C 16/40 Column

Activity of protein is not compatible with solution A and has been irreversibly denatured.

Choose another solution system after testing the salt and pH stability of the protein.

Conditions that are sufficiently effective to elute molecules along the column may irreversibly denature both desorbed substances and the ligand.

Elution should always be carried out in the reverse direction to sample application. Reverse the flow direction through the column by installing it upside down during elution.

Hydrophobic interaction chromatography - Protein is probably denatured by high salt and cannot refold properly upon elution with low salt solution to give active protein.

Try the protein at different salt concentrations and see when activity is lost. Then enter the salt stability limit in Case Data and start a New Case. If this is not successful choose another technique.

Protein probably can not withstand the low pH of the elution solution.

Adjust eluted fractions immediately to a higher pH. If this does not help choose another technique.

Protein is sensitive to the speed of neutralization and is partially denatured.

Use a weaker neutralizing solution of lower pH.

Unstable protein at the pH used for chromatography.

Determine the pH stability profile and start a New Case, or choose another technique.

Lipoprotein may be unstable under the conditions used.

Do not use chaotropic salts if the native structure is to be preserved. Ordinary salts like sodium chloride, sodium sulfate, etc., should be employed.

At sufficiently high ionic strength some proteins may be precipitated from solution. One factor is that the high concentration of salt may remove water of hydration from proteins.

Your protein may be applied at a lower ionic strength than that at which the column is equilibrated provided that precipitation is reversible and that the sample volume is kept to a minimum, 5% to 20% V_t..

Unstable protein at the chosen ionic strength and/or pH resulting in inactivated.

Try changing the ionic strength and/or pH up or down.

Different assay conditions used before and after chromatography.

Use the same conditions for all assays.

Inhibiting component removed during purification.

Happens some times. Take into consideration and try to avoid.

Target molecules transferred from previous run due to poor cleaning between runs.

Evaluate your cleaning procedure

Hydrophobicity of your protein is higher than your current estimate and the lower solubility of hydrophobic proteins can cause decreased recovery.

Use chaotropic salts for elution to increase the recovery of hydrophobic proteins (their lower salting out effect increases sample solubility). However, they might break down protein structure.

Hydrophobicity of your protein is higher than your current estimate and binds more strongly to the chromatography medium than you have expected.

Remedy 1. Add detergents to solution B. Detergents work as displacers of the proteins.

Remedy 2. Change the polarity of solution B. A decrease in the interaction is achieved by adding mild organic solvents such as ethylene glycol (up to 75%) or isopropanol at lower concentration.

Remedy 3. Use chaotropic salts for elution to increase the recovery of hydrophobic proteins (their lower salting out effect increases sample solubility). However, they may break down protein structure.

Ionic strength of solution B may have caused the protein to precipitate onto the column. Some proteins tend to precipitate at high salt concentration due to increased hydrophobic interactions.

Use chaotropic salts in the eluting solution.
However, they may break down protein structure.

Lower solubility of hydrophobic proteins can cause decreased recovery.

Remedy 1. Add detergents to solution B. Detergents work as displacers of the proteins.

Remedy 2. Change the polarity of solution B. A decrease in the interaction is achieved by adding mild organic solvents such as ethylene glycol (up to 75%) or isopropanol at lower concentration.

Remedy 3. Use chaotropic salts for elution to increase the recovery of hydrophobic proteins (their lower salting out effect increases sample solubility). However, they may break down protein structure.

Precipitation of protein on the column may have occurred. Some proteins tend to precipitate at high salt concentration due to increased hydrophobic interactions.

Use chaotropic salts in the eluting solution. However, they may break down protein structure.

Smaller proteins and polypeptides sometimes show lower solubility in salt.

Chaotropic salts may be chosen for elution to give higher recoveries of smaller proteins. These salts allow maximum sample solubility.

Solution A may have caused the protein to precipitate onto the column.

Change the running conditions to be compatible with your sample (e.g. salt concentration, pH).

Unstable protein at salt concentration used for elution.

Change the pH of solution B away from your protein’s pI keeping the salt concentration constant. You are increasing the polarity of the protein which decreases the protein’s interaction with the chromatography medium. If this is not successful choose another technique.

Protein is probably sensitive to low pH.

Neutralize the eluted fractions. Let the protein elute into tubes containing high pH buffer
(e.g. 1 M Tris HCl, pH 7.0 to 8.0) to neutralize the eluted fractions.

