F = k · QE · Po · (1-10[-e·b·c])
where F is the measured fluorescence intensity, k is a geometric instrumental factor, QE is the quantum efficiency (photons emitted/photons absorbed), Po is the radiant power of the excitation source, ε is the wavelength-dependent molar absorptivity coefficient, b is the path length, and c is the analyte concentration (ε, b, and c are the same as used in the Beer-Lambert law).
Expanding the above equation in a series and dropping higher terms gives:
F = k · QE · Po · (2.303 · ε · b · c)
This relationship is valid at low concentrations (<10-5 M) and shows that fluorescence intensity is linearly proportional to analyte concentration.
Determining unknown concentrations from the amount of fluorescence that
a sample emits requires calibration of a fluorimeter with a standard
(to determine K and QE) or by using a working
curve.
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