INdEX 200/500 Column
General-purpose glass columns developed for process development and small-scale production. The columns are simple to use and versatile in application.
FAQ
Surface finish of sealed wetted steel parts: Ra 0.8 µm.
Surface finish of other wetted steel parts: Ra 1.6 µm.
Cleaning-in-place
Cleaning-in-place (CIP) removes precipitated material, strongly bound substances and other contaminants from the column bed without dismantling the column.
Regular CIP of the packed column between production batches ensures proper product quality and maintains expected medium and equipment life. An efficient CIP protocol depends on medium in use and the characteristics of the contaminants. Refer to the instructions for each medium.
Sanitization
Sanitization is the reduction of microbial contamination in the column and related equipment to an acceptable minimum. A specific sanitization protocol should be designed for each process according to the type of contaminants present.
Note: Always ensure that columns, systems and auxiliary components are compatible with the chemicals used.
|
Column |
Inner diameter of tubing (mm) | Column | Inner diameter of tubing (mm) |
| INdEX 70 | 6 | INdEX 100 | 6 |
| INdEX 140 | 6 | INdEX 200 | 6 |
INdEX
Two standard exchangeable tube lengths are available for INdEX columns; 500 mm and 950 mm. Note that you must also replace the tube rods when changing the column tube.
|
Column
|
Min. bed height (cm)
|
Max. bed
height1,2 (cm) |
Min. bed volume (L)
|
Max. bed volume1,2 (L)
|
| INdEX 70/500 |
3
|
32 (41)
|
0.1
|
1.2 (1.6)
|
| INdEX 70/950 |
48
|
61 (79)
|
1.8
|
2.3 (3.0)
|
| INdEX 100/500 |
3
|
32 (41)
|
0.2
|
2.5 (3.2)
|
| INdEX 100/950 |
48
|
61 (79)
|
3.8
|
4.8 (6.2)
|
| INdEX 140/500 |
3
|
32 (41)
|
0.5
|
4.9 (6.3)
|
| INdEX 140/950 |
48
|
61 (79)
|
7.4
|
9.4 (12.2)
|
| INdEX 200/500 |
3
|
32 (41)
|
0.9
|
10.0 (12.9)
|
| INdEX 200/950 |
48
|
61 (79)
|
15.1
|
19.2 (24.8)
|
1Maximum values are based on a slurry concentration of 75% and a compression factor of 1.15. Compression factor is defined as gravity-settled bed volume/packed bed volume.
2 Figures in parentheses are achievable using a packing extension.
The relief pressure must be lower than or equal to the maximum operating pressure.
| Column | Max. operating pressure (bar) | Column | Max. operating pressure (bar) |
| INdEX 70 | 3 | INdEX 100 | 3 |
| INdEX 140 | 3 | INdEX 200 | 3 |
INdEX
In general, the wetted parts of plastics/elastomers have not been tested for biological reactivity.
Column evaluation
The efficiency of a column depends on how well it is packed. A poorly packed column gives rise to uneven flow, resulting in zone broadening and reduced resolution. It is thus important to have a method by which the column can be tested before it is put into operation. Such a method should be simple, quantitative and should not introduce contaminating materials. It is also an advantage if the same method can be used to monitor column performance over its working life, so that it is easy to determine when the medium should be re-packed or replaced.
Avoid methods that use colored compounds such as Blue Dextran. They do not meet the above criteria and cannot be used with ion exchange and affinity chromatography media.
Experience has shown that the best method of expressing the efficiency of a column is in terms of the height equivalent to a theoretical plate, HETP, reduced plate number, h, and the peak asymmetry factor, As. These values can be determined easily by applying a NaCl or acetone solution, to the column (see below).
It is important that the column is properly equilibrated ( >2 column volumes) before evaluating the packing. Ideally, run three test runs to see whether the values are stable. If an initially poor result improves during a later test, the reason can be that the column was not properly equilibrated. To check that the bed is stable, run the column at 70% packing pressure for 20 hours and test it again.
Note that pressure spikes may cause poor packing (cracking). If this happens, fit an air trap and a pressure relief valve between the pump and column. Locate the pressure relief valve between the air trap and the column.
Choice of test sample for columns
The most appropriate material for column testing is, of course, the sample that is to be run in the application, but this is not always practical or economical. As an alternative, a solution of either NaCl or acetone will give a good indication of the column packing quality. The eluate is monitored by measuring conductivity or UV absorption, and the resulting elution profile is used to calculate the HETP value.
