Total Breakthrough Strategy in Hydrophilic Interaction Chromatography for IP‐RPLC × HILIC Separation of Oligonucleotides

TL;DR

This paper introduces a new strategy to solve solvent mismatch in 2D-LC for analyzing oligonucleotides using HILIC as the second dimension.

Contribution

The study proposes a total breakthrough strategy in HILIC to eliminate solvent mismatch effects in IP-RPLC × HILIC separation of oligonucleotides.

Findings

Oligonucleotides exhibit total breakthrough behavior in HILIC under specific conditions.

The IP-RPLC × HILIC configuration improves MS compatibility and avoids solvent mismatch.

The study identifies key factors influencing total breakthrough behavior in 2D-LC.

Abstract

Solvent mismatch between the two dimensions is one of the main limitations in two‐dimensional liquid chromatography (2D‐LC) and presents a significant challenge for method development. Although 2D‐LC provides a powerful means to increase peak capacity for oligonucleotides analysis compared to conventional one‐dimensional LC, solvent incompatibility remains a major obstacle that discourages broader development of such methods. In this work, we investigate a technical solution that can be easily implemented and that eliminates solvent mismatch effects during 2D‐LC analysis of oligonucleotides. This approach is based on the total breakthrough behavior of oligonucleotides, which is a phenomenon that allows the injection of large volumes into the second dimension (2D) without peak distortion. In this work, we showed that under appropriate conditions, oligonucleotides exhibit total breakthrough behavior in HILIC. This behavior in HILIC is particularly advantageous, as the IP‐RPLC × HILIC configuration offers improved mass spectrometry (MS) compatibility compared to IP‐RPLC or HILIC × IP‐RPLC. Assuming an IP‐RPLC × HILIC configuration, we systematically investigated the composition of first‐dimension (1D) fractions and the 2D‐HILIC parameters influencing total breakthrough to identify the key factors promoting this behavior. Our results offer clear guidance for implementing successful IP‐RPLC × HILIC conditions that avoid mismatch effects for oligonucleotides analysis while maintaining a high injection volume in the second dimension. This work demonstrates the potential of the total breakthrough strategy for implementing 2D‐LC methods with HILIC as the second dimension.

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