Analysis of Long-Chain Oligo-DNA with the Shodex™ VN-50 2D HILIC Column

The Application Notebook, The Application Notebook-02-01-2022, Volume 40, Issue S2

Oligonucleotide therapeutics, such as antisense nucleic acid drugs, are promising candidates for genetic, metabolic, and cancer treatments. Generally, oligonucleic acids (oligo-DNAs and oligo-RNAs) around
20 mer are used in approved nucleic acid drugs. However, longer chains are reported by BioNtech and Moderna (1).

The development and quality control of oligonucleotide therapeutics often require highly selective and sensitive analytical methods such as liquid chromatography–mass spectrometry (LC–MS). The most often used method of analysis is reversed-phase chromatography with the use of an ion-pair reagent. However, ion-pair reagents tend to remain in the LC system causing problems. Ion exchange chromatography is another common method. However, this generally requires addition of highly concentrated salt in the eluent, unusable on mass spectrometry detectors.

This technical article outlines the analysis of long-chain oligonucleotides utilizing a simple eluent containing acetonitrile with a relatively low level of salt and no ion-pair reagent.

The LC–MS system used was the Shimadzu Nexera/LCMS-8030 Plus. The column used was the Shodex HILICpak™ VN-50 2D. Dimensions were 2.0 mm I.D. × 150 mm in PEEK housing. The gel material was a multi-porous polyvinyl alcohol support with surface diol functional groups. The packing material particle size was 5 μm and pore size was 100 Å.

A high-pressure linear gradient elution was used. The elution method was optimized during method development. The gradient mixtures of Eluent A (ammonium formate aqueous solution) and Eluent B (acetonitrile) are shown in Figure 1. The column temperature was 40 °C. The mass spectrometer was coupled with UV (260 nm) for detection.

Analysis of 10- to 50-mer oligo-DNAs
Separations of up to 50-mer oligo-DNAs were optimized. The types and sizes of synthetic oligo-DNA in samples 1–5 are listed in Table I. They were dissolved in ultra-pure water and 0.02-mg/mL samples were injected for analysis.

Figure 1 shows the LC–UV/MS chromatograms of 10-, 20-, 30-, 40-, and 50-mer oligo-DNAs. The optimized elution condition was a linear gradient from 65 to 45% acetonitrile over 10 min, and then 45% acetonitrile was held for 10 min. This gradient condition (higher ammonium formate aqueous solution ratio) provided improved separations of longer chain oligo-DNAs.

This application note demonstrates the feasibility of Shodex HILICpak VN-50 coupled with an LC/UV/ESI–MS for the analysis of 10-mer to
50-mer oligo-DNA without chemical modifications.

This mild eluent does not require the ion-pair reagents required for reversed-phase chromatography nor the highly concentrated salts required for ion-exchange chromatography, thus reducing the LC–MS system maintenance burden. Also, the desalting process during purification is simplified since the ammonium formate/acetonitrile eluent mixture is volatile.

Importantly, the polymer-based packing material allows the use of alkaline washing solutions, which helps prevent the non-specific adsorption and carry-over related problems that are often problematic in laboratory- or preparative-scale analyses that demand high precision and accuracy.

The results underscore the importance of the VN-50 2D in the development, quality control, and purification of nucleic acid drugs.


  1. Y. Yamada, Cancers, 13(19), 5002 (2021).

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