The advantages of using NaCl are that it is readily available and can be used safely to test all columns. One disadvantage is that NaCl may interact with the medium matrix, especially ion exchanger matrices, and thus give erroneous results.
Acetone, in contrast, does not interact with the matrix and is detected by UV absorption at 280 nm. Alternatively, you can increase the running buffer concentration 10-fold and use it as test solution.
The figure below shows a UV trace for acetone in a typical BPG column application and gives calculated HETP and As values.
HETP calculation
The sample volume should be approximately 1% of the total bed volume and the concentration 1.0% v/v NaCl, or equivalent when using stronger buffer. Alternatively, use 1.0% v/v acetone. The flow velocity should be between 10 and 30 cm/h depending on the bead size of the chromatography medium. The high flow velocity could be used for small beads whereas large beads only allow low flow velocity. To avoid diluting the sample, apply it as close to the column inlet as possible. If an airtrap is included in the system, by-pass it during sample application to avoid back-mixing. Calculate the HETP value from the conductivity (or UV) curve as follows:
HETP, in its simplest terms, is expressed as:
HETP = L/N
where,
L = Bed height (cm)
N = Number of theoretical plates.
N is defined by the equation:
N = 5.54 (Ve /Wh)2
where,
Ve = Elution volume (ml)
Wh = Peak width at half height (ml)
Ve is measured as the volume passed through the column to the peak maximum.
Wh is measured as the peak width at half-peak height.
From the example in the figure, the HETP value can be calculated from the chromatogram as follows:
 |
Ve (ml) | Wh (ml) | N | N/m | HETP cm |
| Acetone | 18800 | 900 | 2417 | 4203 | 0.024 |
Well-packed columns have low HETP values. However, it is only possible to compare columns that have been packed with the same type of media and that have been tested under identical conditions.
As a general rule-of-thumb, a good HETP value is approximately two to four times the mean bead diameter of the medium in question, provided that the sample does not interact with the medium.
In practice, the correlation between HETP and column performance can only be assessed by the column operator. Once this has been established, a standard can be set to judge the acceptability of a column packing.
For example, the column operator may know from experience that a column packed with Sephadex G-25 gel filtration medium with HETP values above 0.05 cm does not give the required separation. Consequently, the operator will set this value as the maximum permissible i.e. the minimum acceptable quality.
Reduced plate number
Definition of reduced plate number: h = HETP/dp
h = reduced plate number
HETP = above described height equivalent to a theoretical plate
dp = mean particle diameter of the chromatography medium beads
The reduced plate number should be in the range of 2-4 times the mean particle diameter of the chromatography medium beads.
Peak asymmetry factor calculation
The peak asymmetry factor should be as close as possible to 1, and the shape of the peak should be as symmetrical as possible. This is usually the case for gel filtration media, but for certain ion exchange and affinity media, the shape may be asymmetrical due to interaction with the media. A change in peak shape is usually the first indication of column deterioration.
The peak asymmetry factor, As, is calculated from the graph above:
As = b/a
where,
a = distance from peak apex to 10% of the peak height on the ascending side of the peak
b = distance from peak apex to 10% of the peak height on the descending part of the peak
Note: Measuring HETP, h and As values is the best way to judge the condition of the packed column. A packed column can look good, but still need repacking for optimal performance. Always check the column after packing and regularly between runs to ensure best performance.
Sodium hydroxide is widely accepted for cleaning empty equipment, cleaning packed chromatography columns (cleaning-in-place , CIP), sanitizing and storing chromatography media and systems.
For CIP, sodium hydroxide has been extensively used to remove proteins and nucleic acids from ion exchange, hydrophobic interaction and gel filtration media. Its use with affinity media is restricted due to the limited stability of affinity ligands. If lipids are bound to a protein, an increased concentration of sodium hydroxide may be required for effective cleaning.
Sodium hydroxide is also effective for inactivating most viruses, bacteria, yeasts and endotoxins. It is common practice in industrial manufacturing to save time by adding salt, e.g. sodium chloride, to the sodium hydroxide solution to combine cleaning with sanitization as well. Since sodium hydroxide is a bacteriostat, it is also useful for storage.
The benefits of using sodium hydroxide include efficacy and low cost, plus ease of detection, removal and disposal.
Precautions
Always ensure that chromatography media, columns, systems and auxiliary components are compatible with sodium hydroxide at the concentration, time and temperatures used. Also keep in mind that sodium hydroxide may be corrosive to both metal and skin.
