The roots of high performance counter current chromatography lie in the well known process of liquid-liquid extraction. An impure sample is dissolved in a two-phase solvent system, normally in a separating funnel and involving water as one of the two solvents, vigorously shaken then left to settle to allow the two phases to separate. The compound of interest will partition preferentially into one of the phases if the solvents have been selected so that the compound is more soluble in one than the other. This process can, if necessary be repeated by first removing the heavier phase from the separating funnel and mixing with a fresh quantity of lighter phase in a second separating funnel or adding a further quantity of fresh heavier phase to the separating funnel, and performing the procedure again. The compound of interest will partition preferentially again and by these means impurities are removed.
Although quick, simple and effective for the separation of compounds with very different partition ratios it can also be effective for compounds with very similar partition ratios but the process must be repeated hundreds or thousands of times to achieve complete separation (resolution): a time consuming and laborious process. Attempts were made to improve the efficiency labour dependence of carrying out this process in separating funnels. In the late 1940s, Craig and Post designed and produced the first generation of counter current chromatography equipment: the Craig Counter-Current Distribution Apparatus, which mechanised the above process with what, was effectively a series of interconnected separating funnels.
The next progression was the advent of Droplet Counter-Current Chromatography (DCCC), where a series of vertical tubes are connected, top to bottom, by capillaries. The design enables one phase, designated the stationary phase (SP) to be retained in the vertical tubes while the other phase, mobile phase (MP) is then either pumped to the top of each tube if the phase is denser, or to the bottom, if lighter. The MP then passes through the SP as a stream of droplets. Compounds which distribute (partition) preferentially into the MP will pass through the apparatus and be eluted more quickly than those which distribute (partition) less preferentially into the MP and a separation will take place due to this distribution (partitioning) process.
To retain the SP, DCCC used only gravity and so was not very effective. The next development, the first modern CCC instrument, was to use a similar arrangement of capillaries and separation tubes was to use centrifugal force to stabilise the stationary phase and was known initially as centrifugal DCCC but is now known as Centrifugal Partition Chromatography (CPC). The performance of this CPC equipment far exceeds the original gravity stabilised DCCC due to using centrifugal force (circa 200g) but as the illustration shows the construction mimics DCCC and this design concept does have flaws.
CPC machines rotate around only one axis (and are known as hydrostatic instruments); the next development was to rotate the column on two axes (known as hydrodynamic instruments). This construction has become known as the "J" type configuration and instruments built to this design are named High Speed Counter Current Chromatographs (HSCCC). These machines have a continuous length of tubing, the column, helically wound on a bobbin that rotates on its own axis and which itself rotates around a central axis to achieve a planetary motion. This motion sets up an oscillating hydrodynamic force field, which causes a mixing and settling step to occur with each revolution of the bobbin. This hydrodynamic force field also causes phases of differing density to travel to opposite ends of the coil; it is this phenomenon alone that retains the SP. The advantage of this design is it operates at low pressure, which allows higher mobile phase flow rates and hence shorter separation times.
However, despite the hydrodynamic force field the SP retention of these HSCCC machines was poor, compared to CPC machines. Although called high speed, these HSCCC machines only generate a g-level of between 55-80g and as a consequence, only relatively low MP flow rates can be used if high SP retention is to be maintained. These low flow rates inevitably mean that cycle times are still measured in hundreds of minutes. In the early 2000s, Dynamic Extractions designed and built the first true high speed and high g-level machines (240g) that have enabled high performance counter current chromatography (HPCCC) to evolve. It is these latest machines that allow chemists to consider the use of counter current chromatography technology to reliably solve their purification problems with cycle times of minutes or 10s of minutes i.e. cycle times comparable to those of preparative scale HPLC.
