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Countercurrent chromatography (CCC) is a separation technique based on the liquid-liquid partitioning of compounds between the two phases of a biphasic solvent system. Limitations in the separation efficiency and peak capacity of one-dimensional (1D) CCC often result in insufficiently resolved peaks. Therefore, partially resolved peak fractions have to be rechromatographed in order to improve the yield and purity. Additional solvent evaporation steps can be circumvented by the application of two-dimensional CCC (2D CCC). Existing 2D CCC configurations are based on the linking of two CCC instruments which are not readily available in every laboratory. In this study, we introduce a technical improvement which allows performing multiple heart-cuts and 2D CCC separations with one instrument which has up to four independent coils, one pump, and one detector. For this purpose, we modified a commercially available CCC system by the addition of three six-port selection valves and a T-piece. The applicability of the 2D CCC system was shown under overloading conditions with eight alkyl hydroxybenzoates with nonideal conditions. We used two solvent systems which were hydrodynamically compatible and which showed different selectively characteristics with regard to the compounds. Four unresolved compounds in the first dimension were baseline resolved by means of the heart-cut technique, in which between 4 and 9 mL of the flow from coil 1 was transferred to coil 2. Three successive heart-cuts were performed that led to baseline resolution of unresolved compounds in the first dimension. The obtained recovery rates were 94-100%, and the purities of the compounds as determined by GC/MS were 90-100%.

An integrated online column-switching countercurrent chromatography (CCC) with a solid-phase trapping/preconcentration interface is presented. The interface is systematically evaluated in terms of sorbent type, column size, and kinetic factor from the view of the unique CCC process. Results indicate that satisfactory trapping efficiency can be achieved using a 25 mm x 10 mm i.d. column packed with Oasis HLB materials. In addition to the analyte focusing effect, large volume injection is avoided, thereby allowing the use of totally different biphasic liquid systems to enhance the system orthogonality. The present integrated system simply combines the liquid and solid supports and is successfully applied in a one-step preparative separation of four antioxidative compounds from the ethyl acetate extract of traditional Chinese herbal medicine Rubia cordifolia L. (Rubiaceae), exhibiting great advantages in peak resolution, peak capacity, and instrument integration compared with conventional CCC separations.

Elution-extrusion countercurrent chromatography (EECCC) takes full advantage of the liquid nature of the stationary phase in CCC by combining regular chromatographic elution with stationary-phase extrusion. EECCC is shown to be a three-stage process consisting of classical elution (I), sweeping elution (II), and extrusion (III). After only two column volumes of solvent, it rapidly yields a high-resolution chromatogram that covers an extended polarity range of solutes. As hydrophilicity/lipophilicity balance is a crucial discriminatory property of analytes in highly complex mixtures such as metabolomic samples, the precise determination of CCC distribution constants (KD) is vital to the analysis of metabolomes and other complex biological matrixes. This work builds the EECCC concept by performing a full theoretical treatment and providing equations for retention volumes, peak widths, resolution factors, and distribution constants. Experimental validation utilizes natural products standards that resemble the zero to infinity range of the polarity continuum. EECCC extends the "sweet spot" of high resolution in CCC and provides access to the otherwise practically unapproachable high-KD portion of the high-resolution chromatograms in CCC. Its improved capabilities of KD targeting make EECCC a promising tool for the specific analysis of "small" molecules in complex samples such as in metabolomic fingerprinting and footprinting.

Traditionally, chromatograms in countercurrent chromatography (CCC) have been plotted with retention volume or time on the x-axis. However, the distribution constant (K) is a more appropriate, reproducible value for the x-axis, because it is a physicochemical property of a particular analyte in a particular solvent system. Therefore, K is independent of both the total column volume and the stationary-phase volume ratio (SF) of the column. Going one step beyond simple K plots, the reciprocal symmetry (ReS) plot, with K and 1/K positioned on either side of a line of symmetry on the x-axis, represents all K values, zero to infinity. Based on experimental evidence, using a mixture of CCC reference standards, the ReS plot demonstrates both the invertible and "symmetric" nature of CCC, a consequence of the exchange of the mobile and stationary phases by reversing the direction of the flow and the symmetry of the liquid-liquid partitioning process between two immiscible phases, respectively. Moreover, the interval of optimal resolution can be centered on the ReS plot to focus on K values of interest, establishing the reciprocal shifted symmetry (ReSS) plots in CCC. Improved representation of peak shape across the whole CCC polarity range is an added advantage of ReSS plots over both K and classical retention volume plots.