Download Photoluminescence

Photoluminescence
Ashraf M. Mahmoud, Associate professor
Contents
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Principles of photoluminescence
Fluorescence vs phosphorescence
Characteristics of photoluminescence
Excitation and emission spectra
Chemical structure and fluorescence
Fluorescence quenching and inner-filter effect
Laws for relating fluorescence to concentration
Instrumentation of spectrofluorometry
Applications of spectrofluorometry
Photoluminescence
What happens after a molecule has absorbed light ?
Heat (80%)
Exciting light
Excitation
More
Energy
Normal molecule
Excited molecule
Photobleaching
(try to avoid)
Emission of light (20%) (Photoluminescence)
Fluorescence versus Phosphorescence
Definitions:
Photoluminescence: is the emission of an absorbed radiant energy in
the form of light. The emitted light is almost of
wavelength higher than that of the absorbed light.
Fluorescence:
when the emission process occurs very rapidly
after excitation ( l0-6 to 10-9 sec ).
Phosphorescence:
when the light is emitted with a time delay more
than 10-8 sec.
Fluorescence versus Phosphorescence
Vibrational
relaxation
Internal conversion
S2
Intersystem crossing
S1
0-0
Transition
T2
T1
S0
The Electronic levels and Transitions in a fluorescence and phosphorescence
Forbidden transition: no direct excitation of triplet state because
change in multiplicity –selection rules.
Fluorescence – ground state to single state and back.
Phosphorescence ground state to triplet state and back.
Fluorescence
Phosphorescence
10-6 to 10-9 s
10-4 to 10 s
Spins paired
No net magnetic field
Spins unpaired
net magnetic field
Example of
Phosphorescence
0 sec
1 sec
640 sec
Spin multiplicity
S0
.........ground state singlet
S1, S2……excited state singlets
T1, T2….…excited state triplets
The most important selection rule for all systems is that
spin must not change during an electronic transition thus
i.e. multiplicity does not change during an electronic
transition.
In theory therefore, a singlet ground state species can only
transform into a singlet excited state and similarly a triplet
ground state into triplet excited states etc.
Excitation and Emission Spectra
Excitation Spectrum:
very similar in appearance to a typical UV-VIS spectra of the same
molecule.
Emission Spectrum:
It is obtained by measuring fluorescence intensity at varying wavelengths
while the excitation wavelength is constant.
The shape of the emission spectrum is
l1frequently but not always a mirror image
of the excitation spectrum
Fluorescence spectrum is independent of
the wavelength of excitation
STOKES SHIFT
l3
l2
Chemical Structure and Fluorescence
In principle, any molecule that absorbs UV radiation could fluoresce.
• The greater the absorption by a molecule, the greater its fluorescence
intensity.
• Molecules possessing an extensive conjugated double bonds with a
relatively rigid structures have high fluorescence (e.g. Anthracene).
• Electron donating groups (e.g. -OH, -NH2, and -OCH3) enhance the
fluorescence.
• Groups such as -NO2, -COOH, -CH2COOH, -Br, -I and azo groups tend
to inhibit fluorescence. Fluorescein is fluorescent while eosin
(tetrabromofluorescein) is non-fluorescent.
• Polycyclic compounds usually are fluorescent
Chemical Structure and Fluorescence
usually for aromatic compounds (non heteroaromatic)
low energy of p p* transition
Fluorescence increases with number of rings and degree
of condensation.
Examples of fluorescent compounds:
N
H
N
H2
C
O
Zn
N
2
Quinoline
indole
fluorene
8-hydroxyquinoline
Chemical Structure and Fluorescence
The non-fluorescent compound can be converted into a fluorescent
derivative:
• Non fluorescent steroids may be converted to fluorescent compounds
by dehydration using conc. H2SO4.
• Some metals can measured fluorometrically after forming fluorescent
chelates with organic chelating agents.
• Most amino acids do not fluoresce, but fluorescent derivatives are
formed by reaction with dansyl chloride, ninhydrin, ……..etc.
