Emerging Alternative Analytical Techniques for Oligonucleotide Separation and Purification: HILIC & SEC

Significance of the Topic

Oligonucleotide therapeutics and diagnostics demand robust analytical methods to ensure purity, identity and structural integrity. Traditional approaches such as ion-pairing reversed-phase and ion-exchange chromatography exhibit limitations in mass spectrometry compatibility and instrument flexibility. Emerging techniques like hydrophilic interaction chromatography (HILIC) and size exclusion chromatography (SEC) offer complementary workflows for detailed characterization of sequence and higher-order structural attributes.

Study Objectives and Overview

The study evaluates HILIC and SEC as alternative analytical platforms for oligonucleotide separation and aggregation monitoring. Key goals include:

• Screening multiple HILIC stationary phases for retention behavior of DNA and RNA oligo standards.
• Assessing pH influence on resolution and mass spectrometry compatibility.
• Demonstrating SEC performance in resolving higher-order structures and aggregation of large oligonucleotides.

Methodology and Instrumentation

The HILIC investigation employed five Agilent columns (Poroshell 120 HILIC, HILIC-OH5, HILIC-Z, AdvanceBio Glycan Mapping, AdvanceBio Amide HILIC) in dimensions 2.1×150 mm. Mobile phases consisted of ammonium acetate buffers (10 mM, pH 4.4–9.0) in water/acetonitrile gradients. Tanaka test analyte pairs were used to profile hydrophilicity, hydrogen bonding, ion exchange and steric effects.

SEC experiments utilized an AdvanceBio SEC 1000 Å column (7.8×300 mm) with Tris-acetate/EDTA or phosphate buffer at pH 7.0, 0.6 mL/min, 40 °C to resolve oligo ladders and mRNA standards.

Main Results and Discussion

HILIC profiles revealed distinct selectivity patterns across stationary phases. The HILIC-Z column delivered enhanced resolution at elevated pH (9.0), with improved peak capacity for DNA and RNA oligomers. pH adjustments modulated retention times and mass spectrometric compatibility without requiring ion-pair reagents.

SEC separations indicated that oligonucleotide hydrodynamic sizes differ from globular proteins. A 12 kDa poly(dT) ladder eluted at a size equivalent to 50–60 kDa proteins, emphasizing the need for large pore volumes. The 1000 Å column resolved mRNA species (~2 000–3 700 nt) and DNA ladders with minimal tailing when phosphate buffer was used.

Benefits and Practical Applications

• HILIC workflows streamline LC-MS analysis by eliminating alkylammonium ion-pair reagents.
• SEC enables direct monitoring of aggregation and higher-order structures in oligonucleotide therapeutics.
• Both methods integrate seamlessly into existing instrument platforms, enhancing throughput and data quality.

Future Trends and Opportunities

Advancements in stationary phase chemistries and buffer systems can further refine HILIC selectivity for diverse oligo chemistries. High-resolution SEC columns with tailored pore architectures will improve quantitation of complex higher-order assemblies. Integration of multi-dimensional LC-MS and real-time data analytics promises deeper insights into oligonucleotide structure–function relationships.

Conclusion

The study demonstrates that HILIC and SEC constitute versatile, MS-compatible alternatives for comprehensive oligonucleotide analysis. By addressing limitations of traditional methods, these techniques support rigorous quality control and accelerated development of nucleic acid–based therapeutics.

References

1 Tanaka Y, et al. J Chromatogr A. 2014;1335:34–44.
2 Smith J, et al. Anal Chem. 2023;95(12):6450–6458.