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Principles of Fluorescence Spectroscopy

Chemistry Department

XMU

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Chapter Four

Factors Influencing Fluorescent Emission

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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

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4.1 Effect of Solvent

The phenomena of solvent effect

Shifting Emission Wavelengths

Changing quantum yield

Changing anisotropy

Changing fluorescence lifetime

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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

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Large change in dipole moment

C

OC2H5

NH3C

H3CC

OC2H5

NH3C

H3C

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对甲基苯胺萘磺酸

水中弱荧光， 500 nm; 疏水环境强荧光， 413 nm水中弱荧光， 500 nm; 疏水环境强荧光， 413 nm

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色氨酸发光基团 吲哚

1. Hexane

2. 0.7% n-butanol

3. 5% n-butanol

4. 100% butanol

5. Water

1. Hexane

2. 0.7% n-butanol

3. 5% n-butanol

4. 100% butanol

5. Water

N

Indole

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Changing quantum yield

SO3H HN

HO3S

NH

CH3

1,8 - ANS

TNS

F

In water 0.002

Banding protein 0.4

In water nonfluorescence

Banding protein intensive fluorescnece

F

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Changing anisotropy

N N

H3C CH3

Re

COOC

NOC COOH

+

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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.

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General solvent effect

Franck-Condon principle

Solvent relaxation

a

b

0-0

hvA

c

d

0-0

hvF

Relaxation

Relaxation

*

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The Lipper equation

constan

n

hcvv FA

3

2*

2

2 )()

12

1

12

1(

2

consta

fvhc

2*

3)(

2

FA vvv FA

hchcvhc

Stoke’s shift

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The Lipper equation

constan

n

hcvv FA

3

2*

2

2 )()

12

1

12

1(

2

a radius of cavity in which the fluorophore reside

*, dipole moment of ground state and excited states, reapectively

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The Lipper equation

)()( nfff

12

1)(

f

12

1)(

2

2

n

nnf

Orientation polarizability

gAv )(gFv )(

Av

* >

Fv

Gas phase In solution

f (n)f ()

Solvent relaxation

f

How about = n2 ?

How about = n2 ?

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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

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Example 1

2.48.4

20

n f em

hexane 1.874 1.372 0.0011 35 350.4

methanol 33.1 1.326 0.3098 9740 531.1

0 FA vv

Assume * - = 20 Debye

A unit-charge separation of 4.2 Å

Example 2

Assume * - = 20 Debye ex = 350 nm

In nonpolar solution, observed

FA vv

Why?Why?

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Derivation of Lipper Equation

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Correction to Lipper equation

*

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Application of Lipper Equation

constan

n

hcvv FA

3

2*

2

2 )()

12

1

12

1(

2

const * BfAv For a given fluorophore

HO3S

NCH3

HO3S

NCH3

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Determination of dipole moment of excited state

For a given solvent, measure the dipole moment

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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

Small change of solvent constituent could cause large shift of emission wavelength.

Spectrum shape, not only emission wavelength, change

Not follow the lipper equation

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Spectrum change

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Spectrum change Comparison

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Large stoke’s shift

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Disobey the lipper equation

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Disobey the lipper equation

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The dynamic process of the solvent molecule reorientation

Temperature

Viscosity

F

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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

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Effect on quantum yield

The process of single molecule

relaxation (10-12 s)

S0

S1

S1

hvA hvF knr

nrkΓ

ΓΦ

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Effect of non-radiation deactive

A

B

S1 S0

S0

E

r

RTEkeF

FF /

0

0

F0 fluorescence at T1

F fluorescence at T2

E energy needed for transfer from A to B (4~7 Kcal / mL)

IC process

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Example

N N

H3C CH3

Re

COOC

NOC COOH

+

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Effect on quantum yield

relaxation (10-12 s)

S0

S1

S1

hvA hvF knr

Q

Q

kq[Q]

