Fast RP-UHPLC Separation of Ribooligonucleotide Impurities using Evosphere MAX C18/AR Monodisperse Fully Porous Particle Columns without Ion-Pair Reagents for Simplified Oligo Separations

Ribonucleotide synthesis, like any chemical process, can introduce impurities due to various factors inherent in the oligo-synthesis method and the nature of the reagents involved. To mitigate the presence of these impurities in synthesized ribonucleotides, rigorous purification methods, such as high-performance liquid chromatography (HPLC), gel filtration, or solid-phase extraction, to isolate and obtain high-purity products suitable for downstream applications have to be applied.

Ion-pair reagents and inorganic salts are commonly used in RP-UHPLC to enhance the separation of nucleic acids. However, their presence can complicate downstream applications, increase instrument maintenance requirements and reduces the MS sensitivity due to ion suppression.

This research is aimed to develop a simple and efficient ion-pair reagent-free chromatographic method for the separation of ribooligonucleotide impurities utilizing a novel bonded phase chemistry and novel monodisperse particle design packed in an inert coated hardware.

We will show a robust LC-UV method for the separation of ribooligonucleotide impurities using the Evosphere MAX C18/AR stationary phase.

The main advantage of the method is the use of ion-pair-free and inorganic salts-free mobile phases for this challenging separation. This new developed method in conjunction with Copernicus University in Poland eliminates these additives, simplifying the chromatographic system and making the analysis more compatible with downstream applications such as mass spectrometry or biological assays. This approach also reduces the risk of ion suppression effects, which can occur when ion-pair reagents interfere with ionization in mass spectrometry.