SurePac Oligo RP MDi columns

Significance of the topic

The SurePac Oligo RP MDi column addresses a growing need in nucleic acid analytics for robust, high-resolution reversed-phase separations that span a wide range of oligonucleotide and RNA sizes while remaining compatible with MS-friendly mobile phases. Improved particle uniformity, broad pore architecture, inert hardware coatings and high thermal and chemical stability make such columns valuable for applications in oligonucleotide therapeutics development, quality control, and intact RNA characterization where resolution, reproducibility and MS compatibility are critical.

Study objectives and overview

This product manual presents the design rationale, recommended operating practices, method-development guidance and application data for the Thermo Scientific SurePac Oligo RP MDi columns. Key goals are to document column performance across sample types (short oligos, siRNA, sgRNA, tRNA, intact mRNA), to show method transfer and scale-up considerations, and to demonstrate operational stability, lot-to-lot reproducibility, low carryover and compatibility with LC–MS workflows under volatile ion-pairing conditions.

Methods and key methodological recommendations

The stationary phase is a 2.5 µm monodisperse supermacroporous (SMP) polymeric particle featuring a continuum of pore sizes to balance resolving power for short oligonucleotides and mass transfer for long RNA species. The hardware receives a mildly hydrophobic inert coating to reduce metal and surface interactions.

Practical system and method recommendations:

• Mobile phases: TEAA/ACN (LC–UV), HAA/ACN, and volatile alkylamine/HFIP with methanol (e.g., DBA/HFIP/MeOH) for LC–MS compatibility. Optimize ion-pair reagent concentration, pH and organic modifier for target analytes.
• Temperature control: columns are thermally stable to 90 °C; typical oligonucleotide separations use 50–80 °C for denaturing secondary structure and improving resolution. Keep native analyses below melting temperature.
• Pre-column heater: recommended to equilibrate mobile phase and reduce band broadening; shown to increase separation spread and sharpen peaks.
• Flow and gradient: optimize flow rate and gradient slope together. Lower flow increases interaction time and sensitivity; higher flow reduces run time but can reduce resolution. Gradient length trades resolution for speed.
• Column formats and scaling: select length and ID to balance throughput and resolving power. Methods from DNAPac RP can be used as starting points but may require adjustments for loading and mobile phase due to differences in stationary-phase surface area.
• Injection/loading: dynamic capacity depends on analyte molecular weight; injections up to ~2 µL maintained sharp peaks in demonstrated conditions, while ≥5 µL showed onset of overloading. Choose injection volume based on concentration and required resolution.
• Operating ranges: recommended pH 2–12 for 2.1 mm ID formats (2–10 for 0.3 mm ID). Typical temperature range 30–60 °C for many separations; columns tolerate up to 90 °C. Observe column pressure limits per format and ramp flow slowly (~1/3 of max flow ramp rate) to protect the bed.

Instrumentation used

Instrument platforms and devices reported in the manual and used in evaluations include:

• Thermo Scientific Vanquish Horizon and Vanquish Neo UHPLC systems
• Thermo Scientific Orbitrap Ascend Tribrid Mass Spectrometer
• Thermo Scientific Xcalibur, Freestyle and BioPharma Finder software
• Thermo Scientific nanoViper and Viper fittings (recommended small ID tubing for low dead volume)
• Pre-column heater and post-column cooler configurations (as applied in experiments)

Main results and discussion

Performance highlights obtained from the manual's validation experiments:

• Chromatographic performance across a wide oligonucleotide size range: the SMP monodisperse particles provided high resolution for short oligos (12–40 mers) while accommodating very large species (intact mRNA, ~1960 nt).
• Mobile-phase versatility: TEAA/ACN, HAA/ACN and DBA/HFIP/MeOH systems were shown to separate ssDNA mixes, siRNA and sgRNA effectively; DBA/HFIP proved compatible with LC–MS analysis.
• Pre-column heating and elevated column temperature improved peak sharpness, increased separation spread for size variants and reduced viscosity/backpressure enabling faster mass transfer.
• Column format scaling: identical stationary phase chemistry yielded similar selectivity across 2.1 × 20, 50 and 100 mm formats; column length primarily affected efficiency and analysis time.
• Inert coating performance: coated hardware produced high recovery for sensitive analytes (e.g., AMPcP ≥95% across injections), whereas stainless steel hardware showed significant adsorption and poor recovery.
• Carryover: intact 1960 nt mRNA analysis produced low carryover (~0.48%) when a blank run was run immediately afterwards.
• LC–MS compatibility and intact RNA analysis: capillary 0.3 × 50 mm column with DBA/HFIP enabled high-resolution MS of tRNAPhe; multiple intact molecular states were reproducibly detected with monoisotopic mass precision ≤1 ppm and the dominant state showing 0.07 ppm CV.
• Lot-to-lot reproducibility and ruggedness: separations across multiple manufacturing lots showed minimal retention-time and PWHH differences; extended use testing (≥1000 injections) demonstrated stable retention, peak width and system backpressure.

Benefits and practical applications

The SurePac Oligo RP MDi column offers several practical advantages for analytical labs focused on oligonucleotides and RNA:

• Wide molecular-size applicability: one stationary phase supports analysis from short oligos to intact mRNA, simplifying method portfolios.
• MS compatibility: performance with volatile ion-pairing systems enables intact RNA MS characterization and detection of closely related variants.
• Operational robustness: high thermal and pH stability, inert hardware, and demonstrated lots-to-lot consistency support routine QC and method transfer.
• High throughput capability: availability of short and capillary formats and the possibility to operate at elevated temperature and flow allow throughput tuning without sacrificing selectivity when needed.

Future trends and potential uses

Potential developments and applications where this column technology is poised to contribute include:

• Expanded LC–MS intact RNA workflows for therapeutic mRNA and modified oligonucleotides, including automated deconvolution and variant profiling.
• Integration into high-throughput QC pipelines for oligonucleotide therapeutics, leveraging ruggedness and low carryover to reduce downtime and manual passivation steps.
• Method standardization and transfer across labs, enabled by monodisperse particle manufacturing for improved lot-to-lot reproducibility.
• Further optimization of volatile ion-pairing chemistries and microflow/capillary formats to enhance MS sensitivity for low-abundance variants and impurity profiling.

Conclusion

The SurePac Oligo RP MDi column is a purpose-built reversed-phase column for comprehensive nucleic acid separations that balances resolving power for short oligonucleotides with accessibility for very large RNAs. Its monodisperse SMP polymer particles, inert hardware coating, and demonstrated compatibility with both UV and MS-friendly mobile phases make it a versatile tool for researchers and QC labs working with oligonucleotide therapeutics and RNA analytics. Recommendations provided in the manual (mobile-phase selection, temperature control, pre-column heating, careful scaling and loading limits) facilitate robust method development and reliable method transfer.

References

• Thermo Fisher Scientific. SurePac Oligo RP MDi Column Product Manual. Thermo Fisher Scientific, 2026.
• Instrument and software references cited in manual: Thermo Scientific Vanquish Horizon/Neo UHPLC; Orbitrap Ascend Tribrid MS; Xcalibur, Freestyle, BioPharma Finder.