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pH a spin probe with appropriate pK value was used. The time dependence of the EPR
spectrum of the spin probe was monitored at room temperature.
3. RESULTS AND DISCUSSION
Several humic acid samples were prepared and incubated at different pH values. A
detailed pH titration was performed for each nitroxide spin probe and studied with EPR
spectroscopy in the presence and absence of humic acid. In the absence of humic acid,
buffered solutions were used to fix the pH at the desired values. A pKa shift of the spin
probe was observed in the presence of humic acid in two of the three cases studied.
Figure 1 shows a detailed pH titration of a spin probe in the presence and absence of
humic acid. The shift of pKa of the spin probe molecule from pKa = 8.8 to pKa = 9.2 in the
presence of humic acid was observed. This result illustrates the difference of the local pH
value in the hydrophobic interior of humic macromolecule with respect to the pH value of
the aqueous solution.
16.0
15.8
With humic acid
Without humic acid
15.6
15.4
HPF (G)
15.2
15.0
14.8
14.6
14.4
14.2
8.0 8.5 9.0 9.5 10.0 10.5
pH
Figure 1. pH titration of a spin probe in the absence (pKa = 8.8, dashed line) and in the
presence of humic acid (pKa = 9.2, solid line).
This is the first demonstration of the applicability of the pH-sensitive spin probe
method for this type of macromolecules.
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