Development of High-Performance Countercurrent Chromatography and Its Application in the Separation of Bioactive Compounds

Countercurrent chromatography (CCC) is a form of liquid-liquid partition chromatography that eliminates the solid support used in column chromatography. This allows the recovery of all samples subjected to CCC separation of bioactive components without denaturation and adsorption caused by interacting with the column matrix. The CCC apparatus requires numerous and continuous partitioning processes composed of the sufficient mixing of the two-phase solvent system, separating into two liquid phases, and moving the mobile phase. The present paper introduces my studies over 35 years on the development and improvement of CCC including the rotation behavior of the coiled column, column configuration, tube design, and two-phase solvent system followed by the application to the separation of bioactive compounds. Among the CCC instruments developed or improved in those studies, the floor-standing type of cross-axis CCC was first domestically produced and prompted the fabrication of benchtop type small-scale cross-axis CCC to achieve satisfactory separation of proteins and enzymes without loss of their bioactivity using aqueous two-phase solvent systems. The coil satellite centrifuge designed and fabricated in our laboratory enabled sufficient separation using a two-phase solvent system after adjusting the suitable rotation speed combination of the sun axis, planet axis, and satellite axis. Better partition efficiency was achieved using an eccentric coil for the analytical-scale and multilayer coil for the preparative-scale. Notably, the multilayer coil wound with long-pressed locular tubing increased the peak resolution within shortened separation times. The high-performance of the CCC apparatus developed will expand the ability to reveal the mechanisms of cell particles.