Protein is too sensitive to the salt concentration used for elution and can not be recovered by increasing the ionic strength of the eluted fractions.

Choose another technique.

Binding between the protein and the medium is stronger than expected.

Try using urea in solution B.

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.

Precipitation of protein on the column may have occur due to the high ionic strength of solution B.

Try to elute the protein with an increasing pH keeping the salt concentration constant. Determine the salt stability profile and start a New Case, or choose another technique.

Precipitation of protein on the column may have occur due to the low ionic strength or dilution.

The decreased solubility is caused by electrostatic interactions that result in formation of aggregates. To minimize these interactions, use higher concentration of salt in solution A and sample solution.

Precipitation of protein on the column and not dissolved by decreasing the ionic strength.

Combine the decreasing of ionic strength with an increasing ethylene glycol gradient. If this is not successful choose another technique.

Protein precipiated 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.
If the protein is not solubilized by decreasing ionic strength. Combine the decreasing of ionic strength with an increased ethylene glycol gradient. If this is not successful choose another technique.

Protein may exhibit some hydrophobic interactions with the matrix.

If possible decrease the ionic strength, increase pH or introduce an organic (e.g. 24% propanol or 10% ethylene glycol) in solution A in order to minimize hydrophobic interactions.

Too low salt concentration in the elution buffer.

Check and adjust elution buffer composition.

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.

Ionic interactions between the protein and the matrix may cause this behavior.

These effects can be eliminated by the use of solutions with an ionic strength exceeding 0.15 to 0.3 M.

Low ionic strength of start or eluting buffer may have caused precipitation of the protein on the column.

The decreased solubility is caused by electrostatic interactions that result in formation of aggregates. To minimize these interactions, use 10 to 50 mM of salt in start of eluting buffer and sample solution.

Oppositely-charged detergents or other additivies adsorbed to the column.

Clean the column and chromatography medium according to instructions

Precipitation of protein on the column may have occurred.

Change pH and/or add urea.

Develop a procedure with detergents.
If the protein is initially solubilized with special additives, these must be present during chromatography.

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.

Tertiary structure disrupted

Shorten the residence time on the column. Use higher flow velocities to minimize exposure.

Column poorly packed.

Evaluate the packing.
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.

Protein precipitated on the bed supports and/or in the bed.

The column may become discoloured by the precipitated protein.

Clean and regenerate the column and chromatography medium according to instructions

Column over-loaded

Decrease the amount of sample or increase the amount of chromatography medium. I.e. increase the margin of safety.

Column poorly packed.

Evaluate the packing. 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.

Protein precipitated on the bed supports and/or in the bed.

Clean and regenerate the column and chromatography medium according to instructions

Column poorly packed.

Evaluate the packing. 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.

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

Column poorly packed.

Evaluate the packing. 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.

Protein precipitated on the bed supports and/or in the bed.

Clean and regenerate the column and chromatography medium according to instructions

Column poorly packed.

Evaluate the packing. 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.

Protein precipitated on the bed supports and/or in the bed.

Clean and regenerate the column and chromatography medium according to instructions

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.

Column bleeding from previous run.

Check and adjust your cleaning procedure. Clean the equipment and chromatography medium according to instructions.

Contaminated salts and solutions.

Remedy 1
If the component of interest is not co-eluting with the contaminant, ignore it.

Remedy 2
Use pure water Milli-Q or similar quality. Double distilled water may not be pure enough. Make sure acids/bases used to titrate buffers are not contaminated.

Remedy 3
Use a purer or newer reagent to make up the buffer. Some reagents decompose with age (e.g. iminodiacetic acid).

Difference in UV-absorbance between solutions A and B. The difference in ionic strength produces a different ratio between the acid and base form of the buffer. The slope of the baseline is very sensitive to small pH differences.

Gradient baseline drift can be adjusted by small pH changes in solution B.

For ion exchange chromatography - Buffering capacity of buffer is too low. Dissociated ions bind to the charged groups of the chromtography medium.

Make sure that the buffering capacity is sufficient.

Solution not in equilibrium with column.

Continue to flush the system until equilibrium is established.

Unwashed buffer vessels were refilled with fresh solutions.

Prepare new buffers. Make sure the glassware that is used to prepare and store buffers is clean. If plastic bottles are used check that plastic additives are not dissolved in the solution.

Bound substances not removed during cleaning.

