Countercurrent chromatography

Partition chromatography is based on differences in capacity factor,k, and distribution coefficient,Kd.of the analytes using liquid statiionary and mobile liquid phase.(CCC) or Partition Chromatography is a category of liquid-liquid chromatography techniques. Chromatography in general is used to separate components of a mixture based on their differing affinities for mobile and stationary phases of a column. The components can then be analyzed separately by various sorts of detectors which may or may not be integrated into an apparatus. In liquid-liquid chromatography, both the mobile and stationary phases are liquid. In contrast, standard column chromatography uses a solid stationary phase and a liquid mobile phase, while gas chromatography uses a liquid stationary phase on a solid support and a gaseous mobile phase. By eliminating solid supports, permanent adsorption of the analyte onto the column is avoided, and a near 100% recovery of the analyte can be achieved. The instrument is also easily switched between various modes of operation simply by changing solvents. With liquid-liquid chromatography, researchers are not limited by the composition of the columns commercially available for their instrument. Nearly any pair of immiscible solutions can be used in liquid-liquid chromatography, and most instruments can be operated in standard or reverse-phase modes. Solvent costs are also generally cheaper than for HPLC, and the cost of purchasing and disposing of solid adsorbents is completely eliminated. Another advantage is that experiments conducted in the lab can easily be scaled to industrial volumes. When GC or HPLC is done with large volumes, resolution is lost due to issues with surfact-to-volume ratios and flow dynamics; this is avoided when both phases are liquid.

CCC can be thought of as occurring in three stages: mixing, settling, and separation (although they often occur continuously). Mixing of the phases is necessary so that the interface between them has a large area, and the analyte can move between the phases according to its partition coefficient. A partition coefficient is a ratio of the amount of analyte found in each of the solvents at equilibrium and is related to the analyte's affinity for one over the other. The mobile phase is mixing with then settling from the stationary phase throughout the column. The degree of stationary phase retention (inversely proportional to the amount of stationary phase loss or "bleed" in the course of a separation) is a crucial parameter. Higher quality instruments have greater stationary phase retention. The settling time is a property of the solvent system and the sample matrix, both of which greatly influence stationary phase retention.

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Droplet Countercurrent Chromatography (DCCC)

Droplet CCC is the oldest form of CCC. It uses only gravity to move the mobile phase through the stationary phase. In descending mode, droplets of the more dense mobile phase and sample are allowed to fall through a column of the lighter stationary phase using only gravity. If a less dense mobile phase is used it will rise through the stationary phase, this is called ascending mode. The eluent from one column is transferred to another; the more columns that are used, the more theoretical plates can be achieved. The disadvantage of DCCC is that flow rates are low, and poor mixing is achieved for most binary solvent systems, which makes this technique both time-consuming and inefficient.

Centrifugal Partition Chromatography (CPC)

CPC uses centrifugal force to speed separation and achieve greater flow rates than DCCC (which relies on gravity)(e.g., the Kromaton FCPC). The columns are cut into a rotor, oriented out from the middle, and connected by channels. The rotor is filled with the stationary phase, and the mobile phase is pumped through it as the rotor spins. Early CPC instruments had the disadvantage of requiring a rotary seal that frequently needed to be replaced, but a new generation of instruments are now reliable. CPC can also be operated in either descending or ascending mode, where the direction is relative to the force generated by the rotor rather than gravity.

High-Speed Countercurrent Chromatography

The modern era of CCC began with the development by Dr. Yoichiro Ito 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 several books available 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 the most common or type J CCC is discussed here.) 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.

There are numerous potential variants upon this instrument design. The most significant of these is the toroidal CCC (e.g., the Pharma-Tech TCC-1000). This instrument does not employ planetary motion. In some respects it is very like CPC, but retains the advantage of not needing rotary seals. It also 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 the lack of 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.

Modes of Operation

• head to tail: the more dense phase is pumped through as the mobile phase. Derived from terminology for Archimedean screw force.
• tail to head: the less dense phase is used as the mobile phase.
• Dual-Mode: The mobile and stationary phases are reversed part way through the run.
• Gradient Mode: The concentration of one or more components in the mobile phase is varied throughout the run to achieve optimal resolution across a wider range of polarities. For example, a methanol-water gradient may be employed using pure heptane as the stationary phase. This is not possible with all binary systems, due to excessive loss of stationary phase.
• Elution Extrusion Mode (EECCC): The mobile phase is extruded after a certain point by switching the phase being pumped into the system. For example, during the Elution portion of a separation using an EtOAc-water system running head to tail, the aqueous mobile phase is being pumped into the system, . In order to switch to extrusion mode, organic phase is pumped into the system. This can be accomplished either with a valve on the inlet of single pump, or ideally with an integrated system of two or three pumps, each dedicated either to a single phase of a binary mixture, or to an intermediate wash solvent. This also allows for good resolution of compounds with high mobile-phase affinities. It requires only one column volume of solvent and leaves the column full of fresh stationary phase.
• pH Zone Refining: Acidic and basic solvents are used to elute analytes based on their pKa.

External links

• TheLiquidPhase.org - a CCC specific wiki.
• countercurrent.org