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Fluorescence, Phosphorescence, & Chemiluminescence
A) Introduction
1.) Theory of Fluorescence and Phosphorescence:
- Excitation of e- by absorbance of h.- Re-emission of hv as e- goes to ground state.- Use h2 for qualitative and quantitative analysis
10-14 to 10-15 s
10-5 to 10-8 s fluorescence10-4 to 10s phosphorescence
Method Mass detection limit (moles)
Concentration detection limit (molar)
Advantages
UV-Vis 10-13 to 10-16 10-5 to 10-8 Universal
fluorescence 10-15 to 10-17 10-7 to 10-9 Sensitive
For UV/Vis need to observe Po and P difference, which limits detection
10-8 – 10-9s
M* M + heat
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2.) Fluorescence – ground state to single state and back.Phosphorescence - ground state to triplet state and back.
Spins pairedNo net magnetic field
Spins unpairednet magnetic field
10-5 to 10-8 s 10-4 to 10 s
Fluorescence Phosphorescence
0 sec 1 sec 640 sec
Example of Phosphorescence
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3) Jablonski Energy Diagram
S2, S1 = Singlet StatesT1 = Triplet State
Resonance Radiation - reemission at same usually reemission at higher (lower energy)
Numerous vibrational energy levels for each electronic state
Forbidden transition: no direct excitation of triplet state because change in multiplicity –selection rules.
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4.) Deactivation Processes:
a) vibrational relaxation: solvent collisions- emission > excitation (Stokes shift)- vibrational relaxation is efficient and goes to lowest
vibrational level of electronic state within 10-12s or less.- significantly shorter life-time then electronically excited
state- fluorescence occurs from lowest vibrational level of
electronic excited state, but can go to higher vibrational state of ground level.
- dissociation: excitation to vibrational state with enough energy to break a bond
- predissociation: relaxation to vibrational state with enough energy to break a bond
b) internal conversion: not well understood- crossing of e- to lower electronic state.- efficient since many compounds don’t fluoresce- especially probable if vibrational levels of two electronic
states overlap, can lead to predissociation or dissociation.
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c) external conversion: deactivation via collision with solvent (collisional quenching)
- decrease collision increase fluorescence or phosphorescence‚ decrease temperature and/or increase viscosity ‚ decrease concentration of quenching (Q)
agent.
d) intersystem crossing: spin of electron is reversed
- change in multiplicity in molecule occurs (singlet to triplet)- enhanced if vibrational levels overlap- more common if molecule contains heavy atoms (I, Br)- more common in presence of paramagnetic species (O2)
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5.) Quantum Yield (): ratio of the number of molecules that luminesce to the total number of excited molecules.
- determined by the relative rate constants (kx)of deactivation processes
= kf
kf + ki + kec+ kic + kpd + kd
f: fluorescence I: intersystem crossingec: external conversion ic: internal conversionpd: predissociation d: dissociation
6.) Types of Transitions:- seldom occurs from absorbance less than 250 nm
‚ 200 nm => 600 kJ/mol, breaks many bonds
- fluorescence not seen with - typically * or * n
Increase quantum yield by decreasing factors that promote other processes
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7.) Fluorescence & Structure:- usually aromatic compounds
‚ low energy of * transition ‚ quantum yield increases with number of rings
and degree of condensation. ‚ fluorescence especially favored for rigid
structuresfluorescence increase for chelating agent
bound to metal.
N HN
H2C
N
O
Zn
2
Examples of fluorescent compounds:Examples of fluorescent compounds:
quinoline indole fluorene 8-hydroxyquinoline
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8.) Temperature, Solvent & pH Effects:- decrease temperature increase fluorescence- increase viscosity increase fluorescence- fluorescence is pH dependent for compounds with
acidic/basic substituents. ‚ more resonance forms stabilize excited state.
NH H
NH H
NH H
resonance forms of aniline
Fluorescence pH TitrationFluorescence pH Titration
9.) Effect of Dissolved O2:- increase [O2] decrease fluorescence
‚ oxidize compound ‚ paramagnetic property increase intersystem
crossing
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B) Effect of Concentration on Fluorescence or Phosphorescence
power of fluorescence emission: (F) = K’Po(1 – 10 –bc) K’ ~ (quantum yield) Po: power of beam bc: Beer’s law
F depends on absorbance of light and incident intensity (Po)
At low concentrations: F = 2.3K’bcPo
deviations at higher concentrations can be attributed to absorbance becominga significant factor and by self-quenching or self-absorption.
Fluorescence of crude oilFluorescence of crude oil
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C) Fluorescence Spectra
Excitation Spectra – measure fluorescence or phosphorescence at a fixed wavelengthwhile varying the excitation wavelength.
Emission Spectra – measure fluorescence or phosphorescence over a range of wavelengths using a fixed excitation wavelength.
Phosphorescence bands are usually found at longer Phosphorescence bands are usually found at longer (>(>) then fluorescence because excited triple state is ) then fluorescence because excited triple state is lower energy then excited singlet state.lower energy then excited singlet state.
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D) Instrumentation- basic design
‚ components similar to UV/Vis ‚ spectrofluorometers: observe
both excitation & emission spectra.
- extra features for phosphorescence‚ sample cell in cooled Dewar flask with liquid nitrogen‚ delay between excitation and emission
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Fluorometers- simple, rugged, low cost, compact- source beam split into reference and sample beam- reference beam attenuated ~ fluorescence intensity
A-1 filter fluorometer
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Spectrofluorometer- both excitation and emmision spectra- two grating monochromators - quantitative analysis
Perkin-Elmer 204
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E) Application of Fluorescence- detect inorganic species by chelating ion
Ion Reagent Absorption (nm) Fluorescence (nm) Sensitivity (g/ml) Interference
Al3+ Alizarin garnet R 470 500 0.007Be, Co, Cr, Cu, F-,NO3-, Ni, PO4
-3, Th, Zr
F- Al complex of Alizarin garnet R (quenching)
470 500 0.001
Be, Co, Cr, Cu, F-,Fe, Ni,PO4-3, Th, Zr
B4O72- Benzoin 370 450 0.04 Be, Sb
Cd2+ 2-(0-Hydroxyphenyl)-benzoxazole
365 Blue 2NH3
Li+ 8-Hydroxyquinoline 370 580 0.2 Mg
Sn4+ Flavanol 400 470 0.1 F-, PO43-, Zr
Zn2+ Benzoin - green 10B, Be, Sb, colored ions
N
OH
O
O
OH
OH
HO N N
HO
SO3Na
C
O
C
H
OH
8-Hydroxyquinoline flavanol alizarin garnet R benzoin
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F) Chemiluminescence- chemical reaction yields an electronically excited species that emits light as it returns to ground state.- relatively new, few examples
A + B C* C + hExamples:
C
NH
NH
C
NH2 O
O
O2/OH-
NH2
COO-
COO-
+ h + N2 + H2O
1) Chemical systems- Luminol (used to detect blood)
- phenyl oxalate ester (glow sticks)
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2) Biochemical systems- Luciferase (Firefly enzyme)
Luciferin + O2
LuciferaseO C
O O
C R2
R1
SpontaneousCO2 + O C*
R2
R1
Light
S
N
HON
S
O
HO
Luciferin (firefly)
“Glowing” PlantsLuciferase gene cloned into plants