Work

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

Year: 2025

Type: article

Abstract: 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.

Source: Journal of Separation Science

DOI: 10.1002/jssc.70238

Authors: Jakob Haglöf, Jenny M. Nilsson, Jufang Wu Ludvigsson

Institutions: Uppsala University, AstraZeneca (Sweden)

Language: English

Topic: Analytical Chemistry and Chromatography

Subfield: Spectroscopy

Field: Chemistry

Domain: Physical Sciences

Open Access status: hybrid

Estimated APC paid: $4,030

Funder: AstraZeneca