A feasible scaling-up separation of platycosides from Platycodi Radix: From analytical to semi-preparative high performance liquid chromatography coupling with a post-separation flash freezing treatment to obtain highly unstable components

• Nine platycosides were simultaneously separated.
• Linear scale-up of the separation variables.
• Post-HPLC treatment stopped the acetyl migration.
• First time to obtain unstable platycosides by HPLC.

A method of using semi-preparative HPLC coupling with a post-HPLC flash freezing treatment to separate platycosides from Platycodi Radix is developed. Nine platycosides including unstable acetyl platycosides were separated from Platycodi Radix by semi-preparative HPLC. Separation variables of flow rate and loading amount of sample were linearly scaled up from analytical HPLC to semi-preparative HPLC. Variables of semi-preparative HPLC separation including flow rate and loading amount of sample were calculated as 29.8 mL/min and 297.7 mg, respectively. Under the optimized conditions of separation, nine platycosides were simultaneously separated on semi-preparative scale within 1 h. Separation resolutions of the peaks of platycosides were all above 0.85, of which seven platycosides were separated with resolutions above 1.00. With regards to sample pretreatment before chromatography separation, we propose a post-HPLC treatment to the unstable compounds. A post-HPLC treatment by using liquid nitrogen to flash freeze the eluent fractions from semi-preparative HPLC was used to stop the unstable platycosides transformation. By freezing treatment, the pure unstable acetyl platycosides were obtained without transformation. This was the first time that unstable acetyl platycosides were simultaneously separated by preparative HPLC. This method is fast and efficient to separate platycosides from Platycodi Radix, especially for purifying unstable platycosides.