Hydrophobic Regulation of Ion-Exchange Cellulose Microspheres Activating an "Ionic Anchoring-Hydrophobic Locking"...

High separation resolution is essential for ion-exchange chromatography to achieve purification of proteins with similar net surface charge within a limited separation window. Herein, the hydrophobic-regulated design concept was proposed to enhance the separation resolution of ion-exchange cellulose microspheres (CMs, 10-25 μm). Site-targeted hydrophobic modification significantly improved the resolution of quaternary ammonium-functionalized CMs, enabling baseline separation of seven proteins (resolution ≥1.5). The established "ion anchoring-hydrophobic locking" synergistic mechanism elucidates this enhancement: ion anchoring provides charge-selective binding while hydrophobic locking recognizes subtle structural differences of proteins. The hydrophobic-regulated sulfonic acid-functionalized CMs also achieved enhanced resolution, demonstrating the universal applicability of the design concept. Moreover, homogenization-stirring coupled technique (HSCT)-synthesized CMs, featuring small size, efficient mass transfer, facile surface modifiability, and structural stability, underpin the high-resolution separation performance. Practical application capability was evaluated through acid/base tolerance, reusability, protein binding capacity, and real-sample analyses. This study presents a scalable design concept for advanced chromatographic packings, providing a paradigm for separating complex biological systems.