Fluorescence detectors are probably the most sensitive among the existing modern HPLC detectors. It is possible to detect even a presence of a single analyte molecule in the flow cell. Typically, fluorescence sensitivity is 10 -1000 times higher than that of the UV detector for strong UV absorbing materials. Fluorescence detectors are very specific and selective among the others optical detectors. This is normally used as an advantage in the measurement of specific fluorescent species in samples.
When compounds having specific functional groups are excited by shorter wavelength energy and emit higher wavelength radiation which called fluorescence. Usually, the emission is measured at right angles to the excitation.
Roughly about 15% of all compounds have a natural fluorescence. The presence of conjugated pi-electrons especially in the aromatic components gives the most intense fluorescent activity. Also, aliphatic and alicyclic compounds with carbonyl groups and compounds with highly conjugated double bonds fluoresce, but usually to a lesser degree. Most unsubstituted aromatic hydrocarbons fluoresce with quantum yeld increasing with the number of rings, their degree of condensation and their structural rigidity.
Fluorescence intensity depends on both the excitation and emission wavelength, allowing selectively detect some components while suppressing the emission of others.
The detection of any component significantly depends on the chosen wavelength and if one component could be detected at 280 ex and 340 em., another could be missed. Most of the modern detectors allow fast switch of the excitation and emission wavelength, which offer the possibility to detect all component in the mixture.. For example, in the very important polynuclear aromatic chromatogram the excitation and emission wavelengths were 280 and 340 nm, respectively, for the first 6 components, and then changed to the respective values of 305 and 430 nm; the latter values represent the best compromise to allow sensitive detection of compounds.
Figure below shows the optical schematic of a typical fluorescence detector for liquid chromatography. The detectors available on the market differ in the method in which the wavelengths are controlled. Less expensive instruments utilize filters; medium priced units offer monochromator control of at least emission wavelength, and full capability research-grade instruments provide monochromator control of both excitation and emission wavelengths.
Optical schematic of a typical fluorescence detector for liquid chromatography.