Purification of flavonoids from licorice using an off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method

Abstract

An orthogonal (71.9%) off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self-made Click TE-Cys (60 μm) solid-phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE-Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co-eluted in the first dimension were selected for further purification using reversed-phase liquid chromatography. Multiple compounds could be isolated from one normal-phase fraction and some compounds with bad resolution in one-dimensional liquid chromatography could be prepared in this two-dimensional system owing to the orthogonal separation. Moreover, this two-dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off-line two-dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice.

Licocoumarone isolated from Glycyrrhiza uralensis selectively alters LPS-induced inflammatory responses in RAW 264.7 macrophages

“…LC was isolated as our previous report (Fan et al, 2016). In brief, the dried and crushed licorice roots (2.5 kg) were extracted with 95% ethanol (30 L) by refluxing.…”

“…Subsequently, pre-NPLC, pre-RPLC were used in turn to purify LC from ethyl acetate fraction. The purity of LC was determined by HPLC and HRMS analysis, and confirmed to be more than 96% (Fan et al, 2016).…”

Preparative isolation of arylbutanoid‐type phenol [(‐)‐rhododendrin] with peak tailing on conventional C18 column using middle chromatogram isolated gel column coupled with reversed‐phase liquid chromatography

“…Preparative high-performance liquid chromatography (prep-HPLC) is a valuable method for isolating individual constituents from complex systems including biological samples and natural products [14][15][16][17]. This method is extensively employed in numerous areas because of its excellent column efficiency, superior separation reproducibility, online detection, and automatic control [18][19][20][21][22]. However, to protect the stationary phase from contamination and extend its service life, the crude extract cannot usually be directly injected for prep-HPLC separation.…”

Productivity of a preparative high‐performance liquid chromatography isolation of anacardic acids from cashew nut shell liquid

“…A high concentration loading injection as well as robustness, reproducibility and versatility on purification of bioactive compounds make preparative HPLC a primordial approach for natural products isolation . In this regard, several bioactive compounds have been isolated by preparative scale HPLC such as, flavonoids , saponins and other natural compounds from medicinal plants .…”