Comprehensive separation of a wide variety of compounds from olive leaves by counter-current chromatography with three-phase solvent system

The three-phase solvent system counter-current chromatography has been of great research interest, because it can separate compounds with a wide range of polarity. The solvent system of n-hexane/methyl tert-butyl ether/acetonitrile/water (5:5:7:5, v/v) was used for counter-current chromatographic comprehensive separation of olive leaves. The study adopted the normal elution mode. The middle phase and the lower phase (at a volume ratio of 7:3) were pumped into the column simultaneously, followed by eluting with the upper, middle, and lower phases in sequence. The retention rate of the stationary phase measured by the experiment was 73.5%. The upper phase was used to elute the nonpolar compounds, then the mobile phase was switched to the middle phase to elute the moderately hydrophobic compounds, finally, the polar compounds were eluted by the lower phase remaining in the chromatographic column. This method successfully separated eight compounds in one step within 270 min and five compounds were identified. The logP values of these five compounds were 7.44, 7.86, 4.16, -0.11, and 0.96, respectively, covering a wide range of polarities. The present study demonstrated that the three-phase solvent has a strong extraction capacity for ingredients from extremely hydrophilic compounds to extremely hydrophobic compounds.