Liquid Chromatography Detectors - The Fluorescence Detector > Page 55

The Fluorescence Detector

When light is emitted by molecules that are excited by electromagnetic radiation, the phenomenon is termed photoluminescence. If the release of electro-magnetic energy is immediate, or stops on the removal of the excitation radiation, the substance is said to be fluorescent. If, however, the release of energy is delayed, or persists after the removal of the exciting radiation, then the substance is said to be phosphorescent. Fluorescence has been shown to be extremely useful as a detection process and detectors based on fluorescent measurement have provided some of the highest sensitivities available in LC.

When a molecule adsorbs light, a transition to a higher electronic state takes place and this absorption is highly specific for the molecules concerned; radiation of a specific wavelength or energy is only absorbed by a particular molecular structure. If electrons are raised to an upper excited single state, due to absorption of light energy, and the excess energy is not immediately dissipated by collision with other molecules or by other means, light will be emitted at a lower frequency as the electron returns to its ground state and the substance is said to fluoresce. As some energy is always lost before emission occurs then, in contrast to Raman scattering, the wavelength of the fluorescent light is always greater than the incident light. 

Detection techniques based on fluorescence affords greater sensitivity to sample concentration, but less sensitivity to instrument instability, (e.g. sensor temperature and pressure). This is due to the fluorescent light being measured against a very low light background (i.e., against a very low noise level). This is opposite to light absorption measurements where the signal is superimposed on a strong background signal carrying a high noise level. Unfortunately, relatively few compounds fluoresce in a practical range of wavelengths. However, some compounds, including products from foods, drugs, dye intermediates etc., do exhibit fluorescence and can be monitored by fluorescent means. In addition, many substances can be made to fluoresce by forming appropriate derivatives.