HiLoad 26/600 Superdex 75 pg
HiLoad Superdex 75 pg prepacked columns are for high-resolution size exclusion chromatography of recombinant proteins from samples up to 5 mL or 13 mL.
FAQ
All chromatography columns have two types of pressure limits, one limit for the column hardware and one limit for the gel packing.
The pressure affecting the column hardware depends on the backpressure generated after the column by, for example flow cells, flow restrictors and tubing. When the pressure limit for the column hardware is exceeded, the column will start leaking.
Empty chromatography columns from Amersham Biosciences are all supplied with data on the column hardware pressure limit.
The pressure affecting the gel packing is dependent on the flow rate and viscosity of the buffer. When the flow rate is too high and/or a high viscosity buffer is used, the pressure limit for the packed gel bed can be exceeded (the gel pressure limit is the maximum allowable pressure drop over the packed gel bed). When the pressure limit is exceeded, the gel particles are forced to the bottom of the column and cause the backpressure to increase. This leads to a collapse of the packed bed and the risk of poor chromatographic performance.
The pressure limit data referring to the gel packing is found in the column instructions delivered with all prepacked columns from Amersham Biosciences.
What does a flow restrictor do?
A flow restrictor creates a steady backpressure to prevent air bubbles being formed after the column when the pressure created in the column is released. A flow restrictor can be compared to a cork on a bottle of champagne (Fig 1).
The flow restrictor only affects the column hardware pressure limit. The gel packing pressure limit is always unaffected by a flow restrictor. Figure 2 shows an example of how a flow restrictor affects the packed column at a hypothetical flow rate of 1 ml/min. The flow rate generates a pump pressure reading of 0.3 MPa. With no restrictor mounted, this pressure equals the pressure drop over both the gel and the column hardware.
When a flow restrictor generating a backpressure of 0.2 MPa is mounted after the column and the same flow rate is used, the pressure over the column hardware is affected and will be 0.5 MPa. Hence, the pump pressure gauge reading will be 0.5 MPa. However, the pressure drop over the packed gel bed is still 0.3 MPa.
When to use a flow restrictor
It is recommended to use a flow restrictor together with highperformance columns such as prepacked Tricorn™, HR, RESOURCE™, PEEK and steel columns. Flow restrictors are also recommended when using buffer systems containing organic solvents in combination with aqueous buffers.
When to remove
A general rule is that when aqueous buffers are used and backpressure is low (< 0.5–1 MPa), then there is no need to use a flow restrictor. The flow restrictor is not normally used with low-pressure columns such as prepacked HiLoad™, HiTrap™, and HiPrep™ columns or C and XK columns packed with Sepharose™ based media or other soft media such as Sephadex™ and Sephacryl™.
What is the pressure limit of my column?
Prepacked Columns
Please read
Prepacked chromatography columns for ÄKTAdesign systems
Other Columns
| Column |
Maximum pressure (bar)
|
| C 10, C 16 and C 26 |
1
|
| FineLINE Pilot 35 |
20
|
| HiScale |
20
|
| HR (High Resolution) 16 |
30
|
| SR 25 |
1
|
| Tricorn 5 |
100
|
| Tricorn 10 |
50
|
| XK 16 and XK 26 |
5
|
| XK 50 |
3
|
What chemicals are compatible with my column?
Please see the chemical stability in the instructions for prepacked columns, empty columns and media.
The backpressure increases during operation
| 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. |
| Sample and collection vessels at different levels. | Adjust the vessels to approximately the same level. |
| 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 has a gap between the packed bed and adaptor. Can I reuse the column?
| Possible causes | Suggested Remedy |
| Bed support damaged or incorrectly assembled allowing chromatography medium particles to leave the column. | Check the bed support and replace if necessary. Disassemble the column according to instructions. |
| 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. |
| Increased resistance to flow due to blocked bed support compressing the packed bed. | Clean or change the bed support. Disassemble the column and replace the support according to instructions. Preventive action: Pre-filter or centrifuge sample to avoid residues building up. |
| Poorly packed bed (not sufficiently compressed during packing). (Not applicable for prepacked columns) |
Evaluate the packing using recommended methods. 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. If the results are poor, refer to the symptom Poor packing evaluation in the troubleshooting section. |
My column contains air, what can I do?
| Possible causes | Suggested Remedy |
| Unclarified lysates may cause increased air bubble formation during purification. | An attached flow restrictor in the chromatography system can prevent this. If a flow restrictor is attached, it is important to change the pressure limit to 0.5 MPa (5 bar9 on the ÄKTAdesign system (where the column and the flow restrictor give a pressure of 0.3 MPa and 0.2 MPa respectively). |
| 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.
How would I get the best resolution of my column?
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.
How should I prepare my sample prior to loading the column?
Please prepare the sample according to instructions and handbooks
Prepacked column instructions
Please refer to relevant Instruction.
Other columns
Please refer to relevant Handbook.
How much sample, in mg or ml of protein can I load onto my column?
Affinity Chromatography
- The binding capacity values listed in the selection guide below, are typical for the given species. However, there might be considerable deviations in binding capacity for different immunoglobulins derivated from same species, even if they are of the same subclass.
