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Theoretical and experimental studies on erythrocyte partition in aqueous polymer two phase systems

University of British Columbia

Aaueous polymer two phase systems containing dextran T500, PEG 8000, and buffer are widely used to separate and analyse cells and other biological material based on the way they partition between the two phases and their interface. The behaviour of human erythrocytes in such two phase systems was studied in order to characterize some of the physico-chemical interactions important in determining cell partition. Two aspects were studied: the role of electrostatic and affinity ligand effects in determining the relative affinity of the cell for the two phases, and the relationship of this relative affinity to the cell partition. The potential difference produced by the unequal affinity of the buffer cations and anions for each phase was related to the salt partition by a thermodynamic model, which agreed with experimental results obtained in single and mixed salt systems. A thermodynamic theory for the effects of an affinity ligand on the cell surface free energy difference between the phases was derived, and found to agree quantitatively with experimental results using the affinity ligand PEG-palmitate. The change in cell surface free energy difference as a function of potential and ligand concentration was determined by contact angle measurements. This change was very small, based either on previous estimates of the surface charge density, or on the amount of PEG-palmitate bound to the cell surface as determined by adsorption experiments. This was attributed to partial exclusion of the phases from the cell glycocalyx. Cell partition into the upper PEG rich phase increased as this phase was made more positive with respect to the lower phase, or as the amount of an affinity ligand, PEG-palmitate, in the system was increased. Contact angle measurements were used to determine the energy of erythrocyte attachment to the interface between the two phases. The dependence of the cell partition on this parameter showed that thermal energies are far too small to partition cells in these systems. The cell partition was unaffected by the density difference between the phases. This and other results led to the hypothesis that droplet coalescence is the primary process by which large particles (>1 µm dia.) such as cells are distributed between the interface and one of the phases.

Text

2010-06-24

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http://hdl.handle.net/2429/25970

Chemistry

University of British Columbia

Graduate