Clean the chromatography medium according to instructions.

Ligand partially degraded.

Replace the chromatography medium.

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 the Column and media instructions.

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.

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.

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.

Column bleeding from previous run.

Check and adjust your cleaning procedure.
Clean the equipment and chromatography medium according to the Column and media instructions.

Column clogged with denatured proteins and/or lipids.

The column may be discolored by 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.)

Reversed phase and hydrophobic interaction chromatography - Changes in ambient temperature affect retention times.

Keep the temperature constant.

Sample volume is different from earlier runs.

Resolution is dependent on the sample volume. Keep sample volume constant when repeating runs.

Protein properties change with concentration.

Altered stationary phase properties

Confirm by running a known sample. If the change is persistant, replace the chromatography medium.

Blocked bed support creates zones of different flow velocities.

To remove protein: 
Clean-in-place according to media instructions. You may need to extend the time required.

To remove chromatography medium:
Disassemble the column and remove the medium using compressed air, or replace the bed support, according to the column instructions

Cracks in the bed or a gap between the chromatography medium and inlet. The column may have been packed to loosely.

It may be possible to only adjust the adaptor. Otherwise, repack the column with increased packing flow rate or pressure.

After re-packing the column, evaluate the packing, using the recommended method.

Preventive action:
Always consult the packing instructions for the chromatography medium and the specifications for the columns

A crack due to a torn bed support resulting in a jet stream.

Replace the bed support according to instructions.

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.

After repacking the column evaluate the packing

Preventive action:
Always consult the packing instructions for the chromatography medium and the specifications for the columns

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.

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.

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.

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.

Too great a distance between the sample injection and column inlet, or between the column outlet and analyzing instrument

Shorten these distances (minimizes dilution).

Too large sample volume (gives a broad peak).

Use a sample volume that is maximum 2% of the column volume.

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)).

After re-packing the column evaluate the packing.

Preventive action:
Always consult the packing instructions for the chromatography medium and the specifications for the columns

Bed settled too hard during the first step.

Repack the column with decreased packing flow rate for the sedimentation step.

After re-packing the column evaluate the packing.

Preventive action:
Always consult the packing instructions for the chromatography medium and the specifications for the columns

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.

Column not properly equilibrated before packing evaluation

Re-equilibrate the column.
Evaluate the packing again, using recommended methods.

Column not properly equilibrated before packing evaluation

Re-equilibrate the column.
Evaluate the packing again, using recommended methods.

Bed packed too densely or too loosely.

Repack the column. Evaluate the packing again, using recommended methods.

For a leading peak and one-step packing:
Pack the bed with decreased packing flow rate or pressure.

For a leading peak and two-step packing: 
Pack the bed with decreased packing flow rate or pressure during the first step.

For a tailing peak and one-step packing: 
Pack the bed with increased packing flow rate or pressure.

For a tailing peak and two-step packing: 
Pack the bed with increased packing flow rate or pressure during the first step.

Preventive action:
Always consult the packing instructions for the chromatography medium and the specifications for the columns

Bed packed too densely. Cracks present at the beginning get worse.

Repack the column. Evaluate the packing again, using recommended methods.

One-step packing:
Decrease packing flow rate or pressure.

Two-step packing:
Decrease packing flow rate during sedimentation.

Preventive action:
Always consult the packing instructions for the chromatography medium and the specifications for the columns

Bed too loosely packed causing space between chromatography medium and inlet.

Repack the column.
Evaluate the packing again, using recommended methods.

One-step packing:
Increase flow rate or pressure.

Two-step packing:
Increase flow velocity during sedimentation.

Preventive action:
Always consult the packing instructions for the chromatography medium and the specifications for the columns

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:
Always consult the packing instructions for the chromatography medium and the specifications for the columns.

Bed settled too loosely during the first step.

Repack the column with increased packing flow rate for the sedimentation step.

After re-packing the column, evaluate the packing.

Preventive action:
Always consult the packing instructions for the chromatography medium and the specifications for the columns

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.

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-ring. 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.

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.

Column is not properly equilibrated.

Check pH and conductivity of the effluent before applying the sample to make sure the column is properly equilibrated. Continue to equilibrate with start buffer if necessary.

Components in the sample displace the target molecule before elution starts.

Reduce the amount of sample or find more suitable binding conditions.

Flat titration curve of target protein.

Decrease start and elution pH by 0.5 units next time.

