Molecular crowding effect on ion-pair extraction of picric acid and alkylammonium ion

Macromolecular crowding, a highly concentrated environment of macromolecules, can alter chemical equilibria and reaction kinetics. Although molecular crowding has been recognized as a useful strategy for enhancing reaction efficiency, how it affects individual elementary equilibria within a coupled chemical process remains less understood. In this study, we investigated the effects of macromolecular crowding on the ion-pair extraction of picric acid with tetraalkylammonium ions using dextran with molecular weights of 40 kDa (Dex40k) and 200 kDa (Dex200k) as crowding agents. The extraction constant (K_ex), distribution coefficient (K_D), and association constant (K_A) were determined using spectrophotometry. For the Dex40k system, K_ex and K_A increased with dextran concentration, whereas K_D decreased. These changes were analyzed using a thermodynamic model based on the excluded volume and osmotic pressure effects, which are considered the principal origins of molecular crowding in cells. The analysis revealed that the increase in K_ex was primarily driven by the excluded volume effect, whereas the decrease in K_D was primarily governed by the osmotic pressure effect. In contrast, both effects contributed to the enhancement of the K_A. Furthermore, experiments using Dex200k, which forms a polymer-network structure, suggested that the excluded volume effects may remain involved even in polymer-network environments. These results demonstrate that molecular crowding can exert opposite effects on the elementary equilibria involved in ion-pair extraction and clarify the different contributions of excluded volume and osmotic pressure to association and phase distribution.