Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 261
Figure 7.42. Absorption spectra of 2-naphthol in 0.1 M HCl, 0.05 M
NaOH, and at pH 3.
undergone dissociation during the duration of the excited
state. The invariance of the absorption spectrum, under conditions
where the emission spectrum of a reacted state is
observed, is a characteristic feature of an excited-state reaction.
This characteristic of an excited-state reaction is shown
in Figure 7.43. Suppose one measured the fraction of the
signal, absorption or emission, from unionized 2-naphthol.
The absorption measurement would show a decrease in this
fraction at pH 9.2, which is near the ground state pK A . The
fluorescence measurements would show a change near pH
2, near the pK A * value of the excited state. The difference
is due to ionization that occurs during the excited-state lifetime.
Depending on the fluorophore and solvent, the excited-state
reaction can be complete or only partially complete
during the excited-state lifetime. It is also important to note
that the extent of dissociation depends on the buffer concen-
Figure 7.43. Comparison of ground state (dashed) and excited-state
(solid) ionization of 2-naphthol.
tration. For instance, suppose that 2-naphthol is dissolved in
buffer solutions of the same pH, but with an increasing concentration
of the buffer. The extent of ionization will
increase with increasing buffer concentration owing to reaction
of excited 2-naphthol with the weak base form of the
buffer.
The spectral shifts that occur upon dissociation in the
excited state can be used to calculate the change in pK A that
occurs upon excitation. This is known as the Förster
cycle 132–135 (Figure 7.44). The energies of the ground and
excited states depend on ionization. Because the dissociation
constants are small, the dissociated form of the fluorophore
is shown at higher energy. If the pK A value is lower
in the excited state (pK A * < pK A ), then there is a smaller
increase in energy upon dissociation of the excited state
(∆H* < ∆H). If one assumes that the entropy change for dissociation
is the same for the ground and excited states, then
the difference in energy between the ground and excited
states of AH and A – can be related to the change in pK A values
by
∆pK A pK A pK * A E HA E A
(7.17)
where R is the gas constant and T is the temperature (EK).
The energies of the protonated form (E HA ) and of the dissociated
form (E A –) are usually estimated from the average of
the absorption ν A and emission ν F maxima of each species:
E i Nhc ν A ν F
2
2.3RT
(7.18)
where ν A and ν F are in cm –1 , h is the Planck constant, N is
Avogadro's number, and c is the speed of light.
Figure 7.44. Electronic energy levels of an acid AH and its conjugate
base A – in the ground and excited states (the Förster cycle). Reprinted
with permission from [131], the Journal of Chemical Education, Vol.
69, No. 3, 1992, pp. 247–249. Copyright © 1992, Division of Chemical
Education Inc.