Preparation, construction and performance of agarose microspheres prepared with spray cooling method

In this study, an eco-friendly method for preparing agarose gel microspheres was developed based on the spray-cooling technique. The process is simple and avoids the use of organic reagents. Under optimized conditions-aqueous phase flow rate of 0.18 L/min, spray pressure of 0.25 MPa, and spray height of 130 cm for 4% agarose-the resulting microspheres displayed uniform particle size and high reproducibility. Cryo-scanning electron microscopy revealed that spray-cooled microspheres possess a fine and compact porous structure, which results from high curing efficiency and leads to enhanced mechanical strength. Owing to this dense network, the microspheres exhibited superior pressure resistance, as confirmed by pressure-flow analysis and chromatographic evaluation: compared with emulsion-cured counterparts, the spray-cooled 4% agarose microspheres achieved a 22.2% higher linear flow rate under 0.3-0.4 MPa. Furthermore, their tightly packed internal network enabled efficient separation of low-molecular-weight compounds, with particularly outstanding performance for molecules in the 0.1-70 kDa range. These results demonstrate the promising potential of spray-cooled agarose microspheres as chromatographic separation media.