Types of CCC/CPC Experiments and Running Modes

Single-mode CCC/CPC

Single-mode CCC is the most basic type of experiment. This is a one direction separation, and it can be either normal or reversed phase depending on the selection of the mobile phase.

  • In "normal phase" mode, solutes elute in the order of increasing polarity
  • In "reverse phase" mode, solutes elute in the order of decreasing polarity

Dual-mode CCC/CPC

In Dual-mode CCC, the mobile phase is switched in the middle of the run, so the compounds having strong affinity fororiginal stationary phase can also be separated (1).

Gradient CCC/CPC

Gradient CCC employs several mobile phases with slightly different composition to cover compounds with a wider polarity range than can be covered with a single solvent system (2,3).

Elution Extrusion CCC (EECCC)

In EECCC (4), which allows ReS plotting of chromatograms (5), the separation is started in the same manner as in single-mode CCC. However, when the run reaches a certain point (e.g, K=1.0), mobile phase will be switched to extrude the column contents (i.e. the phase initially used as stationary phase will be introduced in the instrument as new mobile phase). The advantages of this method are 1) the peak width of compounds with higher K will be kept narrow, so that the resolution can be improved for otherwise strongly retaining compounds, and 2) the overall run time will be shortened since only one column volume of initial stationary phase (i.e. the new mobile phase) is needed to extrude the entire sample.
Another advantage of the EECCC is that at the end of the experiment the instrument is loaded with stationary phase and ready for another injection.

pH Zone Refining CCC

This method employs basic organic phase and acidic aqueous phase (or vice versa) for the separation. The analytes dissolved in the stationary phase (aka the "retainer acid" in pH zone refining CCC, e.g. HCl) are eluted by mobile phase (aka the "eluter base", e.g. TEA) according to their pKa values and solubility. Two major advantages of this method are its large loading capacity and the high resolution (6,7).


(1) Agnely, M.; Thiebaut, D. Dual-mode high-speed counter-current chromatography: retention, resolution and examples. Journal of Chromatography, A 1997, 790, 17-30.
(2) Berthod, A. Countercurrent Chromatography: The Support-free Liquid Phase; 1st ed.; Elsevier: Amsterdam ; Boston, 2002; Vol. 38.
(3) Ito, Y. High-speed countercurrent chromatography. CRC Crit. Rev. Anal. Chem. 1986, 17, 65-143.
(4) Berthod A, Friesen JB, Inui T, Pauli GF. Elution-extrusion countercurrent chromatography: theory and concepts in metabolic analysis. Analytical Chemistry 79: 3371-3382 (2007); doi: dx.doi.org/10.1021/ac062397g
(5) Friesen JB, Pauli GF. Reciprocal symmetry plots as a representation of countercurrent chromatograms. Analytical Chemistry 79: 2320-2324 (2007); doi: dx.doi.org/10.1021/ac062007q
(6) Ito, Y. pH-peak-focusing and pH-zone-refining countercurrent chromatography. Chemical Analysis 1996, 132, 121-175.
(7) Weisz, A.; Scher, A., L.; Shinomiya, K.; Fales, H., M.; Ito, Y. A new preparative-scale purification technique: pH-zone-refining countercurrent chromatography. J. Am. Chem. Soc. 1994, 116, 704-708.


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Co-ontributors: T. Inui, R.Case, L. Chadwick, B. Friesen