FAQ
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Accessories
Running HiPrep, HisPrep and GSTPrep columns with ÄKTAexplorer, ÄKTAFPLC and ÄKTAprime plus, ÄKTApurifier and ÄKTAxpress.
Make sure you have all connectors and tubing needed for running the column.
Depending on your system configuration and column length you may need extra long tubing to connect your column.
Please do not extend the tubing unnecessarily which results in a lower resolution.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | PEEK Tubing, 2 m, i.d. 0.75 mm, o.d. 1/16" | 18111253 | 96.26 USD |
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|
2 | Fingertight connector 1/16" male, narrow | 28401081 | 108.65 USD |
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3 | Fingertight Connector 1/16" Male for Tubing o.d. 1/16" | 18111255 | 123.16 USD |
Add to cart
|
Running HiPrep, HisPrep and GSTPrep columns with stand alone pump, for instance using HiLoad pump P-50 for step elution.
The figure below shows a complete set up which facilitates bypassing the column, running cleaning-in-place etc.
Make sure you have all connectors and tubing needed for running the column.
Depending on your system configuration and column length, you may need extra long tubing to connect your column.
Please do not extend the tubing unnecessarily which results in a lower resolution.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Tubing cutter, for PEEK, EFTE, and FEP tubing i.d. 0.25, 0.5, 0.75, 1 and 1.6 mm | 18111246 | 99.55 USD |
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|
1 | Unflanged tubing i.d. 1.9 mm, o.d. 2.7 mm | 18820702 | 338.00 USD |
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|
2 | Tubing connectors o.d. 2.7 mm, M6 male | 18465201 | 129.93 USD |
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|
3 | Tubing, 2 m i.d. 0.8 mm, o.d. 1.8 mm, ETFE | 19743501 | 74.52 USD |
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|
5 | Pre-filter 6000 | 18458201 | 1,367.00 USD |
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5 | Filter kit | 18458401 | 156.83 USD |
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5 | Holder Valve PSV-50 | 19199255 | 153.64 USD |
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5 | Solenoid Valve PSV-50 | 19199401 | 819.00 USD |
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|
8 | Fingertight Union 1/16" Male/M6 Female | 18111258 | 135.19 USD |
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|
9 | UV Flow Cell, 2 mm | 18111110 | 2,857.00 USD |
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10 | Filter 215 nm | 11000733 | 808.00 USD |
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|
10 | Filter 260 nm | 11000734 | 781.00 USD |
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10 | Filter 280 nm | 11000735 | 628.00 USD |
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10 | Filter 405 nm | 11000736 | 760.00 USD |
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10 | Empty Filter Holder | 11000738 | 248.00 USD |
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|
10 | Monitor UVis-920 Excluding Filter and Flow Cell | 11000754 | 10,200.00 USD |
Add to cart
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10 | Short optical fibre kit, 200 mm | 18113485 | 970.00 USD |
Add to cart
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10 | Long optical fibre kit, 500 mm | 18113486 | 716.00 USD |
Add to cart
|
10 | UV flow cell, i.d. 8 mm | 28966687 | 3,723.00 USD |
Add to cart
|
10 | O-ring kit | 28969705 | 162.00 USD |
Add to cart
|
12 | Union M6 female - 1/16" male | 18385801 | 299.00 USD |
Add to cart
|
Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
Poor product recovery
Possible cause | Suggested remedy |
---|---|
Column is not clean enough. |
Clean the column according to instructions. |
Columns Column leakage
Possible cause | Suggested remedy |
---|---|
Tubing not compatible with solvents. |
Check chemical resistance with the tubing supplier. |
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. |
Back pressure increases 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 is 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. |
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 |
---|---|
Column is not clean enough. |
Clean the column according to instructions. |
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. |
Decreasing the sample load may improve the resolution significantly. |
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. |
Poor reproducibility
Possible cause | Suggested remedy |
---|---|
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 the Column and media 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. |
Possible cause | Suggested remedy |
---|---|
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 |
---|---|
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. |
Unusual column appearance
Possible cause | Suggested remedy |
---|---|
- |
Reverse the flow direction and pump 100 ml of well degassed binding buffer through the column at a flow rate of 5 ml/min at room temperature. |
The column operates at room temperature after having been stored in a cold room. |
Allow thermal equilibration before use. |
Possible cause | Suggested remedy |
---|---|
Buffer conditions deviate with regard to temperature, conductivity, viscosity, content of organic solvent (reduces surface tension) or other factor. |
Check the buffers and choose more suitable conditions. |