Hyperfine splitting in the electron spin resonance solution spectra of humic substances

Humic substances contain significant quantities of free radicals which have important functions in many biological processes in soil involving electron transfer. Such radicals may be detected by electron spin resonance spectroscopy in both solid and solution state and are usually reported as giving a single unstructured peak. Humic acids from a range of soil types varying in parent material and pH have been examined by electron spin resonance spectroscopy of their solutions in 0.1 M KOH. Spectra with an effective g-value typical of an organic free radical were obtained. Observation of hyperfine structure was highly dependent on the instrument settings used. It was present at a modulation amplitude of 0.0142 mT, but not at 0,101 mT. Similarly, there was a reduction in resolution as microwave power was increased from 0.505-20 mW. This may explain the inability of some investigators to observe structure in soil humic acids. Resolution of hyperfine features was greatly enhanced by recording spectra in the second derivative mode. Using appropriate instrument settings all the humic acids showed hyperfine structure, although the intensity of these components varied by a factor of 100, being much greater in humic acid from highly acidic soils.