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).

Situation	Cause	Remedy
Protein does not bind or elute as expected.	Sample has not been filtered properly. Sample has altered during storage. Sample has wrong pH or buffer conditions are incorrect. Solutions have wrong pH. The column is not equilibrated sufficiently in the buffer. Proteins or lipids have precipitated on the column. Column is overloaded with sample. Microbial growth has occurred in the column. Precipitation of protein in the column filter and/ or at the top of the bed.	Clean the column, filter the sample and repeat. Prepare fresh samples. Use a desalting column to transfer sample into the correct buffer. Calibrate pH meter, prepare new solutions and try again. Repeat or prolong the equilibration step. Clean and regenerate the column or use a new column. Decrease the sample load. Microbial growth rarely occurs in columns during use, but, to prevent infection of packed columns, store in 20% ethanol when possible. Clean the column, exchange or clean the filter or use a new column.
Low recovery of activity, but normal recovery of protein.	Protein may be unstable or inactive in the elution buffer. Enzyme separated from co-factor or similar.	Determine the pH and salt stability of the protein. Collect fractions into neutralization buffer such as 1 M Tris-HCl, pH 9 (60–200 µL per fraction). Test by pooling aliquots from the fractions and repeating the assay.
Lower yield than expected.	Protein may have been degraded by proteases. Adsorption to filter during sample preparation. Sample precipitates. Hydrophobic proteins. Protein is still attached to ligand.	Add protease inhibitors to the sample and buffers to prevent proteolytic digestion. Run sample through a medium such as Benzamidine 4 Fast Flow (high sub) to remove serine proteases. Use another type of filter. May be caused by removal of salts or unsuitable buffer conditions. Use chaotropic agents, polarity reducing agents or detergents.
More activity is recovered than was applied to the column.	Different assay conditions have been used before and after the chromatographic step. Removal of inhibitors during separation.	Use the same assay conditions for all the assays in the purification scheme.
Reduced or poor flow through the column.	Presence of lipoproteins or protein aggregates. Protein precipitation in the column caused by removal of stabilizing agents during fractionation. Clogged column filter. Clogged end-piece or adaptor or tubing. Precipitated proteins. Bed compressed. Microbial growth.	Remove lipoproteins and aggregrates during sample preparation. Modify the eluent to maintain stability. Replace the filter or use a new column. Always filter samples and buffer before use. Remove and clean or use a new column. Clean the column using recommended methods or use a new column. Repack the column, if possible, or use a new column. Microbial growth rarely occurs in columns during use, but, to prevent infection of packed columns, store in 20% ethanol when possible.
Back pressure increases during a run or during successive runs.	Turbid sample. Precipitation of protein in the column filter and/or at the top of the bed.	Improve sample preparation. Improve sample solubility by the addition of ethylene glycol, detergents or organic solvents. Clean using recommended methods. Exchange or clean filter or use a new column. Include any additives that were used for initial sample solubilization in the solutions used for chromatography.
Bubbles in the bed.	Column packed or stored at cool temperature and then warmed up. Buffers not properly de-gassed.	Remove small bubbles by passing de-gassed buffer upwards through the column. Take special care if buffers are used after storage in a fridge or cold-room. Do not allow column to warm up due to sunshine or heating system. Repack column, if possible. De-gas buffers thoroughly.
Cracks in the bed.	Large air leak in column.	Check all connections for leaks. Repack the column if possible.
Distorted bands as sample runs into the bed.	Air bubble at the top of the column or in the inlet adaptor. Particles in buffer or sample. Clogged or damaged net in upper adaptor.	Re-install the adaptor taking care to avoid air bubbles. Filter or centrifuge the sample. Protect buffers from dust. Dismantle the adaptor, clean or replace the net. Keep particles out of samples and eluents.
Distorted bands as sample passes down the bed.	Column poorly packed.	Suspension too thick or too thin. Bed packed at a temperature different from run. Bed insufficiently packed (too low packing pressure, too short equilibration). Column packed at too high pressure.