Stick With Me: The Cool Chemistry of HILIC for Oligonucleotide Analysis
💡Why Use HILIC in the Oligo Revolution?
Oligonucleotides are the workhorses behind gene therapies, diagnostics, and research breakthroughs. But before these tiny powerhouses can work their magic, they need to undergo chromatography analysis to ensure they are fit for work. Therapeutic oligonucleotides are challenging to analyze, but the demand for precise, powerful methods has never been greater. LC-MS leads the charge, with ion-pair reversed-phase LC as the gold standard, yet HILIC is quickly rising as a strong and promising contender. When IP-RP mode hits its limit, HILIC steps in offering sharp separation and strong retention for polar oligonucleotides.
💡How Do Scientists Decode Oligonucleotides with HILIC?
They flip the script on traditional chromatography separation and combine polar normal stationary phase with reversed mobile phase approach! Using a water-loving stationary phase and an organic-rich mobile phase, HILIC enables the analysis of short, modified, or highly polar oligos, without the need for ion-pairing. The trick? Harnessing polar interactions to pull apart even closely related sequences with high resolution and LC-MS compatibility. Figure 1 breaks down the key parameters that drive HILIC success.
Figure 1: Key Parameter of HILIC Mode.
💡Why Do Analytes Stay? The Retention Rules of HILIC?
Hydrophilic Partitioning – The Main Driver Polar analytes like oligos migrate into the thin water layer on the stationary phase, while the organic-rich mobile phase flows by. The more polar the molecule, the stronger the pull into the aqueous zone, and the longer it sticks around (see figure 2).
Hydrogen Bonding - The Subtle Stickiness Functional groups like –OH, –NH₂, and –COOH on analytes and the stationary phase form hydrogen bonds, subtly influencing how long compounds are retained.
Electrostatic Interactions – Charge Matters Slightly charged HILIC surfaces (like pure silica) interact with charged analytes. Depending on pH and buffer conditions, these interactions can either boost or reduce retention.
Dipole-Driven Selectivity – Depth to HILIC Selectivity Strong dipoles or zwitterionic species (e.g., sugars, phosphorylated compounds) engage in dipole–dipole or ion–dipole interactions, adding extra layers of selectivity to your separation.
Figure 2: Hydrophilic Partitioning on Water-enriched Layer.
💡How do you HILIC for Oligo separation?
Like other chromatographic techniques, HILIC follows a stepwise approach, beginning with analyte identification (see Table 1). Choose a polar stationary phase (e.g., amide, zwitterionic) suitable for oligo polarity and charge. Use high acetonitrile content (70–95 %) with a volatile aqueous buffer (e.g., ammonium format/acetate, pH ~ 6–8). Develop a water-increasing gradient to control retention and separate oligos by size or modifications. Match injection solvent closely to starting mobile phase (high acetonitrile) to avoid peak distortion. Control mobile phase ionization and ionic interactions for better peak shape and selectivity. Analyze oligo standards and samples to assess resolution, retention time stability, and reproducibility.
Table 1: HILIC Method Step Procedure for Oligos.
If you evaluate different HILIC columns for oligonucleotide separation, mobile phases with 10 mM ammonium acetate at pH 4.4, 6.8 and 9 (if columns are stable enough at pH 9) can be validated. These pH changes can shift column selectivity and affect both retention and MS ionization. Striking the right balance between buffer pH, separation performance, and ionization efficiency is key to successful HILIC method development with MS detection.
💡 How Mastering HILIC? Tips for Peak Performance?
Water is the key: A small water layer on the stationary phase is essential, as too little water will cause retention to vanish. So, a minimum of 5 % water in mobile phase starting conditions should be applied. Adjust water content to fine-tune retention (see figure 3).
Figure 3: Water Adjustment Principle for HILIC.
High organic content: Mobile phases often start with 70–95 % acetonitrile to promote partitioning. HILIC thrives on high organic to retain polar analytes.
Buffer matters: Always include
a volatile buffer (e.g., ammonium format or acetate, 5–20 mM) at low
concentrations to stabilize pH, reduce peak tailing by control electrostatics
and maintain MS compatibility.
Watch injection solvent: Avoid injecting in high-water solvents, they disrupt the partitioning layer and can cause peak distortion. Instead, use a sample solvent with similar composition to the starting mobile phase (high acetonitrile content).
Column equilibration: HILIC columns need longer equilibration times, at least 10–20 column volumes especially after major gradient or solvent changes. So, it takes longer in HILIC, be patient and allow enough reconditioning time.
Temperature control helps: Operating at 30–50 °C can improve peak shape, reduce viscosity, and help stabilize retention, especially for complex analytes.
💡 From Niche to Game-Changer? Final Thoughts?
HILIC-MS is gaining ground as a powerful, ion-pair-free alternative for oligonucleotide analysis offering growing flexibility and potential, especially with advances in method tuning and miniaturization. While IP-RP with MS detection still leads in sensitivity, HILIC is quickly catching up. So, HILIC isn’t just a niche technique, it’s a growing game-changer for oligonucleotide separations with MS detection. Sure, it plays by different rules we know from reversed phase mode, but once you dial in the conditions, HILIC can handle even your most stubborn oligos with style. Ready to shake up your separation strategy? HILIC might just be your next secret weapon.
If you're planning to set up or optimize your oligonucleotide workflow, feel free to contact us at sales@knauer.net. Stay tuned for more exciting insights into the Oligonucleotide world in our “Oligos Made Easy” series.
For more in-depth discussion or questions, reach out to the author at kindler@knauer.net
