The field of oligonucleotide therapeutics is expanding due to the push towards personalized medicine and continuing research into the development of therapies for rare diseases. The increased demand for oligonucleotides requires easy, cost-effective scale-up from quantities used in research and development settings to commercial needs as oligonucleotides are difficult to produce in high yields with high purity and formulate for efficient delivery and stability.


Irregularities in the oligonucleotide manufacturing process can lead to the formation of common impurities such as Shortmers– oligonucleotides missing one or more nucleotides (N-1, N-2, etc.) and Longmers– oligonucleotides that include more than the intended number of nucleotides (N+1, N+2, etc.).


Accurate characterization of oligonucleotides requires analytical tools and workflow solutions that deliver robust, accurate analytical characterization to confirm identity and determine purity, quality, and strength to improve efficiency and enable regulatory compliance.


HPLC and UHPLC chromatographic methods are part of the typical purification schemes in therapeutic oligonucleotide process development and manufacturing for purity and impurity characterization to satisfy regulatory requirements.

Non-mass spectrometry oligonucleotide applications

For non-mass spectrometry (MS) applications, ion-exchange chromatography is the best way to analyze or purify oligonucleotides. Denaturing conditions obtained with high-pH eluents eliminate Watson-Crick hydrogen bonding and allow resolution of problem sequences such as self-complimentary sequences and poly-G stretches. Anion-exchange chromatography at a high pH has become the preferred approach for oligonucleotide analysis. Thermo Scientific DNAPac PA200 and PA200 RS Columns contain a polymeric resin stationary phase that uses surface anion-exchange ligands as means for separation. These columns are effective at separating various species within a short run due to their ion-exchange capabilities, making them a good choice for complex samples. An example of this type of analysis can be found in the application note Ultra-High-Resolution Separation of Oligonucleotides on Pellicular Anion-Exchange UHPLC Columns. You can also use our Thermo Fisher Scientific HPLC Column Selection Guide to find application appropriate columns and consumables for your bioanalysis.

Separation of deoxythymidine oligodeoxynucleotides, with 84 separate components were resolved in less than 10 minutes on a DNAPac PA200 RS column.

Mass spectrometry oligonucleotide applications

For MS applications, ion-exchange chromatography is not applicable due to the high salt content of the eluting mobile phase. However, reversed phase (RP) ion-pairing analysis is an excellent alternative option for MS applications of oligonucleotides. Using a volatile ion-pairing agent will help to fully retain polar oligonucleotide molecules and be more beneficial for rapid evaporation during mass spectrometry. Thermo Scientific DNAPac RP Columns are reversed phase liquid chromatography columns based on a spherical, supermacroporous 4 µm polymeric resin designed for ion-pair reversed-phase (IP-RP) separations of both oligonucleotides and double-stranded nucleic acids. Their stationary phase is stable over the full pH range (pH 0-14) and displays fewer secondary interactions between its particles and polar oligonucleotides as the particles are polymers. This is in contrast to other columns on the market, which are commonly repurposed C18 columns. By using a polymeric resin stationary phase, higher efficiency and resolution can be achieved as the resulting chromatographic peaks will be more symmetrical.


More information on the analysis of oligonucleotides via HPLC and UHPLC methods can be found on Oligonucleotide HPLC and UHPLC Columns.

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