Here are some questions that have been asked of us:

Q: Is the system adaptable to very small volumes/low concentrations? 

A: If the injection volume of your sample is suitable to the CCC column, separations will be effective no matter how small the injection mass. However, a low injection mass will yield low concentration fractions. In other words, detection is the limiting factor when working with small volumes/concentrations. 

Q: Why settling time need less than 20 sec? (when choosing a solvent system for CCC the rule of thumb is that the two phases should resolve within 20 seconds)

A: CCC can use any two immiscible solvents as long as they are able to resolve within 20-30 seconds. Within the CCC column the two phases mix, settle and separate hundreds of times. If the two solvent don't separate before the next mixing cycle then proper separation fail. Use the 20 second rule as a way to judge the suitability of a solvent system for CCC, but keep an open mind if separation occurs in less that 20 seconds.

Q: What tools are available to help predict my CCC outcome for a given solvent system?

A: TLC can be used to predict outcomes for natural products separated using the HEMWat and ChMWat systems. If you are working with small molecules read the following abstract:

Friesen, J. Brent & Pauli, Guido F. (2005). G.U.E.S.S.—A Generally Useful Estimate of Solvent Systems for CCC. Journal of Liquid Chromatography & Related Technologies, 28 (17), 2777-2806.

The choice of an appropriate solvent system for Countercurrent Chromatography (CCC) is a critical step in the purification of natural products. Targeted towards their high sample diversity, G.U.E.S.S. is a practical approach for the prediction of CCC distribution constants, K values, by standard thin layer chromatography (TLC). G.U.E.S.S. allows a major reduction in workload by direct use of routine TLC information. The separation capability of CCC focuses on an optimal “window of opportunity” that can be described as the “sweet spot” of CCC separation. The sweet spot of optimal CCC performance may be described as an area where compound K values are between 0.4 and 2.5. Two useful CCC solvent systems: hexane/ethyl acetate/methanol/water and chloroform/methanol/water are organized and recommended as the HEMWat and ChMWat methods of solvent system selection. The relationship of (i) P values, measured by the ratio of UV-vis absorption, (ii) TLC R_f values and (iii) CCC retention volumes for over 20 diverse commercially available natural products are described. The HEMWat method characterizes a versatile solvent selection technique. TLC R_f values will often give practical predictions, even with simple single-phase mixtures. Additional information can be acquired from equivalent solvent systems and by calibration with the G.U.E.S.S. standard compounds. The latter will also aid in the important selection of which phase will function as the mobile phase. The choice of normal vs. reverse phase will depend on the polarity of compounds that are desired to be gathered into the sweet spot. In addition, G.U.E.S.S. has been shown to be readily applicable to natural product purification necessary for drug discovery, bioassay guided fractionation, and metabolome analysis.

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