Temperature, Solvent & pH Effects:
- decrease temperature  increase fluorescence (deactivation)
- increase viscosity  increase fluorescence (less collisions)
- fluorescence is pH dependent for compounds with acidic/basic
substituents.
more resonance forms stabilize excited state and fluorescence
Phenol
••
••OH
H
OH -
•• •
••O
•
+
Na
H
N
H
H
N
O
H+
Fluorescent
phenolate anion is not fluorescent
Aniline is not fluorescent
Effect of Dissolved O2:
- increase [O2]  decrease fluorescence
- oxidize compound
- paramagnetic property increase intersystem crossing (spin flipping)
H
H
N
Fluorescence Quenching and Inner-Filter Effect
Fluorescence Quenching:
Decrease the quantum yield (decrease in the efficiency of conversion of
absorbed radiation to fluorescent radiation (e.g. iodide and bromide ions).
Inner-Filter Effect:
A colored species in solution with fluorescent species may interfere by
absorbing the fluorescent radiation (Inner-filter effect).
Potassium dichromate exhibits absorption peaks at 245 and 348 nm,
these overlap with the excitation (275 nm) and emission (350 nm) peaks
for tryptophan and would interfere. The inner-filter effect can also arise
from the too high concentrations of the fluorescent species it self.
Energy source
Inner-filter effect
Concentration and Fluorescence Intensity
The total fluorescence intensity or relative fluorescence intensity (F) is given
by the equation:
F =  Ia
Ia = Intensity of light absorption
 = Quantum yield (constant and a measure for the fraction of absorbed
radiation that is converted into fluorescence radiation. It can be
expressed as:
 = Number of photons emitted / number of photons absorbed
= Quantity of light emitted / quantity of light absorbed
 (Quantum yield) is less than or equal unity, and may be extremely small.
Phosphorescence Quantum Yield
Product of two factors:
- fraction of absorbed photons that undergo intersystem
crossing.
- fraction of molecules in T1 that phosphoresce.
 k isc
P  
 k F  k nr
 k P

 k P  k'nr



knr = non-radiative deactivation of S1.
k’nr = non-radiative deactivation of T1.
Concentration and Fluorescence Intensity
For very dilute solutions, the fluorescence intensity is proportional to both
the concentration and the intensity of the excitation energy:
F = 2.303  Io abC
Factors which result in deviation from the Beer-Lambert’s law can be
expected to have the similar effect in fluorescence.
Deviations at higher
concentrations can be attributed to
either
self-quenching or self-absorption.
Instrument for Fluorometric Analysis: Fluorometer
Sample cuvette
Condensing
lens
Mercury
vapour lamp
Excitation
monochromator
Emission
monochromator
Detector
Amplifier
Meter
Components of Fluorometers
• Light sources
– low pressure Hg lamp → sharp lines energy
• 254, 302, 313 nm lines
– high pressure xenon arc lamp → smooth spectrum
– Lasers
• Wavelength selectors
– Filters
or
monchromators
• Detectors
– photomultipliers
or
cameras
• Cells and sample compartments
– quartz cells or sample cuvettes is fused silica,
transparent from all sides
– light tight compartments to minimize stray light
Fluorometer vs Spectrophotometer
Comparison of this schematic with that of a spectrophotometer shows two
basic differences:
1. The fluorometer contains two monochromators, one before and one
after the sample, whereas a spectrophotometer has only one.
2 . In fluorescence, the detector is placed at right angle to the incident
light to separate the emitted light from transmitted.
Since fluorescence intensity is proportional to the intensity of incident
light, the light source must be very stable. Therefore, two-photocells
(similar in spectral response) instruments are to be used.
Applications of Spectrofluorometry
1. Fluorometry is generally used if there is no spectrophotometric method
sufficiently sensitive or selective for the substance to be determined.
2. Analysis of metals: The most frequent applications are for the
determination of metal ions as fluorescent organic complexes.