Compatibility with chromatography media
The concentration of sodium hydroxide used for CIP and/or sanitization will often depend on the level of contamination. For chromatography media, the ability to withstand stringent sanitizing conditions depends on the functional groups, attachment chemistries and the stability of the base matrices to alkaline conditions.
Table 4 in Application Note “Use of sodium hydroxide for cleaning and sanitizing chromatography media and systems” lists the general stability of a wide range of media as a function of pH. See more on Application notes: Use of sodium hydroxide for cleaning and sanitizing chromatography media and systems
INdEX
23 µm nets are supplied with the column and recommended for beads with an average particle diameter between 70-150 µm. For a diameter less than 70 µm, use 10 µm nets. All are in polymeric material.
Packed bed columns
Packing and running packed bed columns requires a pump that gives a pulse-free flow. Installing a pulse reducer may also be necessary. For high flow velocities and low pressures up to approximately 3 bar, we recommend lobrotor pumps. For higher pressures, use membrane pumps with three pump heads (pump heads reduce pulsations) or screw pumps, although both these have limited flow capacity
Expanded bed adsorption
Use peristaltic pumps for STREAMLINE and STREAMLINE Direct columns
Cracks are mainly due to pump pulsation or a too densely or too loosely packed bed.
Possible cause: Pump pulsation
Remedy: Repack the column with a pulse-free pump or add an air trap and a pressure relief valve to the assembly.
Possible cause: Too densely packed bed
Remedy: Repack the column with reduced packing flow rate or pressure.
Possible cause: Too loosely packed bed
Remedy: It may be possible to only adjust the adaptor. Otherwise repack the column with increased packing flow rate or pressure.
How do I correct a gap between the packed bed and lid/adaptor?
It may be possible to only adjust the adaptor. If not, you have to repack the column.
INdEX
All column parts can be cleaned with the most commonly used agents such as detergents, ethanol, weak acids, sodium hydroxide and high salt concentration. For special cleaning agents, please refer to chemical resistance information in the User Manual supplied with the column.
There are several ways of arranging flow paths to by-pass the column. One is described below.
BPG, INdEX, FineLINE Pilot 35 and FineLINE 70-350
Fit the columns with one 4-port, 4-way valve at the column inlet and two 4-port, 4-way valves at the column outlet.
Spare parts
O-rings and nets should be checked regularly for wear. Worn O-rings may not seal properly and over-used nets can affect distribution. Inspect the O-rings and evaluate the height equivalent to a theoretical plate (HETP) and peak asymmetry factor (As) to prevent poor performance due to old nets. We recommend you to change the nets and O-rings when needed or at least annually. Please keep a set of each on your site, for code numbers refer to the table below.
23 µm nets are supplied with the column and recommended for beads with an average particle diameter between > 70 µm. For an average particle diameter < 70 µm use 10µm nets.
| # | Product Name | Product Code | Price | |
|---|---|---|---|---|
| 1 | O-Ring End Piece 202.8X3.5 EPDM | 18848901 | 74.60 USD |
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|
| 2 | O-ring 7.3 x 2.4 mm | 18110390 | 19.17 USD |
Add to cart
|
| 3 | O-ring 184.1 x 8.4 mm | 18110405 | 107.82 USD |
Add to cart
|
| 4 | Support net, adaptor 200 column | 18025256 | 66.75 USD |
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|
| 5 | Support net, end-piece 200 column | 18025255 | 76.06 USD |
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|
| 6 | Net, 10 μm adaptor 200 column PA | 18025276 | 379.00 USD |
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|
| 7 | Net, 10 μm, end-piece 200 column PA | 18025277 | 365.00 USD |
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|
| 8 | Net, 23 μm adaptor 200 column PP | 18925301 | 387.00 USD |
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|
| 9 | Net, 23 μm, end-piece 200 column PP | 18925401 | 387.00 USD |
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|
| 10 | Sealing ring | 18110409 | 29.48 USD |
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|
IMPORTANT: The following spare parts should be ordered for columns with serial numbers lower than the following serial numbers.
