development studies we found that addition of phosphoric acid
and minimising the initial organic content of the mobile phase
were essential to optimising the retention of gallic acid. Ellagic acid
was strongly retained on the Kinetex C18 stationary phase regardless
of the acid content and required an acetonitrile content in excess
of 65% in the mobile phase in order to be eluted in a timely
fashion. Modifications to the slope of the gradient between the
two extremes were made towards optimising the resolution of
punicalagins A and B from the other matrix components found in
the extract. The finalised method was capable of resolving the targeted
polyphenols in a runtime less than 17 min (Fig. 2A–D).
Fig. 2B–D shows chromatograms extracted at individual wavelengths
and demonstrate that base line resolution of the targeted
compounds was achieved.
overall run time, slope of the gradient, and phosphoric acid composition
of the mobile phase. In the course of our method