Evaluation of ethanol-based aqueous two-phase systems performance for application in centrifugal partition chromatography (CPC)

Abstract

Aqueous two-phase systems (ATPS) have recently been proposed for Centrifugal Partition Chromatography (CPC). This type of biphasic system has several advantages over conventional aqueous-organic liquid–liquid systems, namely, high biocompatibility owing to its large water content. The most commonly used ATPS are polymer-based systems. However, owing to their poor phase separation hydrodynamics compared to aqueous-organic systems, the use of short-length water-miscible alcohols has been evaluated as an alternative to polymers. In this study, an ATPS composed of ethanol and dipotassium hydrogen phosphate (K2HPO4) is proposed to overcome the common hydrodynamic drawbacks of polymer-based ATPS. The Ethanol/K2HPO4 system exhibited a very short settling time (ts = 30.85 ± 0.80 s), high interfacial tension (IFT = 6.38 mN/m), significant density differences between the phases (Δρ = 353.40 kg/m3), and low viscosity of the coexisting phases at 25 °C (maximum µ = 3.79 ± 0.11 mPa.s), making it an ideal choice for implementation in CPC apparatus. A comprehensive study of this biphasic system was conducted using CPC equipment by carefully adjusting the operating parameters, specifically, the mobile phase flow rate (F), rotational speed (ω), and mode of operation, that is, ascending (upper phase is mobile) or descending (lower phase is mobile). In this particular CPC equipment, the use of polymers as phase-forming compounds in ATPS resulted in higher CPC pump pressures compared to ethanol or phosphate salt aqueous solutions, thus confirming the superior performance of the ethanol/salt ATPS. The results obtained using this ATPS demonstrated that high F and ω values lead to lower stationary phase retention (SF) and higher backpressures, respectively. SF remained relatively constant and above 50% for F ≤ 6 mL/min but significantly decreased for F > 6 mL/min. This trend confirms that hydrostatically stable conditions can be achieved by employing the Ethanol/K2HPO4 system in the CPC mode by controlling F (i.e., the hydrodynamics of the mobile phase). The partition of the model solutes gallic acid and L-tyrosine was also evaluated using the ethanol/K2HPO4 system. The critical operating CPC parameters were compared, confirming the significant potential of the ethanol/salt ATPS for successful commercial application of this chromatographic technique in the separation of small value-added biomolecules.