Chemical Sciences Molecular Absorption Spectroscopy Lab Experiments

Demonstration of pH Effect on Fluorescence Excitation and Emission Spectra of a Fluorophore

Fluorescence is a type of optical spectroscopy in which a molecule is first promoted to an electronically excited state by absorption of ultraviolet, visible, or near infrared radiation and then the excited molecule decays back to the ground state (or to a lower-lying excited electronic state) by emission of light. Molecules that fluoresce are called fluorophores. Many fluorophores exist in different molecular and ionic forms depending on the pH of the solution and environment of the fluorophore. The acidic or basic properties of the fluorophore molecule in the excited state differ from that in the ground state due to the redistribution of the electron density upon electronic excitation. These different molecular and ionic forms essentially turns out to be energetically different absorbing and emitting species of the fluorophore. The life times of fluorophores may increase or decrease depending on the change in pH. Collisional quenching by protons usually results in a shortened decay time. These are functions of both the light absorbing group (chromophore) and other chemical functional groups bound to the fluorophore molecule. These make the absorption and fluorescence spectra pH- and environment-dependent. Thus, one can use the fluorescence characteristics of fluorescent (probe) molecules to make inferences about the (acid-base) properties of immediate molecular environment of the probe molecule. Fluorescent probe studies find important applications in biological science.