Two-Dimensional NOESY Nuclear Magnetic Resonance Study of pH-Dependent Changes in Humic Acid Conformation in Aqueous Solution

Two-dimensional homonulcear 1 H nuclear Overhauser effect spectroscopy (NOESY) reveals pH-dependent changes in humic acid conformation in aqueous solution. The lack of cross-peaks spanning aliphatic structures in the pH 13 NOESY spectrum supports a chainlike primary structure for humic aliphatic and oxygen-substituted aliphatic structures. The absence of cross-peaks in the pH 13 NOESY spectrum spanning aromatic structures indicates either negligible cross-linking or branching of humic aromatic structures or a very specific form of cross-linking: extensive aromatic-ring fusion resulting in low proton abundance. In either case, humic aromatic structures exhibit greater rigidity than aliphatic structures, oxygen-substituted or unsubstituted. A dense array of cross-peaks, forming a terrace that spans the full range of both humic aliphatic and oxygen-substituted aliphatic structures, appears at pH 12, revealing a compact folding and intimate mixing of aliphatic and oxygen-substituted aliphatic structures. The folding of humic macromolecules as pH decreases reveals a marked segregation of aromatic and aliphatic structures, as evidenced by the absence of cross-peaks between the two domains: humic aromatic structures do not fold into aliphatic structures as humic macromolecules change conformation.