100 032 50 (INdEX 100/500)
100 013 95 (INdEX 100/950)
200 027 50 (INdEX 200/500)
200 019 95 (INdEX 200/950)
| Pos. | Designation | INdEX 100 | INdEX 200 | Qty |
|---|---|---|---|---|
| 5 | End-piece | 18-1103-81 | 18-1104-01 | 1 |
| 7 | Adaptor | - | 18-1104-00 | 1 |
| 8 | TC-connector, bottom | - | - | |
| 9 | TC-connector | 18-1103-85 | 18-1104-06 | 2 |
| 18 | O-ring 5.1x1.6 EPDM | 18-1107-67 | - | 5 |
| 18 | O-ring 7.1x1.6 EPDM | - | 18-1107-72 |
| # | Product Name | Product Code | Price | |
|---|---|---|---|---|
| 1 | Lid, INdEX 200 column | 18110402 | 454.00 USD |
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|
| 2 | Upper flange | 18110403 | 538.00 USD |
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|
| 3 | Column tube 500 | 18110451 | 3,671.00 USD |
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|
| 3 | Column tube 950 | 18110452 | 6,869.00 USD |
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|
| 4 | Bottom flange | 18110404 | 803.00 USD |
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|
| 5 | End-piece | 18111359 | 546.00 USD |
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|
| 6 | Rod 500 | 18110407 | 101.13 USD |
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|
| 6 | Rod 950 | 18110408 | 150.76 USD |
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|
| 7 | Adaptor | 18111360 | 1,443.00 USD |
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|
| 8 | TC-connector, bottom | 18111361 | 309.00 USD |
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|
| 9 | TC-connector | 18111362 | 150.76 USD |
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|
| 10 | Support net, adaptor 200 column | 18025256 | 66.75 USD |
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|
| 11 | Net, 10 μm adaptor 200 column PA | 18025276 | 379.00 USD |
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|
| 11 | Net, 23 μm adaptor 200 column PP | 18925301 | 387.00 USD |
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|
| 12 | Support net, end-piece 200 column | 18025255 | 76.06 USD |
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|
| 13 | Net, 10 μm, end-piece 200 column PA | 18025277 | 365.00 USD |
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|
| 13 | Net, 23 μm, end-piece 200 column PP | 18925401 | 387.00 USD |
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|
| 14 | Sealing ring | 18110409 | 29.48 USD |
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|
| 15 | Valve 1/4 " | 18110393 | 343.00 USD |
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|
| 16 | O-ring 13.1 x 1.6 mm | 19353701 | 27.12 USD |
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|
| 17 | O-ring 7.3 x 2.4 mm | 18110390 | 19.17 USD |
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|
| 18 | O-ring 7.3 x 2.4 mm | 18110390 | 19.17 USD |
Add to cart
|
| 19 | O-ring 184.1 x 8.4 mm | 18110405 | 107.82 USD |
Add to cart
|
| 20 | O-Ring End Piece 202.8X3.5 EPDM | 18848901 | 74.60 USD |
Add to cart
|
| 21 | O-ring 199.3 x 5.7 mm | 18110710 | 40.03 USD |
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|
| 22 | Washer 8.4 x 25 x 2 mm | 18110398 | 38.60 USD |
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|
| 24 | Nut M8 | 18110542 | 31.25 USD |
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|
| 25 | Grip-nut | 18110399 | 88.25 USD |
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|
| 26 | Hose connector 1/4 " | 18110394 | 68.51 USD |
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|
| 27 | Adjustable Foot | 18112693 | 53.55 USD |
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|
| 28 | Stand for INdEX 140 and INdEX 200 Columns | 18110361 | 1,147.00 USD |
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|
| 31 | Screw | 18111358 | 56.99 USD |
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|
| 31 | Plug for the adaptor shaft | 18112083 | 33.09 USD |
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|
Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
General advice to achieve good performance
Before packing or running the column please make sure that
- the tubing, valves, gaskets etc. have the same inner diameter as the column in and outlets and tubing is not longer than needed.
- the bed supports have a porosity appropriate for your chromatography medium.
- the column has been correctly assembled, verified by passing a leakage test.
- the chromatography medium and equipment are equilibrated in the packing buffer and to ambient temperature of packing environment.
- that the pump is free from pulsations.
- no pressure peaks appear.
- that you have enough slurry volume, taken in account the compression factor of the medium.
- packing is performed at optimal flow velocity.
- the column meets pressure requirement for selected medium and packing procedure.
After completing the packing procedure please evaluate the efficiency of the column in terms of the height equivalent to a theoretical plate (HETP), reduced plate number (h) and the peak asymmetry factor (As).
Between batches it is recommended to perform cleaning-in-place (CIP) to remove precipitated material, strongly bound substances and other contaminants from the column bed without dismantling the column. Regular CIP is needed to ensure proper product quality and expected life time of medium and equipment.
Many issues that face chromatographers are common to all types of columns, while other issues are quite specific.
Select the appropriate equipment.
