Conformational arrangement of dissolved humic substances. Influence of solution composition on association of humic molecules

Both the primary chemical structure and the conformational structure of hum ic substances are still a matter of debate. A traditional assumption is tha t humic substances are large polymers and may:present linear or coiled Conf ormations according to solution properties. We studied the conformational c hanges of humic and fulvic acids of different chemical nature by high-press ure size-exclusion chromatography (HPSEC) after dissolution in mobile phase s differing in composition but constant in ionic strength (l = 0.05). Modif ication of a neutral mobile phase (0.05 M NaNO3, pH 7) by addition of metha nol (4.6 x 10(-7) M, pH 6.97), hydrochloric acid (<2 x 10(-6) M, pH 5.54), and acetic acid (4.6 x 10(-7) M, pH 5.69) produced, in the order, a progres sive decrease in molecular size. Size diminishing was-shown by increasingly larger elution volumes at a refractive index detector and by concomitant r eductions of peaks absorbance at a UV-vis detector. The decrease of molecul ar absorptivity(the phenomenon of hypochromism) proved that size reduction of dissolved humic substances was due more to disruption of an only apparen t high-molecular-size arrangement into several smaller molecular associatio ns than to coiling down of a macromolecular structure. The most significant conformational changes occurred in acidic mobile phases where hydrogen bon dings formation was induced, suggesting that the large and easily disruptab le humic conformation was held together predominantly by:weak hydrophobic f orces. The size of molecular association varied with humic samples indicati ng a close relation between humic chemical composition and stability of con formational structure. Our results show that humic substances in solution a re loosely bound self-association of relatively small molecules, and interm olecular hydrophobic interactions are the predominant binding forces. The s tability of such a conformation in solution is attributed to the entropy-dr iven tendency to exclude water molecules from humic association and thus de crease total:molecular energy. This model of dissolved humic substances bas ed on the reversible self-association of small molecules rather than on the macromolecular random coil represents a new understanding that should cont ribute to predict the environmental behavior of contaminants in association with natural organic matter.