Highly charged protein at the chosen pH and the amount of salt in solution B is not enough to elute the protein. Your protein is more basic than most other proteins.

Increase the salt concentration in solution B to release the protein from the column.

pI of the protein is higher than your current estimate.

Increase start and elution pH by 0.5 units next time

This behavior may be due to ionic interaction between the protein and the chromatography medium.

Keep the ionic strength between 0.15 to 0.3 M to minimize ionic interactions.

Retardation may be due to aromatic and hydrophobic interactions between protein and the matrix.

If possible decrease the ionic strength, increase pH or introduce an organic solvent (e.g. 24% propanol or 10% ethylene glycol) in solution A in order to minimize hydrophobic interactions.

Eluent contains carbon dioxide.

Degas all buffers prior to use.

Eluent composition is incorrect.

Check eluent pH. Make up fresh eluent if necessary.

Column not properly equilibrated.

Re-equilibrate the column.

Sample not properly equilibrated.

Equilibrate sample to correct operating conditions (pH, ionic strength, etc.)

At the running pH, the overall charge of the protein is high and it binds very tight to the column.

The pH stability profile of the protein limits the possibility to decrease the pH. Try to increase the ionic strength of your solution A to obtain an earlier elution, or choose another technique.

pI of the protein is lower than most other proteins.

Decrease start and elution pH by 0.5 units next time.

pI of the protein is higher than most other proteins.

Increase start and elution pH by 0.5 units next time.

Column poorly packed.

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.

Elution strength too low.

Check and adjust the elution strength. Also check gradient, gradient shape or isocratic step.

Carbon dioxide is present.

Degas all buffers prior to use. In alkaline regions, degas water before making up eluent.

Counterions are incorrect.

Use recommended counterions.

Larger sample load applied compare with earlier runs.

Keep mass of sample constant when repeating runs. (High protein 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.

Protein properties change with concentration.

Hydrophobicity of your protein is much higher than your current estimate and binds more strongly to the chromatography medium than you expected.

Decreasing the ionic strength of solution A will save time. Another advantage is that many of the contaminants that would normally bind to the chromatography medium may elute in the void without binding.

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 critical especially in gel filtration.

Reduce the concentration of detergent to below it´s critical micelle concentration (CMC ) value.

Column is bleeding from the previous run.

Check and adjust your cleaning procedure. Clean the equipment and chromatography medium according to instructions

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 amount of sample or find more suitable binding conditions.

Aggregate formation of protein sample.

Use of zwitterions in the solutions, betaine up to 10%, pH 2 to 12, or taurine up to 4%, pH 4 to 6, to inhibit the formation of charge aggregates.

If you protein is a lipoprotein, aggregate formation may have occurred.

Use of zwitterions in the solutions, betaine up to 10%, or taurine up to 4%, may increase resolution and stabilize these complexes during chromatography

Low molecular weight compounds can taken up the charge capacity of the column very quickly.

The resolution may be improved if organic solvents or zwitterions are used in the buffers. However, the solubility of the zwitterions is lower in organic solvents than in water.

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

pH might not be optimal for your separation.

Try running at another pH. It may affect the selectivity.

pI of the protein is close to the pI of some major impurities.

Try to obtain higher resolution by increasing the pH by 0.5 units and/or use a more shallow gradient.

Too much sample has been loaded onto the column.

Decreasing the sample load may improve the resolution or increase the amount of chromatography medium. I.e. increase the margin of safety significantly.

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.

Different counter ions have different elution strengths.

Switching to another counter ion may improve the selectivity.

Try using another counter ion
Anions for example 0.15 M sulfate or 0.50 M chloride.
Cations for example potassium or ammonium.

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.

Gel filtration chromatography - Gel filtration column will resolve only molecules with approximately a two-fold difference in molecular weight.

Use lower flow rate and/or try another technique

Gel filtration chromatography- Sample is too viscous

Dilute the sample with solution A. Reduce the protein concentration two-fold.

Gel filtration chromatography- Sample volume is too large or the sample has been improperly applied.

Decrease sample volume and apply the sample carefully.

Gradient is too steep.

Use a more shallow gradient. A plateau in the gradient where your protein elutes may improve the resolution.

Hydrophobicity of your protein is much lower than your current estimate. The interaction between the protein and the chromatography medium is much weaker than expected.

Apply the sample under conditions than enhance hydrophobic interaction, increase salt concentration and/or change pH towards the pI of your protein.

