How to Navigate Through the Jungle of Columns?
Struggling to Find the Right Column for Your Oligos?
Oligonucleotides are complex, long-chain molecules with a very high charge density and a wide variety of options for their chromatographic separation. Finding the right one for your specific application can be tricky, but we're here to help.
What is the Challenge?
Oligonucleotides have been one of the hottest topics in molecular biology as well as medical and therapeutic research in the last five years. Many applications arise in regulated environments, where quality and safety are key issues, making reliable and precise analysis of oligonucleotides essential.
Oligos pose particular challenges for chromatographic separation. They are highly charged phosphodiesters or phosphorothioates and are therefore extremely polar. Many very similar impurities are produced during synthesis. For example, in terms of length (shortmers/longmers) and side chain modifications. Some differ only by a single base or even a functional group. In our blog, 'Oligos Made Easy: Part 1', we give you an overview on how to synthesize oligonucleotides and what you need to think about to make sure your process is as efficient as possible.
On top of that, residual synthesis reagents, salts, and error sequences can overload the column, reducing performance. When it comes to analytical workflows, the stakes are high: achieving sharp, symmetrical peaks with excellent resolution in minimal time is essential, while maintaining MS compatibility. Repeatable performance, recovery, and minimal sample loss during elution or desalting are equally critical.
In short, the right column must handle high polarity, complex impurity profiles, and challenging matrices, all while delivering reproducible, high-quality results with sufficient peak resolution. Understanding these challenges is the first step toward making informed column choices for efficient oligonucleotide analysis.
Which Column for my Oligonucleotides?
Oligonucleotide separation is a challenging topic, as the extremely polar and often long-chain oligos often differ only by a single base in length or a single modification of synthesis by-products. Preparative LC or SEC are generally used to purify the strands, while UHPLC-MS is the appropriate method for qualitative and quantitative analysis of the products and impurities. Furthermore, special SEC desalting cartridges are ideal for removing interfering salts from a sample prior to purification or analysis. But even if you know whether you want to clean up or analyze, it is often not easy to decide how detailed an analysis actually needs to be, or whether robustness and effectiveness are in the end more important.
“Clearly defining your own chromatographic needs is the first step.” – Dr. Ulrike Krop, Head of Applications & Academy, KNAUER
So, it's important to think about a few things first? Our decision tree in the figures 1 and 2 will help you do this.
Figure 1: Decision tree - Which separation technology for your purification task?
If you want to purify oligos, you first need to be clear about your sample volume. If only a few milligrams need to be purified, small column inner diameters and slightly lower flow rates are usually sufficient. If the focus is also on optimal resolution, IP-RP (ion pairing reversed phase) may be an attractive option. Those who place more value on non-toxic, cost-effective eluents and high loading capacity should decide in favor of AEX (anion exchange chromatography) in the preparative area. If mainly smaller fragments and synthetic reagents are to be separated, SEC (size exclusion chromatography) is an ecological alternative.
In analytical work, the question often arises between maximum resolution and flexibility or high robustness and short analysis times. Those who always have to perform similar separations in high-throughput operation and want to rely on their system for a long time will have to choose between IP-RP and AEX. Due to its high efficiency and excellent resolution, the IP-RP methodology has clearly established itself in this area. Good MS compatibility is another advantage that speaks in favor of this method. Our new Sepapure oliGO columns are optimized precisely for this application and offer a long-lasting solution.
In research and development in particular, efficiency plays a secondary role behind maximum flexibility and sensitivity. If you want to identify a variance in individual side chains between two long oligos that are otherwise identical, you can choose between HILIC (hydrophobic interaction liquid chromatography) and mixed mode columns. Depending on the eluents, these methods are MS-compatible and additionally offer a wide range of adjustment options for sophisticated optimization of the method.
💡Benefits and Limitations at a Glance
Is there any Point in Expensive Bioinert Hardware?
“I have performed countless analyses with a wide variety of oligos and have not noticed any difference between inert and sst (stainless steel) hardware so far. However, I can imagine that this could play a role in very fast 3-minute UHPLC analyses. For most users, the additional costs will probably not be worth it.”
– Marvin Schaek, Product Manager Oligosynthesizer, KNAUER & XNAPharma GmbH
Bioinert hardware is particularly well known in protein purification. The metal ions on the stainless steel surface of the column can interact with histidine and cysteine residues in particular, permanently altering the conformation and thus the function of a protein. To prevent this, the surface must either be passivated, titanium hardware or hardware coated with a layer of glass on the inside can be used. However, this is expensive and not always practical. Since conformation plays a rather minor role in oligonucleotides, the usefulness of bioinert hardware is controversial. If phosphorothioates are present in the backbone, interaction with the surface is likely. There are some cases in the literature where it has been shown that passivation of the column surface can have a positive effect on peak shape in very fast (U)HPLC applications. In classic 10-minute applications, however, this effect is extremely small from our experience.
The Choice is Yours
Anyone entering the exciting field of oligonucleotides cannot avoid purification and suitable analysis by HPLC. Depending on the requirements, there is a suitable method and column for every application.
We have developed a column that is specifically tailored to the needs of oligonucleotide users and is perfectly suited for IP-RP applications from UHPLC to semi-preparative — the Sepapure oliGO is reliable, precise, and cost-effective.
If you're planning to set up or optimize your oligonucleotide workflow, feel free to contact us at sales@knauer.net. Stay tuned for more exciting insights into the Oligonucleotide world in our “Oligos Made Easy” series.
For more in-depth discussion or questions,
reach out to the author at folmert@knauer.net
