Principles of Fluorescence Spectroscopy

1. XMUGXQ PFS0501 Principles of Fluorescence Spectroscopy Chemistry Department XMU

2. XMUGXQ PFS0501 Chapter Five Quenching of Fluorescence

3. XMUGXQ PFS0501 Quenching of fluorescence 5.1 Introduction 5.2 Stern-Volmer equation 5.3 Modified Stern-Volmer equation 5.4 factors influencing quenching 5.5 quenching mechanisms 5.6 Application

4. XMUGXQ PFS0501 5.1 introduction • Fluorescence Quenching Any processes decreasing the fluorescence intensity Excited-state reactions Molecular rearrangements Ground-state complex formation Collision • Quencher Any species causing the decrease in the fluorescence General quencher Specific quencher

5. XMUGXQ PFS0501 hvA hvF S1 Q relaxation (10-12 s) S1 kq[Q] Q  knr S0 Dynamic quenching and static quenching Dynamic quenching Collision quenching

6. XMUGXQ PFS0501 Dynamic quenching Diffusion control Effected by viscosity of solvent In general, without permanent change in the fluorophore No changes in the absorption spectrum Decreasing the lifetime Intensify with temperature increasing Change into

7. XMUGXQ PFS0501 hvA2 hvA1 hvF2 hvF1 S1 S1 relaxation (10-12 s) relaxation (10-12 s) S1 S1   knr knr S0 S0 M MQ Static quenching MQ* → MQ +  MQ* → MQ + hvF2

8. XMUGXQ PFS0501 Static quenching Forming a new species Changing the absorption spectrum How about excitation spectrum? Change? Or not change? Depend on MQ emitting or not No change in the lifetime How about the effect of temperature? Depend on the thermodynamic properties of M and MQ

9. XMUGXQ PFS0501 Quenchers oxygen Causing ISC halogens Aromatic and aliphatic amines Forming excited charge-transfer complexes Carboxyl groups Nitroxides Nitromethane and nitro compounds Heavy atoms more……

10. XMUGXQ PFS0501 5.2 Stern-Volmer eqution F0 and F Fluorescence intensities in the absence and presence of quencher, respectively [Q] Concentration of quencher KSV Stern-Volmer quenching constant, given by kq0 KD dynamic quenching KS static quenching

11. XMUGXQ PFS0501 hvA hvF S1 relaxation (10-12 s) S1 Q kq[Q]  knr Q S0 Dynamic quenching For steady-state measurement In the presence of quencher In the absence of quencher

12. XMUGXQ PFS0501 Dynamic quenching For quantitative measurement Stern-Volmer equation Because Stern-Volmer equation

13. XMUGXQ PFS0501 Dynamic quenching 0 Lifetime in the absence of quencher kq Bimolecular quenching constant Typically, 11010 mol-1 L s-1 kq = f(Q)k0 Quenching efficiency f(Q) The diffusion-controlled bimolecular rate constant k0 R Molecular radius (RF+RQ) collision radius D Diffusion coefficients N Avogadro’s number

14. XMUGXQ PFS0501 Example Oxygen quenches The fluorescence of tryptophan 25°C, O2, Dq = 2.5 10-5 cm2/s; tryptophan, DF = 0.66 10-5 cm2/s The collision radius R = ( RF + Rq ) = 5 Å k0 =1.2  1010 mol-1 L s-1 Thus Measured KD = 32.5 mol-1 L How to measure? Given  = 2.7 ns, Thus kq =1.2  1010 mol-1 L s-1 f(Q) = kq/k0 = 1

15. XMUGXQ PFS0501 The effect of lifetime on quenching Using oxygen as the quencher According to Stern-Volmer equation KD =kq0 Typically, kq = 11010 mol-1 L s-1 Typical fluorescence lifetime 0 = 10-8 s Thus, KD = 102 mol-1 L When Typical phosphorescence lifetime 0 = 10-3 s Thus, KD = 107 mol-1 L When What do these calculations suggest?

16. XMUGXQ PFS0501 Static quenching May or may not fluoresce According to F = Kc Thus

17. XMUGXQ PFS0501 F0/F F0/F F0/F T↑ T↑ T↑ 1.0 1.0 1.0 [Q] [Q] [Q] Stern-Volmer constant KD = kq0 Related with lifetime, controlled by diffusion Increasing with temperature increasing KS Formation constant Endothermal reaction Exothermal reaction

18. XMUGXQ PFS0501 F0/F 1.0 [Q] Combined dynamic and static quenching Violation of Stern-Volmer equation Suggest the combination of dynamic and static quenching

19. XMUGXQ PFS0501 hv F* F* Q* FQ Correction to Stern-Volmer equation the fraction of fluorescence, due to static quenching F0 F FQ the fraction of fluorescence, due to dynamic quenching

