Quantitative Fluorimetry
Introduction
Light
emission from atoms or molecules can be used to quantitate the amount of the
emitting substance in a sample. The relationship between fluorescence intensity
and analyte concentration is:
F = k * QE * Po * (1-10[-epsilon*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, epsilon is the
wavelength-dependent molar absorptivity coefficient, b is the path length, and c
is the analyte concentration (epsilon, 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 * epsilon * 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.
Limitations
Many of the limitations of the
Beer-Lambert law also affect quantitative fluorimetry. Fluorescence
measurements are also susceptible to inner-filter effects. These effects include
excessive absorption of the excitation radiation (pre-filter effect) and
self-absorption of atomic resonance fluorescence (post-filter effect).
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