Comparison of Standard Elution and Displacement Modes in Centrifugal Partition Chromatography for an Efficient Purification of Four Anthocyanins from Hibiscus sabdariffa L

The increasing demand for natural food antioxidants has hastened research to extract the biologically active substances from a variety of raw materials. The consumption of anthocyanin-rich food promotes health by contributing to cardio protection, lowering high blood pressure and by helping to protect against cancer. In this study, centrifugal partition chromatography (CPC) was studied for anthocyanin separation from Hibiscus sabdariffa L. (HS). A simple elution and two displacement protocols were implemented. The displacement modes were strong ion-exchange (SIX) and pH-zone refining (pHZR) method. Among the three protocols, SIX CPC using trioctylmethylammonium chloride (Aliquat 336 ®) as a strong exchanger was the most successful in purifying four anthocyanins from an ethanolic extract of HS, identified as delphinidin-3-O-sambubioside (DS), cyanidin-3-O-sambubioside (CS), delphinidin-3-O-glucoside (DG) and cyanidin-3-O-glucoside (CG). The purification process was carried out on a gram scale using the ternary biphasic system ethyl acetate:n-butanol:water (mobile organic phase: 4.0/4.6/1.4; aqueous stationary phase: 0.5/0.5/9.0) in the ascending mode. Aliquat 336® was used as the anion extractant in the aqueous stationary phase and sodium iodide (NaI) as the cation displacer in the organic mobile phase. From 1 g of crude HS extract, 80 mg of DS, 40 mg of CS, 15 mg of DG, and 4 mg of CG were obtained after 75 minutes. High performance liquid chromatography (HPLC-UV and LC-MS) analysis revealed the identity and the purity of the isolated compounds: 88% for DS, 92% for CS, 93% for DG and 70% for CG. This new methodology for the isolation of anthocyanin mixtures successfully increased the purity and efficiency especially for DG and CG, while maintaining excellent recovery rate and being relatively low cost. The availability of high purity anthocyanin mixtures will facilitate anthocyanin testing studies and will widen the use of anthocyanins in the food, supplements and pharmaceutical industries.