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Demystifying Oligonucleotide Separation

Oligonucleotides are tiny fragments of DNA or RNA. Our genetic information is stored in DNA and RNA. These molecules are also known as nucleic acids. Nucleotides are their basic building blocks. DNA comprises a repeating sequence of nucleotides adenine, cytosine, guanine, and thymine, abbreviated with the letters A, C, G, and T, respectively. RNA comprises a repeating sequence of nucleotides adenine, guanine, cytosine, and uracil, abbreviated with the letters A, C, G, and U, respectively. These molecules can be found as single long chains, single strands, or two long chains or double strands connected by hydrogen bonds. These molecules are also giant and made up of millions of nucleotides.

Oligonucleotides, either single- or double-stranded, are versatile tools in research and therapeutics. They typically contain 13 to 25 nucleotides, but larger fragments are also possible, usually not exceeding 200. The broad range of oligonucleotide applications underscores their importance in various scientific and medical fields.

Separating and purifying synthetic oligonucleotides, a crucial step in their production, poses significant challenges. These challenges stem from the chemical modification of the oligonucleotides and the growing interest in larger fragments, which can affect their analysis and manufacturing.

Chemical modification is crucial to synthetic oligonucleotides, which can significantly alter their properties. For instance, adding phosphorothioate linkages can enhance stability and bioavailability, improving their pharmacokinetic properties in the body. Other modifications, such as the attachment of polyethylene glycol at the terminus and the incorporation of modified nucleosides, can also profoundly affect the behavior of oligonucleotides.

Larger Fragment. Until recently, the size of oligonucleotides for most applications was up to 60 nucleotides. However, oligonucleotide fragments of 100 nucleotides are common for newer clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and non-coding RNA technologies.

Product and process-related oligonucleotide impurities are common impurity types. The chemical modifications and increases in nucleotides present unique separation and purification impurities. Isolating these impurities requires advanced techniques and a deep understanding of the properties of oligonucleotides. These impurities may include failure sequences from the side or incomplete reactions and sequences structurally like the full-length product, sharing many physicochemical characteristics. These impurities may also be formed by incorporating critical impurities in starting materials and reagents and from the degradation of intermediates and the final product [1].

Liquid chromatography techniques such as anion exchange (AEX) chromatography and ion‑pair reversed-phase (IP-RP) chromatography are widely used to separate oligonucleotides and can provide complementary information. The choice of technique depends on the final goal. For example, if the requirement is isolation of the full-length sequence, a purification technique is required to concentrate a purer oligonucleotide. If the goal is characterization analysis of the impurities, a separation technique that will isolate these impurities will be appropriate.

Stationary-phase material type (polymeric, silica C18), size (diameter) and porosity (pore size), mobile phase conditions (pH, salt concentration), and chromatography system parameters (temperature, pressures) affect the separation and purification outcomes. For example, to characterize the impurities as a part of quality control, media with particle size 3 or 5 µm in columns operating at high pressures (10,000 psi) will provide high-resolution separation of the components in the mixture. For the purification of an oligonucleotide, media with particle sizes of 10-15 µm packed into columns operating at low or medium pressures is sufficient.

Fig. 1. Illustration of Anion exchange media particle

Anion exchange chromatography (AEX). AEX employs stationary-phase material or media functionalized with positively charged groups. Quaternary or tertiary amines are the widely used groups. The negatively charged oligonucleotide binds to the positively charged stationary phase during the loading step. The oligonucleotide dissociates when a competing negative charge in the form of salts is introduced to compete with the binding process. It elutes as the salt concentration increases. As oligonucleotide length increases, it has a more negative charge; therefore, more salt concentration is needed to dissociate longer oligonucleotides. Thus, these oligonucleotides elute later. The oligonucleotide elution depends on its sequence and structure.

Modulating pH, temperature, solvent, and eluent salt can control oligonucleotide retention and secondary structures. The ability to control these parameters makes AEX suitable for analyzing single- and double-stranded oligonucleotides.

Ion-pair reversed-phase chromatography (IP-RP). Each phosphodiester linkage on an oligonucleotide strand exhibits one negative above pH 4 in an aqueous solution. Thus, reversed-phase chromatography cannot be directly applied to oligonucleotides due to this charge polarity. Therefore, molecules with the opposite positive charge, ion pairing agents, are added to the mobile phase. These ion pair agents form complexes with the oligonucleotides via electrostatic interaction. The most used ion-pair reagent is triethylamine (TEAA). Other ion-pair reagents could provide alternative selectivity (e.g., tetrabutylammonium bromide (TBAB)) or better compatibility with detection systems such as mass spectrometers (e.g., hexafluoro-2-propanol (HFIP)).

IP-RP chromatography employs organic solvents at high temperatures (unlike AEX). The retention and elution of the oligonucleotide are determined by the charge of the oligonucleotides, the length of the alkyl chain in the ion-pairing reagent, and the proportion of organic solvent in the mobile phase. For example, retention time may increase with the increase in the number of charges in the oligonucleotides and hydrophobicity (longer alkyl chain) in the ion-pairing agent.

Figure 2.  Interaction of an oligonucleotide with an ion-pairing reagent

 Summary and ongoing efforts. Anion exchange (AEX) chromatography and ion‑pair reversed-phase (IP-RP) chromatography, while adequate for oligonucleotides of up to 25 nucleotide fragments, are suboptimal or sparse as the oligonucleotide fragment size increases. The methods are no longer efficient as more nucleotides are added to the oligonucleotide fragment.  Separating longer oligonucleotides is possible, but the separation is no longer size-dependent. The structural changes and compositions of oligonucleotides make it challenging to separate these molecules with single-nucleotide resolution.


Nevertheless, the technologies available for oligonucleotide separation and purification are rapidly evolving. Stationary phase media and separation methods that offer flexible conditions for anion exchange (AEX) and ion-pair reversed-phase (IP-RP) chromatography would be most beneficial. For instance, using stationary phase media that can withstand elevated temperatures (up to 100°C) and an extensive dynamic pH range (pH – 0-14) provides the flexibility to operate in conditions required for high selectivity and resolution. Large pore media could allow long oligonucleotides to diffuse in and out to generate sharper peaks than a small-pore media. This flexibility will enable the development of the most appropriate method to optimize the resolution and selectivity.



[1] Y.A. Fillon, N. Akhtar, B.I. Andrews, D. Benstead, S. Breitler, R.S. Gronke, M. Olbrich, J.A. Stolee, T. Vandermeersch, Determination of Purge Factors for Use in Oligonucleotide Control Strategies, Organic Process Research & Development 26(4) (2022) 1130-1144.

At Millennial Scientific, we develop next-generation products for separation and purification of oligonucleotides and other biologics. Get in touch with us to discuss how we can support your chromatography separation needs. For more information or to request samples, please email us at, call us at 855 388 2800 or fill in our online form.


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