Worrying peak shape
| Possible cause | Suggested remedy |
|---|---|
Column poorly packed |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
Protein precipitated on the bed supports and/or in the bed. |
Clean and regenerate the column and chromatography medium according to instructions. |
| Possible cause | Suggested remedy |
|---|---|
Column over-loaded |
Decrease the amount of sample or increase the amount of chromatography medium. I.e. increase the margin of safety. We recommend a margin of safety of approximately 10 %. |
| Possible cause | Suggested remedy |
|---|---|
Column poorly packed |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
Protein precipitated on the bed supports and/or in the bed |
Clean and regenerate the column and chromatography medium according to instructions. |
| Possible cause | Suggested remedy |
|---|---|
Column poorly packed |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
For reversed phase chromatography - Organic solvents have denatured the protein. |
Use a higher flow velocity and/or an adapted gradient to shorten residence time on the column and thus minimize exposure. |
Gradient slope or isocratic step too shallow |
Increase steepness of gradient or isocratic step. |
Protein precipitated on the bed supports and/or in the bed |
Clean and regenerate the column and chromatography medium according to instructions |
| Possible cause | Suggested remedy |
|---|---|
Column poorly packed |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
Protein precipitated on the bed supports and/or in the bed |
Clean and regenerate the column and chromatography medium according to instructions |
| Possible cause | Suggested remedy |
|---|---|
Column poorly packed |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
Protein precipitated on the bed supports and/or in the bed |
Clean and regenerate the column and chromatography medium according to instructions. |
Strange peaks
| Possible cause | Suggested remedy |
|---|---|
Leakage of non-eluted protein due to poor cleaning-in-place |
Clean and regenerate the column and chromatography medium according to instructions. |
Unwashed buffer tanks |
Clean the tanks and prepare fresh solutions. |
| Possible cause | Suggested remedy |
|---|---|
Air inadvertently injected with the sample. |
Try to remove air bubbles by washing extensively with solution at high flow velocity. Run in upward flow direction. |
Column and/or solution subjected to sudden temperature changes |
Equilibrate the column and solutions to working temperature before use. |
Sample not completely dissolved in buffer |
Ensure that the sample is completely dissolved before applying it to the column. |
System effects, valve-switching, dead volumes, etc. cause uneven buffer velocity |
Minimize if possible. Accept the remaining effects. |
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. Preventive action: |
| Possible cause | Suggested remedy |
|---|---|
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 and test for leakage according to instructions.
|
O-rings and gaskets not compatible with the solvents |
Most standard O-rings and gaskets are made of EPDM (Ethylene propylene diene), which changes characteristics when exposed to some organic chemicals. For repetitive long-term use, use |
For AxiChrom 50, 70, 140 & 200 columns, BPG columns and STREAMLINE columns - Incorrect torque specifications used during assembly of glass columns |
Use the torque and torque wrench setting specified in the instructions. |
End-piece, O-rings and/or flange not properly positioned with respect to the tube |
Disassemble the column and check the position of the end-piece, O-rings and flange. Assemble the column and perform leakage tests according to instructions. |
Poor reproducibility
| Possible cause | Suggested remedy |
|---|---|
Column bleeding from previous run |
Check and adjust your cleaning procedure. Clean the equipment and chromatography medium according to instructions. |
Column clogged with denaturated proteins and/or lipids |
Clean and regenerate the column and chromatography medium according to instructions. |
For reversed phase and hydrophobic interaction chromatography - Changes in ambient temperature affect retention times. |
Keep the temperature constant. |
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 protein concentration can cause protein interaction, resulting in change of elution profile.) |
| Possible cause | Suggested remedy |
|---|---|
Altered stationary phase properties |
Confirm by running a known sample. If the change is persistent, replace the chromatography medium. |
| Possible cause | Suggested remedy |
|---|---|
Bound substances not removed during cleaning |
Clean the chromatography medium according to instructions. |
Ligand partially degraded |
Replace the chromatography medium. |
| Possible cause | Suggested remedy |
|---|---|
Insufficient column regeneration |
Prolong the regeneration. |
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. |
| 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 |
|---|---|
For 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. |
For reversed phase chromatography - Strongly retained contaminants accumulate on the column and saturate active sites on the stationary phase. |
Clean and regenerate the column and chromatography medium according to instructions. |
