Hybrid Large-Pore Superficially Porous Particles for High-Performance Nucleic Acid Separations

Recent advances in oligonucleotide synthesis and their growing role in diagnostics and therapeutics have created a critical need for efficient, high-resolution separation technologies. The increasing complexity of nucleic acid structures—including chemical modifications, phosphate variants, and conjugates—demands robust chromatographic solutions capable of handling longer sequences and diverse chemistries. Our previous work demonstrated that superficially porous particles (SPP, Halo® Fused-Core) with enlarged pore sizes significantly improve reversed-phase HPLC (RP-HPLC) performance for large biomolecules such as intact proteins and conjugates. Building on this, we introduce hybrid SPP technology optimized for nucleic acid separations under ion-pair reversed-phase conditions, which often require elevated pH and temperature for oligonucleotide separations. This study compares hybrid silica SPP columns with 120 Å and 1000 Å pores against fully porous particles (FPP) of varying pore sizes (130 Å and 300 Å) and large-pore polymeric materials. Performance metrics include resolution, column efficiency, and mass transfer kinetics across a range of linear velocities. Large-pore SPP exhibits superior mass transfer properties and enhanced resolution for long single-stranded oligonucleotides (>90–100 bases). Pore size effects influence selectivity for shorter sequences, offering unique advantages for difficult analytical workflows. These findings underscore the value of large-pore hybrid SPP technology in addressing the challenges posed by nucleic acid analysis. Improved separation of oligonucleotides supports emerging applications in biological research and therapeutic development, where sequence length and structural complexity continue to expand. This work highlights a practical path toward faster, more reliable LC/MS-compatible methods for complex nucleic acid characterization.