What is the relationship between intensity and concentration? Is it inverse or direct?
At low concentrations of fluorophore (nanomolar), the relationship is direct. There are several reasons why the relationship becomes nonlinear and can even become inverse as the concentration increases.
1. Saturation of the detector
2. Inner filter effect (self-absorption of light by the fluorophore)
3. Self-quenching (either dynamic or static)
To find the concentration of an unknown sample by fluorescence requires a standard curve consisting of measurements of the fluorescence intensities of a set of solutions of the fluorophore of known concentrations. The sample and standard measurements must be done in the same instrument and under identical conditions. If the sample of the substance is impure, the result is likely to be inaccurate, because impurities may also be fluorescent or may quench fluorescence.
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