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Enabling Large-Volume Injections in Hydrophilic Interaction Chromatography of Oligonucleotides Through In-Line

Joshka Verduin1,2, Luca Tutiš1,2, Antonia Kritsima2,3

  • 1Department of Chemistry and Pharmaceutical Sciences, Division of BioAnalytical Chemistry, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, Amsterdam, Netherlands.

Journal of Separation Science
|February 25, 2026
PubMed
Summary

Hydrophilic interaction chromatography (HILIC) challenges with therapeutic oligonucleotides (ONs) can be overcome. An in-line mixer effectively mitigates breakthrough and peak splitting during large-volume injections of ONs in aqueous solvents.

Keywords:
HILICaqueous injectionmixersnucleobasestherapeutic oligonucleotides

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Area of Science:

  • Analytical Chemistry
  • Chromatography
  • Biopharmaceutical Analysis

Background:

  • Therapeutic oligonucleotides (ONs) are highly polar and typically dissolved in aqueous solutions.
  • Hydrophilic interaction chromatography (HILIC) is suitable for ON analysis but faces challenges due to solvent mismatch.
  • Solvent mismatch in HILIC can lead to breakthrough and peak splitting, impacting separation performance.

Purpose of the Study:

  • To investigate the impact of sample solvent composition and injection volume on HILIC separation of nucleobases and ONs.
  • To evaluate the efficacy of an in-line mixer in mitigating breakthrough and peak splitting in HILIC.
  • To optimize HILIC methods for large-volume injections of therapeutic oligonucleotides.

Main Methods:

  • Analyzed nucleobases and oligonucleotide mixtures using isocratic and gradient HILIC.
  • Varied sample solvent composition (percentage of water) and injection volumes.
  • Employed an in-line mixer between the injector and HILIC column to assess its impact on peak shape and breakthrough.

Main Results:

  • High aqueous injection solvents (e.g., 90% water) caused peak broadening and deformation, exacerbated by larger injection volumes.
  • In gradient HILIC, large-volume injections (up to 20 µL) of ONs in 30% water showed no performance loss.
  • Injecting ONs in 100% water led to severe peak distortion and breakthrough above 2 µL, but an in-line mixer enabled up to 40 µL injections without issues.

Conclusions:

  • Sample solvent composition and injection volume significantly affect HILIC performance for ONs.
  • In-line mixing is a valuable tool for enabling large-volume injections in HILIC, especially for highly aqueous samples.
  • The developed method using in-line mixing successfully analyzed a pharmaceutical antisense oligonucleotide (ASO), including minor impurities.