Principles of Fluorescence Spectroscopy

640 FLUORESCENCE SENSING
Figure 19.34. Top: Excitation spectra of the pH probe 1-hydroxypyrene-3,6,8-trisulfonate
(HPTS) in 0.07 M phosphate buffer at various
pH values. Bottom: Emission spectra of HPTS when excited at
454 nm. Revised from [105].
One possible disadvantage of HPTS is that it undergoes
ionization in the excited state, rather than at ground-state
equilibrium. The fact that HPTS undergoes an excited-state
reaction can be recognized by noting that the excitation
spectra are comparable to the absorption spectra of both the
phenol and phenolate forms, but that there is only a single
long-wavelength emission spectrum (Figure 19.34). The
phenol form emits at shorter wavelengths and is only seen
in highly acidic media. The presence of excited-state ionization
is also indicated by a higher apparent pK A in pure
water than in buffers. 105 It is known that the pK A values of
the hydroxyl group for the ground- and excited-state HPTS
are 7.3 and 1.4, respectively, 106 so that HPTS molecules in
the protonated state will tend to undergo ionization upon
excitation. It seems that any excited-state process will be
dependent on the details of the local probe environment.
Under most conditions excited-state ionization of HPTS is
complete prior to emission, so that only the phenolate emission
is observed. Nonetheless, for sensing purposes we pre-
Figure 19.35. Wavelength-ratiometric pH sensors. Carboxy SNAFL-
2 is a seminaphthofluorescein, carboxy SNARF-6 is a seminaphthorhodafluor,
and CNF is 5-(and 6-)carboxy-naphthofluorescein.
fer probes that display a ground-state pK a near 7.5. One disadvantage
of HPTS has been the relatively short excitation
wavelength, particularly for the acid form. However, availability
of blue light-emitting diodes (Chapter 2) may result
in increased use of HPTS.
SNAFL and SNARF pH Probes: A family of
improved pH probes became available in 1991. 109 These
dyes are referred to as seminaphthofluoresceins (SNAFLs)
or seminaphthorhodafluors (SNARFs). Representative
structures are shown in Figure 19.35. A favorable feature of
these probes is that they display shifts in both their absorption
and emission spectra with a pK A from 7.6 to 7.9 (Figure
19.36). Also, the absorption and emission wavelengths
are reasonably long, so that both forms of the probes can be
excited with visible wavelengths near 540 nm (Table 19.2).