Various countercurrent chromatography (CCC) methods employ two-phase solvent systems for separation of flavonoids. CHCl3:CH3OH:H2O (4:x:2, for which x is between 2.5 and 4 for different samples) may be used for their prefractionation. Two-phase solvent systems can be divided into four types: CHCl3:CH3OH:H2O, CHCl3: CH3OH:n-BuOH:H2O, EtOAc:PrOH:H2O or BuOH: HOAc:H2O, and n-C6H14:EtOAc:CH3OH:H2O, respectively. CCC fractionation permits the use of either stepwise elution or gradient elution, and analytical-scale CCC can also be carried out for separation of flavonoids.

In order to find a versatile high speed counter-current chromatography solvent system that can be used as a general prefractionation system for most alkaloids, the crude extracts of five Chinese traditional medicinal herbs, Cortex phellodendri, Semen strychni, green tea, Sophora flavescens ait, and Datura mete L. were resolved. All separations were performed only with a two-phase system composed of CHCl3-CH3OH-water (4:3:2). The water had different acidities controlled by adding NaH2PO4 or HCl to each sample. The fractionated components were identified by thin-layer chromatography, which confirmed this solvent system was versatile and very useful for the separation of alkaloids.

It is generally believed that multicomponent stationary phases, or mixed stationary phases, show the average effect of individual pure phases in gas chromatography (GC). Investigations have shown the existence of a synergistic effect in some mixed GC stationary phases and about 18 articles have been published since 1996.[1,2] Some chromatographic properties of the mixtures, such as separation factor (a), retention factor (k'), elution order of Grob test regent, and McReynolds constants, are not the simple addition of the chromatographic properties of the two pure phases. These values of mixed stationary phases may be greater or less than either of the pure stationary phases. It is different from general experimental results. The positive synergistic effect is very useful in the separation of some substance pairs.