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

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.