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.

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

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

Possible cause

Remedy

Channeling in column
  • Repack column using thinner slurry of resin. Avoid introduction of air bubbles

View more chromatography troubleshooting tips here

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