This article introduces the use of PL-SAX strong anion-exchange HPLC columns for high-resolution separations of oligonucleotides. After deprotection of the oligonucleotide product, hydrophobicity-based separation can be performed, followed by anion-exchange HPLC for further resolution. The experimental results show that the PL-SAX column can achieve separation of n-1 from n.
This document describes the use of Agilent PL-SAX strong anion-exchange columns for the high-resolution separation and purification of oligonucleotides. It explains how the material's thermal and chemical stability allows for analysis under elevated temperatures and harsh conditions, such as high pH, to resolve failure sequences and modified forms like phosphorothioates.
The note provides specific experimental protocols for three different separation scenarios: separating poly-T oligonucleotides of varying lengths, purifying large synthetic oligonucleotides, and separating complete and incomplete thiolated forms. Each protocol specifies the column dimensions, eluent composition, gradient profiles, flow rates, and temperatures required to achieve baseline separation.
| Column Part Number (50 x 4.6 mm) | PL1551-1802 |
|---|---|
| Column Part Number (150 x 4.6 mm) | PL1551-3802 |
| Particle Size | 8 ym |
| Flow Rate | 1.5 mL/min |
| Operating Temperature | 60 °C |
| Linear Velocity | 360 cm/h |
Eluent A is 7:93 v/v acetonitrile: 0.1 M TEAA, pH 8.5, and Eluent B is 7:93 v/v acetonitrile: 0.1 M TEAA, 1 M ammonium chloride pH 8.5.
The separation uses sodium hydroxide as the eluent with a gradient from 75-100% B for 25 min followed by 0-100% B in 20 min.