Countercurrent chromatography (CCC) is a liquid chromatography technique that uses two immiscible liquid phases and no solid support. One liquid acts as the stationary phase and the other as the mobile phase. The liquid stationary phase is held in place by gravity or by centrifugal force.
- 1 Support-free liquid chromatography
- 2 Techniques
- 2.1 Droplet countercurrent chromatography (DCCC)
- 2.2 Hydrodynamic countercurrent chromatography (CCC)
- 2.3 Hydrostatic or centrifugal partition chromatography (CPC)
- 3 Modes of operation
- 4 See also
- 5 References
Support-free liquid chromatography
Standard column chromatography uses a solid stationary phase and a liquid mobile phase, while gas chromatography (GC) uses a liquid stationary phase on a solid support and a gaseous mobile phase. By contrast, in liquid-liquid chromatography, both the mobile and stationary phases are liquid. By eliminating solid supports, permanent adsorption of the analyte onto the column is avoided, and a high recovery of the analyte can be achieved. The instrument is also easily switched between normal-phase and reversed-phase modes of operation simply by changing the mobile and stationary phases. With liquid chromatography, operation is limited by the composition of the columns and media commercially available for the instrument. Nearly any pair of immiscible solutions can be used in liquid-liquid chromatography provided that the stationary phase can be successfully retained.
Solvent costs are also generally lower than for high-performance liquid chromatography (HPLC), and the cost of purchasing and disposing of solid adsorbents is eliminated. Another advantage is that experiments conducted in the laboratory can be scaled to industrial volumes. When GC or HPLC is carried out with large volumes, resolution is lost due to issues with surface-to-volume ratios and flow dynamics; this is avoided when both phases are liquid.
The CCC separation process can be thought of as occurring in three stages: mixing, settling, and separation of the two phases (although they often occur continuously). Vigorous mixing of the phases is critical so that the interface between them has a large area, and the analyte can distribute between the phases according to its partition coefficient.
The mobile phase mixes with then settles 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. Common factors that influence stationary phase retention are flow rate, solvent composition of the biphasic solvent system, and the G-force created by rotation. The settling time is a property of the solvent system and the sample matrix, both of which greatly influence stationary phase retention.
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 denser 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 inflexible.
Hydrodynamic countercurrent chromatography (CCC)
High-speed countercurrent chromatography (HSCCC)
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 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 apparatus 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 rapidly. 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. 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. See, for instance, the Xanthanolide purification found in.
High-performance countercurrent chromatography (HPCCC)
The operating principle of CCC equipment requires a column consisting of a tube coiled around a bobbin. The bobbin is rotated in a double-axis gyratory motion (a cardioid), which causes a variable gravity (G) field to act on the column during each rotation. This motion causes the column to see one partitioning step per revolution and components of the sample separate in the column due to their partitioning coefficient between the two immiscible liquid phases used.
HPCCC works in much the same way as HSCCC but with one vital difference. A seven-year R&D process that has produced HPCCC instruments that generated 240 G's, compared to the 80 G's of the HSCCC machines. This increase in G-level and larger bore of the column has enabled a tenfold increase in through put, due to improved mobile phase flow rates and a much higher stationary phase retention.
Countercurrent chromatography is a preparative liquid chromatography technique, however with the advent of the higher-G HPCCC instruments it is now possible to operate instruments with sample loadings as low as a few milligrams, whereas in the past 100s of milligrams had been necessary.
Major application areas for this technique include natural products purification and also drug development.
Hydrostatic or centrifugal partition chromatography (CPC)
Centrifugal partition chromatography (CPC) was invented in the eighties by the Japanese company Sanki Engineering Ltd, whose president was the late Kanichi Nunogaki. CPC has been extensively developed in France starting from the late nineties. CPC uses centrifugal force to speed separation and achieves higher flow rates than DCCC (which relies on gravity). This technique is sometimes sold under the name FCPC.
This technique is still based on the principles of liquid/liquid partitioning chromatography: two non-miscible liquid phases are mixed together to form a two-phase system, and are then separated multiple times. The individual solutes are isolated based on the different partitioning coefficients of each compound in this two-phase system. One of the liquid phases of the two-phase system is used as a stationary liquid phase: it is fed into the column (the rotor) while the latter is spinning at moderate rotational speed. The stationary phase is retained inside the rotor by the centrifugal force generated. The second phase of the two-phase system is used as the mobile phase containing the solutes to be extracted. It is fed under pressure into the rotor and pumped through the stationary phase. Both phases are mixed together. It is at that time that the exchange of molecules between the two phases occurs. The separation of the solutes is achieved as a function of the specific partitioning coefficient (Kd) of each solute between the mobile and stationary phases. The mobile phase then decants at each cell outlet thus entering the next cell. The eluted fractions of the mobile and stationary phases are collected over a period of several minutes to several hours. These fractions, or eluates, will contain the individual purified solutes.
