Florescence

1. Fluorescence, Phosphorescence, & Chemiluminescence
A) Introduction
1.) Theory of Fluorescence and Phosphorescence:
- Excitation of e- by absorbance of hn.
- Re-emission of hn as e- goes to ground state.
- Use hn2 for qualitative and quantitative analysis
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

2. Fluorescence, Phosphorescence, & Chemiluminescence
A) Introduction
1.) Theory of Fluorescence and 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
For fluorescence, only observe
amount of PL

3. 2.) Fluorescence – ground state to single state and back.
Phosphorescence - ground state to triplet state and back.
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
Example of
Phosphorescence

4. 3) Jablonski Energy Diagram
S2, S1 = Singlet States
Resonance Radiation - reemission at same l
usually reemission at higher l (lower energy)
Numerous vibrational energy levels for each electronic state
Forbidden transition: no direct excitation of triplet state
because change in multiplicity –selection rules.
T1 = Triplet State

5. 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

6. 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.

7. 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

8. 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)

9. 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

10. 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

11. 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 H
N
H2
C
N
O
Zn
2
Examples of fluorescent compounds:
quinoline indole fluorene 8-hydroxyquinoline

12. 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.
N
H H
N
H H
N
H H
resonance forms of aniline
Fluorescence pH Titration

13. 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

14. 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.
Fluorescence of crude oil

15. 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.

16. 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

17. Fluorometers
- simple, rugged, low cost, compact
- source beam split into reference and sample beam
- reference beam attenuated ~ fluorescence intensity
A-1 filter fluorometer

18. Spectrofluorometer
- both excitation and emmision spectra
- two grating monochromators
- quantitative analysis
Perkin-Elmer 204

19. 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
Cd2+ 2-(0-Hydroxyphenyl)-
benzoxazole
365 Blue 2
NH3
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
C
O
C
H
OH
8-Hydroxyquinoline flavanol alizarin garnet R benzoin

20. 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:
C
NH
NH
C
NH2 O
O
O2/OH-
NH2
COO-
COO-
+ hn + N2 + H2O
1) Chemical systems
- Luminol (used to detect blood)
- phenyl oxalate ester (glow sticks)

21. 2) Biochemical systems
- Luciferase (Firefly enzyme)
Luciferin + O2
Luciferase
O C
O O
C R2
R1
Spontaneous
CO2 + O C*
R2
R1
Light
S
N
HO
N
S
O
HO
Luciferin (firefly)
“Glowing” Plants
Luciferase gene cloned into plants