Partitioning of ecdysteroids using temperature-induced phase separation

Abstract

The partitioning behaviour of ecdysone and 20-hydroxyecdysone in aqueous two-phase systems was characterized. Primary systems were composed of an ethylene oxide-propylene oxide random copolymer, UCON 50-HB-5100, and dextran T500. The ecdysteroids were first partitioned in a two-phase system with a UCON-rich upper phase and a dextran-rich lower phase. After the phases had separated, the upper phase was removed and isolated in a separate container. This UCON solution was placed in a water bath and the temperature increased above the cloud point of the polymer. This resulted in the formation of a new two-phase system with an upper water-buffer phase and a lower UCON phase. Ecdysteroids partitioned mainly to the final upper water phase in this new two-phase system. Therefore, temperature-induced phase separation could be utilized to recover UCON polymer and obtain ecdysteroids in a water-buffer solution. The partitioning behaviour was manipulated by adding ethanol, sodium chloride or sodium sulphate to the primary two-phase systems. The recovery of ecdysteroids increased when ethanol was added to the system. In a two-phase systems with an ethanol concentration of 20%, recovery was 73.6% for ecdysone and 85.6% for 20-hydroxyecdysone.