(e.g. Aluminium forms fluorescent complex with eriochrome blue black).
3. Analysis of non-metallic elements and anion
species: Involve derivatization reactions
leading to ring closure. (e.g. condensation
reaction between boric acid and benzoin.
CH
O
O
C
B
C
O
O
C
4. Analysis of organic compounds (e.g. quinine, riboflavin and thiamine).
The most powerful application of the fluorescence phenomenon is the
quantitative determination of the β-radioactive substances in solutions
Excited solvent molecule
excited fluor
light emitted
Practical Consideration in Spectrofluorometry
Fluorometry is extremely sensitive; limited to very low concentrations,
which have number of problems:
1. Less stable than more concentrated solutions.
2. Adsorption onto the surfaces of the containers is a serious problem.
3. Oxidation of trace substances may be a problem; presence of peroxides
in ether (used as solvent for organic compounds) may cause oxidation of
the test substances.
4. Photodecomposition is more likely to occur at low concentrations and
so these solutions should be protected from light.
Applications of Spectrofluorometry
Introduction of fluorescence in non-fluorescent molecules
Chemical modifications
Addition of
chemical substance
Introduction
of fluorescence
Physicochemical modifications
Changes in
solvent polarity
Processes of
electron transfer
Chemical
treatment
Redox
reactions
Formation
of complexes
Substitution
reactions
Chemoluminescence
It is a 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 of Chemical Systems giving off light:
Direct CL reaction
1. Luminol CL reaction
(used to detect blood)
NH2 O
NH2 O
NH
NH
O
Luminol
Oxidant,OH-
O
O
Catalyst
O
*
Catalyst
Enhancer
Inhibitor
O
+ N2
Excited state
3-Aminophthalate
Oxidant
NH2 O
+
O
O
Ground state
3-Aminophthalate
hn
Mechanism of luminol chemiluminescence
Primary oxidation step
NH2 O
NH2 O
NH
NH
O
Luminol
OH-
NH2 O
NH2 O
N
NH
Oxidant
N
N
N
N
O
O
O
.Luminol monoanion (LH- ) Luminol radical (L ) Diazaquinone (L)
NH2 O
O
N
ON
O
Secondary oxidation step
.
OHO2-
2
NH2 O
hν
O*
N2
NH2 O
O
O
O
Excited state
3-Aminophthalate
NH2 O
N
N
N
ON
OH
Luminol
endoperoxide
O OH
O
Luminol hydroperoxide
Examples of Chemical Systems giving off light:
2. Ruthenium(III) chemiluminescence
Ru(bpy)32+
Oxidation
Ru(bpy)33+ + e-
Ru(bpy)33+ Reductants Ru(bpy)32+*
Ru(bpy)32+*
Ru(bpy)32+ + hu
(lmax = 620 nm)
tris(2,2`-bipyridine)ruthenium(III)
Online Oxidation could be done:
1- Electrically
2- Photochemically
3-chemically
Because Ru (III) is unstable compound
3. KMnO4 chemiluminescence
MnO4-
Reductants
Mn(II) *
Mn (II) + hu
(lmax = 640 nm)
Examples of Chemical Systems giving off light:
Indirect CL reaction
Peroxyoxalate chemiluminescence (PO-CL) reaction
O
Ar O C
O
or
O O
H2O2
C O Ar
Aryloxalate
O
O O
O
O Ar O
Imidazole
Fluorophore
OH
*
Fluorophore
Energy
transfer
Dioxetane derivatives
hn
H 2 O2
Catalyst
Fluorophore
Examples of biological systems giving off light:
Luciferase (Firefly enzyme)
O
O
R2
Luciferase
Luciferin + O2
O
C
2
C
Spontaneous
R
CO2 +
O
Light
C*
R1
R1
N
S
HO
S
N
O
Luciferin (firefly)
HO
“Glowing” Plants
Luciferase gene cloned into plants