Beschreibung:
<jats:p>Gradient elution provides significantly higher peak capacity in comparison to the isocratic elution mode, hence it is very useful in online comprehensive two‐dimensional liquid chromatography (<jats:styled-content style="fixed-case">LC</jats:styled-content>). We compared suitability of five commercial core‐shell columns and one monolithic column for fast gradients in the second <jats:styled-content style="fixed-case">LC</jats:styled-content> dimension, where the time of separation is strictly limited by the fraction cycle time. In two‐dimensional reversed‐phase systems with partially correlated retention, the resolution, the peak capacity, and the regularity of coverage of the second‐dimension retention space can be improved by appropriate adjusting the gradient time and the gradient range to suit the sample properties. We developed a new strategy for adjusting the gradient mobile phase composition range in the second‐dimension, employing the retention data of representative sample standards characterizing the sample properties, which can be calibrated using the reference alkylbenzene series. Optimized second‐dimension gradients with single‐step or segmented profiles covering two or more fraction ranges, employed for the separation of subsequent fractions from the first‐dimension, improve significantly the resolution, the separation time, and the regularity of coverage of the two‐dimensional retention plane. The approach was applied to the two‐dimensional comprehensive separation of phenolic acids and flavonoid compounds occurring as natural antioxidants.</jats:p>