Have you ever seen bubbles in the resin bed of your chromatography column? Or even space between the resin bed and the adaptor?

This is a sign that something is wrong with your column, and it can’t be used with confidence for purifying your protein. But don’t let it get you down! These issues are often easily resolved.

For example, bubbles in the resin bed may be caused by something as simple as the column hardware being inappropriately assembled. And space between the resin bed and the adapter could be caused by improper packing of the column.

These common column problems, and how to fix them, are summarized by Cytiva R&D protein purification experts in the following tables.

Air in the column
Compressed bed

Possible cause

Remedy

Buffers not properly degassed
  • Degas buffers thoroughly. Run degassed equilibration buffer through column to remove air

Inappropriate sample loading or purification method construction
  • Use air sensors to prevent air from entering system

Column hardware inappropriately assembled or mechanically damaged
  • Ensure that column is correctly assembled and free from damage before packing

Blocked or partially blocked inlet filter
  • Change inlet filter or clean if possible

Column packed or stored at cool temperature and then warmed up
  • Remove small bubbles by passing degassed buffer through column
  • Take special care if buffers are used after storage in cold room or refrigerator
  • Do not allow column to warm up in sunshine or by heating system. If possible, repack column

Other restrictions in flow path before pump
  • Check tubing and connections on inlet side

Possible cause

Remedy

Back pressure increase or bed insufficiently packed
  • Turn down adapter to resin surface. Do a column performance test. Repack if needed

Column packing not performed according to instructions
  • Repack according to recommended protocol

Flow rate too high
  • Do not exceed maximum flow rate for resin or prepacked column

Resin bed compressed
  • Repack using lower flow rate
  • Use prepacked columns
  • Check that system back pressure is not too high. Are there any restrictions in system?

Column operated at too high pressure
  • If using recommended flow rates, clean column according to instructions
  • Do not exceed recommended operating pressure for resin or prepacked column
  • For self-packed columns, use ”Column handling” functionality in UNICORN™ system control software to save a defined column with its pressure data. Then select this method to protect this column type from too high pressure

Rapid pressure change
  • Avoid an abrupt change to high flow rate
  • Do not turn valves during flow

View more chromatography troubleshooting tips here