Transcript Slide 1

Fluorescence, Phosphorescence, & Chemiluminescence
A) Introduction
1.)
Theory of Fluorescence and Phosphorescence:
10-5 to 10-8 s fluorescence
10-4 to 10s phosphorescence
10-14 to 10-15 s
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
Fluorescence, Phosphorescence, & Chemiluminescence
A) Introduction
1.)
Theory of Fluorescence and Phosphorescence:
Method
Mass detection
limit (moles)
Concentration Advantages
detection limit
(molar)
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 – ground state to single state and back.
Phosphorescence - ground state to triplet state and back.
10-5 to 10-8 s
10-4 to 10 s
Spins paired
No net magnetic field
Fluorescence
Spins unpaired
net magnetic field
Phosphorescence
Example of
Phosphorescence
0 sec
1 sec
640 sec
3) Jablonski Energy Diagram
S2, S1 = Singlet States
T1 = Triplet State
Numerous vibrational energy levels for each electronic state
Resonance Radiation - reemission at same l
usually reemission at higher l (lower energy)
Forbidden transition: no direct excitation of triplet state
because change in multiplicity –selection rules.
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 (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
ec: external conversion
pd: predissociation
I: intersystem crossing
ic: internal conversion
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 s*  s
- typically p*  p or p*  n
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.
Examples of fluorescent compounds:
H
N
N
H2
C
O
Zn
N
2
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.
Fluorescence pH Titration
H
H
N
H
H
N
resonance forms of aniline
H
H
N
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.
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)
Fluorescence of crude oil
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.
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 usually found at longer
(>l) then fluorescence because excited triple state is
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
Al3+
Reagent
Absorption (nm)
Alizarin garnet R
Fluorescence (nm)
470
Sensitivity (mg/ml)
Interference
0.007
Be, Co, Cr, Cu, F,NO3-, Ni, PO4-3,
Th, Zr
500
Be, Co, Cr, Cu, F,Fe, Ni,PO4-3,
Th, Zr
Al complex of Alizarin
garnet R (quenching)
470
500
0.001
Benzoin
370
450
0.04
2-(0-Hydroxyphenyl)benzoxazole
365
Blue
2
NH3
8-Hydroxyquinoline
370
580
0.2
Mg
Sn4+
Flavanol
400
470
0.1
F-, PO43-, Zr
Zn2+
Benzoin
-
green
10
B, Be, Sb,
colored ions
FB4O72Cd2+
Li+
OH
N
OH
HO
O
HO
N
N
OH
C
C
SO3Na
O
flavanol
O
H
OH
8-Hydroxyquinoline
Be, Sb
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 + hn
Examples:
1) Chemical systems
- Luminol (used to detect blood)
NH2
O
NH2
C
COONH
O2/OH-
+ hn + N2 + H2O
NH
C
COO-
O
- phenyl oxalate ester (glow sticks)
2) Biochemical systems
- Luciferase (Firefly enzyme)
O
O
C
C
R2
Luciferase
Luciferin + O2
O
R2
Spontaneous
CO2 +
O
Light
C*
R1
1
R
“Glowing” Plants
N
S
HO
Luciferase gene cloned into plants
S
N
O
Luciferin (firefly)
HO