Introduction to Fluorescence and Phosphorescence

1 1 Introduction to Fluorescence and Phosphorescence Credits: This notes are a summary from Modern Analytical Chemistry by David Harvey, Molecular Fluorescence : Principles and Applications (Wiley 2001) by Bernard Valeur, and application notes from Perkin Elmer Instruments. Prepared by Jose Hodak for 2302303 Spectroscopy for Chemistry BSAC program 2008. Photoluminescence in the ultraviolet-visible comprises two similar phenomena: Fluorescence and Phosphorescence . Molecules have energy levels determined by the molecular orbitals that hold the molecule bound together. In the case of atoms it is the atomic orbitals what determines the energy levels of the electrons. In this section we will concern ourselves with molecular photoluminescence. We will leave the atomic emission phenomena for the next section. Absorption of an ultraviolet or visible photon promotes a valence electron from its ground state to an excited state with conservation of the electron s spin.

2 For example, a pair of electrons occupying the same electronic ground state have opposite spins (Figure 1) and are said to be in a singlet spin state. Absorbing a photon promotes one of the electrons to a singlet excited state (Figure 1b). This phenomenon is called excitation The ectited states are not stable and will not stay indefinitely. If we observe a molecule in the excited state, at some random moment it will spontaneously return to the ground state. This return process is called decay, deactivation or relaxation. Under some special conditions, the energy absorbed during the excitation process is released during the relaxation in the form of a photon. This type of relaxation is called emission. Emission of a photon from a singlet excited state to a singlet ground state, or between any two energy levels with the same spin, is called Fluorescence .

3 The probability of a fluorescent transition is very high, and the average lifetime of the electron in the excited state is only 10 5 10 8 s. Fluorescence , therefore, decays rapidly after the excitation source is removed. In some cases an electron in a singlet excited state is transformed to a triplet excited state (Figure 1c) in which its spin is no longer paired with that of the ground state. Emission between a triplet excited state and a singlet ground state, or between any two energy levels that differ in their respective spin states, is called Phosphorescence . Because the average lifetime for Phosphorescence ranges from 10 4 to 104 s, Phosphorescence may continue for some time after removing the excitation source. 2 Figure 1 History: The use of molecular Fluorescence for qualitative analysis and semiquantitative analysis can be traced to the early to mid-1800s, with more accurate quantitative methods appearing in the 1920s.

4 Instrumentation for Fluorescence spectroscopy using filters and monochromators for wavelength selection appeared in, respectively, the 1930s and 1950s. Although the discovery of Phosphorescence preceded that of Fluorescence by almost 200 years, qualitative and quantitative applications of molecular Phosphorescence did not receive much attention until after the development of Fluorescence instrumentation. The use of molecular Fluorescence for qualitative analysis and semiquantitative analysis can be traced to the early to mid-1800s, with more accurate quantitative methods appearing in the 1920s. Instrumentation for Fluorescence spectroscopy using filters and monochromators for wavelength selection appeared in, respectively, the 1930s and 1950s. Although the discovery of Phosphorescence preceded that of Fluorescence by almost 200 years, qualitative and quantitative applications of molecular Phosphorescence did not receive much attention until after the development of Fluorescence instrumentation.

5 Photophysical proceses: A molecule in the excited state can decay in several ways. It might also use the extra energy gathered during excitation to carry out processes which it cannot do while it is in the ground state. What else can occur from the excited state? Let s assume that the molecule initially occupies the lowest vibrational energy level of its electronic ground state. The ground state, which is shown in Figure 2, is a singlet state labeled S0. Absorption of a photon of correct energy excites the molecule to one of several vibrational energy levels in the first excited electronic state, S1, or the second electronic excited state, S2, both of which are singlet states. Relaxation to the ground state from these excited states occurs by a number of mechanisms that are either radiationless, in that no photons are emitted, or involve the emission of a photon.

6 These relaxation mechanisms are shown in Figure 2. The most likely pathway by which a molecule relaxes back to its ground state is that which gives the shortest lifetime for the excited state. Radiationless Deactivation One form of radiationless deactivation is vibrational relaxation, in which a molecule in an excited vibrational energy level loses energy as it moves to a lower vibrational energy level in the same electronic state. Vibrational relaxation is very rapid, with the molecule s average lifetime in an excited vibrational energy level being 10 12 s or less. As a consequence, molecules that 3are excited to different vibrational energy levels of the same excited electronic state quickly return to the lowest vibrational energy level of this excited state. Figure 2 Another form of radiationless relaxation is internal conversion, in which a molecule in the ground vibrational level of an excited electronic state passes directly into a high vibrational energy level of a lower energy electronic state of the same spin state.

7 By a combination of internal conversions and vibrational relaxations, a molecule in an excited electronic state may return to the ground electronic state without emitting a photon. A related form of radiationless relaxation is external conversion in which excess energy is transferred to the solvent or another component in the sample matrix. A final form of radiationless relaxation is an intersystem crossing in which a molecule in the ground vibrational energy level of an excited electronic state passes into a high vibrational energy level of a lower energy electronic energy state with a different spin state. For example, an intersystem crossing is shown in Figure 2. between a singlet excited state, S1, and a triplet excited state, T1. Fluorescence Fluorescence occurs when a molecule in the lowest vibrational energy level of an excited electronic state returns to a lower energy electronic state by emitting a photon.

8 Since molecules return to their ground state by the fastest mechanism, Fluorescence is only observed if it is a more efficient means of relaxation than the combination of internal conversion and vibrational relaxation. A quantitative expression of the efficiency of Fluorescence is the fluorescent quantum yield,f , which is the fraction of excited molecules returning to the ground state by Fluorescence . Quantum yields range from 1, when every molecule in an excited state undergoes Fluorescence , to 0 when Fluorescence does not occur. The intensity of Fluorescence , If, is proportional to the amount of the radiation from the excitation source that is absorbed and the quantum yield for Fluorescence ()ff0TI = k P - P (1) 4where k is a constant accounting for the efficiency of collecting and detecting the fluorescent emission.

9 From Beer s law we know that -bCT0P10P = (2) where C is the concentration of the fluorescing species. Solving equation (2) for PT and substituting into equation (1) gives, after simplifying ()-bCff0I = k P 1-10 (3) For low concentrations of the fluorescing species, where ebC is less than , this equation simplifies to ff0I = PbC (4) The intensity of Fluorescence therefore, increases with an increase in quantum efficiency, incident power of the excitation source, and the molar absorptivity and concentration of the fluorescing species. Fluorescence is generally observed with molecules where the lowest energy absorption is a * transition, although some n * transitions show weak Fluorescence . Most unsubstituted, nonheterocyclic aromatic compounds show favorable Fluorescence quantum yields, although substitution to the aromatic ring can have a significant effect on f.

10 For example, the presence of an electron-withdrawing group, such as NO2, decreases f, whereas adding an electron-donating group, such as OH, increases f. Fluorescence also increases for aromatic ring systems and for aromatic molecules with rigid planar structures. A molecule s Fluorescence quantum yield is also influenced by external variables such as temperature and solvent. Increasing temperature generally decreases f because more frequent collisions between the molecule and the solvent increases external conversion. Decreasing the solvent s viscosity decreases f for similar reasons. For an analyte with acidic or basic functional groups, a change in pH may change the analyte s structure and, therefore, its fluorescent properties. Changes in both the wavelength and intensity of Fluorescence may be affected. As shown in Figure 2, Fluorescence may return the molecule to any of several vibrational energy levels in the ground electronic state.