Chapter 5 Flourescence and phosphorescence.pdf
- 1. Chapter 5 Molecular fluorescence spectroscopy
- 2. Fluorescence and Phosphorescence are molecular spectroscopic methods which are based on the phenomenon of emission of radiation by electronically excited species and are collectively termed as photoluminescence methods. ORIGIN OF FLUORESCENCE AND PHOSPHORESCENCE SPECTRA Fluorescence and phosphorescence spectra are originated from radiative relaxation processes associated with electronically excited species. In some of the processes the absorbed energy is given off as heat to the surroundings and in some it is emitted as radiation. When such a relaxation is accompanied by the emission of a radiation it is called luminescence (Fluorescence and phosphorescence).
- 3. There are two types of luminescence phenomena. Fluorescence is the emission of visible light by a substance that has absorbed light of a different wavelength. The emitted photon has a longer wavelength. Phosphorescence is related to fluorescence in emitting a photon, however, a phosphorescent material does not immediately re-emit the radiation it absorbs. As the excitation of the molecule is due to the absorption of a photon (light), these types of luminescence are called photoluminescence. Chemiluminescence is another phenomenon that falls in the category of luminescence. This refers to the emission of radiation during a chemical reaction.
- 4. Jablonski Diagram The Jablonski diagram gives a representation of ground and different excited electronic states of a molecule and the processes associated with absorption and emission (radiative and nonradiative) of energy.
- 5. In the Jablonski diagram two excited singlet states (S1 and S2) and a triplet state (T1) are shown. The absorption and emission of the radiation leads to the activation and deactivation processes. The absorption of a photon of suitable energy causes the molecule to get excited from the ground state to one of the excited states. This process is called as excitation or activation and is governed by Franck-Condon principle. Once the molecule gets excited by the absorption of radiation, it does not stay excited indefinitely; a number of processes bring it back to the ground state. This is called deactivation. The deactivation processes can be broadly categorised into two groups given below. • Nonradiative deactivation • Radiative deactivation In rare occasions, the molecule in the vibrational states of a singlet excited state may cross over to a vibrational level of a triplet state if the two have same energy. This process is called intersystem crossing.
- 6. Fluorescence occurs when i) a molecule returns to the electronic ground state from an excited singlet state by losing its excess energy as a photon. ii) a molecule lowers its vibrational energy by losing its excess energy as a photon. Intersystem crossing refers to i) the reversal of the spin of an excited electron, changing the state of the molecule (from singlet state to triplet state or vice versa). ii) the loss of excess energy by the molecule by emitting a photon. iii) the conversion of the excess electronic energy by the molecule to vibrational energy.
- 7. FLUORESCENCE SPECTRUM A plot of the emitted radiation as a function of wavelength for any given excitation wavelength is known as the emission fluorescence spectrum. The excitation radiation is shown to have caused the transition from S0 to S2 state. Plot of emission wavelength against the wavelength of exciting radiation is known as the excitation spectrum. Fig.: The excitation and emission fluorescence spectra of 9-methylanthracene
- 8. Since a given analyte can fluoresce only after it has absorbed radiation, an excitation spectrum consists of the wavelengths of light that the analyte is able to absorb. The wavelength difference between the absorption and fluorescence maxima is called the Stokes shift. Molecular structure is one of the important parameters in determining the possibility and extent of fluorescence in a chemical species. Besides structure, the fluorescent behaviour of a molecule is affected by the chemical environment in which it exists. Similarly the phosphorescence is also shown by a few species and there is a kind of correlation with the structure and environment. Compounds with fused ring are found to be especially fluorescent, and the extent of fluorescence is found to be directly proportional to the number of rings in the molecule. The structural rigidity in a molecule favours fluorescence.
