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
Haglöf et al. (Sun,) studied this question.