Optimisation and establishment of separation conditions of organic acids from Usnea longissima Ach. by pH-zone-refining counter-current chromatography: Discussion of the eluotropic sequence

• Six organic acids with high purities are successfully separated by pH zone refining CCC.
• Optimization the volume ratio and the concentration of eluter to improve the separation efficiency.
• The discussion of the eluotropic sequence.

The major bioactive constituents of Usnea longissima Ach. are organic acids. However, few recent literatures involve the preparative separation of these organic acids. In the present study, pH zone-refining counter-current chromatography is used to separate organic acids from crude sample of U. longissima Ach. The crude extract was separated with the two-phase solvent system Pet–EtAc–MeOH–H2O (5:5:3:7, v/v) with 10 mM TFA in organic stationary phase and different concentration of the eluter in aqueous mobile phase for the screening of the most suitable separation conditions. From the crude extract (1.2 g), 74.0 mg of orsellinic acid at 92.7% purity, 55.5 mg of 4-O-methylorsellinic acid at 97.7% purity, 353.5 mg of evernic acid at 93.8% purity, 102.0 mg of barbatic acid at 94.8% purity, 19.4 mg of diffractaic acid at 92.2% purity, and 44.9 mg of usnic acid at 95.7% purity were obtained using the selected conditions in which the concentration of TFA in stationary phase was 10 mM and the concentration of NaOH in mobile phase was 10–20 mM. The purities of the separated organic acids were measured by HPLC. And the data of electrospray ionization-liquid chromatography/mass spectrometry (ESI–LC/MS), 1H NMR, and 13C NMR were used for confirming chemical structures.