1. Phosphorescence Quantum Yield Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998. 1

2. Phosphorescence Quantum Yield Product of two factors: - fraction of absorbed photons that undergo intersystem crossing. - fraction of molecules in T 1 that phosphoresce. k nr = non-radiative deactivation of S 1. k’ nr = non-radiative deactivation of T 1. Is phosphorescence possible if k P < k F ? 2

3. Conditions for Phosphorescence Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998. k isc > k F + k ec + k ic + k pd + k d k P > k’ nr 3

4. Luminescence Lifetimes Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998. Emitted Luminescence will decay with time according to: luminescence radiant power at time t luminescence radiant power at time 0 luminescence lifetime ~10 -5 – 10 -8 s ~10 -4 – 10 s 4

5. Fluorescence or Phosphorescence?  –  * transitions are most favorable for fluorescence.   is high (100 – 1000 times greater than n –  *)  k F is also high (absorption and spontaneous emission are related).  Fluorescence lifetime is short (10 -7 – 10 -9 s for  –  * vs. 10 -5 – 10 -7 s for n –  *). 5

6. Luminescence is rare in nonaromatic hydrocarbons. Possible if highly conjugated due to  –  * transitions. Seyhan Ege, Organic Chemistry, D.C. Heath and Company, Lexington, MA, 1989. Nonaromatic Unsaturated Hydrocarbons 6

7. Aromatic Hydrocarbons Ingle and Crouch, Spectrochemical Analysis Low lying  –  * singlet state Fluorescent Phosphorescence is weak because there are no n electrons 7

8. Heterocyclic Aromatics Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998. Aromatics containing carbonyl or heteroatoms are more likely to phosphoresce n –  * promotes intersystem crossing. Fluorescence is often weaker. 8

9. Aromatic Substituents Ingle and Crouch, Spectrochemical Analysis Electron donating groups usually increase  F. Electron donating groups usually increase  F. Electron withdrawing groups usually decrease  F. Electron withdrawing groups usually decrease  F. 9

10. Halogen Substituent Ingle and Crouch, Spectrochemical Analysis Internal Heavy Atom Effect Promotes intersystem crossing.  F decreases as MW increases.  P increases as MW increases.  P decreases as MW increases. 10

11. Increased Conjugation Ingle and Crouch, Spectrochemical Analysis  F increases as conjugation increases.  P decreases as conjugation increases. Hypsochromic effect and bathochromic shift. 11

12. Rigid Planar Structure Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998. Ingle and Crouch, Spectrochemical Analysis  F = 1.0  F = 0.2  F = 0.8 not fluorescent 12

13. Metals Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998. Metals other than certain lanthanides and actinides (with f-f transitions) are usually not themselves fluorescent. A number of organometallic complexes are fluorescent. 13

14. Fluorescence or Phosphorescence? Advantages: Phosphorescence is rarer than fluorescence => Higher selectivity. Phosphorescence is rarer than fluorescence => Higher selectivity. Phosphorescence: Analysis of aromatic compounds in environmental samples. Phosphorescence: Analysis of aromatic compounds in environmental samples.Disadvantages: Long timescale Long timescale Less intensity Less intensity 14 Publications in Analytical Chemistry Fluorescence Phosphorescence 1564 34

15. Solvent Polarity Increasing solvent polarity usually causes a red-shift in fluorescence. http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorescenceintro.html 15

16. Solvent Polarity Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999. 16

17. Temperature Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999. Increasing temperature increases k s 17

18. Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999. Decreasing temperature can induce a blue-shift in fluorescence. 18

19. Shpol’skii Spectroscopy Analytical potential of fluorescence spectroscopy often limited by unresolved band structure (5-50 nm)Analytical potential of fluorescence spectroscopy often limited by unresolved band structure (5-50 nm) homogeneous band broadening – depends directly on radiative deactivation properties of the excited state (usually 10 -3 nm)homogeneous band broadening – depends directly on radiative deactivation properties of the excited state (usually 10 -3 nm) inhomogeneous band broadening – various analyte microenvironments yields continuum of bands (usually few nm)inhomogeneous band broadening – various analyte microenvironments yields continuum of bands (usually few nm) Solution: Incorporate molecules in rigid matrix at low temperature to minimize broadeningSolution: Incorporate molecules in rigid matrix at low temperature to minimize broadening Result: Very narrow luminescence spectra with each band representing different substitution sites in the host crystalline matrixResult: Very narrow luminescence spectra with each band representing different substitution sites in the host crystalline matrix 19

20. Shpol’skii Spectroscopy Requirements: 1.T < 77K with rapid freezing rate 2.Matrix with dimension match 3.Low analyte concentration Instrumentation: 1.Xe lamp excitation 2.Cryogenerator with sample cell 3.High resolution monochromator with PMT Analytes: polycyclic aromatic compounds in environmental, toxicological, or geochemical systems 20 Garrigues and Budzinski, Trends in Analytical Chemistry, 14 (5), 1995, pages 231-239.

21. 21 Garrigues and Budzinski, Trends in Analytical Chemistry, 14 (5), 1995, pages 231-239. Shpol’skii Spectroscopy

22. Luminol Chemoluminescence www.wikipedia.org 22

23. Applications of Luminescence -Luminescence Quenching Sensors FRET -Fluorescence Microscopy Epi-fluorescence Microscopy TIRF PALM 23

24. Quenching Non-radiative energy transfer from excited species to other molecules 24

25. Quantum Yield and Quenching Show that quantum yield in the presence of a quencher is: 25

26. Dynamic Quenching/Collisional Quenching Requires contact between quencher and excited lumophore during collision (temperature and viscosity dependent). Luminescence lifetime drops with increasing quencher concentration. 26 Since fluorescence emission is directly proportional to quantum yield: Stern-Volmer Equation

27. 27 Static Quenching Lumophore in ground state and quencher form dark complex. Luminescence is only observed from unbound lumophore. Luminescence lifetime not affected by static quenching. Dopamine Sensor!

28. 28 Long-Range Quenching/Förster Quenching Result of dipole-dipole coupling between donor (lumophore) and acceptor (quencher). Rate of energy transfer drops with R -6. Used to assess distances in proteins (good for 2-10 nm). Förster/Fluorescence Resonance Energy Transfer Single DNA molecules with molecular Beacons

29. Fluorescence Microscopy Need 3 filters: Exciter Filters Barrier Filters Dichromatic Beamsplitters http://microscope.fsu.edu/primer/techniques/fluorescence/filters.html 29

30. Are you getting the concept? You plan to excite catecholamine with the 406 nm line from a Hg lamp and measure fluorescence emitted at 470 ± 15 nm. Choose the filter cube you would buy to do this. Sketch the transmission profiles for the three optics. http://microscope.fsu.edu/primer/techniques/fluorescence/fluorotable3.html U-MNV 30

31. Fluorescence Microscopy Objectives Image intensity is a function of the objective numerical aperture and magnification: Fabricated with low fluorescence glass/quartz with anti- reflection coatings http://micro.magnet.fsu.edu/primer/techniques/fluorescence/anatomy/fluoromicroanatomy.html 31

32. Fluorescence Microscopy Detectors No spatial resolution required: PMT or photodiode Spatial resolution required: CCD http://micro.magnet.fsu.edu/primer/digitalimaging/digitalimagingdetectors.html 32