Molecular Luminescence

1. Molecular Luminescence • Emission of a photon as an excited state molecule returns to a lower state • Chemoluminescence • Bioluminescence • Crystalloluminescence • Electroluminescence • Photoluminescence • Radioluminescence • Sonoluminescence • Thermoluminescence • Triboluminescence http://www.shef.ac.uk/content/1/c6/01/89/68/luminescence.jpg

2. Jablonski Diagram Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

3. Absorption Selection Rules: DJ = 0, 1 Dv = 1, 2, 3, … DS = 0 (i.e. S  S, T  T) Very Fast  10-14 – 10-15 sec. Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

4. Vibrational Relaxation • Excited molecule rapidly transfers excess vibrational energy to the solvent / medium through collisions. • Molecule quickly relaxes into the ground vibrational level in the excited electronic level. • Non-radiative process • 10-11 – 10-10 sec. Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

5. Internal Conversion • Transfers into a lower energy electronic state of the same multiplicity without emission of a photon. • Favored when there is an overlap of the electronic states’ potential energy curves. • Non-radiative process (minimal energy change) • ~10-12 s between excited electronic states. Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

6. Fluorescence • Radiative transition between electronic states with the same multiplicity. • Almost always a progression from the ground vibrational level of the 1st excited electronic state. • 10-10 – 10-6 sec. • Occurs at a lower energy than excitation. Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

7. Stokes Shift Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

8. Relationship between the shape of the excitation and fluorescence bands. Franck-Condon Factor shift Ingle and Crouch, Spectrochemical Analysis P.R. Callis et. al., Chem. Phys. Lett, 244 (1995), 53-58.

9. External Conversion • Non-radiative transition between electronic states involving transfer of energy to other species (solvent, solutes). • Also referred to as quenching. • Modifying conditions to reduce collisions reduces the rate of external conversion. • Occurs on a comparable time scale as fluorescence. Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

10. Intersystem Crossing • Similar to internal conversion except that it occurs between electronic states with different multiplicities. • Slower than internal conversion. • More likely in molecules containing heavy nuclei. • More likely in the presence of paramagnetic compounds. Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

11. Luminol Chemoluminescence www.wikipedia.org

12. Phosphorescence • Radiative transition between electronic states of different multiplicities. • Much slower than fluorescence (10-4 – 104 s). • Even lower energy than fluorescence. www.wikipedia.org

13. Stokes Shift Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

14. Dissociation Ingle and Crouch, Spectrochemical Analysis

15. Predissociation • Occurs if the molecule enters a vibrational level above the dissociation limit during an internal conversion. • Dissociation and predissociation are more likely in molecules that absorb at low l. Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

16. Quantum Yield Fraction of absorbed photons that are converted to luminescence, fluorescence or phosphorescence photons. May approach unity in favorable cases.

17. Fluorescence Quantum Yield All activation and deactivation processes discussed so far can be described using first order rate constants. nS1, nS0 = population densities of S1 and S0. kA = rate of absorption kF = rate of fluorescence knr = rate of non-radiative deactivation processes.

18. A continuously illuminated sample volume (V) will reach steady-state.

19. FA,p = kAnS0V FF,p = kFnS1V typically ~ 106 – 109 s-1 unitless but describes photons/molecule Fluorescence Quantum Efficiency of a Molecule: kec = external conversion (S1 S0) kic = internal conversion (S1 S0) kisc = intersystem crossing (S1 T1) kpd = predissociation kd = dissociation typically 105-107 s-1 typically 106-109 s-1

20. Time Scales of Processes http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorescenceintro.html

21. FF,p = nS0kAfFV Can put in terms of nS0: Proportional to the number of fluorophores, the rate of absorption (i.e. e), the quantum yield and the volume of the sample measured.

22. Are you getting the concept? For a given fluorophore under steady state conditions, excitation of a 1 cm3 sample volume yields the following first-order rate constants: kf = 5 x 107 s-1, knr = 9 x 105 s-1, and ka = 1 x 1014 s-1 and an overall rate of fluorescence photon emission of 9.8 x 1019 photons/second. What is the molecule number density in the ground electronic state?

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

24. Phosphorescence Quantum Yield • Product of two factors: • fraction of absorbed photons that undergo intersystem crossing. • fraction of molecules in T1 that phosphoresce. knr = non-radiative deactivation of S1. k’nr = non-radiative deactivation of T1. Is phosphorescence possible if kP < kF?

25. Conditions for Phosphorescence kisc > kF + kec + kic + kpd + kd kP > k’nr Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

26. Luminescence Lifetimes 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 Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

27. Quenching 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. Dynamic Quenching/Collisional Quenching Requires contact between quencher and excited lumophore during collision (temperature and viscosity dependent). Luminescence lifetime drops with increasing quencher concentration. 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).

28. Fluorescence Resonance Energy Transfer (FRET) http://www.olympusfluoview.com/applications/fretintro.html

29. Are you getting the concept? Determine the type of quenching being demonstrated in the figures below if the fluorescence lifetime of receptor 1 is unchanged with increasing addition of 3. S. Amemiya et al., Chem. Commun.,1997, 1027.

30. Fluorescence or Phosphorescence? p – p* transitions are most favorable for fluorescence. • e is high (100 – 1000 times greater than n – p*) • kF is also high (absorption and spontaneous emission are related). • Fluorescence lifetime is short (10-7 – 10-9 s for p – p* vs. 10-5 – 10-7 s for n – p*).

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

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

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

34. Aromatic Substituents • Electron donating groups usually increase fF. • Electron withdrawing groups usually decrease fF. Ingle and Crouch, Spectrochemical Analysis

35. Halogen Substituents Internal Heavy Atom Effect Promotes intersystem crossing. fF decreases as MW increases. fP increases as MW increases. tP decreases as MW increases. Ingle and Crouch, Spectrochemical Analysis

36. Increased Conjugation fF increases as conjugation increases. fP decreases as conjugation increases. Hypsochromic effect and bathochromic shift. Ingle and Crouch, Spectrochemical Analysis

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

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

39. Fluorescence and Phosphorescence Which effect is used more regularly? SciFinder Scholar Citations Fluorescence Phosphorescence … Labels/Tags1729 25 … Dyes10612 56 www.wikipedia.org

40. Fluorescence or Phosphorescence?Commercially Available Phosphorescence Labels Erythrosin derivative Eosin derivative http://www.invitrogen.com/

41. Fluorescence or Phosphorescence?Publications in Analytical Chemistry • Fluorescence … Phosphorescence… • 1380 106 • Phosphorescence is rarer than fluorescence => Higher selectivity. • Phosphorescence: Analysis of aromatic compounds in environmental samples.