Multiseparation of anthocyanins and anthocyanidins by high performance liquid chromatography combined with response surface methodology

A method has been developed for the separation of anthocyanins and anthocyanidins with high performance liquid chromatography (HPLC). Experimental designs were applied for multivariate optimization of the HPLC experimental conditions with using response surface methodology (RSM). Three independent factors, namely formic acid concentration, initial ratio of acetonitrile in gradient program and flow rate, were used to design mathematical models. The responses were resolutions of four peak couples which were the most difficult to separate including: pelargonidin-3-glucoside and delphinidin, delphinidin and peonidin-3-glucoside, peonidin-3-glucoside and malvidin-3-glucoside, peonidin and malvidin. Variance analysis proved a chosen model was highly fit and the RSM method yielded good results in improving separation of anthocyanins and anthocyanidins. The optimized HPLC parameters were as follows: C18 column (250 mm × 4.6 mm × 5 µm), mobile phase is FA 10 % and ACN used as a gradient; flow rate 0...