Ion Exchange Chromatography Troubleshooting

Figure 1. The ideal IEX separation: target proteins well resolved by gradient elution

If only certain peaks are of interest in this well-resolved separation, it may be advantageous to transfer to a step elution in order to save time and buffer. The rest of this section focuses on practical problems that may lead to a non-ideal IEX separation.

Figure 2. Sample elutes before salt gradient begins

Ensure that buffers are in the correct containers. Reduce ionic strength of sample by desalting, page 156, or dilution with start buffer. For an anion exchanger, increase buffer pH, for a cation exchanger, decrease buffer pH. If proteins still do not bind at any pH, it is possible that the column has been contaminated by detergent.

Figure 3. Sample still eluting when gradient begins

After sample application the UV trace must return to baseline before elution begins, otherwise proteins that do not bind to the column interfere with the separation. Increase the volume of start buffer (equilibration step) before starting the gradient elution.

Figure 4. Sample elutes during high salt wash

Proteins are binding too strongly. Ensure that buffers are in the correct containers. If using an anion exchanger, decrease buffer pH, if using a cation exchanger, increase buffer pH.

Protein(s) of interest eluting late in gradient

Proteins are binding too strongly. Increase ionic strength of gradient. It is preferable to alter pH if a very high salt concentration is required for elution. For an anion exchanger, decrease buffer pH and for a cation exchanger, increase buffer pH. Refer also to Table 6.

Protein(s) of interest eluting too early in gradient:

Proteins are not binding strongly. Check ionic strength of gradient. Alter pH, for an anion exchanger, increase buffer pH and for a cation exchanger, decrease buffer pH. Refer also to Table 6.

Proteins(s) of interest not sufficiently resolved

Refer to the contents of this chapter to review key parameters for improving resolution. Refer also to Table 6.

Table 6. Troubleshooting.

