Analysis of Oligonucleotides with Ion Exchange Chromatography and Agilent Infinity II UHPLC

Importance of the Topic

Oligonucleotide therapeutics and diagnostics are rapidly advancing fields, driven by FDA-approved mRNA vaccines and gene therapies. Precise characterization of oligonucleotides—from short siRNAs to long mRNA molecules—is essential to ensure product quality, detect impurities, and guide purification processes. Ion exchange chromatography (IEX) offers high resolution for charged biomolecules and complements the more common ion-pair reversed-phase approaches, especially when analyzing large or highly charged nucleic acid species.

Objectives and Overview of the Study

This application note evaluates the performance of Agilent Bio SAX and PL-SAX columns on the Agilent 1290 Infinity II Bio UHPLC platform for analyzing DNA and RNA oligonucleotides. The goals are to establish starting conditions for method development, compare nonporous versus porous stationary phases, assess salt gradient elution with NaCl and NaClO4, and explore the effects of temperature and particle size on separation quality.

Methodology and Used Instrumentation

Reagents and Standards:

• Tris buffers and salts (NaCl, NaClO4) adjusted to pH 8
• Oligonucleotide ladders (DNA and RNA) from Agilent, Invitrogen, Thermo Fisher

Columns and System:

• Agilent 1290 Infinity II Bio UHPLC with high-speed pump
• Bio SAX NP5 PEEK nonporous column (4.6 × 250 mm, 5 µm)
• PL-SAX 4000A porous column (4.6 × 250 mm, 10 µm)

Chromatographic Conditions:

• Mobile phases: 20 mM Tris-HCl (pH 8) with NaCl or NaClO4 gradients
• Flow rate: 1.0 mL/min; temperature range: 20 °C–80 °C
• Detection: UV at 260 nm

Main Results and Discussion

Column Comparison:

• Bio SAX NP5 nonporous columns achieved base-pair resolution up to 10,000 bp and clear separation of DNA/RNA ladders.
• PL-SAX porous columns offered higher loading capacity but slightly lower resolution, suiting preparative or purification tasks.

Salt Elution:

• NaClO4 proved a more potent eluent than NaCl, reducing required salt concentration and enhancing resolution for larger fragments.
• Acetonitrile modulation with NaCl further tailors selectivity.

Temperature Effects:

• Increasing column temperature improved mass transfer and reduced peak broadening for both DNA and RNA standards.
• Optimal separations were achieved near 60 °C–70 °C, balancing retention and resolution.

Gradient and Particle Size:

• Shallower salt gradients (0.25 %/min) enhanced separation of closely sized short-mers (19 nt vs. 20 nt) compared to steeper gradients.
• Unexpectedly, the 10 µm nonporous column outperformed the 5 µm variant under the tested conditions, highlighting the importance of matching gradient and flow settings to particle size.

Benefits and Practical Applications of the Method

High-resolution IEX on the Infinity II UHPLC offers:

• Reliable impurity profiling of gene therapy and oligonucleotide products
• Flexibility to handle a broad size range (15 nt–10,000 bp)
• Robust performance with corrosive salts thanks to inert UHPLC components
• Scalability from analytical to preparative separations using PL-SAX columns

Future Trends and Potential Applications

Advances are expected in:

1. Integration of mass spectrometry for direct oligonucleotide identification under IEX conditions
2. Automated method scouting combining salt and organic modifiers to accelerate method development
3. High-throughput platforms for rapid QC of multiple therapeutic candidates
4. Customized stationary phases to further improve selectivity for modified or conjugated oligonucleotides

Conclusion

This study confirms that Agilent’s UHPLC-based ion exchange solutions can deliver exceptional separations across a wide oligonucleotide size range. Nonporous Bio SAX columns provide unmatched resolution for analytical needs, while porous PL-SAX counterparts enable efficient purification workflows. Salt type, gradient slope, temperature, and particle size must be optimized in concert to achieve the best results for each application.

References

1. Vanhoenacker G. et al. Evaluation of Different Ion-Pairing Reagents for LC/UV and LC/MS Analysis of Oligonucleotides. Agilent Technologies application note, 5994-2957EN, 2021.
2. Duong P. et al. Fast and High-Resolution Reversed-Phase Separation of Synthetic Oligonucleotides. Agilent application note, 5991-6006EN, 2017.
3. Massie J.; Lloyd L. High-Resolution Separations of Oligonucleotides Using PL-SAX Strong Anion-Exchange HPLC Columns. Agilent application note, 5990-8297EN, 2021.
4. Bonilla JV; Srivatsa GS. (Eds.) Handbook of Analysis of Oligonucleotides and Related Products. CRC Press, 2016.
5. LeRouge E. et al. The Investigation of DNA and RNA Structural Differences Using Ultra High Performance Liquid Chromatography. SUNY Albany Honors Thesis, 2016.