HiScreen Capto Phenyl（High Sub）

Gradient slope or isocratic step too shallow.

Increase steepness of gradient or isocratic step.

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.

Column not properly equilibrated

Check the pH and the conductivity of the effluent before applying the sample. Continue to equilibrate with start buffer if necessary

Insufficient column regeneration.

Prolong the regeneration.

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 instructions.

Column clogged with denatured proteins and/or lipids.

The column may be discoloured by 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.

Incorrect pH and/or ionic strength of the solutions.

Check pH and conductivity and adjust if necessary. Calibrate your conductivity and pH meters.

Larger sample mass load applied compared with earlier runs.

Keep mass of sample constant when repeating runs. (High proteins concentration can cause protein interaction, resulting in change of elution profile.)

Sample volume is different from earlier runs.

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

Protein properties change with concentration.

Buffers have wrong pH, ionic strength or solvent concentration.

Check and adjust the buffer conditions. Calibrate your instruments.

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.

Column not properly equilibrated.

Re-equilibrate the column.

Sample not properly equilibrated.

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

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

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.

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.

Slop of gradient too steep.

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

Too much sample has been loaded onto the column.

Decreasing the sample load may improve the resolution significantly.

The column operates at room temperature after having been stored in a cold room.

Allow thermal equilibration before use.

Contaminants accumulating in the column.

1. Optimize the elution conditions
2. Optimize the CIP ( Cleaning-in-place) protocol
3. Perform CIP more frequently

Auxiliary equipment such as manometers and pumps not working properly.

Check the function of all auxiliary equipment. Repair/replace if necessary.

Bent tubing.

Check that the flow path is not restricted.

Buffer viscosity too high.

Check the viscosity of all buffers. Viscosity is a function of temperature. (Lower temperature gives higher viscosity.) Let low-temperature buffer reach operating temperature before starting the run.

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.

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.