Fast and High-Resolution Reversed-Phase Separation of Synthetic Oligonucleotides

Importance of the Topic

Synthetic DNA and RNA oligonucleotides play a critical role in modern biotherapeutic development. Their multistep synthesis yields target sequences alongside shorter failure-products (N-1, N-2), which must be efficiently separated and identified to ensure drug purity, potency, and safety. Traditional anion-exchange methods deliver high resolution but are time-consuming and incompatible with mass spectrometry (MS) due to high salt elution.

Objectives and Study Overview

This application note evaluates high-pH-stable, superficially porous reversed-phase columns (Agilent AdvanceBio Oligonucleotide) for fast, high-resolution separation and MS-compatible identification of deprotected DNA and RNA oligonucleotides. Comparative performance against totally porous hybrid columns is assessed, along with method robustness and throughput potential.

Methodology and Instrumentation

Sample preparation involved Agilent Oligonucleotide Ladder (15–40-mer DNA) and Resolution Standards (14–21-mer RNA). Two mobile phase systems were tested:

• LC/UV: 100 mM triethylammonium acetate (TEAA) in water (A) and acetonitrile (B)
• LC/MS: 400 mM hexafluoroisopropanol:15 mM triethylamine (HFIP:TEA) in water (A) and methanol:A (50:50, B)

Chromatography was carried out at 65 °C on 2.1 × 50 mm, 2.7 µm superficially porous columns at 0.4–0.6 mL/min. Detection modes included UV at 260 nm and negative-ion Q-TOF MS (Agilent 6530) with a 400–1,700 m/z range. Instruments used:

• Agilent 1290 Infinity LC
• Agilent 6530 Accurate-Mass Q-TOF LC/MS

Main Results and Discussion

RNA separation: Four oligos (14–21 mer) were baseline-resolved in under nine minutes; N and N-1 species achieved near-baseline separation. DNA ladder: Six DNA oligos (15–40 mer) eluted within eight minutes with sharp, fully resolved peaks. MS compatibility: A 25-mer DNA sample produced a resolved UV peak in ~3 min and matching total ion chromatogram, enabling deconvolution of main and impurity peaks with 100% recovery. Column comparison: The superficially porous column delivered narrower peaks (0.160 min vs. 0.185 min) at lower backpressure (108 bar vs. 292 bar) than a 1.7 µm totally porous counterpart. Stability: Over ~400 injections, retention times remained stable with negligible drift.

Benefits and Practical Applications

• High throughput separation of oligonucleotides in minutes
• Compatibility with LC/UV and MS workflows
• High-pH stability up to pH 11 for robust operation
• Reduced solvent and salt use, simplifying MS analysis
• Lower backpressure suitable for HPLC and UHPLC platforms

Future Trends and Potential Applications

Advances in superficially porous particle technology will continue to drive faster, higher-resolution oligonucleotide analyses. Integration with automated sample prep and online MS sequencing could further streamline oligo quality control. Expansion into modified and longer oligonucleotides, as well as multiplexed assays, represents an emerging opportunity.

Conclusion

Agilent AdvanceBio Oligonucleotide columns combining superficially porous particles and high-pH tolerance enable rapid, high-resolution reversed-phase separations of synthetic oligonucleotides. They support both LC/UV and LC/MS workflows with robust performance, lower backpressure, and long column lifetime, meeting the demands of biopharmaceutical QC and research.

Reference

Phu Duong et al. Fast and High-Resolution Reversed-Phase Separation of Synthetic Oligonucleotides. Agilent Technologies Application Note 5991-6006EN, 2017.