What’s the point of protein purification? Obtaining pure proteins, of course ☺. If you are having difficulties obtaining the purity you need, these troubleshooting tips may help.

Troubleshooting purity and resolution issues

“Resolution” refers to how well two protein peaks are separated. The closer, or the less well defined your peaks are, the more difficult it is to get your pure protein.  Poor resolution, and thus low purity, can be caused by many different reasons.

A few of these reasons include poorly packed columns, sub-optimal elution conditions, or the wrong sizes and lengths of tubing.

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

Possible cause


Column poorly packed

Large mixing spaces at top of column

  • Adjust top adapter to resin surface if necessary

Elution conditions not optimal (e.g., gradient too steep, flow rate too high)

  • Change elution conditions (e.g., use shallower gradient, reduce flow rate)

Proteins precipitated in column

  • Follow cleaning procedures in instructions
  • HIC*: Reduce salt concentration in buffer, or use existing buffer but apply aliquots of sample that has low salt concentration
  • IEX*: Modify buffer, pH, and/or salt conditions during run to maintain stability

Tubings in chromatography system too long and wide

  • Decrease tubing diameter and minimize length

Separated proteins diluted between column outlet and UV flow cell

  • Minimize volumes after column by decreasing tubing diameter and minimizing length
  • Change to injection and column valves and flow cells with smaller volumes
Possible cause


Sample too viscous

  • Dilute with buffer, but check maximum sample volume. Maintain protein concentration below 50 mg/mL

Sample contains particles

Column is dirty

  • Clean and re-equilibrate

Incorrect SEC resin type

Sample volume too large

  • Check recommendations, and decrease sample volume loaded

Flow rate too high

  • Check recommendations, and reduce flow rate

Sample diluted between injection valve and column inlet, between column outlet and UV flow cell, and/or further to fraction collector

Minimize volumes before and after column by either

  • Decreasing tubing diameter and minimizing length
  • Mounting column directly to UV cell (without column valve)
  • Removing all unnecessary components in flow path
  • Change to injection and column valves and flow cells with smaller volumes

View more chromatography troubleshooting tips here


*IEX = ion exchange chromatography
HIC = hydrophobic interaction chromatography