Fluorescence Spectrophotometry

Fluorescence and phosphorescence are photon emission processes that occur during molecular relaxation from electronic excited states. These photonic processes involve transitions between electronic and vibrational states of polyatomic fluorescent molecules (fluorophores). The Jablonski diagram (Figure 1) offers a convenient representation of the excited state structure and the relevant transitions. Electronic states are typically separated by energies on the order of 10 000 cm . Each electronic state is split into multiple sublevels representing the vibrational modes of the molecule. The energies of the vibrational levels are separated by about 100 cm . Photons with energies in the ultraviolet to the blue-green region of the spectrum are needed to trigger an electronic transition. Further, since the energy gap between the excited and ground electronic states is significantly larger than the thermal energy, thermodynamics predicts that molecule predominately reside in the electronic ground state. The electronic excited states of a polyatomic molecule can be further classified based on their multiplicity. The indistinguishability of electrons and the Pauli exclusion principle require the electronic wave functions to have either symmetric or asymmetric spin states. The symmetric wave functions, also called the triple state, have three forms, multiplicity of three. The antisymmetric wave function, also called the singlet state, has one form, multiplicity of one. To the first order, optical transition couples states with the same multiplicity. Optical transition excites the molecules from the lowest vibrational level of the electronic ground state to an accessible vibrational level in an electronic excited state. Since the ground electronic state is a singlet state, the destination electronic state is also a singlet. After excitation, the molecule is quickly relaxed to the lowest vibrational level of the excited electronic state. This rapid vibrational relaxation process occurs on the time scale of femtoseconds to picoseconds. This relaxation process is responsible for the Stoke shift. The Stoke shift describes the observation that fluorescence photons are longer in wavelength than the excitation radiation. The fluorophore remains in the lowest vibrational level of the excited electronic state for a period on the order of nanoseconds, the fluorescence lifetime. Fluorescence emission occurs as the fluorophore decay from the singlet electronic excited states to an allowable vibrational level in the electronic ground state. The fluorescence absorption and emission spectra reflect the vibrational level structures in the ground and the excited electronic states, respectively. The Frank–Condon principle states the fact that the vibrational levels are not significantly altered during electronic transitions. The similarity of the vibrational level structures in the ground and excited electronic states often results in the absorption and emission spectra having mirrored features. The electronic excited state also has specific polarization properties. Fluorophores are preferentially excited when the polarization of light is aligned along a specific molecular axis (the excitation dipole). Further, the fluorescencephotons subsequently emittedby themolecule will have polarization orientated along another molecular axis (the emission dipole). In general, the excitation and emission dipoles do not coincide. Article