2. Fluorescence, Phosphorescence, & Chemiluminescence
A) Introduction
1.) Theory of Fluorescence and Phosphorescence:
10-14 to 10-15 s
10-5 to 10-8 s fluorescence
10-4 to 10s phosphorescence
10-8 – 10-9s
M* M + heat
- Excitation of e- by absorbance of hn.
- Re-emission of hn as e- goes to ground state.
- Use hn2 for qualitative and quantitative analysis
3. 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
4. 2.) Fluorescence – dari ground state ke posisi single dan kembali.
Phosphorescence - dari ground state ke posisitriplet dan kembali.
Spins paired
No net magnetic field
Spins unpaired
net magnetic field
10-5 to 10-8 s
10-4 to 10 s
Fluorescence Phosphorescence
0 sec 1 sec 640 sec
Contoh
Phosphorescence
5. 3) Diagram Energi Jablonski
S2, S1 = Singlet States
Numerous vibrational energy levels for each electronic state
Radiasi Resonansi - reemissi pada l sama
Biasanya reemisi pada l lebih tinggi (energi rendah)
Transisi terlarang: no direct excitation of triplet state
because change in multiplicity –selection rules.
T1 = Triplet State
6. 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
7. 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.
8. 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
9. 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)
10. 5.) Quantum Yield (f): 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
f = kf
kf + ki + kec+ kic + kpd + kd
f: fluorescence I: intersystem crossing
ec: external conversion ic: internal conversion
pd: predissociation d: dissociation
Increase quantum yield by decreasing factors that promote other processes
Fluorescence probes measuring
quantity of protein in a cell
11. 6.) Types of Transitions:
- seldom occurs from absorbance less
than 250 nm
‚ 200 nm => 600 kJ/mol, breaks many bonds
- fluorescence not seen with s* s
- typically p* p or n p*
12. 7.) Fluorescence & Structure:
- usually aromatic compounds
‚ low energy of p p* transition
‚ quantum yield increases with number of rings and
degree of condensation.
‚ fluorescence especially favored for rigid structures
fluorescence increase for chelating agent
bound to metal.
N HN
H2
C
N
O
Zn
2
EExxaammpplleess ooff fflluuoorreesscceenntt ccoommppoouunnddss::
quinoline indole fluorene 8-hydroxyquinoline
13. 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.
H H
N
H H
N
H H
N
resonance forms of aniline
FFlluuoorreesscceennccee ppHH TTiittrraattiioonn
14. 9.) Effect of Dissolved O2:
- increase [O2] decrease fluorescence
‚ oxidize compound
‚ paramagnetic property increase intersystem
crossing (spin flipping)
Change in fluorescence as a function of cellular oxygen
Am J Physiol Cell Physiol 291: C781–C787, 2006.
15. B) Effect of Concentration on Fluorescence or Phosphorescence
power of fluorescence emission: (F) = K’Po(1 – 10 –ebc)
K’ ~ f (quantum yield)
Po: power of beam
ebc: Beer’s law
F depends on absorbance of light and incident intensity (Po)
At low concentrations: F = 2.3K’ebcPo
deviations at higher concentrations
can be attributed to absorbance becoming
a significant factor and by self-quenching
or self-absorption.
FFlluuoorreesscceennccee ooff ccrruuddee ooiill
16. C) Fluorescence Spectra
Excitation Spectra (a) – measure fluorescence or
phosphorescence at a fixed wavelength
while varying the excitation wavelength.
Emission Spectra (b) – measure fluorescence or
phosphorescence over a range of
wavelengths using a fixed excitation wavelength.
Phosphorescence bands are uussuuaallllyy ffoouunndd aatt lloonnggeerr
((>>l)) tthheenn fflluuoorreesscceennccee bbeeccaauussee eexxcciitteedd ttrriippllee ssttaattee iiss
lloowweerr eenneerrggyy tthheenn eexxcciitteedd ssiinngglleett ssttaattee..
17. 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
19. Spectrofluorometer
- both excitation and emmision spectra
- two grating monochromators
- quantitative analysis
Perkin-Elmer 204
20. E) Application of Fluorescence
- detect inorganic species by chelating ion
Ion Reagent Absorption (nm) Fluorescence (nm) Sensitivity (mg/ml) Interference
Al3+ Alizarin garnet R 470 500 0.007
Be, 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
B4O7
2- Benzoin 370 450 0.04 Be, Sb
benzoxazole 365 Blue 2 NH3
Cd2+ 2-(0-Hydroxyphenyl)-
Li+ 8-Hydroxyquinoline 370 580 0.2 Mg
Sn4+ Flavanol 400 470 0.1 F-, PO4
3-, Zr
Zn2+ Benzoin - green 10 B, Be, Sb,
colored ions
N
OH
O
O
OH
OH
HO N N
HO
SO3Na
O
C
OH
C
H
8-Hydroxyquinoline flavanol alizarin garnet R benzoin
21. 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 + hn
Examples:
NH2 O
C
NH
NH
C
O
O2/OH-NH2
COO-COO-
+ hn + N2 + H2O
1) Chemical systems
- Luminol (used to detect blood)
- phenyl oxalate ester (glow sticks)
22. 2) Biochemical systems
- Luciferase (Firefly enzyme)
Luciferin + O2
Luciferase
O O
O C
C R2
R1
Spontaneous
CO2 + O C*
R2
R1
Light
N
S
HO N
S
O
HO
Luciferin (firefly)
“Glowing” Plants
Luciferase gene cloned into plants
25. Compounds Wavelength of range of
maximum fluorescence
(nm)
Steroids
Aldosterone
Cortisone
Prednisolone
Testersterone
400-450
580
570
580
Vitamins
Riboflavin (B 2)
Cyanocobalamin (B 12)
Tocopherol (E)
565
305
340
Coenzyme
Adenine
Adenozine triphosphate
380
390
Dye
Fluorescene
Methylene blue
510-590
650-700
Editor's Notes
Deaktivasi melalui tumbukan dengan pelarut (pemadaman dengan tumbukan)
Penurunan junlah tumbukan meningkatkan fluoresen dan posporesen
Menurunkan temperatur dan atau menaikkan viskositas
Menurunkan konsentrasi dari reagen yang dapat memadamkan
