Liquid chromatography on soft packing material, under axial compression - Size-exclusion chromatography of polypeptides

The behaviour of Sephadex G-25 gel packing for size-exclusion chromatography under dynamic axial compression in a 100 x 2.5 cm column of original design has been studied. The influence of dynamic axial compression on the bed structure and its chromatographic performance was determined with polypeptide samples of various molecular mass. In the studied range of compression pressure values (1.74-5.22 bar), a considerable reduction in the external bed porosity (from 0.36 to 0.20, respectively), without any collapse, is revealed. Analysis of the pressure drop on the column has shown that, under compression, the bed permeability deviates from the relationship described by the Blake-Kozeny equation. It has been found that packed bed consolidation involves not only a considerable decrease in the retention volumes but also st reduction in the distribution coefficients of substances with intermediate magnitudes of the latter ones. It can be associated with particle deformation. The influence of dynamic axial compression on the efficiency of the packed bed (zone dispersion and height equivalent to a theoretical plate), selectivity and peak resolution was studied at different eluent velocities. Compression has been shown to favour an overall increase in the resolution, with pressure optima observed in some cases. (C) 1997 Elsevier Science B.V.