Pore structure and separation performance of macroporous agarose microspheres fabricated by calcium carbonate,...

This study systematically compares macroporous agarose microspheres fabricated via three templating methods: CaCO₃, starch, and double emulsion. Comprehensive characterization revealed distinct pore structures for each type: CaCO₃-templated microspheres exhibited internally concentrated but non-interconnected pores; starch-templated microspheres showed uniform yet partially closed pores; and double-emulsion-templated microspheres possessed a highly interconnected macroporous network. Functionalized with Q anion-exchange ligands, the microspheres were evaluated for chromatographic performance with bovine serum albumin (BSA). For the double-emulsion formulation at a 15:50 ratio, the specific surface area was 1.60 m²/mL, epoxy content 49.6 μmol/g, ion-exchange capacity 179.8 μmol/g, and dynamic BSA binding capacity 122.7 mg/mL, attributed to the highly interconnected macroporous network enabling efficient mass transfer. All variants achieved sharp peaks, >80% protein recovery, and high stability. The results indicate that double-emulsion-templated microspheres are optimal for macromolecule purification, providing a guideline for selecting pore-engineered agarose media based on application needs.