General Giudelines for CCC/CS

Countercurrent Separation (CS) (syn. Countercurrent Chromatography (CCC)) is a technique that employs two immiscible solvent layers as mobile and stationary phases. To name a few of the biggest advantages of CS over traditional liquid chromatography separations:
(1) sample recovery is theoretically 100%, and
(2) the most efficient polarity range of the separation is more focused and easily modifiable.
It should also be noted that CS gives a completely different pattern of separation (selectivity) compared to the widespread adsorption LC techniques due to its purely partition-based mechanism of separation.

The two most important factors in CCC are K and Sf.

The first one is the Partition Coefficient (K) value). K is a measure of how a compound is distributed between two (mobile and stationary) phases in particular solvent systems. For high-resolution separation, experimental conditions shall be selected such that the compound of interest has a K value between 0.5 and 2.0 (in EECCC, this can be extended to K values up 8, depending on the instrument starting a K values of 0.25). The average K value of the analytes contained in a more complex sample should also be “set” to this range when doing an experiment on mixture of unknowns. The best way to adjust the K value is to modify the two-phase solvent system. One may also change the mode of operation (i.e. switch from normal phase to reverse phase, or vice versa) for this purpose.

The second key parameter is the Stationary Phase Retention (Sf). Sf is the ratio between the stationary phase volume and the total column (system) volume (eq. 1). The larger Sf, the larger the column (system) capacity available for the separation, hence, the better the resolution. In general, higher rotation speeds of the column (system) and lower flow rates of the mobile phase improve Sf. Vmp is equal to the volume of stationary phase eluted during the equilibrium (see below)

(eq. 1)

General Step-By-Step Operation Procedures

1. Choosing the instrument

The column volume of the instrument is the most important factor to be considered when choosing the instrument to use. The CS instrument can separate sample mass from mg to 100 g scale depending on the size of the column. So the instrument is usually chosen in accordance to the size of your sample. As a very rough rule of thumb, 1 g of sample correspond to 100 ml total CS instrument volume. This 1:100 ratio can be substantially higher (e.g., for CPC) or lower (e.g., for solubility limited samples).

2. Optimizing the Solvent System

Probably the most commonly used solvent system families is the HEMWat family (1,2). Thus, it is highly recommended to have stocked hexanes, ethyl acetate, methanol, and DI water. One should also determine the mode of the flow (ascending/descending, or head-to-tail/tail-to-head) and make sure the flow path is configured accordingly. The review article [3] provides further general guidance regarding solvent system optimization.

3. Making the Solvent System

Solvents are thoroughly mixed in a separatory funnel in the ratio determined when optimizing the solvent system. The total volume of the solvent needed for one separation can vary between twice and four times the instrument column volume, depending on the type of experiment you are performing. It is wise to prepare a little more than is anticipated to use, since batches can vary.

4. Stationary phase filling

5. Equilibrate instrument with mobile phase*

Switch the solvent to be pumped from stationary phase to mobile phase and start rotating the instrument. Make sure the instrument setting is correct, i.e. ascending/descending (or H2T/T2H) and rotation speed. Put tubing outlet in graduated cylinder to measure the amount of stationary phase that is being replaced with mobile phase (i.e., the carryover volume). The backpressure of the instrument also needs to be observed and recorded. The pressure should continuously increase while stationary phase is being replaced with mobile phase. Once the instrument reaches equilibrium, no more stationary phase will be eluted and the pressure should stay constant. The pressure should carefully monitored and will be reproducible for the same solvent system with the same instrument. Abnormal pressure levels are often associated with problems, such as clogging, leftover sample/solvent, trapped air, or bad tubing or connections.

*This step can be skipped. This technique is called an “Ito-style injection” or “IBE” (Injection Before Equilibrium).

6. Sample preparation/injection

It is recommended, but not absolutely necessary, to dissolve sample in a mixture of both stationary and mobile phase. In any case all compounds in the sample must be brought in solution by use of one or both phases. The size of the sample loop can be up to 5% or more of the column size. It is possible to dissolve samples in larger volumes if a solubility problem is encountered; however this will cause a loss in resolution. The pressure of the instrument should be monitored while the sample is injected and throughout the run.

7. Performing the separation

Method for this section varies depending on the type of CS experiment one wishes to perform.

8. Cleaning the column

The best solvent for the sample is always the best solvent for cleaning the column. In general, methanol (or another alcohol such as iso-propanol) has been found very suitable for this purpose, in particular when keeping the instrument idle. The wash solvent should be introduced into the column to replace any remaining solvents and samples.

For the detection of possible leaks, hydroalcoholic mixtures (e.g. 70% iso-propanol or methanol) as well as acetone-water mixtures can be useful due to their slower evaporation rate relative to the neat organic solvents.

The instrument should be left with clean solvent in the column, while it is not in use for an extended period of time (few days or more).


(1) Oka, F.; Oka, H.; Ito, Y. Systematic search for suitable two-phase solvent systems for high-speed counter-current chromatography. J. Chromatogr. 1991, 538, 99-108.
(2) Friesen, B.; Pauli, G. F. G.U.E.S.S. to make generally useful estimations of solvent systems in CCC. J. Liq. Chromatogr. Relat. Technol. 2005, 28, 2877.
(3) Pauli GF, Pro S, Friesen JB. Countercurrent separation of natural products. Journal of Natural Products 71: 1489–1508 (2008); doi:

The content and service of this website is offered under the policies of UIC. No reponsibility is assumed for the accuracy of the information on information provided through the links. See disclaimer for further details. (C) 2004-2014
Co-contributors: T. Inui, R.Case, L. Chadwick, B. Friesen, J. Bisson