Strength of the binding between the ligand and the protein varies with the protein type.

Remedy 1
Try another elution method. Decrease the polarity of eluting solution, for instance add dioxane up to 10% or ethylene glycol up to 50% or use deforming agents such as urea up to 8 M or GuHCI up to 6 M. Remove these agents immediately after elution

Remedy 2
Try a nonspecific elution method to increase pH for example use 0.1 M glycine NaOH, pH 10.5, either in a gradient or stepwise elution. Do not forget to neutralize eluted fractions immediately.

Remedy 3
Try a nonspecific elution method increasing ionic strength, either gradient or stepwise elution with for example 1 M NaCl, 4.5 M MgCl₂ or using chaotropic salts often at neutral pH (e.g. 5 M KSCN).
Try the chelating agents EDTA or EGTA.

Remedy 4
Try eluting with 2 M lithium bromide or pH gradient (e.g. from 7.5 to 9.5).

Remedy 5
Try eluting with increasing ionic strength either gradient or stepwise elution, for example with 1 M sodium chloride, 5 M magnesium chloride or 3 M potassium iodide.

Remedy 6
Try elution with an eluting agent that competes for the binding to either the target protein or the ligand.

Remedy 7
Try performing the run at lower pH: equilibrate and wash the column with solution at pH 6.3, without salt and elute with 0.5 M NaCl at pH 6.3.

Buffers have wrong pH.

Calibrate your pH meter, prepare new buffers and try again.

pH of eluting buffer is too high.

Try a lower pH range.

pI of the protein is lower than your current estimate.

Decrease start and elution pH by 0.5 units next time.

pI of the protein is higher than your current estimate.

Increase start and elution pH by 0.5 units next time.

Protein is more acidic than estimated and is therefore highly charged at the suggested pH and is still bound to the column.

If the protein does not elute in the end of the gradient, increase the salt concentration in eluting buffer to release the protein from the column.

At the chosen pH the protein is highly charged and the amount of salt in solution B is not enough to elute the protein. Your protein is more basic than your first estimate.

Increase the salt concentration in solution B to release the protein from the column.

Composition of eluting buffer is incorrect.

Check pH of eluting buffer.

Highly charged protein at the chosen pH and the amount of salt in solution B is not enough to elute the protein. Your protein is more basic than most other proteins.

Increase start and elution pH by 0.5 units next time.

Hydrophobic proteins interact very strongly with the chromatography medium.

Use an additive that decreases hydrophobic interactions. Mild organic solvents such as ethylene glycol, or detergents, or urea will elute tightly bound proteins but may lead to denaturation.

Target protein is more acidic than most other proteins. It's therefore highly charged at the suggested pH.

Decrease start and elution pH by 0.5 units next time.

Column overloaded

Decrease the amount of sample or increase the amount of chromatography medium. I.e. increase the margin of safety.

pI of the protein is lower than most other proteins.

Decrease start and elution pH by 0.5 units next time.

pI of the protein is lower than your current estimate.

Decrease start and elution pH by 0.5 units next time, or enter a new pI estimate for the protein and start a New Case.

pI of the protein is higher than your current estimate.

Increase start and elution pH by 0.5 units next time, or enter a new pI estimate for the protein and start a New Case.

Protein is associated with another component that changes its true charge character.

Use a preliminary clean up to remove these components or use zwitterions in the solutions.

Protein can not bind to the chromatography medium, and may have been precipitated owing the low ionic strength of solution A.

Choose another technique.

Sample dissolved in wrong solution.

Prepare a new solution A, check the pH and conductivity; dissolve your sample in solution A and repeat the run.

Solution A and B have been mixed up.

Check pH and conductivity of the solutions, dissolve the sample in solution A and repeat the run.

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 according to the Media and column 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.

Highly charged protein at the chosen pH and the amount of salt in solution B is not enough to elute the protein. Your protein is more basic than your first estimate.

Increase the salt concentration in solution B to release the protein from the column.

Hydrophobic interaction chromatography- Hydrophobicity of your protein is much lower than your current estimate. The interaction between the protein and the chromatography medium is much weaker than expected.

Apply the sample under conditions that enhance hydrophobic interactions; increase salt concentration and/or change pH towards the pI of your protein.

Very hydrophilic proteins will not adsorb to the chromatography medium, but will elute in the void.

Ionic strength of solution A is too high for binding of the protein.

