Oligos Made Easy _ Part 5 SEC

Size Matters: Mastering Size Exclusion for Oligos

💡Why Use SEC in the Oligo Revolution?

Oligonucleotides are taking center stage in everything from cutting-edge therapeutics to diagnostics and synthetic biology. As the oligo world evolves fast, so does the demand for cleaner, purer, and more precisely characterized oligos. Here’s why Size Exclusion Chromatography (SEC) can be a suitable choice. SEC separates oligos based on size, not chemistry, so your precious oligos come out intact and unaltered, free from truncated sequences, aggregates, and pesky small-molecule contaminants. SEC can handle smaller oligos, but its real strength lies in desalting. When it comes to resolution and precision, AEX or HILIC still remains the method of choice. Keep this in mind and we will help you to purify your oligos like a SEC pro.

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💡How Does SEC Purification Work for Oligos?

Think of SEC as a molecular obstacle course. Your oligonucleotides and all their unwanted companions, salts, solvents, truncated sequences, or aggregates, line up at the starting gate. The column, packed with porous beads, is the course they must run. So, picture a column packed with tiny, porous beads, that are the “inverse” sieves of SEC (see also figure 1). The result? Your large oligo emerges from the column first and purified from smaller contaminants or fragments to be ready for downstream applications. SEC under native conditions keeps oligonucleotides in their natural shape, separating them purely by size with no unwanted dissolution of dimers or oligomers or secondary structures.

Figure 1:   SEC Principle.

💡What are the Rules of the SEC Game?

Oligo Size Matters and big Oligos are sprinters. They’re too bulky to enter the pores of the beads, so they zip around them, taking the fastest route through the column (see also figures 1,2). So, when your oligonucleotide sample runs through:

•  Large molecules (your target oligos, in our example the red ones) can’t enter the pores and come out first.

Small impurities are wanderers.

Salts, small molecules, and degraded fragments slip into the pores, meander through the maze, and take much longer to emerge.

• Smaller impurities (in our example the blue ones) sneak into the beads’ pores, getting trapped ​temporarily and eluting later.

Middle-sized players (like truncated oligos) land somewhere in between, eluting later than your target but earlier than tiny molecules, in the separation range.

Figure 2: SEC Elution.

Let us summarize, SEC for oligonucleotides is like letting your product take the express lane while the impurities get lost in traffic.

💡How to Master Oligo Purification Step by Step?

Choose the right SEC column: 

• Typical oligos range from 10 to 50 nucleotides (~3-15 kDa)
• Select a column with a pore size suitable for this range: ~100 – 150 Å: ideal for short oligos    (10–25 nt), ~200 – 300 Å: better for longer strands (25–50 nt) or aggregates
• Columns with AppliChrom® SEC media or similar SEC media are perfect 

Prepare your sample:  

• Let your samples sit overnight until fully dissolved
• Filter to avoid clogging
• Keep sample concentration moderate (e.g., 0.1–1 mg/mL) for optimal separation

 

Select the mobile phase: 

• A simple aqueous buffer, like 50 mM phosphate or Tris with 100–150 mM NaCl, works well to suppress interaction with column material
• Avoid detergents or organics that can mess with column stability
• Always make sure your column is compatible with the solvent you’re using
• If you’ve run a buffer, flush it afterward with water or even better, water with sodium azide, to keep your column in top shape

Set your flow rate: 

• Slower flows (0.2–0.5 mL/min for analytical columns) improve resolution but take longer
• For preparative purifications, increase flow carefully, balancing speed and resolution
• Always keep an eye on column backpressure limits

Load your sample carefully: 

• Overloading reduces separation quality, stick to recommended injection volumes
• Validate column overload capacities

Collect fractions: 

• Monitor UV absorbance at 260 nm (where oligos absorb strongly)
• Collect the early-eluting peak(s), these should contain your purified oligos
• Later peaks often represent salts or smaller impurities

Analyze your fractions: 

• Run analytical method (e.g., analytical AEX, IP-RP or HILIC HPLC) to confirm purity

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💡Which SEC Column Parameter Make or Break Your Oligo Purification?​

In SEC the gel is the stage, and its properties decide how cleanly your oligos take their bow. So, what matters (see also figure 3)?

