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 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

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. Example of Phosphorescence
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
Spins unpaired
net magnetic field
Fluorescence
Phosphorescence
0 sec
1 sec
640 sec
Example of Phosphorescence

4. 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.

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. kf + ki + kec+ kic + kpd + kd
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. ‚ fluorescence especially favored for rigid structures
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:
quinoline indole fluorene 8-hydroxyquinoline

12. resonance forms of aniline
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
resonance forms of aniline

13. Change in fluorescence as a function of cellular oxygen
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.

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

18. Spectrofluorometer Perkin-Elmer 204
- 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)
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 2
NH3
Li+
8-Hydroxyquinoline
580
0.2 Mg
Sn4+
Flavanol
400
0.1
F-, PO43-, Zr
Zn2+ -
green 10
B, Be, Sb, colored ions
8-Hydroxyquinoline flavanol alizarin garnet R benzoin

20. F) Chemiluminescence Examples:
- 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:
Chemical systems
- Luminol (used to detect blood)
- phenyl oxalate ester (glow sticks)

21. Luciferase gene cloned into plants
2) Biochemical systems
- Luciferase (Firefly enzyme)
“Glowing” Plants
Luciferase gene cloned into plants
Luciferin (firefly)