Development of Enhanced Separation Techniques for Oligonucleotides Utilizing Mixed‐Mode Chromatography and 2D‐LC/UV/MS Analysis

Oligonucleotides are traditionally analyzed using ion‐pair reversed‐phase liquid chromatography. In this project, we explored an alternative approach utilizing a reversed phase‐weak anion exchange mixed‐mode column, which combines separation based on both charge and hydrophobicity. Our findings indicate that the mixed‐mode column provided stronger retention and superior separation compared to the C18 column, eliminating the need for ion‐pairing reagents. Additionally, selectivity can be adjusted using gradient pH and buffer concentration as well as acetonitrile content. However, the use of a phosphate buffer was necessary to ensure adequate retention and separation, despite its incompatibility with MS detection. To address this issue, a 2DLC‐UV‐qTOF setup with multiple loops was established, where the second‐dimension LC, connected to the MS detector, required only an MS‐compatible mobile phase. Using a HILIC‐type LUNA Omega Sugar column in the second‐dimension LC, the MS intensity of the heart‐cut from the first dimension was increased 100‐fold, allowing for the characterization of the main peak.