Geoscience Reference
In-Depth Information
by pH depends on the nature of the acidic or basic substituent (Schulman,
1985
). For
electron withdrawing groups, such as -COOH, protonation shifts fluorescence to longer
wavelengths (red shift) whereas deprotonation shifts the position of fluorescence to shorter
wavelengths (blue shift). The opposite effect happens with electron-donating groups, such
as -OH and -NH
2
. Protonation of groups such as -NH
2
results in shifts to shorter wave-
lengths, whereas deprotonation or dissociation of phenolic -OH groups results in shifts to
longer wavelengths. Changes in intensity and peak positions at different pHs are dependent
on protonation/deprotonation of both the ground and excited states of the fluorophores.
Kelly and Schulman (
1988
) and Sharma and Schulman (
1999
) provide more detailed dis-
cussion of the influence of pH on the fluorescence of aromatic acids and bases.
An excellent example of the complexities associated with pH effects on the ground
and excited states of
o
-hydroxycinnamic acid is presented by Wolfbeis et al. (
1986
). This
molecule contains one carboxyl and one phenolic group. Under basic conditions, the mol-
ecule is highly fluorescent due to deprotonation of the phenolic (p
K
a
9.7) and carboxyl
sites (p
K
a
4.2) in the ground state. Fluorescence intensity is weaker at neutral pH where
only the carboxyl group is deprotonated. Below pH 4, the main peak shifts and a second
fluorophore appears resulting from deprotonation of the phenolic group in the
excited
state.
In the excited state, the p
K
a
for the carboxyl group increased 2.2 units whereas the pK
a
for
the phenolic group decreased 8.3 units, making the phenolic group a much stronger acid
(Wolfbeis et al.,
1986
). Similar results have been reported for other classes of phenolic
molecules, such as anthocyanins (Moreira et al.,
2003
) and coumarins (Fink and Koehler,
1970
).
The effects of pH on the fluorescence behavior of DOM and humic substances have
been examined in a number of studies. Ghosh and Schnitzer (
1980
) reported that the fluo-
rescence of soil humic materials was both ionic strength and pH sensitive. In this study,
fluorescence intensity decreased with greater ionic strength and increased with higher pH
for both humic and fulvic acid. The increasing intensity with higher pH was interpreted as
evidence for the role played by phenolic groups in the fluorescence behavior of humic sub-
stances. Mobed et al. (
1996
) reported both changes in intensity and blue- and red-shifted
portions of the EEMs spectra as a result of collecting spectra under different pH conditions.
The red-shifted portions of the spectra were in the long-wavelength regions and attributed
to the fluorescence characteristics of phenolic fluorophores. Westerhoff et al. (
2001
) also
noted a slight blue shift for a wastewater sample and Suwannee River fulvic acid at pH 3
compared to pH 7. Lowering the pH from 7 to 3 in this study also resulted in 30-40% loss
of intensity for most EEMs peaks, although the greatest change was noted for the peak near
ex 250 nm/em 320 nm. The decreased intensities in this region were hypothesized to be
due to protonation of phenolic fluorophores. The dependencies of different fluorophores on
sample pH may pose a problem for comparing spectra obtained on different water samples
at different pHs. Spencer et al. (2007b) examined the influence of pH over a range of 2-10
for 35 waters from different source areas. The effects of pH were found to be greater at high
and low pH with different responses exhibited at different wavelengths. Given the unknown
structures associated with DOM and humic substances, ascribing structural explanations
