This Application Note describes methods of using the Agilent Capillary Electrophoresis (CE) system to separate single- and double-stranded DNA, single-stranded RNA and antisense oligonucleotides. Oligonucleotides with a chain length of 5 to 80 nucleotides are separated using polymer solution A and polymer solution B. The application note describes how to achieve high reproducibility in separations of oligonucleotides.
Oligonucleotides cannot be successfully separated according to size using conventional capillary zone electrophoresis because they have similar mobilities. To address this, a sieving matrix is required so that smaller oligonucleotides move faster through the capillary than larger ones.
The document details the use of Agilent Capillary Electrophoresis systems equipped with diode array detection and ChemStation software. It outlines specific chromatographic conditions, including buffer types, polymer solutions (A and B), and capillary specifications for analyzing single- and double-stranded DNA, RNA, and antisense oligonucleotides.
| Chain length range | 5 to 80 nucleotides |
|---|---|
| Capillary dimensions | 24.5 cm eff x 100 um id |
| Detection wavelength | 260 nm/8 nm DNA filter |
| Run voltage and temperature | -25 kV, 30 °C |
The system is suitable for the analysis of oligonucleotides in the range 5 to 80-mers.
A PVA coated capillary with a length of 24.5 cm effective and an inner diameter of 100 um.
Polymer solution B, which contains an organic additive, enhances the resolution of the separation.