Separating Compounds with High Partition Coefficients Using Countercurrent Chromatography

Felix Rüttler and Walter Vetter from the University of Hohenheim (Stuttgart, Germany) combined conventional and co-current countercurrent chromatography to optimize how compounds with high partition coefficients can be separated. Their findings were published in the Journal of Chromatography A (1).

Countercurrent chromatography (CCC) is an all-liquid-based analytical method typically used to separate and isolate natural products. The technique is based on the partition of solutes between two liquid phases (mobile phase and stationary phase) that do not get mixed together (2). In CCC, the velocity of transport depends on the compound-specific partition coefficient (K_sm), which is defined as the ratio of the concentrations (c_s/m) of an analyte in the stationary to the mobile phases (K_sm = c_s/c_m). The main task in CCC is to combine solvents in such manner that the resulting biphasic solvent system provides appropriate K_sm values.

With lipophilic compounds, or chemicals that have high affinity for fats, this can be difficult to achieve (3). K_sm is roughly correlated with the logarithmic octanol-water coefficient (log K_OW), and in practice, nearly all lipid compounds (log K_OW 6–22) are much less polar than the least polar organic solvents. In biphasic solvent systems, lipid compounds mostly partition into the least polar phase. Further, lipid extracts of food and other samples are highly complex, and co-elutions can hardly be omitted.

In this research, the scientists aimed to show how these disadvantages can be overcome through combining conventional (CCC) and co-current (ccCCC) countercurrent chromatography. Separations were started in normal CCC followed by fast elution in ccCCC mode (CCC + ccCCC). These experiments were performed using oleic acid methyl ester (18:1n-9-ME) and palmitic acid methyl ester (16:0-ME), which are difficult to separate by CCC and have K_sm values of >10 in n-hexane/acetonitrile (1:1, v/v).

Tests involving different switching times between CCC to ccCCC were used to determine the linear range of CCC+ccCCC runs; this proved crucial for the separation efficiency and stability of the solvent system. By combining simulated data with the obtained experimental data, it was possible to reliably predict the chromatographic parameters in CCC+ccCCC mode. Further, 18:1n-9-ME and 16:0-ME were sufficiently separated with CCC+ccCCC. The yield of pure 18:1n-9-ME and 16:0-ME was improved further by performing CCC+ccCCC in heart-cut mode.

Under optimized conditions, CCC+ccCCC proved itself to be a promising alternative for isolating very non-polar and very polar compounds. As opposed to elution-extrusion mode, the solvent system remained unchanged in a coil, allowing the CCC system to be directly used for future separations. ccCCC also benefitted from additional focusing effects, which slightly improved chromatographic resolution. As opposed to closed-loop recycling CCC, accidental overlapping of analytes with strongly different K_sm values is not a risk to CCC+ccCCC operations. The CCC+ccCCC mode could be a valuable alternative for separating compounds with high K_sm values in complex samples. Stationary phase loss was not decisive in CCC+ccCCC separations, with no changes in the role of the phases, and co-current mode usage stabilizing the solvent system.

This research refined the application of closed-loop recycling and multiple dual mode. The predictable nature of retention volume, peak width, and resolution between analytes in CCC+ccCCC mode using a linear calibration showed the useful nature of the novel CCC+ccCCC mode.

References

(1) Rüttler, F.; Vetter, W. Combination of Conventional and Co-Current Countercurrent Chromatography for the Separation of Compounds with Very High Partition Coefficients. J. Chromatogr. A 2025, 1757, 466104. DOI: 10.1016/j.chroma.2025.466104

(2) Cheriyedath, S. Countercurrent Chromatography. News-Medical and Life Sciences 2023. https://www.news-medical.net/life-sciences/Countercurrent-chromatography.aspx (accessed 2025-7-17)

(3) Lipophilic Substance. ScienceDirect 2001. https://www.sciencedirect.com/topics/earth-and-planetary-sciences/lipophilic-substance (accessed 2025-7-16)