APPLICATION OF HPLC COUPLED WITH HPCCC TO SEPARATION OF MAJOR CHEMICAL COMPONENTS WITH A WIDE RANGE OF POLARITIES FROM THE FLOWERS OF APOCYNUM VENETUM

Abstract

A novel separation method of HPLC coupled with HPCCC was established and applied to the isolation of bioactive components from the extract of Apocynum venetum flowers. Two sets of solvent systems, ethyl acetate–acetonitrile–water–acetic acid (5:0:5:0.05, v:v:v:v) and ethyl acetate–acetonitrile–water–acetic acid (6:3:5:0.05, v:v:v:v) were used as binary solvent systems of HPCCC for the one-step elution. The CCC column was first filled with the upper phase of ethyl acetate–acetonitrile–water–acetic acid (5:0:5:0.05, v:v:v:v) followed by elution with its lower phase to separate the hydrophilic compounds. And then the lower phase of ethyl acetate–acetonitrile–water–acetic acid (5:0:5:0.05, v:v:v:v) (A) and the lower phase of ethyl acetate–acetonitrile–water–acetic acid (6:3:5:0.05, v:v:v:v) (B) were used as the mobile phases to perform a binary gradient elution as follows: 0–60 min, 90% A and 10% B; 60–180 min, from 90% A and 10% B to 0% A and 100% (B), six compounds were eluted continuously. Via HPLC-HPCCC, nine compounds with a wide range of polarities were separated in one-step separation. The purities of the compounds were analyzed by HPLC, and the structures were identified by HPLC-MS, ¹H NMR, and ¹³C NMR.

Keywords:

• Apocynum venetum
• counter-current chromatography
• flavonoids
• flower
• HPLC/HPCCC
• wide range of polarities

ACKNOWLEDGMENT

This project was supported by the National Natural Science Foundation of China (No. 31170326, 30970299) and the National Natural Science Foundation of Jilin Province (No. 20090936, 20110928).

Notes

^a 1: quercetin–3–O–β–D–glucosyl–β–D–glucopyranoside; 2: kaempferol–7–O–β–D–glucopyranosyl–β–D–glucopyranoside; 3: apigenin–7–O–glucoside; 4: avicularin; 5: isoquercetin; 6: vanillic acid; 7: astragalin; 8: quercetin; 9: apigenin.

^a Compound numbers are as provided in Table 1.

^a Relevant abundance.