Bioanalytical Technique Combining Slalom Chromatography and High Vacuum to Outperform Gel Electrophoresis for Large DNA/RNA Analysis in Cell and Gene Therapy

Slalom chromatography (SC) is a separation technique originally discovered in 1988, recently revitalized using bio-inert, ultra-high-pressure liquid chromatography (UHPLC) columns and systems for analyzing large DNA/RNA fragments up to 30 kbp. The first commercial SC column (4.6 × 300 mm, 2.5 μm BEH™) became available in July 2025. The SC separation mechanism is an out-of-equilibrium process governed by the weak entropic elasticity of double-stranded DNA/RNA and the shear forces generated in the interparticle spaces of UHPLC columns. Unlike gel permeation chromatography (GPC), SC elutes smaller DNA fragments first and larger ones last.
However, significant biopolymer extension requires high flow rates (1.0–1.5 mL/min) and back pressures (~10,000 psi), which induce frictional heating (~10 Watt/m), radial temperature gradients (~0.5 K), and reduced separation efficiency.
In this presentation, we investigate a novel approach to enhance SC resolution by operating columns in a high-vacuum environment (10⁻⁶ mbar), effectively eliminating radial heat dissipation and ensuring uniform temperature profiles across the column diameter. The hypothesis was tested by comparing column efficiencies under still-air and high-vacuum conditions for a 1 kbp DNA ladder (1.0–10.0 kbp) and λ-DNA BstEII digest (1.3–8.5 kbp). Results showed an average increase in column efficiency of approximately +30% within a 3-minute runtime.
Ultimately, SC is demonstrated to be a simpler (standard UHPLC method), faster (20×), higher-resolution (2×), more sensitive (~1-5 ng detection limit), and more accurate (±0.5% retention time) alternative to gel electrophoresis for determining the length and size of DNA/RNA fragments >2 kbp. This work introduces SC as a powerful bioanalytical technique for rapid, high-resolution characterization of nucleic acids in plasmid (DNA restriction mapping, PCR amplification, etc..) and mRNA manufacturing workflows.