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Introduction
as a quencher (Q) can reduce the fluorescence intensity of the excited state S ∗ :
Quenching : S ∗ + Q −→ S + Q υq = kq[Q][S ∗ ] (1.5)
The excited fluorophore returns chemically unaltered to the ground state. The
fluorescence quantum yield in the presence of a quencher (φq) is given by:
φq =
kf
kf + kic + kisc + kq[Q]
Using equation 1.4, the ratio of φq/φ is given by:
φq/φ = 1 + τkq[Q] (1.6)
which is the well-known Stern-Volmer equation. Because fluorescence intensity and
lifetime are both proportional to the quantum yield, equation 1.6 can be written as
I0
I = τ 0
τ = 1 + τ 0kq[Q] (1.7)
τ 0 is the lifetime in the absence of a quencher and kq is the bimolecular quenching
constant. The product of τ 0kq is the Stern-Volmer quenching constant and indi-
cates the sensitivity of the fluorophore to the quencher. kq reflects the efficiency of
quenching or the accessibility of the fluorophores to a quencher.
Static Quenching: In some cases, fluorophores can form a stable complex with
another molecule. If this ground-state is non-fluorescent, then the fluorophore has
been statically quenched. In the case of static quenching the lifetime of the sample
will not be reduced since those fluorophores which are not complexed, and hence
are able to emit after excitation, will have normal excited state properties. The
fluorescence of the sample is reduced since the quencher is essentially reducing the
number of fluorophores which can emit (Figure 1.10).
When the complex absorbs light it immediately returns to the ground state
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