Application of stepwise gradients in counter-current chromatography: A rapid and economical strategy for the one-step separation of eight coumarins from Seseli resinosum

• An integrated model of flow-rate gradient and solvent gradient was developed.
• The model efficacy was improved by optimizing the operational parameters.
• The model was applied to CCC for one-step separation of eight coumarins from Seseli resinosum.
• The roles of stepwise gradient, system temperature, and GC-FID were discussed.
• The separation mechanism was discussed on the basis of the final data.

The targeted purification of compounds with a broad polarity range from traditional medicinal plants is a big challenge for counter-current chromatography (CCC). Gradient elution was introduced in CCC to address this problem. However, once a suitable solvent system is selected, the separation process requires optimization of operational parameters. The present study was conducted to optimize various operational parameters to integrate the flow rate and solvent gradients for the rapid isolation of eight coumarins from Seseli resinosum in a single run. An increase in the system temperature from 15 °C to 35 °C increased the stationary phase retention and solubility of the sample, whereas the operation time and viscosity of the system were decreased. The high purity of each compound was ensured by collecting the fractions from the main peaks while all the shoulder peaks were mixed and separated under the same conditions with semi-preparative CCC. GC-FID was used to analyze the components of each phase, which was prepared without presaturation to save the time and solvent consumption. Finally, eight coumarins were purified, including (1) d-laserpitin, (2) (3′S,4′S)-3′-angeloyloxy-4′-hydroxy-3′,4′-dihydroseselin, (3) (+)-samidin, (4) (3′S,4′S)-3′-acetoxy-4′-angeloyloxy-3′,4′-dihydroseselin, (5) deltoin (6), calipteryxin, (7) (3′S,4′S)-3′,4′-disenecioyloxy-3′,4′-dihydroseselin, and (8) (-)-anomalin. The present technique has successfully accomplished the goal of one-step separation of these compounds with high purity and recovery in an economic and time efficient manner.