Quick fixes for retention time issues

Cytiva

Mar 20, 2025

Is your protein eluting at the wrong time? Does it seem to happen too early, too late or maybe not at all? If you are experiencing unexpected retention times, read on to learn how to quickly identify possible causes and how to fix them.

If your protein elutes earlier than expected when using IEX, the cause could be something as simple as incorrect buffer conditions.

If your protein elutes later than expected when using SEC, it could be due to ionic interactions between protein and matrix.

Other common reasons for unexpected retention times, and how to fix them, are summarized by Cytiva R&D protein purification experts in the following tables.

Protein elutes earlier than expected – possible causes and remedies:

Possible cause	Remedy
IEX, HIC: Column equilibration incomplete	Repeat or prolong equilibration step until conductivity and pH are constant
IEX: Ionic strength of sample or buffer too high or pH is incorrect	Decrease ionic strength of sample or buffer Increase pH (anion exchanger) Decrease pH (cation exchanger)
HIC: Salt concentration of sample and buffer too low	Increase salt in sample and buffer

Protein elutes later than expected/not at all – possible causes and remedies:

Possible cause	Remedy
Proteins or lipids precipitated on column or column filter	Clean column and exchange or clean filter
Protein might be unstable or inactive in elution buffer	Determine pH and salt stability of protein
Delivered gradient is distorted	Air bubble caught in pump(s): Purge pumps according to user manual Pump check valve malfunction: Flush check valves at high flow rate and/or clean with ultrasonic bath Worn pump sealing ring: Change sealing rings
SEC:
Ionic interactions between protein and matrix	Maintain ionic strength of buffers above 50 mM (preferably include up to 300 mM sodium chloride)
Hydrophobic interactions between protein and matrix	Reduce salt concentration to minimize hydrophobic interaction. Increase pH. Add suitable detergent or organic solvent (e.g., 5% isopropanol)
IEX:
Incorrect buffer pH	Check pH meter calibration. Use buffer pH closer to pI of protein
Ionic strength too low	Increase salt concentration in elution buffer
Hydrophobic interactions between protein and matrix	Reduce salt concentration to minimize hydrophobic interaction. Increase pH. Add suitable detergent or organic solvent (e.g., 5% isopropanol)
HIC:
Salt concentration too high	Decrease salt concentration in elution buffer
Hydrophobic interactions too strong	Use resin with lower hydrophobicity or lower ligand density Consider using an additive to reduce hydrophobic interaction

Protein elutes before void volume (size exclusion chromatography) – possible causes and remedies:

Possible cause	Remedy
Channeling in column	Repack column using thinner slurry of resin. Avoid introduction of air bubbles

*SEC = size exclusion chromatography
IEX = ion exchange chromatography
HIC = hydrophobic interaction chromatography

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Quick fixes for retention time issues

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