High Performance Liquid Chromatography coupled with Fluorescence Detector is based on the property that certain organic molecules have of being able to emit light radiation after excitation by UltraViolet or Near Visible radiation. When light energy is absorbed by a molecule, it elevates some of the electrons to an excited state. When these electrons return to the ground state, light is emitted and this process is called fluorescence.
In principle we encounter two types of wavelengths with this detector. First of all, the emission wavelength λem (radiation emitted by the molecule) which is different and always higher than the wavelength of the excitation radiation λex. Usually the emission is measured at right angles to the excitation. The angle ensures that the detected radiation only comes from the fluorescence of the sample.
The λex/λem ratio is much more selective than the UV absorption wavelength, allowing the fluorescence detector to perform very specific and much more sensitive analyzes.