A large amount of purified protein ends up in the band-transition zones.

Try adjusting the column you use: Use a longer column with a higher aspect ratio.

The breakthrough curve is distorted.

1. Try running the displacer only to determine an optimal flow rate.

2. Try adjusting the flow rate of the protein: Use a slower flow rate that will not distort the breakthrough curve.

I tried to repeat a displacer breakthrough but was unable to replicate results.

Try modifying your regeneration protocol: The column may not be fully regenerated.

I ran two experiments with displacer alone and got two different breakthrough times.

Try modifying your regeneration protocol: The column may not be fully regenerated.

The results of my experiment appear to be contaminated.

Try modifying your regeneration protocol: After regeneration, run 5 column volumes of a base wash, then 5 column volumes of an acid wash. If lipids could be an issue, you can also run a detergent wash.

I was unable to set up a proper displacement train.

Check for these potential problems in the system:

• Poor column washing and cleaning
• Incomplete column equilibration
• Over-equilibration of the column
• Unintended salt or buffers left in the chromatographic plumbing
• Flow rate too slow or fast
• Loading too low or high
• Displacer concentration too low

The protein binds too weakly to the ion exchange column.

Try adjusting the pH: the pH should be 1.5 units below the pI for cation exchange and 1.5 units above the pI for anion exchange.

The protein seems to not be binding; it all washes through.

For anion exchange, try using a neutral/cation buffer.

I don't seem to get good separations between close-running bands.

Pick a new matrix and suitable pH that leads to better differential binding. Run a quick analytical elution run using similar buffers. If you find good separation there, use the same matrix for displacement.

Too long until breakthrough

Check concentration of protein.

Transition zones are too wide

Check plumbing problems. Column too short. Bad packing.