High performance gel permeation chromatography with silica microspheres

An instrument for high performance gel permeation chromatography (HPGPC) has been assembled with equipment designed for high performance liquid chromatography separations. The pump was a syringe-type giving uniform pulseless flow from 0.05 to 6.0 cm3 min-1 at pressures up to 3000 p.s.i.. Low dead-volume septum and septumless injection heads have been used for single columns and multiple column sets. Two ultraviolet detectors and a refractometer having low cell volumes were used to detect the low weights of solute which were separated. Narrow distribution silica gels of varying particle size and porosity, as supplied by A.E.R.E. Harwell, have been packed into columns. Efficiencies of permeating and non-permeating polystyrenes determined with this HPGPC instrument gave an assessment of chromatogram broadening due to mass transfer as a function of eluent flow rate and polystyrene molecular weight. The results show that fast separations can be obtained in several minutes and that the most precise measurements of polydispersity are accomplished at very slow rates. A further assessment of chromatogram broadening was made by deactivating the surface of the silica particles with a hydrophilic bonded phase and examining the column efficiency and polydispersity of proteins in aqueous buffer. Results show that even after surface modification the composition of the aqueous eluent used must be adjusted, e.g. by careful selection of pH and ionic strength, to minimize interactions. When interactions occur, they can be explained in terms of a thermodynamic representation of a mixed mechanism. A comparison of retention data for proteins, which do not participate in interaction effects with the stationary phase, dextrans, which are considered as "non-ionic" macromolecules, and polystyrenes suggest that solute diameter is a reasonable universal size parameter for the representation of macromolecules separating by a steric exclusion mechanism operating close to equilibrium conditions.