagi, K.; Fujii, T, Res. Chem. Internal. 2003, 29, 485-493. Refrences 23
- 24. Intrinsic structure of a molecule Environment of a molecule 1 2 1) Nature of molecule (conjugation) 2) Nature of substituent group 3) Rigidity of structure 1) Temperature 2) Viscosity 3) Oxygen 4) Effect of pH Factors influencing fluorescence intensity :