Countercurrent chromatography (CCC) is basically the liquid–liquid partition of a sample between two immiscible liquid phases. This is the same separation principle as separating two mixtures using a simple separating funnel experiment. The technique developed from countercurrent distribution, which was investigated in the 1940s to provide separations without a solid chromatographic support. The term "countercurrent chromatography", although generally accepted nowadays, is in fact a misnomer because most systems involve the passage of one phase (the mobile phase) of a biphasic solvent system through a second non-mobile (stationary phase) component of the solvent system.

This article will provide a review of CCC because the technique is at last becoming more widely accepted by the scientific community. Reliable instruments are now being produced and applications in university and industrial circles are increasing.¹

CCC Techniques

There are two basic CCC techniques, each with its own instruments: (a) CCC with coiled tubes and (b) CCC with cartridges or discs.

Figure 1

CCC with coiled tubes: CCC with coiled tubes involves the rapid rotation of a polytetrafluoroethylene (PTFE) or Tefzel tube wrapped around a drum/bobbin (Figure 1) and is known as centrifugal countercurrent chromatography (CCCC) or high-speed countercurrent chromatography (HSCCC). This mainly involves apparatus with a variable gravity field produced by a double-axis gyratory motion and seal-free introduction of solvent onto the column. One of the most important experimental parameters (β) is defined as the ratio of the coil radius (r) to the orbital radius of revolution (R). Almost 100% of the column space is used for the mixing of the two immiscible phases in this hydrodynamic system.

As far as instruments are concerned, Dr Y. Ito from the National Institutes of Health (NIH) in Bethesda, Maryland, USA has invented most of the prototypes² but a variety of instruments are now commercially available.

CCC with cartridges or discs: The CCC technique involving cartridges or discs produces a constant gravity field by single-axis rotation and solvent is supplied through rotating seals. Separation occurs in the cartridges or discs. A hydrostatic equilibrium system results, which can be compared to a static coil. If the disc is filled with the stationary phase of a biphasic solvent system and then the other phase is pumped through the disc at a suitable speed, a point is reached where no further displacement of the stationary phase occurs and the apparatus contains approximately 50% of each of the two phases. Steady pumping-in of the mobile phase results in the elution of mobile phase alone. This basic system uses only 50% of the efficient column space for the actual mixing of the two phases. A range of rotating disc instruments are available commercially.