Phosporesence and spin orbital coupling ppt

Phosporesence and spin orbital coupling

1. Fluorescence, Phosphorescence, & Chemiluminescence
A) Introduction
1.)Theory of Fluorescence and Phosphorescence:
-Excitation of e
-
by absorbance of hn.
-Re-emission of hnas e
-
goes to ground state.
-Use hn
2for 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
-9
s
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
fluorescence10
-15
to 10
-17
10
-7
to 10
-9
Sensitive
For UV/Vis need to observe P
oand P
difference, which limits detection
For fluorescence, only observe
amount of P
L

3. 2.)Fluorescence–ground state to singlestate 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
FluorescencePhosphorescence
0 sec 1 sec 640 sec
Example of
Phosphorescence

4. 3) Jablonski Energy Diagram
S
2, S
1= 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.
T
1= 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
-12
s 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 O
2

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 (O
2)

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 (k
x)of deactivation
processes
f = k
f
k
f+ k
i+ k
ec+ k
ic+ k
pd+ k
d
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
*
por 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
H
2
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 O
2:
-increase [O
2] 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’P
o(1 –10
–ebc
)
K’~ f (quantum yield)
P
o: power of beam
ebc: Beer’s law
F depends on absorbance of light and incident intensity (P
o)
At low concentrations: F = 2.3K’ebcP
o
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
Al
3+
Alizarin garnet R 470 500 0.007
Be, Co, Cr, Cu, F
-
,NO
3-
, Ni, PO
4
-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
B
4O
7
2-
Benzoin 370 450 0.04 Be, Sb
Cd
2+
2-(0-Hydroxyphenyl)-
benzoxazole
365 Blue 2
NH
3
Li
+
8-Hydroxyquinoline 370 580 0.2 Mg
Sn
4+
Flavanol 400 470 0.1 F
-
, PO
4
3-
, Zr
Zn
2+
Benzoin - green 10
B, Be, Sb,
colored ionsN
OH
O
O
OH
OH
HO NN
HO
SO
3Na
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
NH
2 O
O
O
2/OH-
NH
2
COO
-
COO
-
+ hn + N
2 + H
2O
1)Chemical systems
-Luminol (used to detect blood)
-phenyl oxalate ester (glow sticks)

21. 2)Biochemical systems
-Luciferase (Firefly enzyme)Luciferin + O
2
Luciferase
O C
O O
C R
2
R
1
Spontaneous
CO
2 +O C
*
R
2
R
1
Light S
N
HO
N
S
O
HO
Luciferin (firefly)
“Glowing” Plants
Luciferase gene cloned into plants