High-resolution analysis and purification of synthetic oligonucleotides with strong anion-exchange HPLC

A strong anion-exchange high-performance liquid chromatography column, the NucleoPac PA-100, has been developed for high-resolution analysis and purification of synthetic oligonucleotides. Very high-resolution separations of oligonucleotides to 60 bases can be obtained. Separations can be performed under non-denaturing or denaturing conditions because the column can be operated routinely at high temperature or high pH. Analytical methods can be directly transferred to larger NucleoPac PA-100 columns for semi-preparative applications. In this report, methods for the analysis of synthetic oligonucleotides under non-denaturing (pH 8.0) and denaturing (pH 12.4) conditions are presented. A gradient separation for machine-grade DNA at pH 8.0 is developed. Transfer of an analytical method to a 22-mm-diameter semi-preparative column is also demonstrated.