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between the base pairs of double-helical DNA. Their fluorescence emission in water is

very weak and increases about 30-fold upon binding to DNA.

Quenching

In Section 12.3.1, we have seen that the quantum yield of a fluorophore is dependent on

several internal and external factors. One of the external factors with practical implica-

tions is the presence of aquencher. A quencher molecule decreases the quantum yield of

a fluorophore by non-radiating processes. The absorption (excitation) process of the

fluorophore is not altered by the presence of a quencher. However, the energy of the

excited state is transferred onto the quenching molecules. Two kinds of quenching

processes can be distinguished:

• dynamic quenching which occurs by collision between the fluorophore in its excited
  state and the quencher; and


• static quenching whereby the quencher forms a complex with the fluorophore. The
  complex has a different electronic structure compared to the fluorophore alone and
  returns from the excited state to the ground state by non-radiating processes.

It follows intuitively that the efficacy of both processes is dependent on the concen-

tration of quencher molecules. The mathematical treatment for each process is different,

because of two different chemical mechanisms. Interestingly, in both cases the degree of

quenching, expressed asI 0 I^1 , is directly proportional to the quencher concentration.

For collisional (dynamic) quenching, the resulting equation has been named theStern–

Volmer equation(equation 12.4).

I 0

I

 1 ¼kQcQ 0 ð 12 : 4 Þ

I 0

I

 1 ¼KacQ ð 12 : 5 Þ

The Stern–Volmer equation relates the degree of quenching (expressed asI 0 I^1 )tothe

molar concentration of the quenchercQ, the lifetime of the fluorophore 0 , and the rate

constant of the quenching processkQ. In case of static quenching (equation 12.5),I 0 I^1

is related to the equilibrium constantKathat describes the formation of the complex

between the excited fluorophore and the quencher, and the concentration of the

quencher. Importantly, a plot ofI 0 I^1 versuscQyields for both quenching processes a

linear graph with ay-intercept of 1.

Thus, fluorescence data obtained by intensity measurements alone cannot distinguish

between static or collisional quenching. The measurement of fluorescence lifetimes or

the temperature/viscosity dependence of quenching can be used to determine the kind

of quenching process. It should be added, that both processes can also occur simultan-

eously in the same system.

The fact that static quenching is due to complex formation between the fluorophore

and the quencher makes this phenomenon an attractive assay for binding of a ligand

to a protein. In the simplest case, the fluorescence emission being monitored is the

500 Spectroscopic techniques: I Photometric techniques