Isolation of high-purity anthocyanin mixtures and monomers from blueberries using combined chromatographic techniques

• We establish a novel combined chromatography system to purify anthocyanins.
• Three high purity anthocyanin mixtures with purities around 60% are separated.
• The semi-preparative HPLC is successfully used to prepare anthocyanin monomers.
• Three pure anthocyanin monomers with purities higher than 95% are prepared.

Research on the isolation and preparation of anthocyanins has intensified in recent years because of the requirements of quantitative and bioactive analyses. However, simple and effective methods for the scale purification of pure anthocyanins from natural products are rarely reported. In this study, high-purity anthocyanin mixtures and monomers were successfully isolated from wild blueberries using a combination of column chromatography and semi-preparative HPLC. We established an effective elution system to separate high-purity anthocyanin mixtures with aqueous ethanol containing 0.01% HCl first in an Amberlite XAD-7HP column (ethanol/H2O = 35:65) and then in a Sephadex LH-20 column (ethanol/H2O = 25:75). Crude anthocyanin extracts were isolated using the Amberlite column, and a purity of 32% was obtained based on UV–vis analysis. Three fractions of anthocyanin mixtures were isolated from the crude extracts using the Sephadex column with purities ranging from 59% to 68%. Three pure monomeric anthocyanins of malvidin-3-O-glucoside, petunidin-3-O-glucoside, and delphinidin-3-O-glucoside were also isolated by semi-preparative HPLC and identified by HPLC-DAD-ESI–MS/MS. The purities of these anthocyanins were determined by analytical HPLC and estimated to be 97.7%, 99.3%, and 95.4%, respectively. The results of this study may help promote the purification of anthocyanins from most blueberry varieties as well as from other plant materials.