Geoscience Reference
In-Depth Information
the fluorophores themselves, this process is called solute self-absorption. In addition, par-
ticles or colloids in unfiltered samples can bring about the same effect through light scat-
tering. Inner filter effects are an important issue with regard to the fluorescence of natural
samples owing to the complexity of DOM composition. UV-visible absorption spectra of
DOM samples (
Figure 2.1
) indicate that light is absorbed by most samples at the wave-
lengths of interest in fluorescence analyses (approximately 250-550 nm). A number of
authors address this issue with regard to analyses of DOM (Mobed et al.,
1996
; McKnight
et al.,
2001
). Care is needed to ensure that samples are optically dilute (A
254
cm
-1
< 0.2)
before measuring fluorescence of samples that contain DOM (Miller et al.,
2010
). Even for
optically dilute samples, fluorescence data must still be corrected for inner filter effects.
Finally, the relaxation energetics of an excited molecule returning to the ground state
are temperature sensitive (Lakowicz,
2006
). Thermal “quenching” of fluorophores results
at higher temperatures because it is more likely that the excited molecules will return to
the ground state via radiationless pathways; fluorescence intensities are generally enhanced
at lower temperatures. Different fluorophores exhibit different temperature dependencies,
and this phenomenon is useful for providing information about both chemical structure
and reaction pathways in chemistry and biochemistry (Baker,
2005
; Lackowicz, 2006).
Measurement of thermal quenching behavior of DOM samples from a variety of environ-
ments has been explored as a method to provide structural information about DOM (Baker,
2005
). In this approach, the thermal dependency of different fluorophores in DOM was
used to provide structural information about the tryptophan-like groups in untreated and
treated wastewaters. That paper demonstrated that different fluorophores in the samples
exhibited different responses to increasing temperature. An important implication of this
work was that the deployment of
in situ
fluorometers or the acquisition of laser-induced
remote-sensing data may require different compensation equations for samples containing
different DOM compositions.
2.5.2 pH Effects
Excitation emission spectra of most DOM fluorophores are pH sensitive owing to the influ-
ence of either deprotonation or protonation of acidic (-COOH, -OH) and basic (-NH
2
)
functional groups bound directly to aromatic fluorophores. Within the pool of molecules
comprising DOM, the major acidic functional groups are -COOH and -OH, whereas -NH
2
is the major basic functional group. The presence or absence of lone pair electrons on
these functional groups alters the lifetimes of the excited state by influencing the rates of
relaxation. In addition, the properties of acids and bases differ between the ground and
excited states. The equilibrium dissociation constants for -COOH groups are greater in
the excited state (p
K
a
*) than in the ground state (p
K
a
), indicating that -COOH groups
are weaker acids in the excited state. Electron-donating groups (-OH, -NH
2
) are stronger
acids in the excited state relative to the ground state (Sharma and Schulman,
1999
). For
the case of phenols, the excited state may exhibit p
K
a* values as low as 2-3, whereas in
the ground state these compounds are generally weak acids (p
K
a
~ 10). The effect exerted