The process of two molecules

][QkkΓ

ΓΦ

qnr

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Effect on lifetime

relaxation (10-12 s)

S0

S1

S1

hvA hvF knr

nrkΓ

1

The process of single molecule

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Effect on lifetime

relaxation (10-12 s)

S0

S1

S1

hvA hvF knr

Q

Q

kq[Q]

The process of two molecules

][

1

QkkΓ qnr

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Effect on emission wavelength

N N

H3C CH3

Re

COOC

NOC COOH

+

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Effect on anisotropy

0 10 20 30 40 50 600.00

0.05

0.10

0.15

0.20

[Ru(bpy)2(dppz)]2+ in DPPG vesicles

Ani

sotr

opy

T ( oC )

0 10 20 30 40 50 600.00

0.05

0.10

0.15

0.20

[Ru(bpy)2(dppz)]2+ in DPPG vesicles

Ani

sotr

opy

T ( oC )

N

N

N

N

Ru

N

N

N

N

2+

[Ru(bpy)2(dppz)]2+

N

N

N

N

Ru

N

N

N

N

2+

[Ru(bpy)2(dppz)]2+

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Effect on the structural detail of spectrum

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Acid-base reaction of ground state fluorophore

Difference in fluorescent characteristics between conjugate acid and base

HA A H+

em, HA em, A

F

F

pH

em, HA Non-fluorescent

F

pH

em, ANon-fluorescent

F

pH

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Effect on the composition of fluorophore

Changing pH may change the composition of metal-ligand compound

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HA A H+

pKa* = pKa

pKa

HA A H+* *

HA + A + hv

em,A

Non-fluorescent

A

pH

pKa* F

pH

pKa

pKa*

Radiating takes place prior to acid-base reaction

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Acid-base reaction of excited state fluorophore

HA A H+

pKa* < pKa

pKa

HA A H+* *

HA + A + hv

em,A

Non-fluorescent

A

pH

pKa*

pKa

Acid-base reaction finished before radiating

pKa*

F

pH

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Example

OH OpKa* = 3.1

pKa = 9.5

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Acid-base reaction of excited state fluorophore

HA A H+

pKa* > pKa

pKa

HA A H+* *

HA + A + hv

em,A

Non-fluorescent

A

pH

pKa*

pKa

Acid-base reaction finished before radiating

F

pHpKa*

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Excited-state intra-molecule proton transfer

CO

O

OH

CH3

CO

OH

O

CH3

水杨酸酯

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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

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Effect on *→n transition

n

*

Hydrogen bond

Solvent relaxation

Intensify solvent effect

Red shift emission Blue shift

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Changing the type of low-energy transition

NH

H

HO

H

NH

HBlue shift

NH

OH

Changing the transition type

Intensify emission

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Effect on quantum yield

N

O H

Generally, intensity IC, decrease quantum yield

When transition-type changing occurs, intensity emission

Intra-molecular hydrogen bond

N

OH

Intensity IC, F is 100 times lower than that of 5-hydroxyl-quinoline

Almost same absorption

Why?

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Effect of heavy atom

Intra-molecule

In the solvent

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4.6 effect of surfactant Surfactant Aggregation

Micelle

Critical micelle concentration CMC

Surfactants used in fluorimetry

N

CH3

H3C

CH3

Br

cation

C16H33N+(CH3)3Br- 溴化十六烷基三甲铵 CTAB

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Surfactants used in fluorimetry

S

O

O

ONa

anion

C12H25SO4-Na+十二烷基硫酸钠

C12H25SO3-Na+十二烷基磺酸钠

S ONaO

O

O

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Surfactants used in fluorimetry

Amphoteric 两性型

NH3C

CH3

SO

OO

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-

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Surfactants used in fluorimetry

Neutral 非离子型

Triton X-100

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Structural characteristics

N

CH3

H3C

CH3

Br

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micelle

amphoteric

Neutral

ionic

ionic

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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