Quenching by dissolved oxygen
The fluorescence intensity of naphthalene and anthracene decreases with time due to dissolved oxygen. To prevent such quenching, remove dissolved oxygen by N2 gas replacement or vacuum deaeration.
In general, the fluorescence intensity decreases with increasing temperature. Therefore, when performing fluorescence measurements, temperature management is more critical than in the case of absorption measurements.
Fig. 9 Concentration quenching
Quenching by impurities
When the purity of the reagent or solvent used for fluorescence measurements is low, the measurement results may be inaccurate due to quenching by impurities due to reaction with the reagent.
Internal shielding effect
Fig. 10 Excitation spectrum of quinine sulfate solutions with different concentrations (Em: 450 nm)
In UV/VIS spectroscopy, the photometric scale is standardized as 0-100 % transmittance. In fluorescence spectroscopy, there is no reference scale and the relative increase from ‘dark’ is measured. Additionally, the detected fluorescence intensity is the product of the sample’s fluorescence intensity and an instrument function, which is similar to the baseline in UV/VIS spectroscopy. In order to obtain the true spectrum, it is necessary to correct the observed spectrum by removing the effect of the instrument function.