Affinity Chromatography Troubleshooting

This section focuses on practical problems that may occur when running a chromatography column. The diagrams below give an indication of how a chromatogram may deviate from the ideal during affnity purifcation and what measures can be taken to improve the results.

Target elutes as a sharp peak. Satisfactory result

• If it is diffcult or impossible to retain biological activity when achieving this result, either new elution conditions or a new ligand must be found.
• If using low pH for elution, collect the fractions in neutralization buffer (60–200 µL 1 M Tris-HCl, pH 9.0 per mL eluted fraction).

Target is a broad, low peak that elutes while binding buffer is being applied

• Find better binding conditions.

Target elutes in a broad, low peak

• Try different elution conditions.
• If using competitive elution, increase the concentration of the competitor in the elution buffer.
• Stop flow intermittently during elution to allow time for the target molecule to elute and so collect the target protein in pulses (see second figure beneath).
  Note: This result may also be seen if the target protein has denatured and aggregated on the column or if there is non-specifc binding.

Some of the target molecule elutes as a broad, low peak while still under binding conditions

• Allow time for the sample to bind and/or apply sample in aliquots, stopping the flow for a few minutes between each sample application (see second fgure beneath).