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XMUGXQ PFS0401 Principles of Fluorescence Spectroscopy Chemistry Department XMU 20040300 XMUGXQ PFS0401 Chapter Four Factors Influencing Fluorescent Emission 20040300 XMUGXQ PFS0401 Factors Influencing Fluorescent Emission 4.1 4.2 4.3 4.4 4.5 4.6 20040300 Effect of Solvent Effect of Temperature Effect of pH Effect of Hydrogen bond Effect of Heavy atom Effect of Surfactant XMUGXQ PFS0401 4.1 Effect of Solvent The phenomena of solvent effect Shifting Emission Wavelengths Changing quantum yield Changing anisotropy Changing fluorescence lifetime 20040300 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 20040300 XMUGXQ PFS0401 Large change in dipole moment O C2H5 C H3C N O H3C C H3C N H3C 20040300 C2H5 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 20040300 XMUGXQ PFS0401 色氨酸发光基团 吲哚 Indole N 1. Hexane 2. 0.7% n-butanol 3. 5% n-butanol 4. 100% butanol 5. Water 20040300 XMUGXQ PFS0401 Changing quantum yield F SO3H HN In water 0.002 Banding protein 0.4 1,8 - ANS CH3 F NH In water HO3S Banding protein TNS 20040300 nonfluorescence intensive fluorescnece XMUGXQ PFS0401 Changing anisotropy + N N H 3C CH3 Re OC OC 20040300 N CO COOH XMUGXQ PFS0401 General and Specific Solvent effects General solvent effect Solvent properties dielectric constant 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 Solute properties as well as solvent properties Hydrogen bonding 20040300 Complexation Charge transfer Acid-base reaction XMUGXQ PFS0401 General solvent effect Franck-Condon principle Solvent relaxation * b c 0-0 0-0 hvA hvF d 20040300 a XMUGXQ PFS0401 The Lipper equation 2 1 n2 1 ( * )2 v A vF ( 2 ) const 3 hc 2 1 2n 1 a 2f * 2 hcv 3 ( ) const a v v A vF hcv hc A hc F Stoke’s shift 20040300 XMUGXQ PFS0401 The Lipper equation 2 1 n 1 ( ) v A vF ( 2 ) const 3 hc 2 1 2n 1 a 2 a *, * 2 radius of cavity in which the fluorophore reside dipole moment of ground state and excited states, reapectively 20040300 XMUGXQ PFS0401 The Lipper equation f f ( ) f (n) 1 f ( ) 2 1 Orientation polarizability n 1 f ( n) 2 2n 1 2 How about = n2 ? * > (v A ) g (v F ) g vA f (n) Gas phase 20040300 Solvent relaxation f vF f ( ) In solution XMUGXQ PFS0401 Shifting of emission wavelength solvent water ethanol ether hexane 78.3 24.3 4.35 1.89 n 1.33 1.35 1.35 1.37 f 0.32 0.30 0.25 0.001 20040300 XMUGXQ PFS0401 Example 1 20 4.2 Assume * - = 20 Debye 4.8 A unit-charge separation of 4.2 Å Example 2 Assume * - = 20 Debye hexane n f 1.874 1.372 0.0011 methanol 33.1 1.326 0.3098 In nonpolar solution, observed 20040300 ex = 350 nm v A vF em 35 350.4 9740 531.1 vA vF 0 Why? XMUGXQ PFS0401 Derivation of Lipper Equation 20040300 XMUGXQ PFS0401 Correction to Lipper equation * 20040300 XMUGXQ PFS0401 Application of Lipper Equation 2 1 n2 1 ( * )2 v A vF ( 2 ) const 3 hc 2 1 2n 1 a For a given fluorophore * const v Af B N CH3 HO3S 20040300 XMUGXQ PFS0401 Determination of dipole moment of excited state For a given solvent, measure the dipole moment 20040300 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 20040300 XMUGXQ PFS0401 Spectrum change 20040300 XMUGXQ PFS0401 Spectrum change 20040300 Comparison XMUGXQ PFS0401 Large stoke’s shift 20040300 XMUGXQ PFS0401 Disobey the lipper equation 20040300 XMUGXQ PFS0401 Disobey the lipper equation 20040300 XMUGXQ PFS0401 The dynamic process of the solvent molecule reorientation Temperature Viscosity F 20040300 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 20040300 XMUGXQ PFS0401 Effect on quantum yield The process of single molecule Γ Φ Γ k nr S1 relaxation (10-12 s) S1 hvA S0 20040300 hvF knr