Analysis based on fluorescence characteristics of materials by fluroimetric analysis is a highly sensitive technique. At lower concentrations (ng levels or lower) the technique shows higher sensitivity in comparison to UV – Vis spectroscopy. Two major factors contribute to the high sensitivity of fluorimetric determinations.
Limited Number of fluorescent molecules – a large number of molecules absorb significantly in the UV-Visible range but do not show fluorescence appreciably. A relatively small number of molecules exhibit fluorescence. This results in less of interference from compounds showing absorbance in UV-Vis region..
Choice of excitation and emission wavelengths – in comparison to UV – visible spectroscopy where only a single wavelength is available for radiation and absorption fluorescence offers two wavelengths (excitation and emission). This feature is useful for resolving samples having overlapping absorption spectra but show fluorescence at different wavelengths. Similarly two molecules having similar fluorescence spectra can be differentiated by selective choice of excitation wavelengths.
Fluorimetric measurements offer greater sensitivity but have a limitation resulting in decrease of fluorescence intensity due to a number of processes like self- quenching, self- absorption and inner filter effects. Fluorescence quenching is a generic term describing the reduction in intensity on account of interaction of the fluorophore with its neighbouring environment.
Self quenching involves loss of energy through collisions between the excited and ground state molecules. This results in reduction of excited state molecules that relax through fluorescence emissions. Self quenching increases in direct proportion to the rate at which collisions take place.Thus an increase in the concentration of an analyte leads to lowering of fluorescence emissions.
Self absorption or inner- cell effect
In some molecules the absorption wavelength can overlap the wavelength of the emitted fluorescence band. In such situations some emitted photons can be re-absorbed before they are able to leave the solution. In a way self absorption is similar to self- quenching and can result in a non-linear concentration dependence at higher concentrations
Paramagnetic properties of oxygen can result in significant decrease in fluorescence intensity. This has been already discussed earlier. Similarly presence of transition metals with unpaired electrons can result in reduction of fluorescence intensity.
In essence fluorescence is a highly sensitive technique capable of low level determinations of fluorescent species. Dilution of solution proves beneficial as it reduces effects of self quenching and self-absorption. Oxygen quenching can be reduced by prior degassing of sample with a stream of oxygen free nitrogen gas.