Characterization of humic substances by environmental scanning electron microscopy

Environmental scanning electron microscopy (ESEM) is a new technique capable of imaging micron and submicron particles. Here, we have applied it to image and quantify natural aquatic organic matter (standard Suwannee River humic acid, SRHA). Uniquely, we have observed the humic aggregate structures as a function of humidity and pH. Large aggregates of tens of micrometers were observed as the dominant material under all conditions, although much smaller material was also observed. Fractal dimensions (D) were calculated between 1.48 and 1.70, although these values were not statistically different under conditions of low humidity. However, D values calculated at high humidities (85%) during the rehydration phase were significantly lower (1.48+/-0.01) than in the initial dehydration phase (1.69+/-0.01). This hysteresis indicated that full rehydration of the HS was either kinetically slow or irreversible after dehydration. Fractal analysis of ESEM images was also performed to probe the change in aggregate structure as a function of pH. Minimum values were calculated at neutral pHs, rising by 0.1-0.2 at both high and low pHs because of a combination of the physical chemistry of HS and the impacts of the drying regime within the ESEM. Thus, ESEM was an important complementary technique to other analytical methods. At present, ESEM cannot be used to image nonperturbed natural samples. However, the method is an ideal method for probing the changes in colloid structure as function of hydration state and has the potential to perform fully quantitative and nonperturbing analysis of colloidal structure.