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The Principles and Development of Countercurrent Chromatography

Anyone conversant with the techniques of liquid-liquid extraction (using a separatory funnel) and with chromatography (e.g., HPLC) can readily understand the principles of countercurrent chromatography (CCC).

Liquid-Liquid Extraction (“LLX”)

Liquid-liquid extraction provides the chemist a simple means of separating large quantities of materials, using a minimum of solvent. After dissolving the sample into a two-phase solvent system (in a separatory funnel), the steps in performing LLX are as follows:

1. Shake vigorously to thoroughly mix the two phases.
2. Allow the mixture to settle into two phases.
3. Separate the phases from each other.

The chief disadvantage of LLX is that, in the parlance of the chromatographer, it provides only one “theoretical” plate of separation. (Actually, there’s nothing theoretical about it in this case. This is one plate of separation in the original sense from industrial fractional distillation.) Accordingly, either the chemist must design this single-step separation to suit his needs, or he must use multiple LLX’s to increase the separation. The former course is usually taken, due to the difficulties of the latter. (Although multiple LLX’s are often used, the solvent system is generally changed between extractions to enhance the overall efficiency.)

Attempts to Improve upon the Separatory Funnel

Many attempts have been made to improve upon the separatory funnel. The Craig Countercurrent Distribution Apparatus is one such attempt which made some notable inroads. This delicate, elaborate device effectively chained together a series of separatory funnels, then repeatedly used sequential steps,

1. shaking (mixing),
2. settling, and
3. separating,

Droplet Countercurrent Chromatography (“DCCC”)

Another step in the progress was the development of apparatus such as the droplet countercurrent chromatograph. In this device, a series of vertical tubes are connected by capillaries. The liquid stationary phase (SP) remains in the tubes. Mobile phase (MP) is pumped in slowly (from the top if it is denser than the SP, or from the bottom if less dense). As with all chromatography, compounds more soluble in the MP will move more quickly, while those more soluble in the SP will lag behind. A separation is produced. It should be obvious that the smallest possible theoretical plate is the individual tube. Hence, to achieve a significant efficiency, a large number of these tubes must be used. The steps of this separation are as follows:

1. Mixing consists of droplets of MP passing through the motionless SP. There is no shaking. Droplet size and other solvent system parameters limit the exchange of solutes between phases.
2. Settling is accomplished at the end of the tube, where the MP collects after passing through the SP, and before entering the capillary to pass on to the next tube. Too high a flow rate will disturb this settling and destroy the separation.
3. Separating is accomplished by transferring the MP from one tube to the next by means of the capillary.

The chief disadvantages of the DCCC are the low flow rate permissible, and, hence, the long separation times, and the poor mixing of phases, which results in relatively low efficiency.

Centrifugal Droplet Countercurrent Chromatography (“CPC”)

An advancement over DCCC involves the use of a centrifuge to “enhance” gravity. This centrifugal DCCC, more commonly known as the centrifugal planetary chromatograph, uses very small “tubes” and “capillaries” (machined into layers of inert plastic). A practical device contains thousands of “tubes” and may achieve an efficiency of several hundred theoretical plates. The disadvantages of CPC are similar to those of DCCC, though generally less pronounced due to the tremendous advantages of using centrifugal force instead of gravity.

However, CPC has an additional disadvantage: The instrument requires rotary liquid seals for MP inlet and outlet. While these seals may perform well, they are expensive, they do wear out, and they limit the maximum pressure of pumping, and hence the maximum flow rate and/or centrifuge speed.

High-Speed Countercurrent Chromatography (“HSCCC”)

The modern era of CCC began with the development by Dr. Yoichiro Ito (of the National Institutes of Health) of the planetary centrifuge, and the many possible column geometries it can support. These clever devices make use of a little-known means of making non-rotating connections between the stator and the rotor of a centrifuge. (It is beyond the scope of this discussion to describe the method of accomplishing this. Any of the books on CCC discuss it thoroughly.)

Functionally, the high-speed CCC consists of a helical coil of inert tubing which rotates on its (“planetary”) axis and simultaneously rotates eccentrically about another (“solar”) axis. (These axes can be made to coincide, but this discussion centers on the most common, or “type J” CCC.) The effect is to create zones of mixing and zones of settling which progress along the helical coil at dizzying speed. This produces a highly favorable environment for chromatography. Pharma-Tech Research Corporation manufactures two type-J high speed CCC's: the CCC-1000 and the smaller, higher-speed CCC-3000.

There are numerous potential variants upon this instrument design. The most significant of theses is represented by the third instrument in the Pharma-Tech product line: The TCC-1000 toroidal CCC. This instrument does not employ planetary motion. In some respects it is very like CPC, but retains the advantage of not needing rotary seals. In addition, it employs a capillary tube, instead of the larger-diameter tubes employed in the helices of the other CCC models. This capillary passage makes the mixing of two phases very thorough, despite lack of any shaking or other mixing forces. This instrument provides rapid analytical-scale separations, which can nonetheless be scaled up to either of the larger-scale CCC instruments.

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