Hey, ho, oliGO – Comparison of ion-pairing systems for oligonucleotide analysis with HPLC-UV

Importance of the topic

Oligonucleotides are rapidly becoming central molecules in modern molecular biology and therapeutics. Their increasing use in highly specific and personalized drugs places strong demands on analytical workflows in terms of selectivity, robustness and throughput. Reliable HPLC methods that can separate long, highly polar and closely related sequences are therefore essential for quality control, formulation development and batch release of oligonucleotide therapeutics.

Objectives and overview of the study

This work compared two ion-pair reversed-phase (IP-RP) HPLC systems for UV-based analysis of oligonucleotides: triethylamine (TEA) combined with acetic acid (HAc) versus TEA combined with hexafluoroisopropanol (HFIP). The aims were to optimize gradients and ion-pair concentrations, evaluate retention and peak shape across different column lengths, and to probe the On/Off retention mechanism using three synthetic oligonucleotides of 15, 25 and 64 bases with differing modifications (one sample containing a C12 linker). Emphasis was placed on method transferability, run time reduction and maintaining chromatographic performance suitable for high-throughput QC.

Materials and analytical methodology

• Samples: Three synthetic oligonucleotides (15-mer, 25-mer with a C12 linker, and a 64-mer). Concentrations were determined by UV spectrophotometry (NanoDrop) and samples were dissolved in water prior to analysis. A uracil standard was used as dead-time marker.
• Stationary phase: Sepapure oliGO reversed-phase columns (3 µm particles) tested at 50 × 4.6 mm, 30 × 4.6 mm and a 10 × 4.6 mm precolumn/guard.
• Mobile phases: Eluent A = methanol; Eluent B = aqueous solution containing ion-pair reagents (either TEA + HAc or TEA + HFIP). Sandwich injection solvent = methanol:H2O 5:95 (v/v).
• Typical chromatographic conditions: flow 0.75 mL/min, column temperature 60 °C, UV detection at 254 and 260 nm, injection volumes 1–5 µL using sandwich injection mode.
• Ion-pair systems and optimization: For HAc system starting conditions were 20 mM TEA + 20 mM HAc (pH ~8.5), optimized to 10 mM TEA + 10 mM HAc. For HFIP system starting conditions were 7 mM TEA + 100 mM HFIP (pH ~8), with TEA reduced to 4 mM while preserving retention.

Used instrumentation

• Autosampler/Injector: LH 8.1 Liquid Handler with robotic cooler and fast wash station.
• Pump: AZURA P 6.1L HPG (stainless steel, up to 10 mL/min).
• Detector: AZURA DAD 6.1L diode array detector (190–1000 nm) with 10 mm, 2 µL analytical flow cell.
• Thermostat: AZURA CT 2.1 column thermostat (up to 8 columns, 5–85 °C).
• Columns: Sepapure oliGO 50 × 4.6 mm, 30 × 4.6 mm and 10 × 4.6 mm precolumn (3 µm, 120 Å).
• Software: ClarityChrom 10.1 for instrument control and PDA data processing.

Main results and discussion

• Both ion-pair systems produced useful separations of the three test oligonucleotides. After optimization, sufficient retention and resolution were achieved for all samples on the 50 × 4.6 mm column with short run times (separations possible in under 2 minutes), supporting high-throughput workflows.
• HFIP-based mobile phases tended to give slightly shorter retention times, and the TEA concentration could be reduced (to ~4 mM) while maintaining retention in the HFIP system.
• The TEA+HAc system produced improved peak symmetry compared with HFIP, while delivering comparable resolution and overall chromatographic performance. This indicates HAc+TEA as a viable alternative for UV-based analyses where HFIP is often chosen for LC–MS compatibility.
• Column length experiments supported the On/Off retention mechanism typical for oligonucleotides in IP-RP: both ion-pair systems retained functionality when the gradient was transferred to shorter columns, although very short (10 mm) columns produced limited retention for the smallest analyte (15-mer) in the tested HAc method without further adjustment.
• A notable observation: the 25-mer (bearing a C12 linker) eluted later than the 64-mer in some conditions, indicating that lipophilic modifications (e.g., alkyl linkers) can strongly increase reversed-phase interactions and influence elution order independent of sequence length.
• The Sepapure oliGO stationary phase demonstrated good peak symmetry even for the high molecular weight 64-mer, indicating suitability for a wide mass range of oligonucleotide analytes.

Benefits and practical applications of the method

• HAc+TEA provides a cost-effective and chromatographically robust alternative to HFIP+TEA for UV-detection-based assays, with improved peak symmetry and comparable resolution.
• Fast separations (sub-2-minute) are feasible, enabling higher sample throughput for QC and process monitoring in oligonucleotide synthesis and manufacturing workflows.
• Method transfer between columns and between ion-pair chemistries is practicable with limited adjustments, facilitating method scaling and deployment in routine laboratories.
• Awareness of non-sequence modifications (e.g., C12 linker) is critical because they can dominate retention behaviour and affect quantitation and identification strategies.

Future trends and applications

• Further reduction of ion-pair reagent concentrations and evaluation of greener or less volatile alternatives could reduce cost and environmental impact while preserving performance.
• Integration with mass spectrometric detection remains an important direction, especially for identity confirmation and impurity profiling; HFIP is commonly used for LC–MS compatibility, but methods that balance MS-friendliness and chromatographic quality will be valuable.
• Optimization for ultra-short columns and higher flow rates to further increase throughput, combined with automated sample handling, will support industrial QC demands.
• Method standardization and robustness testing aligned with regulatory expectations for oligonucleotide therapeutics will be increasingly important as more products reach clinical and commercial stages.

Conclusion

Both TEA+HAc and TEA+HFIP ion-pair systems are effective for rapid IP-RP-HPLC separation of oligonucleotides with UV detection. HFIP offered marginally faster retention while HAc delivered better peak symmetry; overall resolution and practicality were comparable. The investigated Sepapure oliGO columns supported high-quality separations across a broad size range and demonstrated method transferability to shorter columns, confirming the feasibility of fast, high-throughput oligonucleotide analysis for QC applications.

Reference

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