Chitosan macromolecules with different molecular weights and degrees of acetylation were dissolved in carbonic acid solutions saturated with carbon dioxide at high pressure and were adsorbed from such solutions onto model mica substrate. Their conformation on the substrate was revealed by means of atomic force microscopy. The results of the observations indicate that there are adsorbed nanosized stable aggregates with peculiar, regular, and reproducible geometry, which are formed in carbonic acid solutions. The aggregates appear as rather persistent rod-like structures and consist of a few individual chitosan macromolecules. This effect can be explained by the competition of the Coulomb repulsion of charged monomer units with the attraction between segments of polymer chains. There are several factors contributing to the total intra-and interchain attraction of different monomer units: hydrogen bonding, hydrophobic interaction, and dipole−dipole attraction caused by the formation of ion pairs due to counterion condensation. The uncompensated charges prevent further growth of the aggregates and therefore stabilize their size at the nanoscale range. The experimental data are supported by theoretical calculations and computer simulations.

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