Photoluminescence Ashraf M. Mahmoud, Associate professor

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

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)

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 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

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

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 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

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: 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 • 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)

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.

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. 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 Excitation monochromator Mercury vapour lamp 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. 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 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

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

* 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

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

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 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 * Aryloxalate h H2O2 Imidazole Fluorophore Energy transfer Dioxetane derivatives H2O2 Fluorophore Catalyst

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

Good Luck Ashraf M. Mahmoud