Pressure-induced retention variations in reversed-phase alternate-pumping recycle chromatography.

The progressions of peak width and peak separation in reversed-phase alternate-pumping (AP) recycle chromatography are found to be inconsistent with conventional chromatographic theory. These discrepancies are explained by subtle pressure-induced variations of solute retention that become amplified by AP recycling. The presence of these retention variations is demonstrated by multiply injecting a single solute into an AP system at offset times. As the serially injected peaks are recycled, the separation time between the peaks is shown to vary significantly, indicating that the retention of the solute is dependent upon the position of the peak. A new model of chromatographic retention that appropriately accounts for this variable retention is presented. When this retention model is applied to an AP system for the binary separation of phenylalanine and a pentadeuterated phenylalanine, the model accurately describes the experimentally observed progressions of peak width and peak separation. Furthermore, the retention model predicts that the improvement of resolution in AP recycling closely matches the expectations of conventional theory, so the effectiveness of AP recycling is not significantly compromised by the variations in retention.