Page 74 - Tandem Techniques

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Fluorescence Spectroscopy

Fluorescence spectroscopy has two unique advantages over UV spectroscopy for solute identification in
tandem systems. Firstly, the spectrometer has an order of magnitude greater sensitivity. Secondly
florescence spectra often show greater detail than UV spectra and are consequently more reliable for
identification purposes by spectra matching. In addition, for a given compound, fluorescence spectra
can be obtained over a large number of different excitation wavelengths, each providing a unique
spectrum that improves the confidence of identification.

Fluorescence is a specific type of luminescence. When molecules are excited by electromagnetic
radiation to produce luminescence, the emitted light is called photoluminescence. If the release of
electromagnetic energy is immediate, or stops on the removal of the exciting radiation, the substance is
said to be fluorescent. If the release of energy is delayed, or persists after the removal of the exciting
radiation, then the substance is said to be phosphorescent. When light is adsorbed by a molecule, and a
transition to a higher electronic state occurs, the wavelength at which this happens will be determined
by the structure of the particular substance. When electrons are raised to an upper excited single state as
a result of the adsorption of light energy, the transitions produce the characteristic UV or visible
adsorption spectrum of the respective compound. If the excess energy of the molecule in the excited
state is not dissipated rapidly by collisions with other molecules, or by other means, the electron will
return to the ground state with the emission of electromagnetic radiation in the form of fluorescence. As
some energy is inevitably lost before the emission occurs, the emitted fluorescent light is always of a
longer wavelength than that absorbed on excitation. In due course Raman spectroscopy will be
discussed and it will be seen the two processes have considerable similarity. However, the wavelength
of the emitted light is different from that produced by fluorescence.

Discussions on the theoretical basis of fluorescence have been given by Guilbault [3], Udenfriend [4]
and Rhys Williams [5].