Resolving Solvent Incompatibility in Two-Dimensional Liquid Chromatography with In-Line Mixing Modulation

Abstract

Two-dimensional liquid chromatography (2D-LC) is a powerful technique used to characterize complex samples such as synthetic polymers, biomacromolecules, and hybrid modalities (conjugates, oligonucleotides, nanoparticles, etc., which fall between traditional small molecules and large molecules). Characterizing such molecules often requires a highly orthogonal 2D-LC workflow to resolve structurally similar impurities. However, it remains a challenge to achieve truly orthogonal 2D-LC coupling due to incompatibility of the chromatographic conditions used in each dimension. In this work, we present a facile strategy of connecting an in-line mixer, in-line mixing modulation (ILMM), to realize challenging 2D-LC workflows: (1) coupling gel permeation chromatography (GPC) with reversed-phase liquid chromatography (RPLC) for hydrophobic oligomer analysis and (2) coupling ion-pair reversed-phase (IPRP) with hydrophilic interaction liquid chromatography (HILIC) for polar antisense oligonucleotide (ASO) analysis. Compared with the state-of-the-art commercially available active solvent modulation (ASM), engaging the ILMM significantly reduces the peak distortion in GPC-RPLC, allowing an at least 67% higher transfer volume from the primary to secondary dimension, and resolves the ASO sample breakthrough in selective comprehensive IPRP×HILIC. Also remarkably, ILMM demonstrated superiority in comprehensive RPLC×RPLC analysis in comparison with ASM, suggesting its potential in broader 2D-LC applications. In addition to chromatography improvement, ILMM offers several advantages over benchmark modulation approaches in regard to alleviating the need of an additional dilution flow and a simple as well as flexible system configuration, opening many opportunities to establish innovative and versatile multidimensional workflows for characterizing compounds with increasing complexity.

Impact of conditioning runs on hydrophilic interaction chromatography repeatability and its application as a second dimension in online comprehensive two‐dimensional liquid chromatography

“…In addition, sometimes large volumes are injected. The effects of the incompatibility of mobile phases between HILIC and RPLC on peak shape have already been studied [46, 47]. Here, we focused on the impact of the injection process on the repeatability of retention times, a relationship that has never been previously studied to our knowledge.…”

“…In addition, sometimes large volumes are injected. The effects of the incompatibility of mobile phases between HILIC and RPLC on peak shape have already been studied [46,47]. Here, we focused on the impact of the injection…”

Minimize Precolumn Band Broadening with Immiscible Solvent Sandwich Injection

“…This also holds true for 2D-LC, where the modulation between the two dimensions requires considerable connection tubing and, in optimized cases, also mixing. 16 T h i s c o n t e n t i s ECBB is well known to adversely affect column efficiency and sensitivity in liquid chromatography, particularly for early eluters in small-i.d. columns, such as 2.1 mm analytical columns and microscale columns with even smaller diameters.…”

“…A further decrease in the extra-column band broadening (ECBB) is necessary. This also holds true for 2D-LC, where the modulation between the two dimensions requires considerable connection tubing and, in optimized cases, also mixing …”