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A) Introduction
1.)
10-14 to 10-15 s
10-8 10-9s
M* M + heat
- Excitation of e- by absorbance of h.
- Re-emission of h as e- goes to ground state.
- Use h2 for qualitative and quantitative analysis
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
2.)
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
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 dont 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.
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 (): 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
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
6.)
Types of Transitions:
- seldom occurs from absorbance less
than 250 nm
200 nm => 600 kJ/mol, breaks many bonds
- fluorescence not seen with
- typically * or * n
7.)
H
N
H2
C
O
Zn
N
2
quinoline indole
fluorene
8-hydroxyquinoline
8.)
Fluorescence pH Titration
H
N
H
N
H
N
9.)
= KPo(1 10 bc)
K ~ (quantum yield)
C) Fluorescence Spectra
D) Instrumentation
- basic design
components similar to UV/Vis
spectrofluorometers: observe
both excitation & emission spectra.
Fluorometers
- simple, rugged, low cost, compact
- source beam split into reference and sample beam
- reference beam attenuated ~ fluorescence intensity
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
Al
3+
Reagent
Absorption (nm)
Alizarin garnet R
Fluorescence (nm)
470
Sensitivity (g/ml)
Interference
0.007
500
Al complex of Alizarin
garnet R (quenching)
470
500
0.001
Benzoin
370
450
0.04
Be, Sb
2-(0-Hydroxyphenyl)benzoxazole
365
Blue
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
F-
B4O72Cd2+
Li+
OH
N
OH
O
HO
HO
N
SO3Na
flavanol
OH
OH
8-Hydroxyquinoline
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 + h
Examples:
1) Chemical systems
- Luminol (used to detect blood)
NH2
NH2
COONH
O2/OH-
+ h + N2 + H2O
NH
C
COO-
2) Biochemical systems
- Luciferase (Firefly enzyme)
Luciferin + O2
Luciferase
O
C
1
R2
Spontaneous
R2
CO2 +
Light
C*
R1
Glowing Plants
N
S
HO
S
N
O
Luciferin (firefly)
HO