The Chromatographic Impact of Hydrophobic and Steric Modifications in siRNAs

Did you see this month’s newsletter? This month’s HALO® Hint explores the chromatographic impact of hydrophobic and steric modifications in siRNAs, including why these changes matter when optimizing siRNA analysis, separation performance, retention, and selectivity. Read the full feature below.

Separating siRNAs can be challenging because their retention behavior is strongly influenced by the chemical modifications commonly incorporated to improve stability and in vivo performance. Many of these modifications change the hydrophobicity of the oligonucleotide, which directly affects retention in reversed-phase or ion-pair reversed-phase separations. Hydrophobic groups such as 2′-O-methyl, 2′-O-methoxyethyl, 2′-fluoro substitutions, and phosphorothioate linkages increase the analyte’s interaction with the stationary phase, often producing longer retention times and more pronounced separation between strands and impurities. In contrast, other types of modifications alter the steric environment of the siRNA rather than its polarity. Bulky or rigid structures, such as locked nucleic acids, large 2′ sugar modifications, or terminal linkers, can reduce the effective surface contact between the siRNA and the stationary phase or hinder access to the ion-pairing reagent. These steric effects may decrease retention, shift selectivity, or change the elution pattern of duplex versus single-stranded species.

The structural state of the siRNA adds another layer of complexity. Duplex formation reduces exposed hydrophobic surface area compared to the individual strands, meaning that a fully annealed siRNA often elutes earlier than expected from the sum of its components. Modifications that stabilize the duplex can amplify this effect, while destabilizing modifications or impurities may lead to broader or later-eluting peaks. Understanding how hydrophobicity and steric hindrance interplay within modified siRNAs allows analysts to better predict chromatographic behavior and optimize conditions to achieve clean resolution. These considerations are especially important when separating heavily modified therapeutic candidates, where even small shifts in polarity or structure can lead to substantial differences in retention.