Theoretical investigation of the optimum particle size for the resolution of proteins by size-exclusion chromatography

Abstract

A theoretical discussion is presented of the possibilities of the dramatic acceleration of the size-exclusion chromatographic separation of proteinsas a result of a marked decrease in the diameter of the gel particles used to pack the column. The use of very small particles, in the range 1–2 μm, is made compatible with the use of moderate inlet pressures by the requirement of a low mobile phase velocity, due to the small diffusion coefficient of high-molecular-weight compounds. Thus, conditions regarding (i) the suppression of eluent flow in the intra-particle pores, (ii) the avoidance of shear degradation of proteins because of excessive viscous stress and (iii) the avoidance of size exclusion of protein molecules from some interstitial channels can be met for proteins with molecular weights up to several hundred thousand. The optimum particle diameter is discussed, and it is shown that efficiencies of a few tens of thousands plates, permitting peak capacities of up to 30–40, could be generated by using 20–30 cm long columns packed with 1- or 2-μm particles. The analysis time depends on the molecular weight range but, with these columns, it would be of the order of 1 h. The results of this discussion are supported by experimental data, illustrating the practical possibilities of this approach.