The centrifugal partition chromatograph is constituted with a unique rotor (=column). This rotor rotates on its central axis (while HSCCC column rotates on its planetary axis and simultaneously rotates eccentrically about another solar axis). With less vibrations and noise, the CPC offers a wider rotation speed range (from 500 to 2000 rpm) than HSCCC. That allows a better decantation and retention for unstable biphasic system (e.g., aqueous aqueous systems or butanol/water systems).
The CPC rotor is constituted by the superposition of disks engraved with small cells connected by head / tail ducts. These cells, where the chromatographic separation takes place, can be compared to lined-up separate funnels.
The rotor is filled with the stationary phase, which stays inside the rotor thanks to the rotation speed, while the mobile phase is pumped through. CPC can be operated in either descending or ascending mode, where the direction is relative to the force generated by the rotor rather than gravity. According to the fast and permanent evolution of the cells design, the efficiency and flow rate with low back pressure are improved.
Advantages over other approaches
The CPC offers now the direct scale up from the analytical apparatuses (few milliliters) to industrial apparatuses (some liters) for fast batch production.
Modes of operation
- Reversed-phase - Aqueous phase mobile - The aqueous phase is pumped through as the mobile phase.
- Normal-phase - Organic phase mobile - The non-aqueous 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 ethyl acetate-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.
- Berthod, Alain; Maryutina, Tatyana; Spivakov, Boris; Shpigun, Oleg; Sutherland, Ian A. (2009). "Countercurrent chromatography in analytical chemistry (IUPAC Technical Report)". Pure and Applied Chemistry 81 (2): 355–387. doi:10.1351/PAC-REP-08-06-05. ISSN 0033-4545.
- Ito, Y.; Bowman, RL (1970). "Countercurrent Chromatography: Liquid-Liquid Partition Chromatography without Solid Support". Science 167 (3916): 281–283. Bibcode:1970Sci...167..281I. doi:10.1126/science.167.3916.281. PMID 5409709.
- Ian A. Sutherland (2007). "Recent progress on the industrial scale-up of counter-current chromatography". Journal of Chromatography A 1151: 6–13. doi:10.1016/j.chroma.2007.01.143.
- Hao Liang, Cuijuan Li, Qipeng Yuan and Frank Vriesekoop (2008). "Application of High-Speed Countercurrent Chromatography for the Isolation of Sulforaphane from Broccoli Seed Meal". J. Agric. Food Chem. 56: 7746–7749. doi:10.1021/jf801318v.
- Yoichiro Ito (2005). "Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography". Journal of Chromatography A 1065: 145–168. doi:10.1016/j.chroma.2004.12.044.
- Ito, Y; Bowman, R L. (1970). "Countercurrent chromatography: liquid-liquid partition chromatography without solid support". Science 167 (1–2): 281–283. doi:10.1126/science.167.3916.281. PMID 5409709.
- Berthod, A (2002). Countercurrent Chromatography: The support-free liquid stationary phase. Wilson & Wilson's Comprehensive Analytical Chemistry Vol. 38. Boston: Elsevier Science Ltd. pp. 1–397. ISBN 978-0-444-50737-2.
- Conway, Walter D. (1990). Countercurrent Chromatography: Apparatus, Theory and Applications. New York: VCH Publishers. ISBN 9780895733313.
- Conway, Walter D.; Petroski, Richard J. (1995). Modern Countercurrent Chromatography. ACS Symposium Series #593. ACS Publications. doi:10.1021/bk-1995-0593. ISBN 9780841231672.
- Ito, Yoichiro; Conway, Walter D. (1995). High-speed Countercurrent Chromatography. Chemical Analysis: A Series of Monographs on Analytical Chemistry and Its Applications (Book 198). New York: John Wiley and Sons. ISBN 978-0471637493.
- Mandava, N. Bhushan; Ito, Yoichiro (1988). Countercurrent Chromatography: Theory and Practice. Chromatographic Science Series, Vol. 44. New York: Marcel Dekker Inc. ISBN 978-0824778156.
- Menet, Jean-Michel; Thiebaut, Didier (1999). Countercurrent Chromatography. Chromatographic Science Series Vol 82. New York: CRC Press. ISBN 978-0824799922.
- Pinel B, Audo G, Mallet S, et al. (June 2007). "Multi-grams scale purification of xanthanolides from Xanthium macrocarpum. Centrifugal partition chromatography versus silica gel chromatography". J Chromatogr A 1151 (1–2): 14–9. doi:10.1016/j.chroma.2007.02.115. PMID 17433347.
- Hacer Guzlek et al (May 2009). "Performance comparison using the GUESS mixture to evaluate counter-current chromatography instruments". Journal of Chromatography A 1216 (19): 4181–4186. doi:10.1016/j.chroma.2009.02.091. PMID 19344911.
- Wataru Murayama, Tetsuya Kobayashi, Yasutaka Kosuge, Hideki Yano1, Yoshiaki Nunogaki, and Kanichi Nunogaki (1982). "A new centrifugal counter-current chromatograph and its application". Journal of Chromatography A 239: 643–649. doi:10.1016/S0021-9673(00)82022-1.
- Foucault, Alain P. (1994). Centrifugal Partition Chromatography. Chromatographic Science Series, Vol. 68. CRC Press. ISBN 978-0824792572.