Methanol linear gradient counter-current chromatography for the separation of natural products: Sinopodophyllum hexandrum as samples

• Published in: Journal of Chromatography A 2019-10, Vol.1603, p.251-261
• Main Authors: Yang, Zhi, Guo, Peipei, Han, Rui, Wu, Dingfang, Gao, Jin-Ming, Wu, Shihua
• Format: Article
• Language: English
• Subjects: Counter-current chromatography; Flavones; Gradient elution; Podophyllotoxins; Sinopodophyllum hexandrum
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2019.06.055

•A new methanol linear gradient CCC was introduced based on HepEMWat solvent systems.•Tedious preparations of solvent systems and waste of solvent was avoided.•This method can increase the range of K values separated in a single separation.•12 compounds were highly enriched from Sinopodophyllum hexandrum in one-step separation. Counter-current chromatography (CCC) is a unique, liquid-liquid partition chromatography process. Both the mobile and stationary phases are liquids, so no solid support matrix is used. CCC has gained wide acceptance as a preparative technique in a variety of fields. Because the mobile and stationary phases are both liquids, gradient elution is difficult to perform with CCC. Phase equilibrium must be maintained, so any change in the composition of one phase may induce a compositional change in the other. In this work, a new linear gradient elution method was developed for CCC. Biphasic solvent systems containing heptane, ethyl acetate, methanol, and water (HepEMWat) in various ratios were prepared and used to optimize both isocratic and linear gradient CCC separation with methanol. We first separated a test mixture of four standard compounds with partition coefficients ranging from 0.8 to 7.8. The separation resembled a reversed-phase process, and elution was performed while progressively decreasing the polarity of the mobile phase. Target molecules with small partition coefficients eluted first in the lower phase of the optimized HepEMWat solvent system. Elution of constituents with large partition coefficients was quite slow under isocratic conditions. Separation time was significantly reduced when elution was performed with a linear gradient using methanol and the optimal HepEMWat system. Elution with a 3:7:4:6 (v/v/v/v) HepEMWat system took approximately 200 min. This included an 80-min isocratic step, followed by gradient elution with methanol from 0% to 30%. The optimized methanol linear gradient CCC method was then used to separate a complex mixture of natural products isolated from Sinopodophyllum hexandrum (Royle) Ying roots. Twelve compounds with a wide range of polarities were well-resolved in a single separation. We have developed a convenient and cost-effective strategy for the separation of complex mixtures. No tedious mobile phase preparation step is required. The volume of unused mobile phase is minimal, so little solvent is wasted. The method is an important advance for the separation of mixtures that contain many co