Isolation and Purification of Anthocyanins from Blood Oranges by Column Chromatography

In this study, isolation and purification of anthocyanins from blood oranges by column chromatography were investigated, and then the anthocyanins of blood orange were identified. The behaviors of static adsorption and desorption, dynamic adsorption and desorption of 12 kinds of resins were compared. The results indicated that NKA-9 macroporous resin was optimum for isolation of blood orange anthocyanins, and the optimal elution reagent was 50% ethanol with citric acid (pH 2.5). Toyopearl TSK HW-40S column was employed to separate and purify the anthocyanin extracts from blood orange. The best separation of Toyopearl TSK HW-40S column was obtained using a mobile phase of 35% methanol with 2% formic acid at a flow-rate of 0.6 mL min−1. Three kinds of anthocyanins were purified from blood orange. Then, the anthocyanins of blood orange were identified by HPLC-ESI/MS analysis. The results showed that cyanidin-3-glucoside (35.2%) and cyaniding-3-(6″-malonyl) glucoside (42.9%) were the major anthocyanins of blood orange. Furthermore, cyanidin-3-(3″-malonyl) glucoside, cyanidin 3-(6″-dioxalyl) glucoside and cyanidin-3-glucoside adduct:4-vinylcatechol were identified in blood orange. The combination of NKA-9 macroporous resin and Toyopearl TSK HW-40S column chromatography for isolation and purification of blood orange anthocyanins was an effective method, and HPLC-ESI/MS analysis was a convenient, rapid and effective method for identification of anthocyanins from blood orange.