# Enabling Large‐Volume Injections in Hydrophilic Interaction Chromatography of Oligonucleotides Through In‐Line Mixing

## TL;DR

This paper shows how in-line mixing improves HILIC separations for oligonucleotides by preventing peak distortion and breakthrough during large-volume injections.

## Contribution

The study demonstrates that in-line mixing enables large-volume injections in HILIC without compromising separation quality for oligonucleotides.

## Key findings

- In-line mixing prevents analyte breakthrough and peak splitting during large-volume injections of oligonucleotides in HILIC.
- Gradient HILIC with in-line mixing allows up to 40 µL injections of oligonucleotides dissolved in 100% water without separation loss.
- Large-volume injection with in-line mixing enables detection of main compounds and minor impurities in pharmaceutical antisense oligonucleotides.

## Abstract

Hydrophilic interaction chromatography (HILIC) is an attractive separation mode for the analysis of therapeutic oligonucleotides (ONs). ONs are very polar compounds that are commonly dissolved in highly aqueous media, whereas HILIC eluents often comprise a high percentage of organic solvent. This solvent mismatch can cause breakthrough and peak splitting. In this study, we investigated the effects of the sample solvent composition and injection volume on the HILIC separation of nucleobases and ONs, and to what extent an in‐line mixer between injector and column can mitigate breakthrough and peak splitting. Using isocratic HILIC with nucleobases as medium‐polar, less‐retentive test compounds, we illustrated that an injection solvent of 90% water results in peak broadening, which deteriorates with increasing injection volume, leading to serious peak deformations and asymmetries. Here, in‐line mixing did not improve peak shapes, as the mixer volume adds to band broadening and the homogenization of the injection solvent with the HILIC eluent does not lead to on‐column focusing conditions. When analyzing an ON mixture (15/16/17‐mer) dissolved in a weak solvent (30% water) by gradient HILIC, injection volumes could be increased up to 20 µL (5.7% of the column volume) without losing separation performance. However, when the ON mixture was dissolved in 100% water, injection volumes above 2 µL caused severe peak distortion and extensive analyte breakthrough in gradient HILIC. When injecting the ONs in 100% water with a 100‐µL in‐line mixer, no analyte breakthrough and peak splitting were observed for injection volumes up to 40 µL, while maintaining baseline separation of the ONs. The usefulness of in‐line mixing in HILIC was demonstrated by the analysis of a pharmaceutical antisense ON (ASO). Large‐volume injection of this ON in 100% water allowed for the separation and detection of the main compound and adjacent minor impurities, without breakthrough or severe peak distortion.