Segregative phase separation in aqueous mixtures of polydisperse biopolymers

Keywords: biopolymer, gelatine, dextran, PEO, phase separation, polydispersity, molar mass distribution, SEC-MALLS, CSLM The temperature-composition phase diagram of aqueous solutions of gelatine and dextran, which show liquid/liquid phase segregation, were explored at temperatures above the gelation temperature of gelatine. It turned out that the compositions of coexisting phases show practically no dependence on temperature between 40oC and 80oC. Also, the total polymer concentration at which phase separation occurred was found to be nearly independent on temperature. These observations suggest an entropy driven phase separation. Phase separation is found to be accompanied by strong fractionation of the molar mass distribution in the two phases. Molar mass distributions in coexisting phases were investigated using Size Exclusion Chromatography with MultiAngle Laser Light Scattering. The molar mass of the native material and concentration appeared to be the only factors that affected the final molar mass distributions, temperature having no effect. The results show that in the molar mass range where fractionation is the strongest, i.e. roughly below the maximum in the distribution, fractionation is governed by a Boltzmann factor , where DG denotes the free energy involved in transferring a polymer with a certain length from the enriched to the depleted phase, and in this case turns out to be proportional to the molar mass. The phase behaviour of aqueous mixtures of poly(ethylene oxide) (PEO) and dextran is also studied. From the temperature dependence we conclude that the phase separation between PEO and dextran is partly caused by sterical interactions. From the equilibrium phase volumes of the phase separated mixture and the shape of the temperature-composition phase diagram of PEO and dextran we conclude that also the decrease of solvent quality of water for PEO at increasing temperatures is involved. It is suggested that the characteristics of the PEO-water interaction can affect the degree of fractionation. This suggestion is based on the observation that the degree of fractionation is not a simple exponential function of the molar mass. Adsorption of high molar mass dextran at the interface between gelatine and dextran during the phase separation of aqueous mixtures of these two polymers is also studied. This adsorption is observed after macroscopic phase separation as well as during the phase separation. In the last case, the system is studied with Confocal Scanning Light Microscopy. For this purpose, the dextran is covalently labelled with fluorescein 5-isothiocyanate (FITC). It turns out that the adsorption of high molar mass dextran is highly affected by the labelling. The adsorption of labelled dextran leads to a stable film between the two coexisting phases, thus preventing dextran droplets to coalesce. If the degree of labelling increases above a certain level, the labelled dextran does not lead to a stable film, but is preferentially present in the gelatine-rich phase.