Achieving High‑Resolution Separation of Oligonucleotides with Novel PRP‑Z2 HPLC Columns
Oligonucleotide therapeutics and advanced nucleic acid tools have rapidly transitioned from niche research reagents to key components of commercial drug development pipelines. Consequently, contract development and manufacturing organizations (CDMOs) and institutional research cores face increasing demand for sophisticated chromatographic support, including high-resolution impurity profiling, length ladder characterization, and robust system suitability testing across a broad range of oligonucleotide sizes and chemical modifications.
These expanding requirements place significant stress on conventional reversed-phase LC platforms. Silica-based stationary phases, which dominate small molecule separations, are inherently limited by pH and temperature instability. Furthermore, silica phases are prone to hydrolytic degradation and progressive loss of efficiency under typical oligonucleotide separation conditions (>60°C and ion-pairing agents). The exposure of residual silanols to the separation conditions, leaves active metal sites from degraded silica, which can introduce secondary interactions with the anionic phosphate backbone of oligonucleotides, leading to peak tailing, variable recoveries, and difficulties in quantifying low-level impurities.
Additionally, many widely adopted oligonucleotide methods rely on high concentrations of ion-pairing reagents such as triethylammonium acetate (TEAA) or hexylammonium acetate (HAA) during gradient elution. While effective for generating retention and resolution, these high salt loads complicate integration with mass spectrometry (MS), increase the burden of fraction cleanup, and accelerate fouling of chromatographic systems.
Polymeric reversed-phase packings based on crosslinked polystyrene–divinylbenzene (PS–DVB) offer a promising solution by providing a silica-free, pH-independent matrix with exceptional chemical and thermal stability. However, for widespread adoption in oligonucleotide workflows, these materials must demonstrate not only robustness but also consistent, high-efficiency separations across a range of sequence lengths and mobile phase systems.
In this work, we evaluate a PS–DVB-based reversed phase material (PRP-Z2) for the separation of model oligonucleotide systems: a deoxycytidine homopolymer ladder (d(C)12–d(C)18), a 10–60 nucleotide (nt) ladder, and a 20–100 nt ladder. Using TEAA- and HAA-based ion-pair systems at elevated temperatures (60, 80 and 85°C) and column formats ranging from 50 × 2.1 mm to 150 × 4.6 mm, we assess chromatographic performance, robustness, and scalability. Emphasis is placed on the ability of the polymeric phase to support organic-rich gradients and moderate ion-pairing conditions, thereby reducing non-volatile salt exposure while maintaining resolution and peak shape.
