DIRECT MEASUREMENTS OF CONVECTIVE FLUID VELOCITIES IN SUPERPOROUS AGAROSE BEADS

Superporous agarose beads contain two sets of pores, diffusion pores a nd so-called superpores or flow pores, in which the chromatographic fl ow can transport substances to the interior of each individual bead [G ustavsson and Larsson, J. Chromatogr. A 734 (1996) 231]. The existence of pore flow may be proven indirectly by the chromatographic performa nce of beads but it has never been directly demonstrated in a chromato graphic bed. In this report, pore flow was directly measured by follow ing the movement of micro-particles (dyed yeast cells) in a packed bed . The passage of the micro-particles through the superpores and throug h the interstitial pores was followed by a microscope/video camera foc used on beads which were situated four layers from the glass wall. The video recordings were subsequently used to determine the convective f luid velocities in both the superpores and the interstitial pores. Exp eriments were carried out with three different bead size ranges, all o f which contained superporous beads having an average superpore diamet er of 30 mu m. The superpore fluid velocity as % of interstitial fluid velocity was determined to be 2-5% for columns packed with 300-500-mu m beads (3% average value), 6-12% for columns packed with 180-300 mu m beads (7% average value) and 11-24% for columns packed with 106-180- mu m beads (17% average value). These data were compared to and found to agree with theoretically calculated values based on the Kozeny-Carm an equation. In order to observe and accurately measure fluid velociti es within a chromatographic bed, special techniques were adopted. Also , precautions were made to ensure that the experimental conditions use d were representative of normal chromatography runs. (C) 1998 Elsevier Science B.V.