- Protein binding capacity is protein-to-protein dependent. The given capacities are to be considered as starting values.
- For optimal separation use approximately one fifth of the total binding capacity
For more information refer to:
Affinity chromatography columns and media selection guide
Glutathione Sepharose selection guide
Ni Sepharose and IMAC Sepharose selection guide
Desalting
- In group separation (desalting) the sample volumes can be up to 30% of the column volume.
- Regarding sample volumes for prepacked columns, please refer to Sample preparation for analysis of proteins, peptides and carbohydrates selection guide.
Gel filtration chromatography
- Choose a sample volume of < 0.5% of the column volume for media with average particle size in the range of 10-15 µm and < 5% of the volume for media with average particle size in the range of 30-100 µm.
- In group separation (desalting) the sample volumes can be up to 30% of the column volume.
- Regarding sample volumes for prepacked columns, please refer to selection guide below:
Sample preparation for analysis of proteins, peptides and carbohydrates selection guide.
Gel filtration columns and media selection guide and product profile selection guide.
Hydrophobic interaction chromatography
- The binding capacity values in the media and column instructions, there are examples and to be considered as starting values. Protein binding capacity is protein-to-protein dependent.
- For optimal separation use approximately one fifth of the total binding capacity.
Ion exchange chromatography
- The binding capacity values in the Ion exchange columns and media product profile, please refer to Ion exchange columns and media product profile selection guide, there are examples and to be considered as starting values. Protein binding capacity is protein-to-protein dependent.
- For optimal separation use approximately one fifth of the total binding capacity.
Reversed phase chromatography
- The binding capacity values in the media and column instructions, please refer to the related document, there are examples and to be considered as starting values. Protein binding capacity is protein-to-protein dependent.
- For optimal separation use approximately one fifth of the total binding capacity.
How do I store my column?
Prepacked columns
Please store the column according to instructions.
Other columns
Please follow the recommendations in the media instructions.
My column is clogged and/or discoloured. How can I clean my packed column?
Prepacked columns
Please clean the column according to instructions.
Other columns
Please follow the recommendations in the media instructions.
If the issue persists after cleaning you have to replace the column with a new one (prepacked columns) or repack the column with fresh medium.
How can I check the functionality of my column?
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.
Can I run two columns in series to increase resolution or capacity?
Gel filtration chromatography
Running columns in series increases the resolution
Affinity chromatography, Hydrophobic interaction, Ion exchange chromatography
Reverse phase chromatography
Running columns in series increases the capacity but may have a bad impact on the resolution
due to increased dead volume.
Please note that back pressure may increase.
All chromatography columns have two types of pressure limits, one limit for the column hardware and one limit for the gel packing.
The pressure affecting the column hardware depends on the backpressure generated after the column by, for example flow cells, flow restrictors and tubing. When the pressure limit for the column hardware is exceeded, the column will start leaking.
Empty chromatography columns from Amersham Biosciences are all supplied with data on the column hardware pressure limit.
The pressure affecting the gel packing is dependent on the flow rate and viscosity of the buffer. When the flow rate is too high and/or a high viscosity buffer is used, the pressure limit for the packed gel bed can be exceeded (the gel pressure limit is the maximum allowable pressure drop over the packed gel bed). When the pressure limit is exceeded, the gel particles are forced to the bottom of the column and cause the backpressure to increase. This leads to a collapse of the packed bed and the risk of poor chromatographic performance.
The pressure limit data referring to the gel packing is found in the column instructions delivered with all prepacked columns from Amersham Biosciences.
What does a flow restrictor do?
A flow restrictor creates a steady backpressure to prevent air bubbles being formed after the column when the pressure created in the column is released. A flow restrictor can be compared to a cork on a bottle of champagne (Fig 1).
The flow restrictor only affects the column hardware pressure limit. The gel packing pressure limit is always unaffected by a flow restrictor. Figure 2 shows an example of how a flow restrictor affects the packed column at a hypothetical flow rate of 1 ml/min. The flow rate generates a pump pressure reading of 0.3 MPa. With no restrictor mounted, this pressure equals the pressure drop over both the gel and the column hardware.
When a flow restrictor generating a backpressure of 0.2 MPa is mounted after the column and the same flow rate is used, the pressure over the column hardware is affected and will be 0.5 MPa. Hence, the pump pressure gauge reading will be 0.5 MPa. However, the pressure drop over the packed gel bed is still 0.3 MPa.
When to use a flow restrictor
It is recommended to use a flow restrictor together with highperformance columns such as prepacked Tricorn™, HR, RESOURCE™, PEEK and steel columns. Flow restrictors are also recommended when using buffer systems containing organic solvents in combination with aqueous buffers.
When to remove
A general rule is that when aqueous buffers are used and backpressure is low (< 0.5–1 MPa), then there is no need to use a flow restrictor. The flow restrictor is not normally used with low-pressure columns such as prepacked HiLoad™, HiTrap™, and HiPrep™ columns or C and XK columns packed with Sepharose™ based media or other soft media such as Sephadex™ and Sephacryl™.
