Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
Worrying peak shape
Possible cause | Suggested remedy |
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Gradient slope or isocratic step too shallow. |
Increase steepness of gradient or isocratic step. |
Poor reproducibility
Possible cause | Suggested remedy |
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Affinity, hydrophobic interaction and ion exchange chromatography - Continuous build-up of contaminants has altered the selectivity of the chromatography medium. |
Clean the chromatography medium according to instructions. |
Possible cause | Suggested remedy |
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Column not properly equilibrated |
Check the pH and the conductivity of the effluent before applying the sample. Continue to equilibrate with start buffer if necessary |
Insufficient column regeneration. |
Prolong the regeneration. |
Possible cause | Suggested remedy |
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Protein properties change with concentrations |
Dilute or concentrate the sample to minimize effects. |
Proteins precipitate at high concentration. |
Reduce sample concentration and/or binding capacity. |
Possible cause | Suggested remedy |
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Column bleeding from previous run. |
Check and adjust your cleaning procedure. |
Column clogged with denatured proteins and/or lipids. |
Clean and regenerate the column and chromatography medium according to instructions. |
Incomplete equilibration of the column. |
Check pH and conductivity of the effluent before applying the sample. Continue to equilibrate if necessary. |
Incorrect pH and/or ionic strength of the solutions. |
Check pH and conductivity and adjust if necessary. Calibrate your conductivity and pH meters. |
Larger sample mass load applied compared with earlier runs. |
Keep mass of sample constant when repeating runs. (High proteins concentration can cause protein interaction, resulting in change of elution profile.) |
Sample volume is different from earlier runs. |
Resolution is dependent on the sample volume. Keep sample volume constant when repeating runs. |
Unsatisfactory elution
Possible cause | Suggested remedy |
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Buffers have wrong pH, ionic strength or solvent concentration. |
Check and adjust the buffer conditions. Calibrate your instruments. |
Possible cause | Suggested remedy |
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The column not properly equilibrated |
Check the pH and conductivity of the effluent before applying the sample. Continue to equilibrate with start buffer if necessary. |
Possible cause | Suggested remedy |
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Column not properly equilibrated. |
Re-equilibrate the column. |
Sample not properly equilibrated. |
Equilibrate sample to correct operating conditions (pH, ionic strength, etc.) |
Possible cause | Suggested remedy |
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Column not properly equilibrated. |
Check the pH and conductivity of the effluent before applying the sample. Continue to equilibrate with start buffer if necessary. |
Components in the sample displace the target molecule before elution starts. |
Reduce the concentration of detergent to below it´s critical micelle concentration (CMC) value. |
Possible cause | Suggested remedy |
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Target protein not stable under the chosen conditions and partly degrades |
Find better ways to stabilize the protein, e.g. shorten the process time. |
The detergent has formed micelles with the protein, thereby increasing its size and changing its elution position. |
Reduce the concentration of detergent to below it´s critical micelle concentration (CMC) value. |
Possible cause | Suggested remedy |
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Dead volume in chromatography systems is high. |
Minimize dead volume in the chromatography system by decreasing capillary length and dimensions between injector and detector. Bypass unused system components e.g. column valves from the flow path. |
Flow velocity too high. |
Run the separation at a lower flow velocity. This is especially important for adsorbents that bind several substances and where selectivity is low. |
Slop of gradient too steep. |
Use a more shallow gradient. A plateau in the gradient where your protein elutes may improve the resolution. |
Too much sample has been loaded onto the column. |
Decreasing the sample load may improve the resolution significantly. |
Unusual column appearance
Possible cause | Suggested remedy |
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The column operates at room temperature after having been stored in a cold room. |
Allow thermal equilibration before use. |
Poor product recovery
Possible cause | Suggested remedy |
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Contaminants accumulating in the column. |
1. Optimize the elution conditions |
Back pressure increases during operation
Possible cause | Suggested remedy |
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Auxiliary equipment such as manometers and pumps not working properly. |
Check the function of all auxiliary equipment. Repair/replace if necessary. |
Bent tubing. |
Check that the flow path is not restricted. |
Buffer viscosity too high. |
Check the viscosity of all buffers. Viscosity is a function of temperature. (Lower temperature gives higher viscosity.) Let low-temperature buffer reach operating temperature before starting the run. |
Microbial growth in buffers. |
Check buffers, especially those with phosphate, for microbial growth. Replace with fresh buffer if necessary. |
Sample and collection vessels at different levels. |
Adjust the vessels to approximately the same level. |
The prefilter might be blocked. |
Check the prefilter. |
Valve not fully open. |
Check all valves. Open any that is not fully open. |