Back pressure increases during operation
| Possible cause | Suggested remedy |
|---|---|
Air trapped in the bed supports |
Get rid of the air by wetting and washing the bed supports with 20% ethanol. If necessary disassemble the column according to the instructions. |
Pre-filter blocked |
Check the pre-filter and remove the blockage. |
Protein precipitated on the column |
Clean and regenerate the column and chromatography medium according to instructions. |
Sample and collection vessels at different levels |
Adjust the vessels to approximately the same level. |
Valve flow-paths too narrow |
Check all valves. Replace if necessary with valves that have a correct (wider) inner diameter. |
Valve not fully open |
Check all valves. Open any that is not fully open. |
Auxiliary equipment such as manometers and pumps not working properly |
Check the function of all auxiliary equipment. Repair/replace if necessary. |
Bed packed too densely |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
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 bed supports blocked by protein or medium |
To remove protein: To remove chromatography medium: Preventive action: |
Equipment in general (valves, flow cells, gaskets, etc.) has internal diameters too narrow for the flow rates used. |
Ensure that the internal diameter of all equipment before and after the column is the same as the column inlet and outlet. |
For hydrophobic interaction chromatography - Protein precipitated on the column due to incorrect ionic strength. |
Check and adjust conductivity to the correct value. Clean and regenerate the column and chromatography medium according to instructions. |
Microbial growth in buffers |
Check buffers, especially those with phosphate, for microbial growth. Replace with fresh buffer if necessary. |
Unusual column appearance
| Possible cause | Suggested remedy |
|---|---|
Column and chromatography medium not correctly cleaned |
Clean the column and chromatography medium according to instructions. Replace the bed supports with new ones if necessary. |
Column not stored under bacteriostatic conditions |
Include a bacteriostatic agent in the storage solution, e.g. 20% ethanol or 10 mM NaOH. |
Connectors not correctly tightened |
Check and tighten if necessary. |
Top adaptor sealing loose and/or supernatant above the seal no longer contains 20% ethanol. |
Always ensure that the seal is tight and that the supernatant above the seal contains 20% ethanol. |
| Possible cause | Suggested remedy |
|---|---|
Bed support damaged or incorrectly assembled allowing chromatography medium particles to leave the column |
|
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: |
Poorly packed bed (not sufficiently compressed during packing) |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
| Possible cause | Suggested remedy |
|---|---|
Buffer volume of the air trap inadequate |
The column must generally be repacked. However, you can remove a small amount of air trapped between the column top bed support and the top adaptor head by pumping a solution with a temperature a few degrees centigrade higher than the chromatography medium through the column. Use upward flow. If the flow has been reversed, re-evaluate the packing prior to use. Evaluate the packing using recommended methods. If the results are poor, refer to the symptom `Poor packing evaluation´ Preventive action: Check that the size of the air trap corresponds to the flow velocity. Also check that the buffer volume in the air trap is adequate. |
Buffers and column at different temperatures |
The column must generally be repacked. However, you can remove a small amount of air trapped between the column top bed support and the top adaptor head by pumping a solution with a temperature a few degrees centigrade higher than the chromatography medium through the column. Use upward flow. If the flow has been reversed, re-evaluate the packing prior to use. Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ` Poor packing evaluation´ Preventive action: Equilibrate column and solutions to the working temperature before use. |
Connectors not correctly tightened or not compatible with each other |
The column must generally be repacked. However, you can remove a small amount of air trapped between the column top bed support and the top adaptor head by pumping a solution with a temperature a few degrees centigrade higher than the chromatography medium through the column. Use upward flow. If the flow has been reversed, re-evaluate the packing prior to use. Evaluate the packing using recommended methods. If the results are poor, refer to the symptom "Poor packing evaluation" Preventive action: Tighten connectors prior to use. Also check that they are compatible with each other. Please see the connector guides. |
The column operates at room temperature after having been stored in a cold room |
Allow thermal equilibration before use. |
Valves that should be closed are not shut tightly |
The column must generally be repacked. However, you can remove a small amount of air trapped between the column top bed support and the top adaptor head by pumping a solution with a temperature a few degrees centigrade higher than the chromatography medium through the column. Use upward flow. If the flow has been reversed, re-evaluate the packing prior to use. Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. Preventive action: |
