Instrument: Edinburgh Instruments FLS980-xs2
Like other spectroscopic techniques fluorescence spectroscopy uses the result of interaction of electromagnetic radiation with matter to provide insight into the chemical composition of the matter. In the case of fluorescence spectroscopy, ultraviolet or visible light is used to excite a sample and the absorbance or emitted light is used to characterise the fluorophore present.
The technique can be used to quantify an analyte of interest as well as to observe changes to a fluorescent molecule.
Excitation of electrons within an atom or molecule by absorption of radiation can lead to the electron existing in a higher energy state. Since this excited state is unstable, the absorbed energy is dissipated either by decomposition, reacting with other species or re-emission.
Fluorescence arises when the absorbed energy is re-emitted, typically in the form of visible light. The light emitted is of lower energy (longer wavelength) than the incident light since some energy is lost during vibrational relaxation of the molecule via collisions.
The energies (wavelengths) at which a molecule will absorb/ emit radiation are dependent on the chemical composition of the molecule. Thus, the absorption/ emission spectra provides a chemical “fingerprint” for molecules which fluoresce.
Structural changes in compounds which fluoresce can be detected using fluorescence spectroscopy, which can be useful for monitoring changes during processing/reaction of species of interest.
Aromatic amino acids such as tryptophan fluoresce, which means that fluorescence spectroscopy can reveal structural information about proteins (protein conformation). In addition, changes in fluorescent intensity can reveal information about chemical structure following binding of a fluorophore with another species.
Quantitative analysis of a species of interest is also possible by fluorescence spectroscopy.
Samples are typically dissolved in a suitable solvent, such as water or ethanol. Samples should contain a fluorophore, that is, a region within a molecule that will re-emit absorbed light.
Generally, molecules that contain fluorophores have one or more aromatic group. Some molecules which don’t normally fluoresce can be made fluorescent by the addition of a fluorescent label thus rendering them suitable for the studies outlined above.
The solvent used should be chosen carefully as it can affect the fluorescence spectrum and even prevent fluorescence by quenching (excited energy dissipated by means other than re-emission).