The Chromatography Forum

of Delaware Valley

Purification of Oligonucleotides using Reverse Phase and Anion Exchange Chromatography

Chromatographic separation is routinely used for oligonucleotide (oligo) purification, with the most widely employed techniques being reverse-phase high performance liquid chromatography (RP-HPLC) and anion exchange chromatography (AIEX). The hydrophobic dimethoxytrityl (DMT) functional group resulting from synthesis can be utilized in efficient DMT-On RP-HPLC purification. AIEX uses resins with cationic charges to separate product from impurities based on charge and sequence length, usually after DMT removal. In both processes, the elution outcome can be examined closely through fraction analysis, also using HPLC. This presentation will highlight purification studies performed on various oligonucleotide modalities in DMT-On and DMT-Off modes through various approaches. It will highlight a new anion exchange resin which exhibits a relatively higher loading capacity and compare its oligonucleotide purification performance to other AIEX resins. Finally, the presentation will showcase the role of the ion-pairing agent in the purification outcome of longer oligonucleotides and its impact on the resulting yields and purities.