- 9. Factors affecting Fluorescence and Phosphorescence The fluorescence spectrum and intensity of a molecule often depend strongly on the molecule’s environment. The common factors affecting the fluorescence are as follows. Temperature pH Dissolved oxygen Solvent Temperature: A rise in temperature is almost always accompanied by a decrease in fluorescence. The change in temperature causes the viscosity of the medium to change which in turn changes the number of collisions of the molecules of the fluorophore with solvent molecules. The increase in the number of collisions between molecules in turn increases the probability for deactivation by internal conversion and vibrational relaxation.
- 10. pH: Relatively small changes in pH can sometimes cause substantial changes in the fluorescence intensity and spectral characteristics of fluorescence. In the molecules containing acidic or basic functional groups, the changes in pH of the medium change the degree of ionisation of the functional groups. This in turn may affect the extent of conjugation or the aromaticity of the molecule which affects its fluorescence. Dissolved oxygen: The paramagnetic substances like dissolved oxygen and many transition metals with unpaired electrons dramatically decrease fluorescence and cause interference in fluorimetric determinations. The paramagnetic nature of molecular oxygen promotes intersystem crossing from singlet to triplet states in other molecules. The longer lifetimes of the triplet states increases the opportunity for radiationless deactivation to occur. Presence of dissolved oxygen influences phosphorescence too and causes a large decrease in the phosphorescence intensity.
- 11. FLUORESCENCE QUENCHING The fluorescence emission is quite sensitive to the presence of impurities and other species in the sample. These cause a decrease in the intensity of fluorescence emission. This decrease in the fluorescence intensity arising out of the interaction of the excited state of the fluorophore with its surroundings is called quenching. For example, the quinine fluorescence is quenched by the presence of halide ions. One of the mechanisms of quenching involves collisions between excited and ground state molecules leading to an increase in the amount of radiationless relaxation. It is called self-quenching and it alters the ratio of excited molecules that relax via the fluorescence pathway. Since self-quenching depends on the rate at which collisions occur, it increases with an increase in the concentration of the analyte. Due to self-quenching, the photoluminescence efficiency varies with the concentration.
- 12. Another mechanism that leads to the decrease in fluorescence intensity is called self-absorption or the inner-cell effect. It is observed in the molecules in which the absorption band overlaps with the wavelength of the emitted (fluoresced) photon. The intermolecular electronic energy transfer from the excited molecule to a quencher molecule is one of the common ways by which the fluorescence quenching occurs. The process can be represented as follows. where, the excited analyte molecule (M*) transfers its excitation energy to a quencher molecule Q, whereby it gets de-excited to M forming an excited quencher molecule, Q*.
- 13. Mathematically, the quenching generally follows the Stern–Volmer equation given below.
- 14. Quantum Yield A number of processes contribute towards the deactivation of the excited state. Only a certain fraction of the excited molecules relax through the fluorescence pathway i.e., by emission of radiation. This fraction is quantified in terms of a parameter called quantum efficiency or quantum yield. Mathematically, the quantum yield is defined as the quotient of the number of photons that are emitted and the number of photons that are absorbed. It can be written as the following equation.
- 16. Instrumentation for Fluorescence Measurement All fluorescence instruments use essentially the same components as are used in absorption spectrophotometers. However, the geometric arrangement of the components is somewhat different. This is due to the reason that any transmitted radiation is not measured along with the fluorescence. You know that the absorption and transmission of radiant energy occur only along the direction of the incident light whereas the fluorescence radiation emanates in all directions. The detection of transmitted radiation is avoided by placing the detector at a right angle to the transmitted beam, as shown in Fig.
- 17. Fig.: The relative location of the light source and the detector in a fluorescence measurement set up
- 18. The essential components of an instrument used to measure fluorescence of the sample are: • Excitation light sources – more intense • Filters or monochromators • Sample holder • Detector • Readout device Currently, two broad categories of commercial instruments are being manufactured. The low cost instruments needed for routine measurements are based on filters as wavelength selectors whereas the more sophisticated spectrofluorometers use monochromators.
- 19. Fig.: A schematic layout of a fluorimeter