Influence of temperature on the separation performance in solid support-free liquid-liquid chromatography.

Abstract

In a biphasic liquid system, temperature affects the phase composition, so that a change in temperature can even turn the system into a single-phase system. Furthermore, temperature influences the physical properties of the phases, such as density, viscosity and interfacial tension. In this work, temperature was evaluated as an influencing factor for the separation performance in solid support-free liquid-liquid chromatography, i.e. countercurrent chromatography and centrifugal partition chromatography. To this end, two biphasic systems were selected: the non-aqueous solvent system n-hexane/ethyl acetate/acetonitrile (11.2/2.0/5.3 v/v/v at 25\u2009°C) and the aqueous-organic system n-heptane/ethyl acetate/methanol/water (1/1/1/1 v/v/v/v at 25\u2009°C). The effect of elevated temperature on the separation was systematically analyzed between 25\u2009°C and 40\u2009°C. The results were related to the changes in the liquid-liquid equilibria of the solvent systems and physical properties of the phases. The temperature influence on the stationary phase retention, column efficiency and separation resolution was analyzed. An appropriate temperature control of the column was shown to be essential in order to avoid destabilizing the biphasic system and enable a satisfactory separation for temperature sensitive systems. In this context, the influence of different column and mobile phase inlet temperature on the separation performance was investigated by pulse injections. In addition, the stability and settling time of the biphasic systems were evaluated as indicators whether temperature susceptibility needs to be considered during method development in order to prevent a decrease in separation resolution.