Situation	Cause	Remedy
Reduced or no flow through the column.	Outlet closed or pumps not working.	Open outlet. Check pumps for signs of leakage (if using a peristaltic pump, check tubing also).
	Blocked filter, end-piece, adaptor or tubing.	Remove and clean or replace if possible. Always filter samples and buffer before use.
	Lipoproteins or protein aggregates have precipitated.	Remove lipoproteins and aggregates during sample preparation, (Appendix 1). Follow cleaning procedures, Appendix 10.
	Protein precipitation in the column.	Modify buffer, pH and/or salt conditions during the run to maintain stability. Follow cleaning procedures, Appendix 10.
	Protein precipitation in the column caused by removal of stabilizing agents during separation.	Modify eluent to maintain stability.
	Microbial growth has occurred in the column.	Store in the presence of 20% ethanol to prevent microbial growth when not in use. Always filter buffers. Follow cleaning procedures, Appendix 10.
Peak of interest is poorly resolved from other major peaks.	Sample applied incorrectly.	Check bed surface and top filter for possible contamination.
	Large mixing spaces at top of or after column.	Adjust top adaptor to surface of medium if necessary. Reduce all post-column volumes.
	Incorrect buffer pH and/or ionic strength.	Check pH and ionic strength to ensure that column was re-equilibrated after previous run. Check conditions required. Prepare new solutions.
	Sub-optimal elution conditions e.g. incorrect pH, gradient too steep, flow rate too high.	Alter elution conditions: alter pH, use shallower gradient, reduce flow rate (listed in priority order).
	Sample is too viscous.	Dilute with buffer. Maintain protein concentration below 50 mg/mL.
	Column is poorly packed.	Check column efficiency (Appendix 3). Repack if needed. Use prepacked columns.
	Column overloaded.	Decrease sample load.
	Lipoproteins or protein aggregates have precipitated.	Remove lipoproteins and aggregates during sample preparation (Appendix 1).
	Precipitation of proteins in the column.	Modify buffer, pH and/or salt conditions during the run to maintain stability.
	Microbial growth has occurred in the column.	Store in the presence of 20% ethanol to prevent microbial growth. Always filter buffers. Follow cleaning procedures, Appendix 10.
Proteins do not bind or elute as expected.	Proteins or lipids have precipitated on the column or column filter.	Clean the column and exchange or clean the filter. Check pH and salt stability of sample.
	Sample not filtered properly.	Clean the column, filter the sample and repeat.
	Sample has changed during storage.	Prepare fresh samples.
	Protein may be unstable or inactive in the elution buffer.	Determine the pH and salt stability of the protein.
	Column equilibration incomplete.	Repeat or prolong the equilibration step until conductivity and pH are constant.
	Incorrect buffer pH and/or ionic strength.	Check conditions required. Prepare new solutions.
	Proteins are forming aggregates and binding strongly to the medium.	Use urea or zwitterions, betaine up to 10%, taurine up to 4%.
	Sample or buffer conditions are different from previous runs	Check sample and buffer conditions.
	Microbial growth has occurred in the column.	Store in the presence of 20% ethanol to prevent microbial growth when not in use. Always filter buffers. Follow cleaning procedures, Appendix 10.
Protein elutes later than expected or not at all.	Incorrect buffer pH.	Check pH meter calibration. Use a buffer pH closer to the pI of the protein.
	Ionic strength too low.	Increase salt concentration in elution buffer.
	Ionic interactions between protein and matrix.	Maintain ionic strength of buffers above 0.05 M.
	Hydrophobic interactions between protein and matrix.	Reduce salt concentration to minimize hydrophobic interaction. Increase pH. Add suitable detergent or organic solvent, e.g. 5% isopropanol.
Protein elutes earlier than expected (during the wash phase).	Ionic strength of sample or buffer is too high.	Decrease ionic strength of sample or buffer.
	Incorrect pH conditions.	Increase pH (anion exchanger). Decrease pH (cation exchanger).
	Column equilibration incomplete.	Repeat or prolong the equilibration step until conductivity and pH are constant
Leading or very rounded peaks in chromatogram.	Channeling in the column.	Repack column using a thinner slurry of medium. Check column packing (Appendix 3).
	Column overloaded.	Decrease sample load and repeat.
	Column contaminated.	Clean using recommended procedures.
Peaks are tailing.	Incorrect start buffer conditions, sample is not binding to column.	Adjust pH. Check salt concentration in start buffer.
	Sample too viscous.	Dilute in application buffer.
	Column packing too loose.	Check column efficiency (Appendix 3). Repack using a higher flow rate. Use prepacked columns.
Peaks have a leading edge.	Column packing compressed.	Check column efficiency (Appendix 3). Repack using a lower flow rate. Use prepacked columns.
Medium/beads appears in eluent.	Column packing compressed.	Check column efficiency (Appendix 3). Repack using a slower flow rate. Use prepacked columns.
	Bed support end piece is loose or broken.	Replace or tighten.
	Column operated at too high pressure.	Do not exceed recommended operating pressure for medium or column.
	Medium has been damaged during column packing.	Do not use magnetic stirrers when equilibrating loose medium
Low recovery of activity, but normal recovery of protein.	Protein may be unstable or inactive in the buffer.	Determine the pH and salt stability of the protein.
	Enzyme separated from co-factor or similar.	Test by pooling aliquots from the fractions and repeating the assay.
Protein yield lower than expected.	Protein may have been degraded by proteases.	Add protease inhibitors to the sample and buffers to prevent proteolytic digestion. Run sample through a medium such as Benzamidine 4 Fast Flow (high sub) to remove rypsin-like serine proteases.
	Adsorption to filter during sample preparation	Use another type of filter.
	Sample precipitates.	Check pH and salt conditions, adjust to improve sample solubility.
	Hydrophobic proteins.	Add denaturing agents, polarity reducing agents or detergents. Add 10% ethylene glycol to running buffer to prevent hydrophobic interactions.
	Non-specific adsorption.	Reduce salt concentration to minimize hydrophobic interaction. Add suitable detergent or organic solvent e.g. 5% isopropanol. If necessary, add 10% ethylene glycol to running buffer to prevent hydrophobic interactions.
Peaks too small.	Sample absorbs poorly at chosen wavelength.	If appropriate, check absorbance range on monitor. If satisfactory, use a different wavelength, e.g. 214 nm instead of 280 nm.
	Different assay conditions have been used before and after the chromatographic step.	Use same assay conditions for all assays.
	Excessive band broadening.	Check column packing. Repack if necessary.
More sample is recovered than expected.	Protein co-eluting with other substances.	Optimize conditions to improve resolution. Check buffer conditions used for assay before and after the run. Check selection of medium.
More activity is recovered than was applied to the column.	Different assay conditions have been used before and after the chromatography step.	Use same assay conditions for all assays.
	Removal of inhibitors during separation.
Back pressure increases during a run or during successive runs.	Bed compressed.	If possible repack the column or use a new column. Check sample preparation.
	Microbial growth.	Store in the presence of 20% ethanol to prevent microbial growth. Always filter buffers. Follow cleaning procedures, Appendix 10.
	Turbid sample.	Improve sample preparation (Appendix 1). Improve sample solubility: add betaine (max. 10% w/v at 25 °C), taurine (max. 4% w/v at 25 °C, below pH 8.5) or glycerol (1–2 %). For hydrophobic samples, add ethylene glycol, urea, detergents or organic solvents.
	Precipitation of protein in the column filter and/or at the top of the bed.	Clean using recommended methods. If possible, exchange or clean filter or use a new column. Include any additives that were used for initial sample solubilization in the running buffer.
	Incorrect pH is causing precipitation.	Calibrate pH meter, prepare new solutions And try again. Change pH.
	Precipitation of lipoproteins at increased ionic strength.	Lipoproteins may be removed prior to chromatography by the addition of 10% dextran sulfate (final 0.2%) and 1 M calcium chloride (final 0.5 M).
Air bubbles in the bed. Cracks in the bed.	Buffers not properly degassed.	Degas buffers thoroughly.
	Column packed or stored at cool temperature and then warmed up.	Remove small bubbles by passing degassed buffer through the column. Take special care if buffers are used after storage in a fridge or cold-room. Do not allow column to warm up due to sunshine or heating system. Repack column, if possible (Appendix 3).
Cracks in the bed.	Large air leak in column.	Check all connections for leaks. Repack the column if possible (Appendix 3).
Negative peaks at solvent front.	Refractive index effects.	Exchange the sample into start buffer.
Unexpected peaks in chromatogram.	Buffer impurities.	Clean the buffer by running it through a precolumn. Use high quality reagents.
Peaks appear on gradients.	Incomplete elution of previous sample	Wash the column according to recommended blank methods.
Spikes in chromatogram.	Air bubble trapped in UV monitor flow cell.	Always use degassed buffers.
UV baseline rises with gradient.	Micelle formation as salt concentration changes.	Work below or above the critical micelle concentration of any detergents being used or change the gradient so that the increase in UV absorption does not occur while the samples are eluting.
	Buffer impurities.	Use high quality reagents.

*Polar organic solvents such as methanol, ethanol, isopropanol and acetonitrile can be used at concentrations from 0–20%, but remember that some proteins may irreversibly lose their biological activity in the presence of organic solvents. Check sample and buffer solubility, buffer pH and chemical stability of the medium before running a column.
Note that back pressure may increase when working with organic solutions.