Ultrahigh-Resolution Separations of Oligonucleotides

The Thermo Scientific^™DNAPac^™PA200 and PA200 RS are strong anion-exchange columns developed to provide unsurpassed high-resolution analysis and purification of synthetic oligonucleotides. Both supports are designed to resolve full length from n–1, n+1, and other failure sequences not possible with other columns. Retention times and selectivity can be controlled by choice of salt, pH, and solvent. Therefore, the separation can be tailored to the requirements of many different oligonucleotide analysis challenges.

• Achieve n, n-1 resolution for oligonucleotides.

• Resolve oligonuleotide linkage and stereoisomers.

• Assay phosphorothioate purity.

• Attain selectivity control with eluent pH, salt, and solvent.

• Resolve RNA with aberrant (2’–5’) links from normal SSRNA.

The DNAPac PA200 is packed with a pellicular anion-exchange resin composed of an 8 μm diameter nonporous polymeric substrate to which quaternary amine-functionalized Thermo Scientific^™Dionex^™MicroBeads^™are bound. The rapid mass transport characteristics of this resin result in high-resolution oligonucleotide separations.

The DNAPac PA200 RS is smaller, with 4 μm diameter resin for bio-UHPLC separations. These rapid separation (RS) columns are designedfor use with the Thermo Scientific^™Dionex^™UltiMate^™3000 BioRS system, with an operational backpressure limit of 10,000 psi. Columns packed with smaller particles improve resolution and offer better performance.

The DNAPac PA200 and PA 200 RS can be operated under denaturing conditions, such as high temperature (up to 85 °C), high-pH eluents (up to pH 12.5), or by the inclusion of chaotropic agents (such as Urea).

These columns offer the highest-quality phase stability over a broad pH range, deliver exceptional resolution, and support high-throughput separations. DNAPac PA200 and PA 200 RS columns support resolution of normal-length oligonucleotides (8- to 30- mer), extended-length oligonucleotides (30- to 70-mer), linear double-stranded DNA, and supercoiled versus nicked/linear DNA.

Analytical separations on the DNAPac PA200, 8µm, 4 mm diameter column can be scaled directly to larger diameter columns so preparative methods can be conveniently developed using small samples. Scaling the flow rate and sample size up for the 9 × 250 mm and 22 × 250 mm column yields essentially identical chromatography.

Resolution of oligonucleotides using DNAPac PA200 column (8µm).

Separation of oligonucleotides by length using DNAPac PA200 column (8µm).

Influence of pH on Oligonucleotide Retention: NaCl Eluent ±20% CH₃CN.

Improved Stability to Alkaline Conditions: Comparison of Capacity upon Exposure to pH 12.4 at Elevated Temperature.

Improved oligonucleotide sensitivity using the 4µm DNAPac PA200 RS column.

Fast separation of 46 oligonucleotides using the 4µm, DNAPac PA200 RS column.

Comparison of 4µm, DNAPac PA200 RS column throughput.

Separation of three identical sequence oligonucleotides harboring zero, one, or two 2',5'-linkages using the 4µm, DNAPac PA200 RS columns of different lengths.

Thermo Scientific DNAPac PA200 HPLC (8µm) and DNAPac PA200 RS (Rapid Separation) UHPLC (4µm) columns for high-resolution separations of ssDNA and RNA.