Electron spin resonance characteristics of humic acids from a wide range of soil types

Electron spin resonance (ESR) spectroscopy was applied to humic acids in solid and solution states obtained from a wide range of soil types. The relationships between the relative free-radical concentration and the degree of humification expressed as A600/C and log(A400/A600) and the aromatic C concentration estimated by cross-polarization magic-angle spinning 13C nuclear magnetic resonance spectroscopy were analyzed. The hyperfine structure observed in solution ESR spectra was also compared among the humic acids. The free-radical concentration varied up to 147-fold among 42 humic acids, with a sample from a buried Umbric Andosol (Ando soil) having the greatest concentration and that from an upland field soil (Dystric Cambisol) the least. The linewidth of the radical signals ranged between 0.32 and 0.55 mT and showed an inverse correlation with the free-radical concentration, suggesting a decrease in the heterogeneity of free-radicals with their increase. The free-radical concentration was correlated (P < 0.005) positively with the A600/C and the aromatic C concentration, whereas it varied in inverse proportion to the log(A400/A600) value. Regression analysis suggested that the electron-conjugated systems are more developed in the humic acids with greater aromatic C concentration, which help stabilize free-radicals. Hyperfine splitting observed in the ESR spectra of a range of Ando soil humic acids dissolved in 0.1 M KOH was similar, while the peak strength decreased with increasing degree of humification. Although continuous application of manure to upland field soils scarcely affected the free-radical concentration in their humic acids, their hyperfine structure was weakened. The solution ESR spectra of the green fraction, which contains a chromophore related to 4,9-dihydroxyperylene-3,10-quinone (DHPQ) structure, was composed of basic features with an additional hyperfine structure superimposed on them. Since the basic features were consistent with the hyperfine structure of humic acids from Ando soils and the H layer of a Podzol which do not contain the green fraction, it was considered that the additional hyperfine structure is associated with DHPQ and thus the influence of DHPQ on the ESR spectra of humic acids is minor.