Tingyue Gu, Yesong Gu, Yizhou Zheng, Paul E. Wiehl, John J. Kopchick
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Phase separation of acetonitrile-water mixture in protein purification
The removal of acetonitrile (ACN) from reversed-phase high performance liquid chromatography (RP-HPLC) effluent fractions often presents a problem. High concentrations of ACN place a tough demand on the equipment in terms of solvent resistance. Energy cost is also a concern when ACN is removed by evaporation or freeze-drying. This work shows that a phase separation occurs for ACN-water solutions at -17°C. The top phase contains 88 (volume)% ACN, and the bottom phase is 65% water. Since the bottom phase contains 35% ACN, it is not frozen. Surprisingly, proteins such as human growth hormone and its analogs remain in the bottom phase 99% or more after a phase separation. This appears to be an easy and energy efficient method to remove the majority of ACN after RP-HPLC.
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