Protein separation via association with confined polyelectrolytes: Coacervation and chromatography

The protein binding properties of a cationic polyelectrolyte, poly(diallyldimethylammonium chloride) (PDADMAC), were used for protein separation. The polyelectrolyte was used in two confined states: as a separate dense, viscous, aqueous phase (coacervate), or adsorbed on siliceous substrates. Polyelectrolyte-protein coacervation may be suitable for large-scale separations. Coacervation selectivity and efficiency was studied as a function of polyelectrolyte MW, ionic strength (I), protein-polymer mixing ratio (r), and pH, using bovine serum albumin (BSA), ribonuclease (RNAse), β-lactoglobulin, and γ-globulin. Coacervate structure was studied via fluorescence recovery after photobleaching (FRAP) and rheology. FRAP showed the diffusivity of BSA in coacervates is the same as for a non-interacting probe of equal size. The coacervate microviscosity was found to be two orders of magnitude lower than its (Newtonian) macroviscosity. Protein-polyelectrolyte “bonds” in coacervates are therefore loose and non-specific. PDADMAC was also electrostatically bound to a chromatographic packing material, controlled porous glass (CPG), or to silica capillaries. The selectivity of PDADMAC for BSA vs. β-lactoglobulin, seen in coacervation was preserved after its immobilization on both surfaces. The pH, ionic strength, and mixing time for polyelectrolyte adsorption on CPG all affect subsequent protein binding, presumably via the molecular properties of the adsorbed polyelectrolyte layer. Size-exclusion chromatography and quasi-elastic light scattering studies were used to establish a correlation between the configuration of adsorbed polyelectrolytes and subsequent protein binding. Measurements of electroosmotic flow as a function of polymer MW and ionic strength were interpreted in terms of the structure of adsorbed PDADMAC. Enhancement of protein separations with treated capillaries was demonstrated