FAQ
Replace the column.
See Product Data or Instruction for the product.
Possible causes | Suggested Remedy |
The column operates at room temperature after having been stored in a cold room. | Allow thermal equilibration before use. |
- | Reverse the flow direction and pump well degassed water through the column. For recommended volumes and flow rates for your specific column, please refer to media and column instructions. |
More causes and remedies might be presented in the troubleshooting section.
For stand alone columns please assemble the monitor to the column outlet.
If the column is connected to a system, connect the column as close as possible to the monitor.
Gel filtration chromatography
Running columns in series increases the resolution
Please note that back pressure may increase.
See Product Data or Instruction for the product.
Please see the chemical stability in the instructions for prepacked columns.
Leading and tailing peaks as defined according to the following figures:
Leading peak | Tailing peak |
Experience shows that the best method of expressing the efficiency of a packed column is in terms of its height equivalent to a theoretical plate (HETP), reduced plate number (h) and its peak asymmetry factor (As).
Please be aware of that for small columns, less than 10 ml bed volume, the system dead volume has an impact on the column evaluation values.
See Product Data or Instruction for the product.
See Product Data or Instruction for the product.
Leakage around connectors
Possible causes | Suggested Remedy |
Connectors not compatible with each other. | Check compatibility. |
Connectors not compatible with solvents. | Check chemical resistance with the connector supplier. |
Connectors poorly positioned or not tightened. | Check the connectors. |
Gaskets worn out. | Gaskets lose flexibility with time and need to be replaced regularly. Inspect and replace if necessary and at least annually. |
Leaking tubing
Possible causes | Suggested Remedy |
Tubing not compatible with solvents. |
Check chemical resistance with the tubing supplier. Preventive action: Always check tubing solvent compatibility prior to packing or running the column. |
Possible causes | Suggested Remedy |
Auxiliary equipment such as manometers and pumps not working properly. | Check the function of all auxiliary equipment. Repair/replace if necessary. |
Column is clogged. | Clean the column according to instructions. Choose the more rigorous cleaning protocol when available. See media and column instructions. |
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. The buffer normally become opalescent due to microbial growth. |
Check buffers, especially those with phosphate, for microbial growth. Replace with fresh buffer if necessary. |
The prefilter might be blocked. | Check the prefilter. Preventive action: Prefilters are not meant to substitute sample treatment. |
Valve not fully open. | Check all valves. Open any that is not fully open. |
Find more causes and remedies in the troubleshooting section.
My column is clogged and/or discoloured. How can I clean my packed column?
Please clean the column according to instructions.
If the issue persists after cleaning you have to replace the column with a new one.
If the column still is free from bacterial growth it might be possible to reuse the column. It could be worth trying.
Remove the air by running liquid slowly from bottom to top of a vertical placed column at a pressure close to maximum column pressure. The size of air bubbles decreases with increased pressure and facilitate the bubbles escape from the column.
If you don't succeed in removing air from the column, the column must be replaced.
Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
Unusual column appearance
Possible cause | Suggested remedy |
---|---|
The column operates at room temperature after having been stored in a cold room. |
Allow thermal equilibration before use. |
Column leakage
Possible cause | Suggested remedy |
---|---|
Connectors not compatible with each other. |
Check compatibility. |
Connectors not compatible with solvents. |
Check chemical resistance with the connector supplier. |
Connectors poorly positioned or not tightened. |
Check the connectors. |
Gaskets worn out. |
Gaskets lose flexibility with time and need to be replaced regularly. Inspect and replace if necessary and at least annually. |
Possible cause | Suggested remedy |
---|---|
Tubing not compatible with solvents. |
Check chemical resistance with the tubing supplier. |
General advice to achieve good performance
Before using the column make sure that:
- Correct system has been selected in UNICORN System Control
- Correct wavelength has been set for UV/UPC monitor
- All tubing has been properly connected and tubing is not longer than needed
- All connectors are free from leakage, verified by passing a leakage test
- No tubing is folded or twisted
- Online filter, if used, is changed on a regular basis
- Correct buffers are used for the chosen columns and proteins
- All inlet tubing has been immersed in correct buffer solutions
- Enough buffer has been prepared
- Buffers have been equilibrated to the environment temperature
- Buffers/eluents have been degassed if necessary (e.g., in RPC runs)
- Suitable columns have been selected for the target proteins
- Column meets pressure requirements for selected medium
- Columns have been cleaned and prepared according to column instructions
- Samples have been clarified by centrifugation and/or filtration prior to sample loading
- Samples have been adjusted to binding buffer conditions
- Auto sampler (if used) has been prepared according to user manual
- The fraction collector has been filled with appropriate number of microtiter plates or tubes
- Appropriate arrangement for waste handling has been prepared
Unsatisfactory elution
Possible cause | Suggested remedy |
---|---|
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. |
Too much sample has been loaded onto the column. |
Decrease the amount of sample. |
Possible cause | Suggested remedy |
---|---|
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. This effect is especially critical in gel filtration. |
Reduce the concentration of detergent to below it´s critical micelle concentration (CMC ) value. |
Back pressure increase during operation
Possible cause | Suggested remedy |
---|---|
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. |
Column might be clogged. |
Clean the column according to instructions. |
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. |