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Fluorescence, Phosphorescence, & Chemiluminescence
Fluorescence, Phosphorescence, & Chemiluminescence
A) Introduction
1.) Theory of Fluorescence and Phosphorescence:
- Excitation of e- by absorbance of h.- Re-emission of h 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
10-8 – 10-9s
M* M + heat
Fluorescence, Phosphorescence, & Chemiluminescence
A) Introduction
1.) Teori Fluorescence dan Phosphorescence:
Metode limit deteksi (mol)
Konsentrsi limit deteksi (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
For fluorescence, only observe amount of PL
2.) Fluorescence – dari ground state ke posisi single dan kembali.Phosphorescence - dari ground state ke posisitriplet dan kembali.
Spins pairedNo net magnetic field
Spins unpairednet magnetic field
10-5 to 10-8 s10-4 to 10 s
Fluorescence Phosphorescence
0 sec 1 sec 640 sec
Contoh Phosphorescence
3) Diagram Energi Jablonski
S2, S1 = Singlet States
Radiasi Resonansi - reemissi pada samaBiasanya reemisi pada lebih tinggi (energi rendah)
Numerous vibrational energy levels for each electronic state
Transisi terlarang: no direct excitation of triplet state because change in multiplicity –selection rules.
T1 = Triplet State
4.) Deactivation Processes:
a) vibrational relaxation: solvent collisions- 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
4.) Deactivation Processes:
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.
4.) Deactivation Processes:
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.
Quenching of Ru(II) Luminescence by O2
4.) Deactivation Processes:
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)
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
Increase quantum yield by decreasing factors that promote other processes
Fluorescence probes measuring quantity of protein in a cell
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 *
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
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 (spin flipping)
Am J Physiol Cell Physiol 291: C781–C787, 2006.
Change in fluorescence as a function of cellular oxygen
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
C) Fluorescence Spectra
Excitation Spectra (a) – measure fluorescence or phosphorescence at a fixed wavelengthwhile varying the excitation wavelength.
Emission Spectra (b) – 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.
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
Fluorometers- simple, rugged, low cost, compact- source beam split into reference and sample beam- reference beam attenuated ~ fluorescence intensity
A-1 filter fluorometer
Spectrofluorometer- both excitation and emmision spectra- two grating monochromators - quantitative analysis
Perkin-Elmer 204
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
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)
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
Contoh senyawa Contoh senyawa
Compounds Wavelength of range of maximum
fluorescence (nm)Aromatic hydrocarbon naphthalene Anthracene Pyrene 1-Benzopyrene
300-365370-460370-400400-450
Heterocyclic compound Quinoline Quinoline sulfate
380-490400-500
Coenzyme Adenine Adenozine triphosphate
380390
Drugs Aspirin Codeine Phenobarbital Procaine
335350440345
Compounds Wavelength of range of maximum
fluorescence (nm)Steroids Aldosterone Cortisone Prednisolone Testersterone
400-450580570580
Vitamins Riboflavin (B 2)
Cyanocobalamin (B 12)
Tocopherol (E)
565305340
Coenzyme Adenine Adenozine triphosphate
380390
Dye Fluorescene Methylene blue
510-590650-700