Poor product recovery
| Possible cause | Suggested remedy |
|---|---|
Irreversible adsorption to matrix |
First try to modify the elution conditions. If this doesn’t help, clean and re-generate the column and chromatography medium according to instructions. |
| Possible cause | Suggested remedy |
|---|---|
Tertiary structure disrupted |
Shorten the residence time on the column. Use higher flow velocities to minimize exposure. |
| Possible cause | Suggested remedy |
|---|---|
Target molecules transferred from previous run due to poor cleaning between runs. |
Evaluate your cleaning procedure. |
Inhibiting component removed during purification |
Happens some times. Take into consideration and try to avoid. |
Different assay conditions used before and after chromatography |
Use the same conditions for all assays. |
| Possible cause | Suggested remedy |
|---|---|
A co-factor used to define recovery has been removed, thus causing lower activity |
Check the integrity of the enzyme or enzyme/co-factor complex. |
Column not equilibrated properly |
Check the pH and the conductivity of the effluent before applying the sample to make sure the column is properly equilibrated. Continue to equilibrate with start buffer if necessary. |
Column over-loaded |
Decrease the amount of sample or increase the amount of chromatography medium. I.e. increase the margin of safety. We recommend a margin of safety of approximately 10 %. |
For ion exchange chromatography - Ionic strength of the sample too high or pH incorrect |
Check and adjust the ionic strength and/or pH. Calibrate your conductivity and pH meters. |
Incorrect buffer pH |
Check and adjust the pH. Calibrate your pH meter. |
Oppositely-charged detergents or other additives adsorbed to the column |
Clean the column and chromatography medium according to instructions |
Protein precipitated on the column |
If the protein is initially solubilized with special additives, ensure that these are present during chromatography. Also check pH and ionic strength. |
Too low salt concentration in the elution buffer |
Check and adjust elution buffer composition. |
Drifting baseline
| Possible cause | Suggested remedy |
|---|---|
Unwashed buffer vessels were refilled with fresh solutions |
Prepare new solutions. Make sure that vessels/containers used to prepare and store solutions are clean. If you use plastic containers, ensure that additives do not dissolve in solution. |
Solution not in equilibrium with the chromatography medium |
Continue to flush the system until equilibrium is established. |
For ion exchange chromatography - Buffering capacity of buffer is too low. Dissociated ions bind to the charged groups of the chromatography medium. |
Make sure that the buffering capacity is sufficient. |
Column bleeding from previous run. |
Check and adjust your cleaning procedure. Clean the equipment and chromatography medium according to instructions |
| Possible cause | Suggested remedy |
|---|---|
At a certain salt concentration, the detergent forms micelles, resulting in a sharp increase in UV absorption. |
Work above the critical micelle concentration (CMC) or change the gradient so that the increase in UV absorption does not occur while the samples are eluting. |
Poor packing evaluation result
On this troubleshooting site, we refer to 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).
| Possible cause | Suggested remedy |
|---|---|
Cracks in the bed or a gap between the chromatography medium and inlet. The column may have been packed too loosely |
It may be possible to only adjust the adaptor. Otherwise, repack the column with increased packing flow rate or pressure. Preventive action: |
Blocked bed support creates zones of different flow velocities |
To remove protein: |
| Possible cause | Suggested remedy |
|---|---|
Cracks in the bed due to pump pulsation or a too densely packed bed. The cracks are mainly vertical. |
Repack the column with a pulse-free pump or add an air trap and a pressure relief valve. Preventive action: |
A crack due to a torn bed support resulting in a jet stream. |
Replace the bed support according to instructions |
| Possible cause | Suggested remedy |
|---|---|
Test molecule is repelled by the chromatography medium under the conditions used, e.g. salt for ion exchange chromatography, or chromatography medium in water as running buffer |
Use appropriate test molecule and conditions, e.g. 800 mM salt as sample in 400 mM salt buffer for ion exchange media, or use acetone. |
| Possible cause | Suggested remedy |
|---|---|
Test molecule interacts with the chromatography medium under the conditions used, e.g. acetone interacts with SOURCE media |
Use appropriate test molecule and conditions, e.g. salt for SOURCE media. |
| Possible cause | Suggested remedy |
|---|---|
Too large sample volume (gives a broad peak) |
Use a sample volume that is maximum 2% of the column volume. |
Too great a distance between the sample injection and column inlet, or between the column outlet and analyzing instrument |
Shorten these distances (minimizes dilution). |
Large liquid volume in tubing, valves, connectors, etc |
Minimize dilution by reducing the liquid volume. For example, if the inner diameters of the tubing, valves, connectors, etc. are wider than the column inlet and outlet, change them to the same diameter. |
