Salt-induced phase separation can effectively remove the acetonitrile from the protein sample after the preparative RP-HPLC

Acetonitrile (ACN)–water system is one of the most commonly used mobile phases in practical reverse-phase high-performance liquid chromatography (RP-HPLC). However, a higher concentration of ACN (normally greater than 60% (v/v)) is required to elute the target protein from the RP-HPLC column in which, further steps to remove the ACN from the protein samples are demanded. It has been demonstrated that the phase separation occurring under the sub-zero temperature could easily remove the majority of ACN from the effluent of RP-HPLC. Recently, we found that the comparable phase separation could be achieved by adding a small amount of proper salts, such as K2HPO4 and KH2PO4, and the phase separation could take place effectively at 4 °C where the protein-purification processes were usually carried out. In addition, the pH value of the solution could be maintained properly by using potassium phosphate buffer (pH 7.0). With an optimized condition for this salt-induced phase separation, we demonstrated that greater than 60% of ACN could be easily removed; on the other hand, more than 90% of water-soluble protein could be successfully recovered within five hours.