Phase behavior and protein partitioning in aqueous two-phase systems of cationic--anionic surfactant mixtures.

Cationic-anionic surfactant mixtures can form aqueous two-phase systems. Such aqueous surfactant two-phase systems (ASTP systems) can be used for separation and purification of biomaterials. In this work we investigated the phase behavior and the partitioning of BSA and lysozyme in the ASTP system formed by mixtures of dodecyltriethylammonium bromide and sodium dodecylsulfate (SDS). The pseudo ternary phase diagram of these mixtures at low total surfactant concentrations contains two narrow two-phase regions, which represent two kinds of different ASTP systems formed when cationic and anionic surfactants are in excess, respectively (called ASTP-C and ASTP-A). The phase separation is associative, one phase is surfactant-rich, and the other phase is surfactant-depleted. Mechanisms behind the phase behavior are discussed. The phase behavior, especially phase separation time and phase volume ratio, is strongly influenced by total concentration and molar ratio of mixed surfactants. The effect of molar ratio is strong, which enables one to get desired phase systems also at very low total concentration by tuning the molar ratio of the surfactants. It was shown that the marked differences of surfactant concentration between the phases makes proteins distribute with different partitioning coefficients. The charges on the micellar surface, which can be adjusted by tuning the molar ratio of cationic surfactants to anionic surfactants, enhance the selectivity of protein partitioning by electrostatic effects. At pH 7.1, in the ASTP-C systems, negatively charged BSA is concentrated in the surfactant-rich phase and positively charged lysozyme in the surfactant-depleted phase, while in ASTP-A systems, a totally opposite partitioning was observed. It was shown that lysozyme could retain activity in ASTP systems.