Application NoteImproved Chromatographic Analysis of Oligonucleotides with ACQUITY PREMIER Oligonucleotide BEH C18 Columns

Importance of the Topic

Therapeutic oligonucleotides such as antisense sequences, siRNA and mRNA vaccines are emerging in drug development and diagnostics. Accurate chromatographic analysis ensures reliable quality control, potency assessment and method repeatability.

Study Objectives and Overview

This work evaluates non-specific adsorption of oligonucleotides on metallic LC hardware and demonstrates how ACQUITY PREMIER Oligonucleotide BEH C18 Columns with MaxPeak HPS surfaces eliminate sample loss and improve analytical performance.

Methodology and Instrumentation

The study comprised:

• MISER experiments with 2.1 mm stainless steel frits and PEEK unions to quantify adsorption of a 25-mer phosphorothioate oligonucleotide at varying mobile phase pH values.
• Ion-pair reversed-phase UPLC separations of 15 to 35 mer oligodeoxythymidine standards using hexylammonium acetate mobile phases at pH 6–8.5.
• Comparative analysis on conventional stainless steel columns versus PREMIER columns under identical gradient and temperature conditions.

Key instrumentation:

• ACQUITY UPLC H-Class Bio system conditioned with oligodeoxythymidine.
• ACQUITY PREMIER Oligonucleotide BEH C18 Columns, 130 Å, 1.7 µm, 2.1×50 mm.
• Empower 3.0 chromatography data software.
• UV detection at 260 nm with titanium 5 µL flow cell.

Main Results and Discussion

Experiments revealed significant oligonucleotide adsorption on stainless steel frits, with up to 29 % recovery loss even after 50 injections at neutral pH. Acidic conditions (pH 5) exacerbated adsorption, while alkaline pH (7–8) reduced but did not eliminate losses. Transient conditioning was observed: repeated injections temporarily saturate metal binding sites but prolonged mobile phase flushing causes de-conditioning and peak variability. PREMIER columns equipped with MaxPeak HPS hardware achieved near-complete recovery from the first injection across pH 6–8.5, demonstrating permanent mitigation of surface adsorption.

Benefits and Practical Applications

This approach offers:

• Elimination of extended column conditioning protocols.
• Consistent, high-recovery analysis for trace and preparative oligonucleotide samples.
• Improved reproducibility and quantitation accuracy in LC-UV and LC-MS workflows.

Future Trends and Possibilities

Advancements in surface chemistry and HPS technologies may extend to other analytes prone to metal interactions, such as phosphopeptides and organophosphates. Integration with high-throughput LC-MS platforms and further miniaturization could enhance sensitivity in genomic and clinical applications.

Conclusion

Non-specific adsorption on metallic LC components poses a significant challenge in oligonucleotide analysis. Elevating mobile phase pH offers limited relief. MaxPeak HPS hardware in ACQUITY PREMIER columns provides a robust solution, delivering reliable, out-of-the-box performance and increasing confidence in quantitative oligonucleotide assays.

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