Superporous agarose a new material for chromatography

Abstract: Agarose, the most commonly employed chromatography base material in industrial purification processes for biomolecules – proteins, has almost ideal properties. The major disadvantage of the agarose support is its poor mechanical strength which excludes its use in high speed separations where small particles are needed in order to achieve improved chromatographic efficiency. This drawback can be reduced by cross-linking the agarose. A different approach, exploited in this work, is to introduce the concept of intraparticle convective flow to be able to use comparatively large agarose beads which tolerate higher flow rates and still achieve high efficiency. Thus, this thesis describes superporous agarose as a support material for chromatography. Superporous agarose beads in sizes varying between 50-500 µm were prepared. Apart from normal diffusion pores, these beads contain large flow pores (about 5-50 µm) in which chromatographed substances are transported by flow to the interior of each bead. This intraparticle convective flow significantly reduces the distance which must be covered by the slow diffusion process giving an improved mass transport. Superporous agarose beads were derivatized with different functional groups and their performance compared with corresponding homogeneous agarose beads in ion exchange-, affinity- and hydrophobic interaction chromatography separations of proteins. In these chromatographic modes superporous beads performed considerably better than homogeneous beads. In a separate study, convective fluid velocities inside superporous agarose beads were directly measured for the first time by following the movement of micro particles in a packed bed. The fluid velocity data obtained were compared with and found to agree with theoretically calculated values based on the Kozeny–Carman equation. Superporous agarose can also be prepared as continuous gels in the form of cast beds, fibres and thick membranes. Applications of such continuous gels are demonstrated and discussed.