A linear relationship between concentration and fluorescence intensity is seen as long as solutions being quantified have an absorbance of:

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Linearity between concentration and fluorescence intensity is maintained as long as the concentration of the solution being measured is maintained at a sufficiently dilute concentration so as to have an absorbance of less than 0.100.Solutions with concentrations above 0.100 are concentrated to the point where all portions of the solution do not absorb light equally.As a result, all portions of the solution do not fluoresce equally.This will cause the relationship between concentration and fluorescence intensity to become nonlinear.This phenomenon is known as the inner filter effect.

A Linear Relationship Between Concentration and Fluorescence Intensity Is Seen