Fluorescence Spectrometry
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where, I o and I are the fluorescence intensities in the absence and presence of a
quencher, respectively. The value of the Stern-Volmer constant (K SV = k q τ o ), will
be equivalent to the slope of a linear plot between the I o /I as a function of quencher
concentration [Q]. The I o /I is expected to be linear on the scale of quencher concentration; however, if two different classes of fluorophore populations are present,
and one class is not available to quencher for interaction, then the Stern-Volmer plot
deviates toward the x-axis. In case, all classes of fluorophore populations are available to the quencher for interaction, then the Stern-Volmer plot deviates toward the
y-axis. The τ o = 1/[k r + k i + k isc ] and τ = 1/[k r + k i + k isc + k q (Q)], respectively, are
the average fluorescence lifetime of the fluorophore in the absence and the presence
of a quencher. In dynamic quenching, both fluorescence intensity and lifetime are
affected and it increases with increasing temperature.
Though, the collisional quenching and dynamic quenching look alike; however,
they have certain differences. The first difference between the collisional and dynamic
quenching can be observed in the Förster resonance energy transfer (FRET), where
a fluorophore and a quencher are attached to the same molecule and diffuse together.
This energy transfer takes place over a distance up to 10 nm, and therefore, does
not require collisions with any other molecule. In a second observation, iodide
is quenched via the promotion of intersystem crossing (ISC) during a collision
with a fluorophore. Thus, this process refers to the collisional quenching and not
to the dynamic quenching. In general, collision-driven mechanisms of quenching
are termed as dynamic quenching, but not all dynamic quenchings are known as
collision-mediated quenching.
1.4.2 Static Quenching
The mechanism of static quenching is analogous to the formation of a nonfluorescent complex and its instantaneous electronic transition to the ground state without
emitting any photon after absorbing the light. This phenomenon is known as static
quenching and Stern-Volmer equation can be described by the following equation:
I o
I
= 1 + K a [Q]
(13)
where, K a is the association constant or static quenching constant. The plot I o /I
versus [Q] exhibits a linear nature and the slope of the plot provides the value of
static quenching constant.
Static quenching only affects fluorescence intensity and it decreases with increasing temperature. The linear I o /I versus [Q] plots are not always ascribed to the
occurrence of the dynamic quenching. One can differentiate between the static and
dynamic quenching by analyzing their dependence on temperature, viscosity, and
lifetimes.
The dynamic and static quenching mechanisms and their temperature dependence
are illustrated in Fig. 6. It is observed that a fluorophore can be quenched both by
443
where, I o and I are the fluorescence intensities in the absence and presence of a
quencher, respectively. The value of the Stern-Volmer constant (K SV = k q τ o ), will
be equivalent to the slope of a linear plot between the I o /I as a function of quencher
concentration [Q]. The I o /I is expected to be linear on the scale of quencher concentration; however, if two different classes of fluorophore populations are present,
and one class is not available to quencher for interaction, then the Stern-Volmer plot
deviates toward the x-axis. In case, all classes of fluorophore populations are available to the quencher for interaction, then the Stern-Volmer plot deviates toward the
y-axis. The τ o = 1/[k r + k i + k isc ] and τ = 1/[k r + k i + k isc + k q (Q)], respectively, are
the average fluorescence lifetime of the fluorophore in the absence and the presence
of a quencher. In dynamic quenching, both fluorescence intensity and lifetime are
affected and it increases with increasing temperature.
Though, the collisional quenching and dynamic quenching look alike; however,
they have certain differences. The first difference between the collisional and dynamic
quenching can be observed in the Förster resonance energy transfer (FRET), where
a fluorophore and a quencher are attached to the same molecule and diffuse together.
This energy transfer takes place over a distance up to 10 nm, and therefore, does
not require collisions with any other molecule. In a second observation, iodide
is quenched via the promotion of intersystem crossing (ISC) during a collision
with a fluorophore. Thus, this process refers to the collisional quenching and not
to the dynamic quenching. In general, collision-driven mechanisms of quenching
are termed as dynamic quenching, but not all dynamic quenchings are known as
collision-mediated quenching.
1.4.2 Static Quenching
The mechanism of static quenching is analogous to the formation of a nonfluorescent complex and its instantaneous electronic transition to the ground state without
emitting any photon after absorbing the light. This phenomenon is known as static
quenching and Stern-Volmer equation can be described by the following equation:
I o
I
= 1 + K a [Q]
(13)
where, K a is the association constant or static quenching constant. The plot I o /I
versus [Q] exhibits a linear nature and the slope of the plot provides the value of
static quenching constant.
Static quenching only affects fluorescence intensity and it decreases with increasing temperature. The linear I o /I versus [Q] plots are not always ascribed to the
occurrence of the dynamic quenching. One can differentiate between the static and
dynamic quenching by analyzing their dependence on temperature, viscosity, and
lifetimes.
The dynamic and static quenching mechanisms and their temperature dependence
are illustrated in Fig. 6. It is observed that a fluorophore can be quenched both by