Agilent AdvanceBio Oligonucleotide Columns and Oligonucleotide Standards

Importance of the Topic

Synthetic oligonucleotides have become critical therapeutic agents in areas such as antiviral and oncological treatments. These molecules, including antisense oligonucleotides, siRNAs and aptamers, require precise removal of synthesis-related and product-derived impurities to ensure efficacy and safety.

Objectives and Study Overview

This work evaluates the chromatographic performance of Agilent AdvanceBio Oligonucleotide columns for high-resolution separation of deprotected oligos on HPLC and UHPLC platforms. The study also assesses column longevity under high pH conditions and demonstrates methods for impurity profiling.

Methodology

Ion-pair reversed-phase chromatography was employed using trityl-off, deprotected oligonucleotide samples. Mobile phases included triethylammonium acetate (TEAA) and a volatile combination of triethylamine and hexafluoroisopropanol (TEA-HFIP) to enable compatibility with UV and mass spectrometric detection. Separation performance was monitored through multiple injection cycles and standard mixtures.

Instrumentation Used

• Agilent AdvanceBio Oligonucleotide Columns, 2.1 x 50 mm, 2.7 µm superficially porous C18, 100 Å pores, endcapped, pH stability 3–11, 65 °C temperature limit
• Agilent 1260 Infinity LC System and 1260 Infinity Bio-inert Quaternary LC System
• Agilent 1290 Infinity II LC System for UHPLC applications
• UV detection at 260 nm and electrospray ionization mass spectrometry

Main Results and Discussion

The AdvanceBio columns maintained consistent retention times and narrow peak widths through over 300 injections in a high pH TEAA environment, demonstrating extended lifetime and reliable performance. Single nucleotide resolution was achieved, separating full-length oligos from N-1 species, confirmed using an oligonucleotide resolution standard. Application of TEA-HFIP mobile phases enabled clean interface with mass spectrometry, facilitating accurate mass-based sequence confirmation.

Benefits and Practical Applications

• High-resolution separations enable accurate impurity identification and quantitation
• Robust silica-based columns resist dissolution in alkaline mobile phases, reducing replacement frequency
• Compatibility with both HPLC and UHPLC systems enhances laboratory flexibility
• Volatile mobile phase options support direct LC/MS workflows for structural characterization

Future Trends and Potential Applications

The demand for advanced oligonucleotide analytics will grow with the expansion of nucleic acid therapeutics. Integration with high-resolution MS and automation platforms could accelerate method development and quality control. Emerging chemistries and higher throughput requirements will drive further column innovations.

Conclusion

Agilent AdvanceBio Oligonucleotide columns offer a balanced combination of high resolving power, chemical stability under alkaline conditions and compatibility with modern detection methods. These attributes support reliable, cost-effective, and flexible workflows for oligonucleotide analysis in research and quality control environments.

References

• Agilent Technologies 2015, Agilent AdvanceBio Oligonucleotide Columns and Oligonucleotide Standards, Publication 5991-5845EN