DETERMINATION OF SIZE LIMITS OF MEMBRANE SEPARATION IN VESICLE CHROMATOGRAPHY BY FRACTIONATION OF POLYDISPERSE DEXTRAN

A recently described vesicular chromatographic packing material (VP), consisting of purified plant cell walls with vesicular and cellular morphology, was characterized with respect to the minimum Stokes' diameter necessary for complete exclusion (size limit of exclusion = SLE) and the maximum Stokes' diameter permitting permeation into the whole stationary liquid volume (size limit of permeation = SLP). Using vesicle chromatography, the size fractionation of a polydisperse dextran preparation with a defined size distribution was carried out to determine the percentages of completely excluded (P(ex)) and completely permeable (P(perm)) dextran molecules. SLE may be derived from P(ex) and SLP from P(perm) taking into account the molecular size range of the fractionated polydisperse dextran sample. Values determined for the size limits of the vesicle membrane with the help of dextran 35 (calibrated on Sephadex G-200) were nearly equal to those determined with the help of dextran 15 (calibrated on Sephadex G-75). The SLP of the standard VP is 5.6 nm. However, negatively charged proteins with a Stokes' diameter slightly below the SLP (pepsin and ovalbumin) are excluded from the VP. The method may be applied in controlling the separation limits of VPs with altered ultrafiltration properties.