20. XMUGXQ PFS0501 F0 F FQ hv F* F* Q* FQ Correction to Stern-Volmer equation fs fS.fD

21. XMUGXQ PFS0501 Correction to Stern-Volmer equation Kapp apparent Stern-Volmer constant

22. XMUGXQ PFS0501 KD+KS [Q] Correction to Stern-Volmer equation KDKS

23. XMUGXQ PFS0501 Modified Stern-Volmer equation in interpreting “sphere of action” Where  is the volume of the sphere. The radius of the sphere is slightly larger than the sum of the radii of the fluorophore and the quencher. There exists a high probability that quenching will occur before these molecules diffuse apart.

24. XMUGXQ PFS0501 • 0/ x F0/F Example 1 Oxygen quenching of tryptophan

25. XMUGXQ PFS0501 • F0/F • F0/FQ ■0/  Example 2 Acrylamide(丙烯酰胺）quenching of N-acetyl-L-tryptophan-amide(N-乙酰-L-色氨酸酰胺）

26. XMUGXQ PFS0501 Example 3 From JRL. P.245 Acrylamide quenching of dihydroequilenin (DHE，二氢马萘雌甾酮) in buffer containing 10% sucrose（蔗糖） at 11°C

27. XMUGXQ PFS0501 Example 4 10-methylacridinium chloride quenching of guanosine-5’-monophosphate （鸟嘌呤核苷-5‘- 单磷酸）

28. XMUGXQ PFS0501 Example 4

29. XMUGXQ PFS0501 5.4 factors influencing quenching • Steric effect Example 1

30. XMUGXQ PFS0501 The ethidium bromide-DNA complex Oxygen quenching of ethdium bromide fluorescence Why smaller than 11010?

31. XMUGXQ PFS0501 Example 2

32. XMUGXQ PFS0501 F Charge effect I O2

33. XMUGXQ PFS0501 Example 1 Copolymer 1 1% tryptophan + 99% glutamic acid Copolymer 2 3% tryptophan + 97% lysine At neutral pH glutamic acid nagatively charged Lysine positive charged What happens to the fluorescence of tryptophan in the presence of oxygen and iodide, respectively?

34. XMUGXQ PFS0501 Example 1

35. XMUGXQ PFS0501 氯化十二烷基三甲铵 十二烷基硫酸钠 Example 2

36. XMUGXQ PFS0501 Micro-environment Fluorescence quenching of trypsinogen 胰蛋白酶原荧光猝灭

37. XMUGXQ PFS0501 F0/F 1.0 [Q] Fex Fin Different in micro-environment Downward-curving Stern-Volmer plot Partial quenching I In the absence of quencher F0 = Fin,0 + Fex,0

38. XMUGXQ PFS0501 Modified Stern-Volmer equation in interpreting the difference in micro-environment In the presence of quencher Total fluorescence

39. XMUGXQ PFS0501 Deriving modified equation

40. XMUGXQ PFS0501 Deriving modified equation Let Then Modified Stern-Volmer equation

41. XMUGXQ PFS0501 1 / fex Deriving modified equation 1 /(KDfex) 1/[Q]

42. XMUGXQ PFS0501 Example Native protein Iodide quenching of tryptophan fluorescence in lysozyme(溶菌酶） Denatured protein Native protein 1/fex = 1.5, fex = 0.66 Denatured protein 1/fex = 1.0, fex = 1.0

43. XMUGXQ PFS0501 example

44. XMUGXQ PFS0501 Localization of membrane-bound fluorophores

45. XMUGXQ PFS0501 Localization of membrane-bound fluorophores

46. XMUGXQ PFS0501 5.5 quenching mechanisms 5.5.1 Due to energy transfer • Nonradiative energy transfer, due to dipole-dipole interaction of donor and acceptor D donor A acceptor Rate of energy transfer depends Overlap of spectra Relative orientation Distance between A and D

47. XMUGXQ PFS0501 Principle of energy • Rate of energy transfer D quantum yield of D D lifetime of D r distance between A and D n refrcative index N Avogadro’s number k orientation factor J overlap integral

48. XMUGXQ PFS0501 Overlap integral The corrected fluorescence intensity of D in -d, the total intensity normalized to unity The extinction coefficient of A at  The unit is (mol / L)-1 cm3

49. XMUGXQ PFS0501 Orientation factor randomize by rotational diffusion prior to energy transfer k = 2/3

50. XMUGXQ PFS0501 S1 relaxation (10-12 s) hvA hvF S1 A(S1)  knr A(S0) S0 Förster distance R0 The distance between A and D when R0 > r, energy transfer decay dominate R0 < r, usual radiative and nonradiative decay dominate