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

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

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

Target elutes in a broad, low peak
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

Some of the target molecule elutes as a broad
  • 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).
Materials
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