The osmotic behaviour of Sephadex and its effects on chromatography

Abstract

1. Sephadex in bead form shows reversible changes of inner volume when immersed in solutions of a non-penetrating solute. These changes are in accordance with the theory of Flory (1953) for the swelling of gels. This makes possible the use of single beads for measuring the osmotic pressures of polymer solutions, up to or beyond 1kg./cm.(2). 2. Measurements of the inner volume of Sephadex G-200 by equilibrium dilution, at various concentrations of dextran 500, gave values in agreement with those obtained from the dimensions of single beads. 3. Measurements by chromatography of the inner volume of Sephadex G-200 gave values that differed slightly but significantly from those obtained by the other methods. Similar small disagreements were found between the dilution and chromatographic values for bovine serum albumin measured in the presence of various concentrations of dextran 500. The major factor that affects the dependence of the chromatographic elution volume (of dextran 500) on concentration is shown to be the change of the inner volume of the Sephadex, with dynamic factors playing a minor role. 4. Contrary to the findings of Ackers (1964), the equilibrium dilution and chromatographic methods gave closely agreeing values for the distribution coefficient, K(av.), of bovine serum albumin on Sephadex G-200; this cast doubt on his explanation of the nature of chromatographic separations on Sephadex.

The osmotic second virial coefficient for protein self-interaction: Use and misuse to describe thermodynamic nonideality

“…Although positive concentration dependence of elution volume for nonassociating proteins was observed in the early days of frontal gel chromatography on Sephadex [57], that effect has been traced to osmotic shrinkage of the gel beads in response to the difference between protein concentrations (and therefore pressures) in the mobile and gel phases [58]. However, the advent of high-performance…”

The molecular compression of dextran

“…The results on the macroscopic shrinkage of Sephadex to be used in the present comparative study are those reported by Edmond et al (1968) and are given in Table 2. The shrinkage behaviours of Sephadex grades G-75, G-100 and G-200 are reported (columns 3, 5 and 7 of Table 2).…”

“…The shrinkage behaviours of Sephadex grades G-75, G-100 and G-200 are reported (columns 3, 5 and 7 of Table 2). The corresponding osmotic pressure of the solutions of this dextran can be calculated from xr/RT= c/1.53 x 10'+ 3.41 x 10-40+3.35 x 10-3c3 (Edmond et al, 1968) and are given in column 2 of Table 2. It is necessary, in turn, to find the concentrations of 'matched' dextrans that correspond to these osmotic pressures.…”

“…Approaches are developed describing the molecular shrinkage (i) as arising from intra-and inter-molecular forces, (ii) based on the molecular characteristics of the dextran, and (iii) as estimated by viscosity measurements. Comparison with the macroscopic shrinkage of cross-linked dextran (Sephadex) beads [Edmond, Farquhar, Dunstone & Ogston (1968) Biochem. J.…”

“…It has been shown (Edmond et al, 1968) that the shrinkage of cross-linked dextran (Sephadex) beads, when immersed in solutions of a non-penetrating solute, can be expressed empirically as V' r=l+kc+k'c2+*vv (10) where VI is the bead volume in solvent and V' the volume in a solution of non-penetrating solute of concentration c.…”