Optical Spectroscopy Instrumentation Design
181
of sample preparation and handling. Trace levels or contaminants are enough to destroy
measurement quality. Insufficient cleaning of the cuvette and related glassware, leaching
from plastic parts in contact with samples, dirty pipettes, fingerprints, vacuum grease from
degassing stations, impure solvents and solutes, and old stock solutions are just some of the
opportunities for contaminated samples and all lead to erroneous fluorescence signals.
5.4.16 Background Signals: Rayleigh and Raman Scattering
Rayleigh and Raman scattering effects can be useful for providing reference signals to
enable comparison between samples; however, they can often be the cause of signal interference too. Rayleigh scattering is associated with elastic scattering from small molecules
or particles and can be seen by scanning the emission monochromator over the spectral
region of the excitation wavelength. This invariably will produce intense optical signals
and is normally to be avoided if possible, as such intense signals can “blind” 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 wavelengths). In many solvents, the Raman scattered signal may overlap with observed fluorescence 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 internal 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. (5.13):
F
k I
cl k c
exc
=
=
2 303
0
.
(
)
λ
′
ε
φ
(5.13)
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 singularly 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
181
of sample preparation and handling. Trace levels or contaminants are enough to destroy
measurement quality. Insufficient cleaning of the cuvette and related glassware, leaching
from plastic parts in contact with samples, dirty pipettes, fingerprints, vacuum grease from
degassing stations, impure solvents and solutes, and old stock solutions are just some of the
opportunities for contaminated samples and all lead to erroneous fluorescence signals.
5.4.16 Background Signals: Rayleigh and Raman Scattering
Rayleigh and Raman scattering effects can be useful for providing reference signals to
enable comparison between samples; however, they can often be the cause of signal interference too. Rayleigh scattering is associated with elastic scattering from small molecules
or particles and can be seen by scanning the emission monochromator over the spectral
region of the excitation wavelength. This invariably will produce intense optical signals
and is normally to be avoided if possible, as such intense signals can “blind” 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 wavelengths). In many solvents, the Raman scattered signal may overlap with observed fluorescence 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 internal 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. (5.13):
F
k I
cl k c
exc
=
=
2 303
0
.
(
)
λ
′
ε
φ
(5.13)
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 singularly 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