Unexpected SEC Results – Can It Be Improved?

Sometimes your size exclusion chromatography (SEC) results don’t look like you want them to. Fix issues like poor resolution, peak tailing and fronting with the tips in this FAQ.

Q: How can I optimize the running conditions to optimize resolution in my SEC runs?

A: After selecting a resin that has the right fractionation range, lowering the flow rate and sample volume will improve the resolution. Buffer conditions might also affect resolution as the environment for the protein is important to avoid interaction with the resin or other compounds in the sample.

It is also important to configure the system you are using. For maximum resolution, system volumes should be kept at a minimum. Short and narrow capillaries should be used and unnecessary system components should be bypassed.

You can learn more from the white paper Protein analysis with size exclusion chromatography

Q: What causes peak tailing?

A: Peak tailing could depend on different factors:

• Poorly packed column. Carry out a performance test to check column conditions (see protocol in the column instructions).
• Column is contaminated. Clean the column using recommended procedures.
• Buffer conditions of the sample are unfavorable. Adjust pH and check salt concentration in start buffer.
• Sample is too viscous. Dilute the sample in buffer.
• Band broadening due to large volumes in the system. Minimize system volumes in system valves and detectors, tubing, and connections.

Need more troubleshooting tips? Download the Size exclusion chromatography handbook.

Q: What causes peak fronting?

A: Fronting peaks could depend on different factors:

• An excessively large sample volume has been applied. Decrease sample volume and perform a new run.
• Poorly packed column. Carry out a performance test to check column conditions (see protocol in the column instructions).
• Column is contaminated. Clean the column using recommended procedures.

Need more troubleshooting tips? Download the Size exclusion chromatography handbook.

Q: I do not get the resolution I need with my SEC column. What to do?

A: Several factors affects the resolution in SEC. Possible causes for poor resolution:

• Sample is too viscous. Dilute the sample in buffer, but check maximum sample volume for the column at hand. Maintain protein concentration below 50 mg/mL.
• Sample contains particles. Re-equilibrate the column, filter sample with a low protein binding filter (e.g., Whatman SPARTAN filters), and repeat.
• Column is contaminated. Clean the column using recommended procedures.
• Incorrect SEC resin type. Check selectivity curve in available selection guides.
• Sample volume too large. Check recommendations and decrease sample volume to be applied.
• Flow rate too high. Check recommendations and lower flow rate.
• Sample might be diluted in the system before or after the column. Minimize volumes in the system, for example, decrease tubing diameter and/or length, mounting column directly to UV cell, change injection valve and/or column valve to valves with smaller internal volumes.

Need more troubleshooting tips? Download the Size exclusion chromatography handbook.

For more application-related questions, we recommend the compiled questions and answers from the live session of the webinar Protein characterization using size exclusion chromatography