Macromolecular separations by liquid exclusion chromatography

Abstract

High-performance separations of macromolecules by liquid exclusion chromatography have been performed with silica microspheres (particle diameter ≈ 8 µm) having a bonded phase resulting from reaction with γ-glycidoxypropyltrimethoxysilane. Experimental retention data for dextrans and several proteins, e.g., ovalbumin and myoglobin, in aqueous media suggest that the separation mechanism is steric exclusion. For other proteins secondary mechanisms, which may be either adsorption or partial exclusion owing to ionic repulsion, occur and may be interpreted in terms of a thermodynamic representation of a mixed mechanism. When interactions between solute and stationary phase are not involved, solute diameter is a reasonable universal size parameter for dextrans and proteins in aqueous media and for polystyrenes in tetrahydrofuran. Experimental plate-height data for proteins and polystyrenes indicate a considerable increase in the contribution from solute mass-transfer in the stationary phase as the eluent flow-rate is raised. Diffusion coefficients for macromolecules in the stationary phase determined from experimental plate-height data by a method proposed for high polymers are far below (ca. one tenth) the values for the solutes in free solution. The same method also permits the evaluation of the polydispersity from experimental plate-height data, demonstrating that ovalbumin may be regarded as monodisperse.