Affinity Chromatography - Department of Molecular and Cellular ...

Situation Cause Remedy
Back pressure increases Turbid sample. Improve sample preparation (see Appendix 1).
during a run or during
Improve sample solubility by the addition of
successive runs.
ethylene glycol, detergents or organic solvents.
Precipitation of protein in the column Clean using recommended methods. Exchange
filter and/or at the top of the bed. or clean filter or use a new column.
Include any additives that were used for initial
sample solubilization in the solutions used for
chromatography.
Bubbles in the bed. Column packed or stored at Remove small bubbles by passing de-gassed
cool temperature and then
buffer upwards through the column. Take
warmed up.
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, (see Appendix 3).
Buffers not properly de-gassed. De-gas buffers thoroughly.
Cracks in the bed. Large air leak in column. Check all connections for leaks. Repack the
column if possible (see Appendix 3).
Distorted bands as sample Air bubble at the top of the Re-install the adaptor taking care to avoid air
runs into the bed. column or in the inlet adaptor. bubbles.
Particles in buffer or sample. Filter or centrifuge the sample. Protect buffers
from dust.
Clogged or damaged net in
Dismantle the adaptor, clean or replace the net.
upper adaptor.
Keep particles out of samples and eluents.
Distorted bands as Column poorly packed. Suspension too thick or too thin. Bed packed at
sample passes down
a temperature different from run.
the bed.
Bed insufficiently packed (too low packing
pressure, too short equilibration). Column
packed at too high pressure.
24