Replacement of Acetonitrile with Ethanol in the Determination of Anthocyanins by Reversed-Phase High-Performance Liquid Chromatography

It was found experimentally that the replacement of acetonitrile with ethanol upon the acidification of the mobile phase with phosphoric acid is an effective version for replacing acetonitrile. At that, the order of elution of glycosides of the same type of the five main anthocyanidins (delphinidin, cyanidin, petunidin, peonidin, and malvidin) does not change in eluents convenient for analysis, and the range of retention times for a complete set of such anthocyanins becomes noticeably narrower in going from acetonitrile to ethanol. To determine the effect of aglycone structure on the retention of derivatives of the same anthocyanidin (cyanidin), a mixture of six glycosides was used: one monoglycoside (3-glucoside), two 3-diglycosides (sophoroside and sambubioside), and two 3-triglycosides (2"-glucosylrutinoside and 2"-xylosylrutinoside). This mixture is a real set of anthocyanins, requiring the careful selection of the mobile phase for separating all components. In this work, based on an analysis of separation maps, the composition of a mobile phase based on ethanol is determined that ensures the achievement of the effective separation of these anthocyanins.