Systematic optimization of gel-free capillary zone electrophoresis for phosphodiester oligonucleotide separations at acidic pH

Reliable analysis of oligonucleotides is essential for therapeutic development and biomarker studies, yet conventional methods often rely on gels or high-salt buffers that hinder mass spectrometry compatibility. MicroDNA and microRNA are commonly analyzed using liquid chromatography or capillary gel electrophoresis (CGE). In a recent study, we introduced a gel-free capillary zone electrophoresis (CZE) method for rapid, high-resolution separation of oligonucleotides at acidic pH. Here we optimized a gel-free capillary zone electrophoresis (CZE) method operating at acidic pH using a PVA-coated capillary and malonic acid-ammonia buffer. Adjustments to buffer composition, cation type, and temperature (up to 60 °C) enhanced resolution and reduced current, enabling rapid, high-resolution separation of phosphodiester oligonucleotides ranging from 18 to 25 nucleotides with excellent repeatability (<0.5% mobility variation). This gel-free CZE approach provides a fast and reproducible platform, facilitating detailed characterization of phosphodiester oligonucleotides, microDNA, and microRNA in pharmaceutical and bioanalytical contexts. • Oligonucleotides are analyzed using acidic pH gel-free capillary zone electrophoresis • High resolution separations of same length oligonucleotides based on nucleobase pKa • Systematic optimization of the separation system for reproducible analyses