Since some salt is needed in solution A, try decreasing the pH of solution A to obtain binding of the protein.

Anionic detergents or other additives are adsorbed to the column.

Clean the column according to the Media and column instructions.

Ionic strength of the sample is too high.

Remove salt in the sample by gel filtration in Polybuffer.

pI of the protein is higher than most other proteins.

Increase start and elution pH by 0.5 units next time.

Protein is associated with another component which changes its true charge character.

Use a preliminary cleanup to remove these components or use zwitterions in the buffers.

Sample is dissolved in wrong buffer.

Prepare new buffers, check the pH and conductivity, dissolve your sample in start or eluting buffer and repeat the run.

Start pH is too low.

Choose a start buffer with a higher pH.

Buffers have wrong pH, ionic strength or solvent concentration.

Check and adjust the buffer conditions. Calibrate your instruments.

Air trapped in the bed supports.
You may discover air in the packed bed.

Get rid of the air by wetting and washing the bed supports with 20% ethanol. Fill a syringe with 20% ethanol and inject the liquid into the bottom of the column. Trapped air in the top net should be avoided by inject 20 % ethanol to the top adaptor before assembling the adaptor. Make sure that the ethanol passes the nets.

Preventive action:
Always wet and wash bed supports with 20% ethanol before use.

Microbial growth in buffers.


The buffer normally become opalicent due to microbial growth.

Check buffers, especially those with phosphate, for microbial growth. Replace with fresh buffer if necessary.

Precipitation of protein on the column.

The column may be discoloured by precipitated proteins.

Remedy 1
Change pH and/or add urea. Develop a procedure with detergents.

Remedy 2
Clean and regenerate the column and chromatography medium according to instructions supplied with the column. If this does not help replace with a new one.

Precipitation of proteins on the column at high salt concentration resulting in increased hydrophobic interaction.

Use chaotropic salts in solution B. However, if the protein structure is destroyed choose another technique.

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.

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.

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.

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.

The bed support may become discoloured if blocked by proteins

To remove protein:
Clean-in-place according to chromatography medium instructions. You may need to prolong the exposure time.

To remove chromatography medium:
Disassemble the column. Clean the bed support using compressed air or replace it according to instructions.

Preventive action:
Always wet and wash bed supports with 20% ethanol before use.

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.

Ionic strength of solution may have caused the protein to precipitate in the column.

Remedy 1
Determine the stability of your protein and run a new case. Clean the column according to the instructions supplied with the column.

Remedy 2
The decreased solubility is caused by electrostatic interactions which results in the formation of aggregates. To minimize these interactions, use 10 to 50 mM salt in start or eluting buffer and equilibrated sample solution.

Remedy 3
Use chaotropic salts in solution B. However, if protein structure is destroyed, please choose another technique.

Remedy 4
Change pH and/or add urea. Develop a procedure with uncharged detergents, they are usually mild and less likely to cause protein precipitation.

Lipoproteins are present and may clog the column.

Lipoproteins may be precipitated prior to chromatography by the addition of 10% dextran sulfate and 1 M calcium chloride to final concentrations of 0.2 % and 0.5 M respectively. Remove precipitate by centrifugation.

Buffers and column at different temperatures.

The column must generally be repacked. However, you can remove a small amount of air trapped at the column top by pumping a solution with a temperature a few degrees centrigrade higher than the chromatography through the column. Use upward flow. If the flow has been reversed, re-evaluate the packing prior the use.

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.

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 at the column top by pumping a solution with a temperature a few degrees centrigrade higher than the chromatography through the column. Use upward flow. If the flow has been reversed, re-evaluate the packing prior the use.

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.

Preventive action:
Tighten connectors prior to use. Also check that they are compatible with each other.

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 at the column top by pumping a solution with a temperature a few degrees centrigrade higher than the chromatography through the column. Use upward flow. If the flow has been reversed, re-evaluate the packing prior the use.

Evaluate the packing.
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.

Preventive action:
Check the valves and replace if necessary.

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).

Evaluate the packing.
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.

Some carbon dioxide gas bubbles might develop with a carbonate buffer during ligand coupling.

It does not affect the coupling result.

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.

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

Unwashed buffer tanks.

Clean the tanks and prepare fresh solutions.

Leakage of non-eluted protein due to poor cleaning-in-place.

Clean and regenerate the column and chromatography medium according to instructions

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.