Simulation of peptide retention on dual reversed phase: strong cation exchange functionalized stationary phase...

Mixed-mode and multi-column chromatographic methods are important separation strategies for resolving complex biological matrices, but an alternative approach involves utilizing a continuous stationary phase gradient within a single column housing. This study investigates the latter strategy, focusing on its application for the separation of peptides. In this work, a multimodal system combining C4 (reversed-phase) and ion exchange (IEX) functionalities was explored. Experimental retention data for four representative peptides (valine-tyrosine-valine, methionine enkephalin, leucine enkephalin, and angiotensin II) were collected using uniform C4 and strong cation exchange columns to establish accurate fit parameters for the linear solvent strength and ionic exchange models, respectively. These parameters were subsequently used in an in-house simulation program to predict the retention behavior of the analytes on theoretical stationary phase gradient columns for different IEX fractions and column orientations. To explore the interplay between the stationary phase gradient and the mobile phase, simulations were run using two primary mobile phase approaches: a salt gradient at a fixed organic composition, and an organic gradient at a fixed salt composition. Separation quality was evaluated based on the total analysis time and the resolution of the least-resolved pair. Variables deemed to be most important were the fraction of IEX in the stationary phase and the orientation of the stationary phase relative to the detector. Simulations revealed that placing the stationary phase functionalization most affected by the mobile phase gradient at the column inlet generally resulted in higher retention times but better resolution compared to placing it at the outlet. Notably, the best resolution results were consistently achieved when using a relatively small fraction of the functionalization affected by the mobile phase gradient. For the conditions tested, the resulting analysis confirmed that a resolution of 2.5 for all critical pairs could be achieved within 15 min across various gradient combinations. The resulting simulations indicate a strong potential for utilizing small, strategic segments of functionalization on stationary phase gradients to significantly enhance separations using traditional mobile phase gradients.