Thermo Scientific DNAPac columns - Ultra-high resolution oligonucleotide separation

Significance of the Topic

High-resolution separation of oligonucleotides is essential for assessing purity, detecting synthesis failures, and ensuring quality of therapeutic and diagnostic nucleic acids. Ultra-high-performance liquid chromatography (UHPLC) columns with advanced chemistries enable rapid, high-capacity, and robust analyses that support research, quality control, and manufacturing workflows.

Objectives and Study Overview

This application note reviews the evolution and performance characteristics of Thermo Scientific™ DNAPac™ and DNASwift™ columns for oligonucleotide separation. Key goals are to summarize column chemistries, demonstrate separation efficiency of target and truncated sequences, and highlight practical benefits for analytical and semi-preparative workflows.

Methodology and Instrumentation

The report covers six column families designed for anion-exchange and reversed-phase separations of oligonucleotides:

• DNAPac PA100: Original anion-exchange UHPLC columns introduced in 1990
• DNAPac PA200 (2004) and PA200RS (2013): High-resolution charge-based separations of full-length vs. failure sequences
• DNAPac RP (2016): Ion-pair reversed-phase columns compatible with LC-MS, pH 1–14, and temperatures up to 110 °C, resolving fragments up to 10 000 bp
• DNASwift SAX-1S (2009): Monolithic anion-exchange columns offering high loading capacity for laboratory-scale purification
• DNAPac semi-preparative (2024): Scale-up formats for higher throughput

Chromatographic conditions include short 2.1 × 50 mm UHPLC formats, high-capacity polymeric stationary phases, and gradients optimized for speed and resolution.

Main Results and Discussion

Separation of a 22-mer DNA target (n) from its 21-mer failure sequence (n–1) was achieved in under 2 minutes using both:

• DNAPac RP 2.1 × 50 mm, 4 µm
• DNAPac PA200RS 2.1 × 50 mm, 4 µm

UV absorbance traces demonstrate baseline resolution of truncated sequences with minimal peak broadening. The high surface-charge density of PA200 chemistries and optimized pore sizes (50–2000 Å) in RP columns combine rapid analysis with ultra-high resolution. Table summaries of column dimensions, particle sizes, and ID formats illustrate broad availability for analytical and preparative scales.

Benefits and Practical Applications

• Exceptional resolution of full-length oligos from n±1 impurities
• Compatibility with LC-MS, extreme pH, and high temperature supports diverse analytical workflows
• Rapid UHPLC formats reduce cycle times and solvent consumption
• Monolithic and semi-preparative options enable scale-up from method development to production
• Reliable performance over hundreds of injections ensures reproducibility in QA/QC environments

Future Trends and Opportunities

Advances in stationary-phase design and instrument control will further accelerate oligonucleotide analysis. Emerging areas include:

• Integration of AI-driven method optimization for gradient and buffer selection
• Next-generation monolithic and core–shell particles for even higher throughput
• Enhanced coupling with high-resolution mass spectrometry for impurity profiling
• Automated purification workflows using semi-preparative formats for oligo manufacturing

Conclusion

Thermo Scientific DNAPac and DNASwift columns deliver industry-leading resolution, speed, and robustness for oligonucleotide analysis. Their range of chemistries and formats meets the demands of research, quality control, and production, ensuring accurate characterization of therapeutic and diagnostic nucleic acids.

References

No formal literature citations were provided in the source document.