Oligonucleotide Analysis with Ion-Pair Reversed-Phase Chromatography and Agilent 1260 Infinity II Prime LC

Importance of the Topic

High-quality characterization of oligonucleotides is essential for the development and quality control of gene therapies and RNA-based biopharmaceuticals. Ion-pair reversed-phase chromatography (IPRP) offers the resolution needed to detect sequence variants and n-1 short-mer impurities across a wide range of oligonucleotide lengths.

Study Objectives and Overview

This application note evaluates the performance of Agilent AdvanceBio oligonucleotide columns on an Agilent 1260 Infinity II Prime LC. It aims to compare common ion-pairing reagents (TEAA, DBAA, HAA) and to optimize gradient profiles and column temperatures for improved separation of ssDNA and ssRNA standards.

Methodology and Instrumentation

• Reagents: Triethylamine acetate (TEAA), dibutylamine acetate (DBAA), hexylamine acetate (HAA), acetic acid, acetonitrile.
• Standards: ssDNA oligo-dT ladder (15–40 nt) and ssRNA resolution mix (14, 17, 20, 21 nt).
• Instrument: Agilent 1260 Infinity II Prime LC, AdvanceBio oligonucleotide column 2.1×150 mm, 2.7 µm; UV detector at 260 nm; flow rate 0.6 mL/min; sampler at 4 °C; column oven nominal at 60 °C.
• Mobile phases: 100 mM ion-pair agent in water (A) and in acetonitrile (B), both adjusted to pH 7; typical gradients from 10 to 90 % B over 19 min.
• Software: Agilent OpenLab CDS 2.6.

Results and Discussion

• Ion-pairing comparison: HAA achieved the highest resolution for ssDNA (Rs = 3.9 between 19 and 20 nt) and ssRNA (Rs = 2.8 between 20 and 21 nt), outperforming TEAA and DBAA.
• Gradient optimization: Slower gradients (0.25 % B/min) enhanced resolution (up to Rs = 4.32 for ssDNA) but extended run times and reduced peak heights to 40 % of those at 1.25 % B/min.
• Temperature effects: ssDNA resolution improved steadily with higher temperatures (up to Rs = 4.32 at 80 °C), while ssRNA retention times decreased significantly without resolution gain.

Practical Benefits and Applications

This optimized IPRP method enables precise impurity profiling and sequence confirmation in oligonucleotide manufacturing and QC. The flexibility in reagent choice, gradient slope, and temperature allows tailoring of analysis time, sensitivity, and resolution to specific workflow demands.

Future Trends and Opportunities

• Integration of volatile ion-pair reagents for seamless LC–MS analysis.
• Design of specialized stationary phases for high-throughput oligonucleotide screening.
• Automation of method development and coupling with synthetic oligo workflows.
• Extension to duplexes, modified bases, and longer mRNA constructs.

Conclusion

The Agilent 1260 Infinity II Prime LC combined with AdvanceBio oligonucleotide columns delivers high-resolution separations when using longer-chain ion-pairing agents and shallow gradients. Temperature control further enhances ssDNA selectivity. These findings support robust method development for diverse oligonucleotide analyses.

Reference

• Gerd V.; Sonja S. Evaluation of Different Ion-Pairing Reagents for LC/UV and LC/MS Analysis of Oligonucleotides. Agilent Technologies Application Note. 2021.
• Phu D.; Brain A. Fast and High-Resolution Reversed-Phase Separation of Synthetic Oligonucleotides. Agilent Technologies Application Note. 2017.
• Massie J.; Lloyd L. Use Temperature to Enhance Oligonucleotide Mass Transfer and Improve Resolution in Ion-Pair RP HPLC. Agilent Technologies Application Note. 2011.
• Bonilla JV; Susan GS. Handbook of Analysis of Oligonucleotides and Related Products. Taylor & Francis Group. 2011.
• Alexander J. Investigation of Pore Size Effects at Separation of Oligonucleotides Using Ion-Pair RP HPLC. Faculty of Health, Science and Technology. 2019.
• Lingzhi G.; James SO. Comparing Ion-Pairing Reagents and Sample Dissolution Solvents for Ion-Pairing Reversed-Phase LC/ESI-MS Analysis of Oligonucleotides. Rapid Commun. Mass Spectrom. 2014;28:339–350.
• Evanna L.; Maria B.; Srivathsan V. Investigation of DNA and RNA Structural Differences Using Ultra-High Performance Liquid Chromatography. Anthropology. 2016;21.