Ion-pair HPLC of anthocyanins in the flavylium form with alkanesulfonates
DOI:
https://doi.org/10.17308/sorpchrom.2026.26/14177Keywords:
ion-pair, reversed-phase, HPLC, alkanesulfonates, retention patternsAbstract
The chromatographic behavior of selected anthocyanins under ion-pair HPLC conditions with alkanesulfonate additives in the mobile phase was investigated while the anthocyanins existed in the flavylium (positively charged) form. It was shown that starting from a mobile phase with zero concentration of the ion-pair additive, the retention of all anthocyanins increases with increasing alkanesulfonate concentration in the mobile phase, with a small change in the separation selectivity of the stationary phase. The increase in retention times in this case is described by a convex curve associated with an increase in the proportion of ion pairs in the mobile phase. An equation is proposed to describe the dependence of retention time on sodium heptanesulfonate concentration, allowing all its parameters to be determined by fitting. In reality, however, the retention mechanism of anthocyanins in this case appears to be an ion-exchange reaction on an in situ converted reversed phase into a cation-exchange phase due to the sorption of alkanesulfonates by the grafted C18 layer, which is confirmed by the decrease in retention of myricetin-3-rhamnoside (uncharged under the experimental conditions) with increasing concentration of the ion-pair additive. This leads to an additional change in the retention mechanism of anthocyanins, the "float" mechanism, which involves the insertion of the flavylium part of anthocyanins into the grafted layer, is replaced by adsorption ion exchange. This hypothesis is supported by a number of experimental findings. First, on the separation map in terms of relative retention, the trend lines in the left part of the map for anthocyanins no longer group according to the number of hydroxyl groups in the B ring (as occurs when retention changes with increasing acetonitrile concentration). Second, on the efficiency map, the dependence of the square of the peak width on the product of the retention factor and the retention factor plus one does not fit the experimental points to a straight line, indicating a change in the sorption mechanism. Finally, for the relative retention of various cyanidin glycosides, the unique HPLC dependence of the slope of the lines on the number of monosaccharides in the glycosidic radical disappears because the glycosidic radical loses its function as a float for the flavylium part of anthocyanins inserted into the grafted layer. At the same time, the separation selectivity of all components also changes little with increasing concentration of the ion-pair additive in the mobile phase. Consequently, ion-pair reversed-phase HPLC is a good alternative to traditional reversed-phase HPLC with predictable separation selectivity for anthocyanins.
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