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20040300 XMUGXQ PFS0401 Principles of Fluorescence Spectroscopy Chemistry Department XMU.

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Presentation on theme: "20040300 XMUGXQ PFS0401 Principles of Fluorescence Spectroscopy Chemistry Department XMU."— Presentation transcript:

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 SolventEffect of Solvent 4.2 Effect of TemperatureEffect of Temperature 4.3 Effect of pHEffect of pH 4.4 Effect of Hydrogen bondEffect of Hydrogen bond 4.5 Effect of Heavy atomEffect of Heavy atom 4.6 Effect of SurfactantEffect of Surfactant

4 XMUGXQ PFS 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 cyclohexane Chlorobenzene Dimethylformamide Ethanol Water 6-propionyl-2-(dimethylamino)naphthalene 6- 丙酰基 -2- (二甲基氨基)萘 solvent

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 色氨酸发光基团 吲哚 1.Hexane % n-butanol 3. 5% n-butanol % butanol 5. Water 1.Hexane % n-butanol 3. 5% n-butanol % butanol 5. Water Indole

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

10 XMUGXQ PFS0401 Changing anisotropy

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

12 XMUGXQ PFS0401 General solvent effect Franck-Condon principle Solvent relaxation a b 0-0 hv A c d 0-0 hv F 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 The Lipper equation Orientation polarizability  * >  Gas phaseIn solution f (n) f (  ) Solvent relaxation ff How about  = n 2 ?

16 XMUGXQ PFS0401 Shifting of emission wavelength solventwaterethanoletherhexane  n ff

17 XMUGXQ PFS0401 Example 1  n ff em hexane methanol Assume  * -  = 20 Debye A unit-charge separation of 4.2 Å Example 2 Assume  * -  = 20 Debye ex = 350 nm 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. Specific solvent effect: changing to an new species that fluoresces differently, duo to the reaction between fluorophore and the solvent molecule. Discrimination Electronic polarizability, molecular polarizability S mall 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 The dynamic process of the solvent molecule reorientation Temperature Viscosity F

29 XMUGXQ PFS 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 Effect on quantum yield The process of single molecule relaxation ( s) S0S0 S1S1 S1S1 hv A hv F  k nr

31 XMUGXQ PFS0401 Effect of non-radiation deactive A B S1S1 S0S0 S0S0 E r F 0 fluorescence at T 1 F fluorescence at T 2  E energy needed for transfer from A to B (4~7 Kcal / mL) IC process

32 XMUGXQ PFS0401 Example

33 XMUGXQ PFS0401 Effect on quantum yield relaxation ( s) S0S0 S1S1 S1S1 hv A hv F  k nr Q Q k q [Q] The process of two molecules

34 XMUGXQ PFS0401 Effect on lifetime relaxation ( s) S0S0 S1S1 S1S1 hv A hv F  k nr The process of single molecule

35 XMUGXQ PFS0401 Effect on lifetime relaxation ( s) S0S0 S1S1 S1S1 hv A hv F  k nr Q Q k q [Q] 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 Effect of pH Acid-base reaction of ground state fluorophore Difference in fluorescent characteristics between conjugate acid and base em, HA em, A F F pH em, HA Non-fluorescent F pH em, A Non-fluorescent F pH

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

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

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

43 XMUGXQ PFS0401 Example

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

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

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

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

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

51 XMUGXQ PFS effect of surfactant Surfactant Aggregation Micelle Critical micelle concentration CMC Surfactants used in fluorimetry cation C 16 H 33 N + (CH 3 ) 3 Br - 溴化十六烷基三甲铵 CTAB

52 XMUGXQ PFS0401 Surfactants used in fluorimetry anion C 12 H 25 SO 4 - Na + 十二烷基硫酸钠 C 12 H 25 SO 3 - Na + 十二烷基磺酸钠

53 XMUGXQ PFS0401 Surfactants used in fluorimetry Amphoteric 两性型 Sulfobetaine, SB-12 N- 十二烷基 -N,N- 二甲基铵 -3- 丙烷 -1- 磺酸 C 12 H 25 N + (CH 3 ) 2 (CH 2 ) 3 SO 3 -

54 XMUGXQ PFS0401 Surfactants used in fluorimetry Neutral 非离子型 Triton X-100

55 XMUGXQ PFS0401 Structural characteristics

56 XMUGXQ PFS0401 micelle amphoteric Neutral ionic

57 XMUGXQ PFS0401 Application of surfactant  Sensitize fluorescence intensity Decrease the quenching of oxygen Decrease the quenching due to collision Increase the solubility of fluorophore in water Decrease the interference from the other species Micelle-sensitized fluorimetry Micelle-stabilized room temperature phosphorimetry  Simulating membranous micro-environment


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