Pore size: The pore properties of the size exclusion gels have a major effect on the elution of analytes. Big molecules are excluded, and small molecules explore the pores. The result is a clean size-based elution.

Zero interactions: The ideal gel surface doesn’t grab, stick, or interfere, it avoids ionic charges and hydrophobic patches altogether. Instead, it stays completely inert and non-interactive, ensuring that size, not chemistry, determines how molecules separate.

Built to last: SEC gels are the quiet workhorses of purification. They hold their shape under pressure, shrug off aggressive buffers, and keep their cool even at elevated temperatures, all without dissolving or breaking down. No matter how tough the conditions, a reliable gel stays solid and keeps the separation running smoothly. However, rapid changes between conditions should be avoided, regardless of whether these are flow rate, temperature, or eluent composition.

Water-friendly: Hydrophilic by design, SEC gels are made to thrive in water-based separations. Built from trusted materials like crosslinked dextran, agarose, polyacrylamide, or hydroxylated methacrylate, they welcome aqueous buffers, making them a god choice for oligonucleotide purification.

Figure 3: SEC Column Gel Parameter Characterization.

The size-based SEC mechanism is completely non-interactive: no charge effects, no hydrophobic sticking, and no harsh solvents. Just pure physics will realize that molecules are sorted by their ability to take shortcuts. So, the perfect SEC column is like a good referee, it stays neutral, stands strong, and lets size decide who wins.

💡 SEC Hacks for Perfect Oligos?

Sample contaminants: Avoid pre-purified samples with desalting columns or spin filters if your sample’s heavy on salts.

Temperature control: Room temperature is usually fine but keep consistent to avoid retention shifts, our recommendation is use a column thermostat.

pH value: Avoid extreme pH values generally, keep between 6.5–8.0 to protect both oligos and gel stability.

Buffer exchange: Use the mobile phase you want as the final product buffer to simplify downstream steps.

Injection volumes: Keep <2–5% of column volume for analytical runs to maintain peak shape, for preparative SEC, injection volumes are typically much larger and prioritizes throughput over perfect resolution, so inject 5–10% of column volume if the oligo mass load is within resin capacity.

Flow rate: Avoid abrupt changes in pressure to prevent column bed collapse.

Fraction Collection: Use peak width and symmetry to decide cut points, don’t rely solely on retention time and collect slightly earlier than the main peak tail to maximize purity (see also figure 4).

Figure 4:  Preparative SEC with Fraction Collection. 

SEC column maintenance & care: Keep SEC columns healthy by using clean, filtered samples, flushing with recommended buffers, storing in preservative (e.g., 20–30% ethanol), avoiding overload, and monitoring performance. Gentle care ensures long-lasting, reproducible oligo separations (see also figure 5).

Figure 5:  SEC Column Maintenance.

💡 Final Thoughts, Strengths and Shortcomings in the Real World? 

Size Exclusion Chromatography (SEC) often gets praised for being “gentle” and straightforward, no harsh gradients, no fancy additives, just separation by size. For oligonucleotides, that sounds ideal. But is SEC always the best tool in the box? Not quite. 

So, where does SEC truly shine? In desalting, buffer exchange, and quick quality checks, it’s fast, robust, and non-destructive. But for high-resolution, in-depth characterization, other chromatographic methods will usually outperform SEC.

Bottom line: SEC is a reliable workhorse for oligos, but not the silver bullet. Knowing when to use SEC and when to switch to AEX, IP-RP or HILIC, is the real key to smarter oligo purification.

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 kindler@knauer.net