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Principles of Fluorescence Spectroscopy

Principles of Fluorescence Spectroscopy. Chemistry Department XMU. Chapter Four. Factors Influencing Fluorescent Emission. Factors Influencing Fluorescent Emission. 4.1 Effect of Solvent 4.2 Effect of Temperature 4.3 Effect of pH 4.4 Effect of Hydrogen bond

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Principles of Fluorescence Spectroscopy

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  1. XMUGXQ PFS0401 Principles of Fluorescence Spectroscopy Chemistry Department XMU

  2. XMUGXQ PFS0401 Chapter Four Factors Influencing Fluorescent Emission

  3. XMUGXQ PFS0401 Factors Influencing Fluorescent Emission 4.1 Effect of Solvent 4.2 Effect of Temperature 4.3 Effect of pH 4.4 Effect of Hydrogen bond 4.5 Effect of Heavy atom 4.6 Effect of Surfactant

  4. XMUGXQ PFS0401 4.1 Effect of Solvent The phenomena of solvent effect • Shifting Emission Wavelengths • Changing quantum yield • Changing anisotropy • Changing fluorescence lifetime

  5. XMUGXQ PFS0401 Shifting emission wavelengths 6-propionyl-2-(dimethylamino)naphthalene 6-丙酰基-2-（二甲基氨基）萘 solvent Water Ethanol Dimethylformamide Chlorobenzene cyclohexane

  6. XMUGXQ PFS0401 Large change in dipole moment

  7. XMUGXQ PFS0401 对甲基苯胺萘磺酸 水中弱荧光，500 nm; 疏水环境强荧光，413 nm

  8. XMUGXQ PFS0401 Indole 色氨酸发光基团 吲哚 • Hexane • 2. 0.7% n-butanol • 3. 5% n-butanol • 4. 100% butanol • 5. Water

  9. XMUGXQ PFS0401 Changing quantum yield F In water 0.002 Banding protein 0.4 1,8 - ANS F In water nonfluorescence Banding protein intensive fluorescnece TNS

  10. XMUGXQ PFS0401 Changing anisotropy

  11. XMUGXQ PFS0401 General and Specific Solvent effects General solvent effect  dielectric constant Solvent properties n refractive index Reflect the freedom of motion of the electrons in the solvent molecules, and the dipole moment of these molecules. Specific solvent effect Specific chemical interaction Hydrogen bonding Solute properties as well as solvent properties Complexation Charge transfer Acid-base reaction

  12. XMUGXQ PFS0401 Relaxation b c 0-0 hvF 0-0 hvA d Relaxation a General solvent effect Franck-Condon principle Solvent relaxation * 

  13. XMUGXQ PFS0401 The Lipper equation Stoke’s shift

  14. XMUGXQ PFS0401 The Lipper equation a radius of cavity in which the fluorophore reside *,  dipole moment of ground state and excited states, reapectively

  15. XMUGXQ PFS0401 * >  Solvent relaxation f (n) f f () Gas phase In solution The Lipper equation Orientation polarizability How about  = n2 ?

  16. XMUGXQ PFS0401 Shifting of emission wavelength

  17. XMUGXQ PFS0401 Example 1 Assume * -  = 20 Debye A unit-charge separation of 4.2 Å Example 2 ex = 350 nm Assume * -  = 20 Debye In nonpolar solution, observed Why?

  18. XMUGXQ PFS0401 Derivation of Lipper Equation

  19. XMUGXQ PFS0401 *   Correction to Lipper equation

  20. XMUGXQ PFS0401 Application of Lipper Equation For a given fluorophore

  21. XMUGXQ PFS0401 Determination of dipole moment of excited state For a given solvent, measure the dipole moment

  22. XMUGXQ PFS0401 Specific solvent effect General solvent effect: the effect of the properties of solvent on the emitting behavior of fluorophore. Electronic polarizability, molecular polarizability Specific solvent effect: changing to an new species that fluoresces differently, duo to the reaction between fluorophore and the solvent molecule. Discrimination • Small change of solvent constituent could cause large shift of emission wavelength. • Spectrum shape, not only emission wavelength, change • Not follow the lipper equation

