Geoscience Reference
In-Depth Information
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0
0.02
0.04
0.06
0.08
0.1
[Q] (
M
)
Figure 7.1. An idealized example of fluorescence quenching presented in a Stern-Volmer plot.
F
0
is
the initial fluorescence and
F
is the fluorescence after the quencher, Q, has been added.
Static quenching
occurs when the fluorophore forms a stable, nonfluorescent complex
with the quencher in the ground state. The newly formed complex may have a different
absorption spectrum from the fluorophore alone, which can be used to detect the complex.
In some cases, self-quenching occurs when the fluorophore quenches itself (in most cases,
concentration dependent). Metal cations are very effective static quenchers because they
form coordination complexes with the fluorescent ligands with the DOM pool (see
Section
7.6
). Similarly, protons can be effective quenchers (Senesi,
1990
); both transition metals
and protons are effective electron scavengers, likely underlying their quenching effect by
forming coordination complexes.
DOM fluorescence quenching is traditionally observed using a Stern-Volmer plot, in
which the fractional change in loss of fluorescence (ratio of initial fluorescence,
F
0
to
fluorescence after addition of the quencher,
F
) is plotted against the concentration of the
quencher,
Q
(
Figure 7.1
). The slope of the line describing the linear fit is the quenching
constant (
K
SV
) from the Stern-Volmer equation:
FF
/
=+
1
KQ
[
]
=+
1
kQ
τ
[
]
=
τ
/
(7.2)
0
SV
q
0
0
where
τ
0
and
τ
are the fluorescent lifetimes in the absence and presence of the quencher,
Q
, respectively, and
k
q
is the rate constant for the quenching process. Although they may
have an impact on quenching within short time domains, fluorescence lifetimes are not
discussed in detail in this chapter.
In studying humic substance electrostatic quenching properties, Green et al. (
1992
)
found that both fulvic (FA) and humic (HA) acids' anionic components were the fluoro-
phores that may be quenched. Presumably, this was due to the formation of stable com-
plexes between metals and ligands, such as deprotonated carboxyl groups. They found
higher
K
SV
for cationic quenchers than for neutral quenchers due to Coulombic cation
