Platform Ion Pairing RPLC Method for Oligonucleotides Using High Throughput 20 mm Length Columns

Significance of the Topic

Ion-pair reverse-phase liquid chromatography (IP-RPLC) is a cornerstone analytical technique for oligonucleotide characterization, offering superior resolution and compatibility with LC-MS. As oligonucleotide-based therapeutics and diagnostics proliferate, rapid and robust separation methods are essential for quality control and method development.

Study Objectives and Overview

This work proposes a generic platform method using ultra-short ACQUITY™ Premier Oligonucleotide BEH™ C18 columns (2.1×20 mm, 1.7 µm) to separate size variants of oligonucleotides (10–100-mers) in under three minutes. The authors apply a non-linear (logarithmic-like) gradient to achieve uniform selectivity across homologous series.

Methodology and Instrumentation

• System: ACQUITY UPLC™ H-Class Bio with Binary Solvent Manager or equivalent Premier system.
• Detection: UV at 260 nm.
• Column: ACQUITY Premier Oligonucleotide BEH C18, 300 Å, 1.7 µm, 2.1×20 mm at 70 °C.
• Mobile phases: 100 mM HFIP + 10 mM DIPEA in water (A) and in 1:1 MeCN:water (B), pH ∼8.4.
• Flow rates: 0.8 mL/min (3 min gradient) or 1.5 mL/min (1.33 min gradient).
• Sample: Oligo dT and ssDNA ladders (10–100-mer).

Main Results and Discussion

1. Column length effect: Modeling and experiments show that a 20 mm column matches the separation power of a 150 mm column for oligonucleotides, with a 7.5× reduction in analysis time and minimal resolution loss.
2. Ion-pair reagent comparison: Four IP systems (TEA, DIPEA, hexylamine, octylamine) display logarithmic retention vs. chain length (“homologue rule”). Stronger hydrophobic reagents increase absolute retention but do not alter relative selectivity; choice depends on LC-MS compatibility and sequence vs. size separation.
3. Platform gradient design: A logarithmic-like (concave) multi-segment gradient derived from inverse retention modeling yields uniform peak spacing for 10–100-mer mixtures. A 3–5 segment approximation enables 1–3 minute separations with high resolution.

Benefits and Practical Applications

• Ultra-fast run times (1–3 min) accelerate routine QC and high-throughput workflows.
• Adjustable non-linear gradients maximize selectivity across broad size ranges.
• Short columns reduce solvent consumption and instrument wear without sacrificing performance.

Future Trends and Applications

• Integration with LC-MS for direct mass confirmation of oligonucleotide species.
• Automated gradient optimization tools to further streamline method development.
• Extension to modified and conjugated oligonucleotides and other large biomolecules.

Conclusion

By exploiting the on-off elution mechanism of oligonucleotides and applying logarithmic-like gradients on ultra-short columns, this platform method achieves rapid, high-resolution separations of 10–100-mers. The approach supports routine QC, method development, and high-throughput analysis in academic and industrial laboratories.

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