Introduction to Affinity Coupling
Activated media enable successful, convenient immobilization of ligands without the need for complex chemical syntheses or special equipment. We have developed a wide range of high-capacity media with a variety of coupling chemistries for fast, easy, and safe immobilization through a chosen functional group.
The correct choice of an activated medium is dictated by both the group available in the ligand molecule, and by the nature of the binding reaction with the substance to be purified. To ensure minimal interference with the normal binding reaction, immobilization should be attempted through the least critical region of the ligand.
Our activated media allow design of reliable affinity adsorbents to match individual applications.
High selectivity and high capacity make the technique ideally suited to the isolation of specific components of complex biological mixtures. Extremely high purity can be achieved in a single step.
Because it is the most successful medium for immobilizing biologically active molecules, Sepharose™ (beaded agarose) forms the matrix for most of the affinity media. The hydroxyl groups on the agarose sugar residues can be easily derivatized for covalent attachment of ligands. Sepharose™ exhibits low nonspecific adsorption and its beaded structure provides excellent flow properties.
| Activated gel | Group to be coupled |
| Activated CH Sepharose™ 4B | -NH2 |
| Activated Thiol Sepharose™ 4B | -SH |
| CNBr-activated Sepharose™ 4B | -NH2 |
| NHS-activated Sepharose™ 4 Fast Flow | -NH2 |
| CNBr-activated Sepharose™ 4 Fast Flow | -NH2 |
| EAH Sepharose™ 4B | COOH |
| ECH Sepharose™ 4B | -NH2 |
| Epoxy-activated Sepharose™ 6B | -NH2,-OH or -SH |
| HiTrap™ NHS-activated HP Columns | -NH2 |
| Thiopropyl Sepharose™ 6B | -SH |
My column is leaking buffer, what shall I do?
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. |
Leakage around end-pieces ( Not applicable for the pre-packed columns )
| Possible causes | Suggested Remedy |
| End-piece and O-rings not properly positioned with respect to the tube. | Disassemble the column and check the position of the end-piece and O-rings. Assemble the column according to instructions and perform leakage tests. |
| O-rings worn out | O-rings loose their flexibility with time and need to be replaced regularly. Disassemble the column and inspect the O-rings. Replace if necessary and at least annually. Assemble the column according to instructions and test for leakage. Preventive action: Replace O-rings when needed or at least annually. |
My column has run dry. Can I reuse the column?
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 (prepacked columns) or repacked.
Spare parts
| # | Product Name | Product Code | Price | |
|---|---|---|---|---|
| 1 | Net for Columns, i.d 26 mm, 10 µm | 18876001 | 76.88 USD |
Add to cart
|
| 1 | Net for Columns, i.d 16 mm, 10 µm | 18876101 | 76.88 USD |
Add to cart
|
| 2 | Support Screen for Columns, i.d. 26 mm | 18937701 | 76.88 USD |
Add to cart
|
| 2 | Support Screen for Column, i.d. 16 mm | 19065101 | 52.28 USD |
Add to cart
|
| 3 | O-Ring, 10.3 × 2.4 mm | 19016301 | 60.47 USD |
Add to cart
|
| 3 | O-Ring 20.3x 2.62 mm | 28978227 | 83.02 USD |
Add to cart
|
| 11 | Fingertight connector 1/16" male, narrow | 28401081 | 108.65 USD |
Add to cart
|
| 12 | Stop Plug Female, 1/16" | 11000464 | 103.52 USD |
Add to cart
|
Accessories
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 | ETFE Tubing, 1.5 m, i.d. 0.75 mm, o.d. 1/16" | 18111974 | 80.73 USD |
Add to cart
|
| 2 | Fingertight connector 1/16" male, narrow | 28401081 | 108.65 USD |
Add to cart
|
| 3 | Union 1/16" female-1/16" female | 11000339 | 170.07 USD |
Add to cart
|
| 4 | Stop Plug Female, 1/16" | 11000464 | 103.52 USD |
Add to cart
|
Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
Poor reproducibility
| Possible cause | Suggested remedy |
|---|---|
Column not properly equilibrated |
Check the pH and the conductivity of th effluent before applying the sample. Continue to equilibrate with start buffer if necessary |
Insufficient column regeneration. |
Prolong the regeneration. |
| 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 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. |
Sample volume is different from earlier runs. |
Resolution is dependent on the sample volume. Keep sample volume constant when repeating runs. |
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 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. |
Column is clogged. |
Clean the column according to 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. |
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. |
Unusual column appearance
| Possible cause | Suggested remedy |
|---|---|
- |
Close the outlet tubing with the domed nut and then disconnect the inlet tubing. Slacken the O-ring slightly by turning the adjusting knob counter-clockwise and push or screw the adaptor down until it touches the medium surface. Tighten the O-ring. To maintain an airtight system, reconnect the inlet tubing immediately. |
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. |
| 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. |
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 |
|---|---|
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 |
|---|---|
Column is not clean enough. |
Clean the column according to instructions. |
Column is overloaded. |
Decrease the amount of sample i.e. increase the margin of safety. |
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. |