DNA-induced inter-particle cross-linking during expanded bed adsorption chromatography. Impact on future support design.

We have investigated the effects of adsorbent size, ionic capacity and surface immobilised polymers on dynamic capacity and changes occurring to beds of anion-exchangers during the binding of DNA. During application of low concentrations of "3-20 kilobase" calf thymus DNA feeds to expanded beds of anion-exchangers, the bed heights dropped progressively as DNA molecules physically cross-linked neighbouring adsorbent particles together, to form severely aggregated fluidised beds. In plots of dynamic binding capacities and absolute changes in bed porosity at maximum contraction, against the inverse of the mean hydrated particle radii, the anion-exchangers were observed to split into three distinct, but different clusters in each case. The highest index of surface packing of DNA was observed for two prototype pellicular supports, one derivatised with highly charged high molecular mass polyethyleneimine (Mr approximately 50,000) and the other with long dextran (Mr approximately 500,000) chains weakly derivatised with DEAE. However, the ability of the surfaces of these two matrices to bring about bed contraction, was strikingly different. The highly charged surface afforded by coupling of polyethyleneimine exhibited a three-fold higher tendency to interact with neighbouring particles in the presence of DNA than that of the dextran DEAE support. The implications of these findings on the design of future expanded bed materials for separation of both proteins and nucleic acids are discussed.

Fluidisation and dispersion behaviour of small high density pellicular expanded bed adsorbents.

The fluidisation and dispersion properties of various agarose-based expanded bed matrices--small high density stainless steel cored prototypes and standard commercial types--were studied in 1-cm diameter expanded bed contactors in which fluid entering the column base is locally stirred. In all cases, fluidisation behaviour was poorly predicted from the Richardson-Zaki correlation, with experimentally determined values of the expansion index being considerably higher than the theoretical values. The resons for these discrepancies are discussed in detail and the validity of applying this widely used correlation for characterisation of expanded bed systems is questioned. Residence time distribution studies using acetone tracers, demonstrated that in comparison to existing commercial supports, the small pellicular prototype materials generally possessed far superior hydrodynamic properties, which augurs well for their future employment in expanded bed chromatographic separations.