Analysis of Oligonucleotide Standard 6 Mix by Liquid Chromatography-UV

by | Jan 26, 2024

Explore the cutting-edge world of oligonucleotide analysis with Chromolith® columns.

This article from issue 15 of the Analytix Reporter discusses the analysis of the Oligonucleotide Standard 6 mix by liquid chromatography-UV. The study focuses on the challenges in analyzing oligonucleotides and presents the experimental procedure using Chromolith® RP-18e columns. The results and discussions cover topics such as the impact of phosphate group linkage, ion-pairing additive concentration tests, and the evaluation of Chromolith® HighResolution RP-18e columns. The findings demonstrate the suitability of Chromolith® columns for efficient separation and analysis of oligonucleotides, offering insights for high-throughput assays and LC-MS applications.

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With the COVID-19 pandemic, oligonucleotides (Oligos) have proven their importance in diagnostic and therapeutic applications.  Currently, 11 oligonucleotide drugs crossing many disease areas have been approved by the FDA. Obstacles preventing quicker development of oligonucleotide therapeutics include the challenges of unfavorable absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies for many clinical trials. Some strategies have been developed to tackle the challenges, such as chemical modification to improve drug delivery.

Synthetic oligonucleotides are typically small, single- or double-stranded modified nucleic acids. There are many established techniques to analyze and characterize oligonucleotides, including capillary gel electrophoresis (CGE), ion exchange chromatography (IEX), and ion pair reversed-phase liquid chromatography (IP-RPLC). Generally, liquid chromatography is very challenging due to the similarity of oligonucleotide structures, very polar characteristics, presence of truncated and/or modified oligos, ease of self-association into a variety of conformations, and affinity for metal surfaces. This application describes the separation of an internally produced oligonucleotide standard (Oligo Standard 6) mix, which includes six oligonucleotides, on Chromolith® RP-18e column from the Supelco® portfolio.


Oligo Standard 6 is an internal (in-house) system suitability mix for HPLC-UV evaluation of oligonucleotide separations. It contains six components with molecular weights of 3588.3 Da (Oligo 1), 4157.93 Da (Oligo 2), 7580.83 Da (Oligo 3), 10014.35 Da (Oligo 4), 6116.97 Da (Oligo 5), and 4395.8 Da (Oligo 6) following their elution order on Chromolith® RP-18e columns tested here.

See the full article for reagent and sample preparation specifications.


With the linkage of phosphate groups, oligonucleotides tend to stick to metal surfaces present in stainless steel column hardware and the LC system, resulting in reduced sensitivity and inaccurate quantitation. Researchers have made a variety of efforts to mitigate this adsorption inside instrumentation, such as treatment of the system with EDTA, high pH mobile phase, or utilizing bio-inert HPLC system components. Conventional HPLC columns are typically packed in metal columns, exposing the metal surfaces with the positive charge, which can adsorb acidic molecules, such as oligonucleotides containing phosphate groups. Chromolith® HPLC columns are made of highly porous monolithic rods of silica. These columns have an innovative, bimodal pore structure and are packed in metal-free PEEK (polyetheretherketone) columns. This attribute makes them a good candidate for oligonucleotide analysis. 

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In the application note, the separation of Oligo Standard 6, an internally created HPLC-UV system suitability mix, was demonstrated on standard Chromolith® and Chromolith® High-Resolution RP-18e columns. Flow-rates up to 3 mL/min were evaluated on the standard Chromolith® with excellent separation of the six oligos, indicating that it is ideal for high throughput assays. The effects of ion-pairing reagent, TEAA, on Oligo Standard 6 separation were discussed to provide optimization guidance in oligo analysis. Chromolith® High-Resolution RP-18e columns were evaluated with a typical LC-MS flow-rate, demonstrating this column is suitable for oligonucleotide analysis by mass spectrometry. In addition, the polymeric column housing can be used as part of a metal-free, or bio-inert HPLC system.

*The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.

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