High-performance anion-exchange chromatography of oligonucleotides

Several types of high-performance silica-based supports have been found to be effective in the separation of polynucleotides. The principal difference in these materials is the type of bonded phase and the method by which it is attached to the silica support. One approach is the coupling of stationary-phase groups to the surface through siloxane bonding. This technique is simple and produces a material of high capacity and resolution, but it suffers from poor bonded-phase stability. An alternative approach is the adsorption of low-molecular-weight polyethylene imines (PEI) that are crosslinked into a surface film. The stationary phase is held in place by adsorption of the film at multiple sites. A previous report on this material showed the resolution of oligonucleotides containing up to 30 bases. This paper reports further optimization of the PEI bonding chemistry in the preparation of HP-IEC columns for oligonucleotides and tRNA species. Quaternization of the ion-exchange matrix was found to increase resolution of oligonucleotides from 30 to 50 bases. The same support was found to be capable of resolving multiple tRNA species. Separations were achieved on small (0.42 X 5 cm) columns, using a 60- to 120-min ammonium sulfate gradient. The initial solvent was 15% acetonitrile in 0.05 M potassium phosphate (pH 5.9). The addition of 1 M ammonium sulfate to the initial solvent was used to prepare the final solvent.