  23. XMUGXQ PFS0401 Spectrum change

  24. XMUGXQ PFS0401 Spectrum change Comparison

  25. XMUGXQ PFS0401 Large stoke’s shift

  26. XMUGXQ PFS0401 Disobey the lipper equation

  27. XMUGXQ PFS0401 Disobey the lipper equation

  28. XMUGXQ PFS0401 F  The dynamic process of the solvent molecule reorientation Temperature Viscosity

  29. XMUGXQ PFS0401 4.2 Effect of temperature • Effect on quantum yield • Effect on lifetime • Effect on emission wavelength • Effect on anisotropy • Effect on structural detail of spectrum

  30. XMUGXQ PFS0401 hvA hvF S1 relaxation (10-12 s) S1  knr S0 Effect on quantum yield The process of single molecule

  31. XMUGXQ PFS0401 E S1 S0 B A S0 r Effect of non-radiation deactive F0 fluorescence at T1 F fluorescence at T2 E energy needed for transfer from A to B (4~7 Kcal / mL) IC process

  32. XMUGXQ PFS0401 Example

  33. XMUGXQ PFS0401 hvA hvF Q S1 relaxation (10-12 s) kq[Q] S1 Q  knr S0 Effect on quantum yield The process of two molecules

  34. XMUGXQ PFS0401 S1 relaxation (10-12 s) S1 hvA hvF  knr S0 Effect on lifetime The process of single molecule

  35. XMUGXQ PFS0401 hvA hvF Q S1 relaxation (10-12 s) kq[Q] S1 Q  knr S0 Effect on lifetime The process of two molecules

  36. XMUGXQ PFS0401 Effect on emission wavelength

  37. XMUGXQ PFS0401 Effect on anisotropy

  38. XMUGXQ PFS0401 Effect on the structural detail of spectrum

  39. XMUGXQ PFS0401 F F  F pH F pH pH Effect of pH • Acid-base reaction of ground state fluorophore Difference in fluorescent characteristics between conjugate acid and base em, HA em, A em, HA Non-fluorescent em, A Non-fluorescent

  40. XMUGXQ PFS0401 Effect on the composition of fluorophore Changing pH may change the composition of metal-ligand compound

  41. XMUGXQ PFS0401 F A pH pH * pKa * * * pKa Acid-base reaction of excited state fluorophore pKa* = pKa pKa pKa HA +  A + hv Radiating takes place prior to acid-base reaction Non-fluorescent em,A

  42. XMUGXQ PFS0401 A pH * F pKa * * * pKa pH Acid-base reaction of excited state fluorophore pKa* < pKa pKa pKa HA +  A + hv Acid-base reaction finished before radiating Non-fluorescent em,A

  43. XMUGXQ PFS0401 Example

  44. XMUGXQ PFS0401 A pH * F pKa * * * pKa pH Acid-base reaction of excited state fluorophore pKa* > pKa pKa pKa HA +  A + hv Acid-base reaction finished before radiating Non-fluorescent em,A

  45. XMUGXQ PFS0401 Excited-state intra-molecule proton transfer 水杨酸酯

  46. XMUGXQ PFS0401 * Hydrogen bond n  Blue shift absorption Effect of hydrogen bond Ground state: changing absorption as well as emission spectrum Excited state: changing emission spectrum • Effect on n→* transition

  47. XMUGXQ PFS0401 Hydrogen bond Solvent relaxation * n Blue shift  Intensify solvent effect Red shift emission Effect on *→n transition

  48. XMUGXQ PFS0401 Changing the type of low-energy transition Blue shift Changing the transition type Intensify emission

  49. XMUGXQ PFS0401 Effect on quantum yield Generally, intensity IC, decrease quantum yield When transition-type changing occurs, intensity emission • Intra-molecular hydrogen bond Intensity IC, F is 100 times lower than that of 5-hydroxyl-quinoline Almost same absorption Why?

  50. XMUGXQ PFS0401 Effect of heavy atom Intra-molecule In the solvent

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