2. Photoluminescence
Ashraf M. Mahmoud, Associate professor

3. Contents 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

4. Photoluminescence What happens after a molecule has absorbed light ?
Heat (80%)
Exciting light
Normal molecule
Excited molecule
Photobleaching
(try to avoid)
More
Energy
Excitation
Emission of light (20%) (Photoluminescence)

5. 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 sec.

6. Fluorescence versus Phosphorescence
Vibrational relaxation
Internal conversion S2 S1
Intersystem crossing
0-0
Transition T2 T1
Phosphorescence
(10-4 –10 sec)
(10-9 –10-6 sec)
Fluorescence
Absorption
(10-15 sec) S0
The Electronic levels and Transitions in a fluorescence and phosphorescence

7. Afterglow in 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
0 sec
1 sec
640 sec
Example of Phosphorescence
Afterglow in phosphorescence

8. Spin multiplicity
S 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.

9. 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.

10. The shape of the emission spectrum is frequently but not always a mirror image of the excitation spectrum
Fluorescence spectrum is independent of the wavelength of excitation l1
STOKES SHIFT l2 l3

11. 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

12. 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:
Quinoline indole fluorene hydroxyquinoline

13. 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.

14. 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 • Na +
Phenol
phenolate anion is not fluorescent
Fluorescent
Aniline is not fluorescent
Effect of Dissolved O2:
- increase [O2]  decrease fluorescence
- oxidize compound
- paramagnetic property increase intersystem crossing (spin flipping)

15. Fluorescence Quenching and Inner-Filter Effect
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.

16. Energy source
Inner-filter effect

17. 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.

18. Phosphorescence Quantum Yield
Product of two factors:
fraction of absorbed photons that undergo intersystem crossing.
fraction of molecules in T1 that phosphoresce.
knr = non-radiative deactivation of S1.
k’nr = non-radiative deactivation of T1.

19. 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 =  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.

21. Instrument for Fluorometric Analysis: Fluorometer
Sample cuvette
Condensing lens
Excitation monochromator
Mercury vapour lamp
Emission
monochromator
Detector
Amplifier
Meter

22. 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

23. 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.

24. 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.
4. Analysis of organic compounds (e.g. quinine, riboflavin and thiamine).

25. 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

26. 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.

27. Applications of Spectrofluorometry
Introduction of fluorescence in non-fluorescent molecules
Chemical modifications
Physicochemical modifications
Introduction
of fluorescence
Chemical treatment
Addition of chemical substance
Changes in solvent polarity
Processes of electron transfer
Redox reactions
Formation of complexes
Substitution reactions

28. Chemoluminescence Relatively new, few examples A + B  C*  C + hn
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

29. * Examples of Chemical Systems giving off light: Direct CL reaction h
1. Luminol CL reaction
+ N2 *
Luminol + h
Oxidant,OH-
Catalyst
(used to detect blood)
Excited state
3-Aminophthalate
Ground state
Oxidant Catalyst
Enhancer Inhibitor

30. Mechanism of luminol chemiluminescence
Primary oxidation step
Luminol
Diazaquinone (L)
Luminol monoanion (LH- )
Luminol radical (L.- )
OH-
Oxidant
O2-.
HO2-
Secondary oxidation step
Luminol hydroperoxide N2
Luminol endoperoxide hν *
Excited state
3-Aminophthalate

31. Examples of Chemical Systems giving off light:
2. Ruthenium(III) chemiluminescence
Ru(bpy)32+
tris(2,2`-bipyridine)ruthenium(III)
Oxidation
Ru(bpy)33+ + e-
Reductants
Ru(bpy)33+
Ru(bpy)32+*
Ru(bpy)32+ + hu (lmax = 620 nm)
Ru(bpy)32+*
Online Oxidation could be done:
1- Electrically Photochemically chemically
Because Ru (III) is unstable compound
3. KMnO4 chemiluminescence
MnO4-
Reductants
Mn(II) *
Mn (II) + hu (lmax = 640 nm)

32. * Examples of Chemical Systems giving off light: Indirect CL reaction
Peroxyoxalate chemiluminescence (PO-CL) reaction *
Aryloxalate h
H2O2
Imidazole
Fluorophore
Energy
transfer
Dioxetane derivatives
H2O Fluorophore
Catalyst

33. Luciferase gene cloned into plants
Examples of biological systems giving off light:
Luciferase (Firefly enzyme)
Luciferin (firefly)
“Glowing” Plants
Luciferase gene cloned into plants

34. Good Luck
Ashraf M. Mahmoud