Mitigation of Non-Specific Adsorption During Column Conditioning for an Oligonucleotide HILIC Analysis

Significance of the Topic

Oligonucleotides are key tools in genetic research and therapeutics, yet their analysis is often compromised by non-specific adsorption on metal surfaces. Hydrophilic Interaction Liquid Chromatography (HILIC) provides a more sustainable alternative to ion-pair reversed-phase methods by using less toxic solvents and reducing costs, but adsorption issues can degrade sensitivity and reproducibility.

Objectives and Study Overview

This application note evaluates how MaxPeak High Performance Surfaces (HPS) technology mitigates non-specific adsorption during HILIC analysis of oligonucleotides. A comparative study was conducted across four configurations combining standard stainless-steel and inert MaxPeak HPS-coated HPLC systems with corresponding columns.

Methodology

Recovery was assessed by measuring peak area ratios before and after passivation. Each test sequence comprised six injections of a 40 pmol oligonucleotide standard, 20 injections of a 160 pmol passivation solution, and six repeat standard injections. Percent recovery was calculated from the peak area of the first injection post-passivation. The four system/column combinations under HILIC conditions allowed direct attribution of performance differences to HPS technology.

Instrumentation

• HPLC Systems: Waters Alliance iS (stainless steel) and Alliance iS Bio (MP35N, corrosion resistant) with MaxPeak HPS technology.
• Columns: XBridge BEH Amide and XBridge Premier BEH Amide, 2.5 µm, 4.6 × 100 mm.
• Detector: Tunable UV at 260 nm, 5 Hz sampling.
• Mobile Phases: Water (A), acetonitrile (B), 50 mM ammonium acetate pH 6.7 (C).
• Temperatures: Column 60 °C; sample manager 10 °C.
• Flow Rate: 1.0 mL/min; injection volumes: 10 µL (standard), 20 µL (passivation).
• Data System: Empower 3.7.0.

Results and Discussion

The standard stainless-steel system and column showed low initial recoveries due to significant non-specific adsorption, improved only after time-consuming passivation. Pairing the stainless-steel HPLC with a MaxPeak HPS column increased column recovery (~57%) but did not address system adsorption. Conversely, the inert Alliance iS Bio system with a MaxPeak HPS column achieved an average initial recovery of 98%, stable peak areas (RSD < 2%), and superior peak shape without any passivation step, clearly demonstrating the combined benefit of system and column surface treatment.

Benefits and Practical Applications

• Immediate high recoveries of oligonucleotides without passivation or ion-pair reagents.
• Enhanced method robustness and reproducibility critical for bioanalytical and QC workflows.
• Use of less toxic solvents aligns with green laboratory initiatives and lowers operational costs.

Future Trends and Applications

Advances in inert surface technologies will further streamline analysis of metal-sensitive biomolecules, including longer or modified oligonucleotides. Integration with high-throughput and automated workflows promises improved data integrity for pharmaceutical development and personalized medicine.

Conclusion

MaxPeak HPS technology in both HPLC system and column effectively eliminates non-specific adsorption under HILIC conditions, delivering out-of-the-box high recoveries, reduced method development time, and robust performance for oligonucleotide analysis in research and pharmaceutical environments.

References

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