Isolation of three polyoxins by reversed-phase liquid chromatography with pure aqueous mobile phase

Developing methods for the isolation of highly polar compounds from complex samples is of great significance. In this study, three polyoxins were successfully isolated from a complex sample (PN1-1^# ) by preparative high-performance liquid chromatography. Separation was carried out on five polar reversed-phase stationary phases, using pure aqueous as mobile phase, where the C18HC column can provide the best performance for PN1-1^# . Next, the effects of the mobile phase composition were studied. It was found that adding NaClO₄ can enhance the retention and resolution, and adding NaH₂ PO₄ was beneficial to maintain good peak shapes when the sample loading increased. Therefore, the optimized mobile phase consisting of 20 mmol NaH₂ PO₄ and 20 mmol NaClO₄ (adding H₃ PO₄ to adjust pH 2) was used to separate PN1-1^# . This method of using 100% aqueous phase can effectively improve both the retention and the solubility of polar samples. Eventually, through further purification, three compounds, namely, polyoxins B, D, and G, were obtained. This paper provided an effective and eco-friendly strategy for the preparative-scale separation of polar samples.