Molecular Fluorescence and Phosphorescence Spectra
To appreciate the origin of molecular fluorescence and phosphorescence, we must consider what happens to a molecule following the absorption of a photon. Let’s as- sume that the molecule initially occupies the lowest vibrational energy level of its electronic ground state. The ground state, which is shown in Figure 10.43, is a sin- glet 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. These relaxation mechanisms are shown in Figure 10.43. 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.
One form of radiationless deactivation is vibra- tional 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 are excited to different vibrational energy levels of the same excited elec- tronic state quickly return to the lowest vibrational energy level of this excited state.
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. 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 compo- nent 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 10.43 between a singlet excited state, S1, and a triplet excited state, T1.
Fluorescence occurs when a molecule in the lowest vibrational en- ergy level of an excited electronic state returns to a lower energy electronic state by emitting a photon. Since molecules return to their ground state by the fastest mech- anism, 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, Φf, which is the fraction of excited molecules returning to the ground state by fluores- cence. Quantum yields range from 1, when every molecule in an excited state un- dergoes 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
where k is a constant accounting for the efficiency of collecting and detecting the fluorescent emission. From Beer’s law we know that
where C is the concentration of the fluorescing species. Solving equation 10.30 for PT and substituting into equation 10.29 gives, after simplifying
For low concentrations of the fluorescing species, where εbC is less than 0.01, this equation simplifies to
The intensity of fluorescence therefore, increases with an increase in quantum effi- ciency, incident power of the excitation source, and the molar absorptivity and con- centration of the fluorescing species.
Fluorescence is generally observed with molecules where the lowest energy ab- sorption is a π → π* transition, although some n → π* transitions show weak fluo-rescence. Most unsubstituted, nonheterocyclic aromatic compounds show favorable fluorescence quantum yields, although substitution to the aromatic ring can have a significant effect on nf. For example, the presence of an electron-withdrawing group, such as —NO2, decreases Φ whereas adding an electron-donating group, such as —OH, increases Φf. 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 vari- ables such as temperature and solvent. Increasing temperature generally decreases Φf because more frequent collisions between the molecule and the solvent increases external conversion. Decreasing the solvent’s viscosity decreases for similar rea- sons. 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 10.43, fluorescence may return the molecule to any of several vibrational energy levels in the ground electronic state. Fluorescence, therefore, occurs over a range of wavelengths. Because the change in energy for fluorescent emission is generally less than that for absorption, a molecule’s fluorescence spec- trum is shifted to higher wavelengths than its absorption spectrum.
A molecule in the lowest vibrational energy level of an excited triplet electronic state normally relaxes to the ground state by an intersystem cross- ing to a singlet state or by external conversion. Phosphorescence is observed when relaxation occurs by the emission of a photon. As shown in Figure 10.43, phospho- rescence occurs over a range of wavelengths, all of which are at a lower energy than the molecule’s absorption band. The intensity of phosphorescence, Ip, is given by an equation similar to equation 10.32 for fluorescence
where Φp is the quantum yield for phosphorescence.
Phosphorescence is most favorable for molecules that have n → π* transitions, which have a higher probability for an intersystem crossing than do π → π* transitions. For example, phosphorescence is observed with aromatic molecules contain- ing carbonyl groups or heteroatoms. Aromatic compounds containing halide atoms also have a higher efficiency for phosphorescence. In general, an increase in phos- phorescence corresponds to a decrease in fluorescence.
Since the average lifetime for phosphorescence is very long, ranging from 10–4 to 104 s, the quantum yield for phosphorescence is usually quite small. An improve- ment in Φp is realized by decreasing the efficiency of external conversion. This may be accomplished in several ways, including lowering the temperature, using a more viscous solvent, depositing the sample on a solid substrate, or trapping the molecule in solution.
Photoluminescence spectra are recorded by measuring the intensity of emitted radiation as a function of either the excitation wavelength or the emission wavelength. An excitation spectrum is obtained by monitoring emission at a fixed wavelength while varying the excitation wavelength. Figure 10.44 shows the excitation spectrum for the hypothetical system described by the energy level diagram in Figure 10.43. When corrected for variations in source intensity and detector response, a sample’s excitation spectrum is nearly identical to its absorbance spectrum. The excitation spectrum provides a convenient means for selecting the best excitation wavelength for a quantitative or qualitative analysis.
In an emission spectrum a fixed wavelength is used to excite the mol- ecules, and the intensity of emitted radiation is monitored as a function of wavelength. Although a molecule has only a single excitation spectrum, it has two emission spectra, one for fluorescence and one for phosphores- cence. The corresponding emission spectra for the hypothetical system in Figure 10.43 are shown in Figure 10.44.
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