Geoscience Reference
In-Depth Information
Axis of Incident Light
Axis of detection
The volume of
sample that is
“sensed”
Figure 5.8. The sensing volume.
defined sampled volume is collected via optical components and then detected and ana-
lyzed. The overlap of the excitation and detection volumes
defines the sensing volume
of
the system (
Figure 5.8
). The chemical, physical, and molecular nature of the sample within
this volume will dictate the contribution to the fluorescence signal. At the same time the
measured fluorescence intensity is also dependent on the fluorophore concentration, the
absorption coefficient, and its quantum yield.
In general, fluorescence measurements are made at rather dilute concentrations, for
example, with
εcl
<0.01. An increasing concentration of fluorophore usually increases the
measured fluorescence signal levels; however, there comes a point where the increased
concentration results in a decrease in the fluorescence signal. This is termed
the inner filter
effect
, which manifests itself either by reabsorption of the fluorescence signal by the sam-
ple or its matrix (for example, where the emission from one fluorophore is reabsorbed by
another fluorophore molecule) or at the same time the sensing volume changes because the
light intensity in the excitation is not constant throughout the defined sensing volume. This
can result in little or no excitation light reaching the detection volume. This phenomenon
is a direct result of the Beer-Lambert law. A high concentration of fluorophore means high
attenuation of light at the cuvette wall and hence less light through the defined sensing vol-
ume or pathlength to the cuvette center, as demonstrated by
Figure 5.9
. These inner filter
effects can change not only the intensity of the emitted light but also the emission spectrum
and can be a significant source of error. Elimination of these effects by suitable sample
geometry or correction but other means (Parker,
1968
) must be seriously considered if high
concentration measurements are to be performed.
