Weak to strong ion-pair gradients to expand the selectivity of oligonucleotide separations in reversed phase liquid chromatography - A proof of concept

• A dual gradient approach is proposed to enhance the separation of oligonucleotides. • Running a weak to strong ion pair gradient significantly expands the elution window. • Combination of dual gradient with concave gradient program shows further benefits. • The degree of freedom can be significantly improved to extend the design space. This study introduces a novel ion-pair reversed-phase liquid chromatography (IP-RPLC) method using a dual gradient approach to enhance the separation of oligonucleotides. By combining a weak ion-pairing (IP) agent in the starting (weaker) mobile phase with a strong IP agent in the final (stronger) mobile phase, this method widens the elution window, improving the resolution and selectivity for complex ON mixtures. The approach outperforms traditional single IP systems, particularly in separating sequence and size variants of ONs. Experimental results demonstrate that the weak-to-strong gradient yields better peak separation and higher selectivity compared to conventional methods. This innovative gradient strategy, combined with concave gradients, offers significant potential for optimizing ON separations in the development of oligonucleotide-based therapeutics.