Strategies of solvent system selection for the isolation of flavonoids by countercurrent chromatography.

Flavonoids form a large class of important naturally occurring bioactive compounds. Their isolation and purification from natural sources can sometimes be very difficult and time-consuming when traditional phytochemical techniques are used. Countercurrent chromatography (CCC), a support-free liquid-liquid partition chromatography technique, is very useful for the isolation of polar compounds and its use is increasing in the natural products field. In this paper, we propose strategies of solvent system selection for the isolation of flavonoids by CCC, based on data from the literature, plus incorporation of own practical experiences. The selected references report the isolation of over 300 different flavonoid compounds from more than 100 plant species, using 40 different solvent systems, showing the versatility of this technique. The solvent system hexane-ethylacetate-methanol-water is proposed as a starting point for the separation of samples containing free flavonoids, as it was cited in more than 60% of the papers. A "fine tuning" step is proposed at each level of this solvent family. Other modifications include exchanging the alcohol in the system as well as introducing a fifth solvent. The solvent system ethyl-acetate-butanol-water is proposed as the starting point for glycosylated flavonoids. Other solvent systems are also discussed. The use of gradients is proposed for samples containing both free and glycosylated flavonoids, as the polarity window is larger in these cases. High-speed countercurrent chromatography was used in 89% of the reviewed data.