Flow velocity too high or too low |
Flow velocity should be in the range 15-60 cm/h depending on particle size. The smaller the particle, the higher the velocity. |
| Possible cause | Suggested remedy |
|---|---|
Bed packed too densely |
Repack the column with decreased packing flow rate or pressure (prepare a pressure/flow curve and use the data to define the correct packing flow rate, i.e. approx. 70% of maximum flow (plateau)). |
| Possible cause | Suggested remedy |
|---|---|
Bed settled too hard during the first step |
Repack the column with decreased packing flow rate for the sedimentation step. |
| Possible cause | Suggested remedy |
|---|---|
Extra volume in the system, e.g. new tubing (longer, shorter or different diameter) affects peak position and HETP |
Ensure the equipment is the same or identical to your test routine or SOP. |
| Possible cause | Suggested remedy |
|---|---|
Column not properly equilibrated before packing evaluation |
Re-equilibrate the column. Evaluate the packing again, using recommended methods. |
| Possible cause | Suggested remedy |
|---|---|
Column not properly equilibrated before packing evaluation |
Re-equilibrate the column. Evaluate the packing again, using recommended methods. |
| Possible cause | Suggested remedy |
|---|---|
Bed packed too densely or too loosely |
Repack the column. Evaluate the packing afterwards, using recommended methods. For a leading peak and one-step packing: For a leading peak and two-step packing: For a tailing peak and one-step packing: For a tailing peak and two-step packing: Preventive action: |
| Possible cause | Suggested remedy |
|---|---|
Bed packed too densely. Cracks present at the beginning get worse |
Repack the column. Evaluate the packing again, using recommended methods. One-step packing: Two-step packing: Preventive action: |
| Possible cause | Suggested remedy |
|---|---|
Bed too loosely packed causing space between chromatography medium and inlet |
Repack the column. Evaluate the packing again, using recommended methods. One-step packing: Two-step packing: Preventive action: |
| Possible cause | Suggested remedy |
|---|---|
Bed not packed densely enough |
Repack the column with increased packing flow rate or pressure (prepare a pressure/flow curve and use the data to define the correct packing flow rate, i.e. approx. 70% of maximum flow (plateau)). After re-packing the column evaluate the packing. Preventive action: |
| Possible cause | Suggested remedy |
|---|---|
Bed settled too loosely during the first step |
Repack the column with increased packing flow rate for the sedimentation step. Preventive action: |
Unsatisfactory elution
| Possible cause | Suggested remedy |
|---|---|
Sample not properly equilibrated |
Equilibrate sample to correct operating conditions (pH, ionic strength, etc.) |
Column not properly equilibrated |
Re-equilibrate the column. |
| Possible cause | Suggested remedy |
|---|---|
Column poorly packed |
Evaluate the packing using recommended methods. If the results are poor, refer to the symptom ‘Poor packing evaluation’. |
Elution strength too low |
Check and adjust the elution strength. Also check gradient, gradient shape or isocratic step. |
| Possible cause | Suggested remedy |
|---|---|
Column is bleeding from the previous run |
Check and adjust your cleaning procedure. Clean the equipment and chromatography medium according to instructions |
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. |
| Possible cause | Suggested remedy |
|---|---|
Components in the sample displace the target molecule before elution starts |
Reduce the amount of sample or find more suitable binding conditions. |
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 |
|---|---|
Column overloaded |
Decrease the amount of sample or increase the amount of chromatography medium. I.e. increase the margin of safety. We recommend a margin of safety approximately 10 %. |
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. |
Microbial growth in the column |
Microbial growth rarely occurs in a column during use. However, always take steps to prevent infection of packed columns and solutions. Store the column according to instructions. |
Slope of gradient too steep |
Use a shallower gradient (larger gradient volume) |
| Possible cause | Suggested remedy |
|---|---|
Buffers have wrong pH, ionic strength or solvent concentration |
Check and adjust the buffer conditions. Calibrate your instruments. |
| Possible cause | Suggested remedy |
|---|---|
Column overloaded |
Decrease the amount of sample or increase the amount of chromatography medium. I.e. increase the margin of safety. We recommend a margin of safety of approximately 10 %. |
For ion exchange chromatography - Ionic strength of the sample too high or pH incorrect |
Check and adjust the ionic strength and/or pH. Calibrate your conductivity and pH meters. |
For ion exchange chromatography - Reduced net charge due to complex between the component in the sample and the target molecule. |
Check the composition of the sample. Remove any interfering compounds such as nucleic acids. |
Oppositely –charged detergents or other additives adsorbed on the column. |
Clean the column and chromatography medium according to instructions. |
pH of the sample incorrect |
Check and adjust the pH. Calibrate your pH meter. |
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. |