Strong Anion-Exchange HPLC for Size-Range and Topology-Resolved Analysis of Nucleic Acids

Significance of the topic

Strong anion-exchange (AEX) chromatography is increasingly important for analysis of nucleic-acid therapeutics and manufacturing intermediates because these workflows must handle a very wide molecular-weight and structural range: short oligonucleotides, long linear double-stranded DNA (dsDNA), and topologically distinct plasmid forms. A single, robust LC method that can size-resolve short fragments and distinguish plasmid topoisomers would simplify QC, enable fraction-compatible workflows, and provide an alternative to electrophoresis for routine, automatable separations.

Objectives and study overview

• Determine whether a single strong AEX method can separate nucleic acids across size regimes (short ssDNA oligos to large dsDNA and plasmid topoisomers).
• Identify the mechanistic origins of retention and selectivity across size ranges and whether flow rate becomes a selectivity parameter for large DNA.
• Demonstrate practical conditions (mobile phase, gradient, column) that deliver broad-range separations with high recovery and low carryover.

Methodology

• Column: Protein-Pak Hi Res Q, nonporous quaternary amine packing, 5 µm, 4.6 × 100 mm.
• Eluents: A = 20 mM Tris, pH 9.0, containing 5% (v/v) urea; B = eluent A + 1.0 M NaCl.
• Detection: UV at 260 nm.
• General ladder separations: 0.5 mL/min, 35 °C.
• Mechanistic studies: linear salt gradients (60–95% B over a fixed 7.5 mL) run at varied flow rates (0.1, 0.3, 0.5, 0.8 mL/min) with pressure-control experiments to separate shear-flow from static-pressure effects.
• Ladders tested included single-stranded DNA ladders (heteropolymer and homopolymer Poly dT) and a 1 kb plus dsDNA ladder covering 100 bp to ~15 kbp; plasmid topoisomers were also examined.
• Method optimization included addition of 5% urea to reduce secondary-structure effects and improve peak shape and recovery.

Main results and discussion

• Low-size regime (short ssDNA): Strong AEX with salt-gradient elution resolved discrete single-stranded DNA length variants. Separations of heteropolymeric ssDNA ladders demonstrated charge-dominated retention across a wide short-oligo range (reported on different ladder sets from ~10–100 nt, with clear resolution of 15–35 nt fragments in focused conditions). Poly dT homopolymer ladders showed excellent resolution of failure sequences.
• Intermediate-size regime: Compared with agarose gel electrophoresis, AEX selectivity compressed at intermediate dsDNA sizes and then broadened again for the largest fragments, indicating a transition from pure charge-density control to a secondary retention contribution.
• Large dsDNA and plasmids: AEX separated dsDNA fragments from ~100 bp up to 15 kbp. For large linear dsDNA and plasmid topoisomers, retention behavior could not be explained solely by classical ion-exchange (charge-density) models.
• Dual-mechanism interpretation: Retention proceeds via classical AEX adsorption and salt-gradient–mediated release for small and moderate fragments. For sufficiently large molecules, after release from the stationary phase the molecules undergo flow-induced extension and interaction with interparticle flow fields, producing an additional, flow-dependent retardation termed a slalom mechanism.
• Flow-rate as a selectivity parameter: Increasing flow rate selectively increased retention of large, extension-prone species (notably linear plasmid forms) without reproducing the effect by simply raising static pressure. Pressure-control experiments showed that raising pressure at constant flow did not shift selectivity; raising flow at similar inlet pressures increased retention of the largest dsDNA fragments, demonstrating shear-driven (not pressure-driven) selectivity.
• Plasmid topology resolution: Higher flow rates selectively retarded linear forms more than supercoiled or open-circular forms, improving separation between oc/l and l/sc topoisomers. Thus flow rate is a practical tuning knob for topology-resolved separations in the extension-sensitive size regime.
• Effect of urea and carryover: Inclusion of 5% urea improved peak shape, valley definition, and reduced structure-dependent chromatographic behavior. Recovery tests with the 1 kb plus ladder showed effectively complete recovery and no observable A260 carryover on blank injections.

Practical benefits and applications

• Unified method: A single strong AEX-HPLC method can be used to connect analyses spanning short ssDNA oligonucleotides through large dsDNA fragments and plasmid topologies, supporting streamlined QC and method transfer.
• Tunable selectivity: For large DNA, flow rate is an actionable selectivity parameter in addition to gradient shape and salt concentration; this enables optimization for plasmid impurity profiling (topoisomers) without changing chemistry.
• High recovery and low carryover: The method with 5% urea provided high recovery and practically no carryover, facilitating fraction collection and downstream processing.
• Compatibility with automation: LC-based AEX separations are more amenable to routine, automated workflows and fraction-compatible operations compared with electrophoresis-based approaches.

Used instrumentation

• Analytical column: Protein-Pak Hi Res Q, 5 µm, 4.6 × 100 mm (nonporous quaternary amine packing).
• LC system configured to control flow precisely (tested flows 0.1–0.8 mL/min) and to measure inlet pressure; detectors used: UV at 260 nm.
• Temperature control: separations reported at 35 °C for ladder runs.

Future trends and potential applications

• Method transfer to QC and regulated environments: the demonstrated broad-range separations and good recovery make AEX a candidate for routine plasmid and oligonucleotide QC workflows.
• Instrument and column optimization: exploration of particle size, pore architecture, and interparticle geometry to control slalom effects and extend the topology-selective regime.
• Mechanistic modeling: development of quantitative models that combine ion-exchange thermodynamics with flow-field–induced extension (slalom) to predict retention and guide method development.
• Broader biopolymer applications: application of the dual-mechanism concept to other large, flexible polyanions (e.g., long RNA, chromatin fragments) and to preparative separations with scaled geometry.
• Integration with orthogonal techniques: combining AEX-based topology sizing with mass-sensitive or single-molecule methods to confirm identity and to quantify low-level impurities.

Conclusions

• Strong AEX chromatography provides high-resolution separations across a wide nucleic-acid size range, resolving short ssDNA length variants and dsDNA fragments up to ~15 kbp, and distinguishing plasmid topological forms.
• Retention mechanisms are dual: classical AEX adsorption and salt-gradient release dominate for small-to-moderate fragments, while large dsDNA and linear plasmids experience an additional, flow-dependent slalom retardation after release.
• Flow rate becomes a true selectivity parameter in the extension-sensitive size and topology regime; this offers an experimental lever for optimizing plasmid topology separations.
• Practical method elements (Protein-Pak Hi Res Q column, Tris buffer with 5% urea, salt gradient to 1 M NaCl) delivered good peak shape, high recovery, and negligible carryover, making the approach suitable for routine analysis and method transfer.

References

1. Finny AS; Addepalli B; Lauber MA. High-Resolution Separations of Single and Double-Stranded Nucleic Acids Using Strong Anion-Exchange Chromatography. Waters Application Note 720009136, 2025.
2. Gritti F; Fekete S; Finny AS. Dual retention mechanisms in DNA separation: Relevance of slalom chromatography in anion-exchange gradients. Journal of Chromatography A 2025, 1763, 466457. doi:10.1016/j.chroma.2025.466457.
3. Finny AS; Gritti F; Fekete S; Addepalli B; Lauber MA. Slalom-Aided Anion Exchange Chromatography for Enhanced Analysis of Plasmid DNA Topological Impurities. Waters Application Note 720008928, 2025.