XMUGXQ PFS0401 Effect of non-radiation deactive F0 F E / RT ke F0 E S1 S0 F0 fluorescence at T1 B F fluorescence at T2 E energy needed for transfer from A to B (4~7 Kcal / mL) A S0 r IC process 20040300 XMUGXQ PFS0401 Example + N N H 3C CH3 Re OC OC 20040300 N CO COOH XMUGXQ PFS0401 Effect on quantum yield The process of two molecules Γ Φ Γ knr kq [Q] S1 relaxation (10-12 s) S1 hvA S0 20040300 hvF knr Q kq[Q] Q XMUGXQ PFS0401 Effect on lifetime The process of single molecule 1 Γ k nr S1 relaxation (10-12 s) S1 hvA S0 20040300 hvF knr XMUGXQ PFS0401 Effect on lifetime The process of two molecules 1 Γ knr kq [Q] S1 relaxation (10-12 s) S1 hvA S0 20040300 hvF knr Q kq[Q] Q XMUGXQ PFS0401 Effect on emission wavelength + N N H 3C CH3 Re OC OC 20040300 N CO COOH XMUGXQ PFS0401 Effect on anisotropy 2+ N 0.20 N N N N N Ru 2+ [Ru(bpy)2(dppz)] in DPPG vesicles N Anisotropy 0.15 N 0.10 [Ru(bpy) 2(dppz)] 2+ 0.05 0.00 0 10 20 30 40 o T( C) 20040300 50 60 XMUGXQ PFS0401 Effect on the structural detail of spectrum 20040300 XMUGXQ PFS0401 Effect of pH Acid-base reaction of ground state fluorophore Difference in fluorescent characteristics between conjugate acid and base F F HA A + H em, HA em, A pH F em, HA Non-fluorescent pH Non-fluorescent 20040300 em, A F pH XMUGXQ PFS0401 Effect on the composition of fluorophore Changing pH may change the composition of metalligand compound 20040300 XMUGXQ PFS0401 Acid-base reaction of excited state fluorophore pKa* = pKa pKa HA A A + H pKa * HA pK * a F * A + H pK*a HA + Nonfluorescent 20040300 pH A + hv em,A pH Radiating takes place prior to acidbase reaction XMUGXQ PFS0401 Acid-base reaction of excited state fluorophore pKa* < pKa A pKa HA A + H pKa * HA pKa* F * A + H pK*a HA + Nonfluorescent 20040300 pH A + hv em,A pH Acid-base reaction finished before radiating XMUGXQ PFS0401 Example OH pKa* = 3.1 pKa = 9.5 20040300 O XMUGXQ PFS0401 Acid-base reaction of excited state fluorophore pKa* > pKa A pKa HA A + H pKa * HA pKa* pH F * A + H pK*a HA + Nonfluorescent 20040300 A + hv em,A pH Acid-base reaction finished before radiating XMUGXQ PFS0401 Excited-state intra-molecule proton transfer O CH3 O C O C OH OH O 水杨酸酯 20040300 CH3 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 20040300 XMUGXQ PFS0401 Effect on *→n transition * Hydrogen bond Solvent relaxation n Intensify solvent effect Red shift emission 20040300 Blue shift XMUGXQ PFS0401 Changing the type of low-energy transition H O H N H H H N H H Changing the transition type O H N 20040300 Intensify emission Blue shift XMUGXQ PFS0401 Effect on quantum yield Generally, intensity IC, decrease quantum yield When transition-type changing occurs, intensity emission Intra-molecular hydrogen bond OH N O H Intensity IC, F is 100 times lower than that of 5-hydroxylquinoline 20040300 N Almost same absorption Why? XMUGXQ PFS0401 Effect of heavy atom Intra-molecule In the solvent 20040300 XMUGXQ PFS0401 4.6 effect of surfactant Surfactant Aggregation Micelle Critical micelle concentration CMC Surfactants used in fluorimetry cation C16H33N+(CH3)3BrCH3 Br H3C N CH3 20040300 溴化十六烷基三甲铵 CTAB XMUGXQ PFS0401 Surfactants used in fluorimetry anion 十二烷基硫酸钠 C12H25SO4-Na+ O Na O S O O 十二烷基磺酸钠 O Na O S O 20040300 C12H25SO3-Na+ 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 C12H25N+(CH3)2(CH2)3SO3- O S O O H3C N CH3 20040300 XMUGXQ PFS0401 Surfactants used in fluorimetry Neutral 非离子型 Triton X-100 20040300 XMUGXQ PFS0401 Structural characteristics CH3 Br H3C N CH3 20040300 XMUGXQ PFS0401 micelle ionic Neutral ionic amphoteric 20040300 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 20040300
