Oligonucleotide analysis demands chromatographic performance capable of resolving closely related impurities and increasingly long, complex sequences. The HALO® OLIGO C18 column range from Advanced Materials Technology has been purpose-designed to meet these demands, combining the proven efficiency of Fused-Core® particle technology with chemistries and hardware optimised specifically for oligonucleotide separation. The result is a robust, high-performance solution that delivers speed, resolution and reliability across a wide range of oligo lengths – from short sequences to large, therapeutically relevant molecules.
With both 120 Å and 1000 Å pore size options available, HALO® OLIGO C18 columns provide analysts with the flexibility to tackle everything from routine separations to advanced applications such as impurity profiling, failure sequence identification and long-chain oligonucleotide characterisation.
Figure 1: HALO® OLIGO C18 pore sizes: 120 Å for ≤60-base oligonucleotides and 1000 Å for high-efficiency separations up to and beyond 100 bases.
HALO® 120 Å OLIGO C18: Precision and Speed for Shorter Oligos
The HALO® 120 Å OLIGO C18 column is designed for high-efficiency separations of oligonucleotides up to approximately 60 bases in length. Built on 2.7 µm Fused-Core® particles, it delivers exceptional resolving power and fast analysis times without the high backpressure typically associated with fully porous sub-2 µm materials.
This column demonstrates excellent stability under the high pH and elevated temperature conditions commonly used in oligonucleotide methods. Its surface-modified, alkaline-resistant organo-silane chemistry ensures consistent performance, while surface-passivated hardware minimises unwanted interactions that can lead to sample loss or distorted peak shapes.
In comparative studies, the HALO® 120 Å OLIGO C18 column has shown faster separations than 1.9 µm fully porous particle (FPP) Oligo C18 columns, while maintaining comparable efficiency at more than half the backpressure (Figure 2). Enhanced resolution between critical peak pairs further supports confident impurity identification and quantitation, making this column an excellent choice for routine oligonucleotide analysis in both UHPLC and LC-MS workflows.
Figure 2: Demonstration of Fused-Core® Particle Technology for Oligonucleotide Separations using column p/n P2A62-402. Full method conditions in application note [1].
HALO® 1000 Å OLIGO C18: Unlocking Performance for Long-Chain Oligonucleotides
As oligonucleotide therapeutics continue to develop in size and complexity, conventional column technologies can struggle to deliver adequate resolution and peak capacity. The HALO® 1000 Å OLIGO C18 column has been specifically developed to address this challenge, offering superior performance for oligonucleotides up to – and beyond – 100 bases in length.
The large 1000 Å pore size allows improved mass transfer for long-chain molecules, resulting in narrower peaks, increased peak capacity and lower backpressure compared with competitor wide-pore columns. This enables the use of longer column lengths and broader gradients without compromising analysis time or system limits, supporting scalable and flexible method development.
As shown in Figure 3, under commonly used TEA/HFIP mobile phase conditions, the HALO® 1000 Å OLIGO C18 column exhibited a broader gradient range between the 20- and 100-base oligonucleotides, contributing to higher peak capacity, along with lower backpressure. When using the more hydrophobic DiBA/HFIP mobile phase, peak shapes were further improved for the larger oligonucleotides. These characteristics are particularly valuable for advanced applications such as therapeutic oligonucleotide development and CRISPR-related research, where accurate impurity profiling and high sequence resolution are essential.
Figure 3: Separation of 20- to 100-base oligonucleotides using HALO® 1000A OLIGO C18 p/n P2762-602 compared to fully porous sub 2 µm competitor columns. Full method conditions in application note [2].
Inert, Surface-Passivated Hardware: Maximising Recovery and Peak Quality
Beyond stationary phase chemistry, column hardware plays a crucial role in oligonucleotide performance. Oligonucleotides are known to interact non-specifically with stainless steel surfaces, leading to adsorption, peak tailing and reduced recovery. HALO® OLIGO C18 columns address this issue through the use of inert, surface-passivated hardware.
Comparative data clearly demonstrates the benefits of this design, showing significantly increased peak areas and reduced tailing factors compared with conventional stainless steel columns (Figure 4). By minimising metal–analyte interactions, retention times are shortened and reproducibility is improved, ensuring accurate and reliable results – particularly for low-level or highly polar oligonucleotide samples.
Figure 4: Improvements in peak shape and recovery using Inert hardware HALO® OLIGO C18 column p/n P2762-402 compared to stainless steel hardware. Full method conditions in application note [3].
Conclusion: A Complete Column Solution for Modern Oligonucleotide Analysis
The HALO® OLIGO C18 column range from Advanced Materials Technology represents a next-generation solution for oligonucleotide separations. By combining Fused-Core® particle technology, optimised surface chemistry and inert hardware, these columns deliver outstanding efficiency, resolution and robustness across a broad range of oligo lengths. Whether analysing short sequences or tackling complex, long-chain therapeutics, HALO® OLIGO C18 columns provide the performance and confidence required for modern oligonucleotide research and development.
Resources
[1] Application 387 – Advantage of Fused-Core® Particle Technology for Oligonucleotide Separations
[2] Application 408 - Advantage of HALO 1000 Å over Competitor Columns for Oligonucleotides
[3] Application 390 - Advantage of Inert Hardware with HALO® OLIGO C18
