Geoscience Reference
In-Depth Information
photomultiplier detectors. Even so, there are situations where this signal will be present in
both first and second orders of the analyzing monochromator, as is evident in excitation-
emission matrices.
Raman scattering, on the other hand, is
inelastic
in nature (Raman and Krishnan,
1929
),
and observed signals are generally wavelength shifted to lower energies (longer wave-
lengths). In many solvents, the Raman scattered signal may overlap with observed fluo-
rescence signals. Depending on one's application needs, the presence of these signals can
be advantageous or a nuisance. For many applications this Raman signal can be used as a
reference intensity to compare a fluorescence signal against, or to determine the day-to-day
“stability” of an instrument (Mosier-Boss,
1995
). In relation to aquatic fluorescence, the
water Raman signal is often used as both a measure of instrument stability and as an inter-
nal normalization standard. It is usual to perform a water Raman test using deionized water
in a sealed cuvette to minimize contaminants interfering with the measurement.
5.4.17 Spectral Irradiance of the Excitation Channel
The fluorescence emission signal is dependent on the excitation light intensity. In general,
for dilute samples and assuming that photo-bleaching of the sample is not a problem, the
fluorescence signal can be expressed as shown in
Eq. (5.12)
:
F
=
2 303
.
kI
(
λ
)
ε φ
clkc
=
(5.12)
exc
0
The excitation channel contains a light source in which intensity varies with wavelength,
a monochromator where transmission efficiency varies with wavelength, and bandpass
adjustment that can change the wavelength resolution. All of these effects, either singu-
larly or in combination, introduce a different amount of light onto the sample. Because the
fluorescence intensity is directly proportional to the incident light intensity it is therefore
difficult to determine if a change in observed fluorescence signal is attributable to changes
in the light source or the sample. Therefore, it is necessary to characterize the excitation
channel in terms of how much light, in relative terms, is incident upon the sample. For this,
the excitation channel of a fluorimeter is calibrated to ensure that the excitation spectrum
exhibits the correct spectra in terms of intensity and wavelength positions. Excitation inten-
sity levels can change by more than two orders of magnitude and there are several dips and
intensity spikes visible that will cause measurement errors.
Figure 5.20
shows a typical
uncorrected
excitation intensity profile at the sample position of a fluorimeter.
Most modern fluorimeter instruments have a means to monitor the excitation intensity
as a function of wavelength and over time. This task is usually performed using a beam
splitter to separate a small proportion of the excitation light and record that signal using
some form of photon detector such as a photodiode, photomultiplier, or a quantum counter.
Originally, such reference detectors used a quantum counter approach in which a concen-
trated dye solution, often Rhodamine B, would absorb all photons incident upon it and
whose emission spectrum and emission intensity are not excitation wavelength dependent.
