Online-storage recycling counter-current chromatography for preparative isolation of naphthaquinones from Arnebia euchroma (Royle) Johnst

Counter-current Chromatography (CCC) has gradually become a popular method for preparative separation, especially in natural product isolation. As an effective separation method, one-dimensional (1D) CCC often results in insufficiently resolved peaks, due to limitations in the separation efficiency and peak capacity in an equipment. Therefore, two dimensional (2D)/multi-dimensional (multi-D) CCC strategies with recycling elution mode were developed to achieve successful separation of target compounds. However, the reported 2D or multi-D CCC approaches lead to experimental costs, complicated procedures, higher requirements for equipment, and increased time consumption. In this study, an online-storage recycling (OSR) CCC strategy was designed to achieve sequential recycling elution for multi-fractions of effluent in non-stop separation with single instrument using three 6-port valves and two storage loops, which would be realized by introducing 2D or multi-D CCC method before. In this non-stop separation system, the fraction C of effluent was subjected to recycling separation while the other fractions (A and B) were storing online, following which these two fractions were subjected to subsequent recycling separations in order, after the completion of the previous recycling elution. Then, six natural occurring naphthaquinone analogues, namely, shikonin (1), propionylshikonin (2), deoxyshikonin (3), isobutyrylshikonin (4), β, β-dimethylacrylshikonin (5) and isovalerylshikonin (6), were isolated from the crude extract of Arnebia euchroma in single run. The purities of all compounds were > 95.0% as determined by HPLC, and their structures were determined by means of UV, MS, 1H NMR, 13C NMR, and optical rotatory dispersion (ORD).