Development of Enhanced Separation Techniques for Oligonucleotides Utilizing Mixed-Mode Chromatography and
Jakob Haglöf1, Jenny M Nilsson1, Jufang Wu Ludvigsson2
1Department of Medicinal Chemistry, Uppsala Biomedical Centre, Uppsala University, Uppsala, Sweden.
Journal of Separation Science
|August 5, 2025
Summary
This study introduces a novel mixed-mode chromatography method for analyzing oligonucleotides, offering superior separation without ion-pairing reagents. A two-dimensional liquid chromatography setup enabled sensitive mass spectrometry detection.
Area of Science:
- Analytical Chemistry
- Chromatography
- Biochemistry
Background:
- Traditional oligonucleotide analysis relies on ion-pair reversed-phase liquid chromatography (IP-RP-LC).
- IP-RP-LC often requires specialized reagents that can complicate downstream analysis.
- There is a need for alternative, more efficient, and MS-compatible methods for oligonucleotide separation.
Purpose of the Study:
- To explore a reversed-phase weak anion exchange mixed-mode column as an alternative for oligonucleotide analysis.
- To evaluate the separation performance of the mixed-mode column compared to traditional C18 columns.
- To develop a two-dimensional liquid chromatography (2DLC) method for enhanced MS detection of oligonucleotides.
Main Methods:
- Utilized a reversed-phase weak anion exchange mixed-mode column for oligonucleotide separation.
- Investigated the impact of pH, buffer concentration, and acetonitrile content on selectivity.
- Established a 2DLC-UV-quadrupole time-of-flight (qTOF) setup with a HILIC-type LUNA Omega Sugar column for MS detection.
Main Results:
- The mixed-mode column demonstrated stronger retention and superior separation of oligonucleotides compared to C18 columns, eliminating the need for ion-pairing reagents.
- Selectivity was tunable via pH, buffer concentration, and acetonitrile gradients.
- A 100-fold increase in MS intensity was achieved using the 2DLC setup, enabling characterization of the main peak.
Conclusions:
- Reversed-phase weak anion exchange mixed-mode chromatography is a viable alternative for oligonucleotide analysis, offering improved separation and eliminating ion-pairing reagents.
- The developed 2DLC-UV-qTOF method successfully overcomes the MS incompatibility of phosphate buffers, enabling sensitive and accurate oligonucleotide characterization.
- This approach provides a powerful tool for the analysis of oligonucleotides, particularly when coupled with mass spectrometry.
Keywords:
2D liquid chromatographymass spectrometrymixed‐mode chromatographyoligonucleotidereversed‐phase/weak anion‐exchangeMore Related Videos
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