Bio-Rad UNO® Monolith Cation Exchange Columns - Manual (Page 7)

concentration (CMC). As the salt concentration (i.e. the counter-ion concentration)
increases, the CMC drops and eventually micelles will form. This may cause a sud-
den increase in the UV baseline as the micelles themselves scatter light. We rec-
ommend using a concentration of detergent above the CMC during gradient elution.
If subsequent chromatographic steps (e.g. hydrophobic interaction) or other bio-
chemical manipulations will be affected by the presence of the detergent, then the ini-
tial choice of detergent may be influenced by the availability of a protocol for its
removal.
See Reference 2 for a more detailed explanation of the characteristics and use of
detergents in biology and biochemistry and their removal from biological samples.
Chromatography Pre-Runs
Prior to loading the sample, it is recommended running a blank gradient to check
that the column is clean. In addition, detergents and other buffer components will
themselves elute as sharp, UV-absorbing peaks during the gradient, complicating
subsequent analysis of the chromatogram.
Column Hygiene
Careful preparation (especially filtration) of the sample and the buffers will
maintain the column performance and lifetime. Normally, washing with 1.0 M NaCl
or KCl will remove most bound components. However, if there is a significant
decrease in column performance (i.e. increasing backpressures or a significant drop
in resolution) then a more extensive cleaning protocol as described below (steps
1–5) should be used. Always reverse the flow during this procedure so tightly-
bound substances at the top of the column are quickly removed. This process
should be performed at 0.5 ml/min.
1. Wash with 2 ml of deionized water. Elevated backpressures may occur when
washing with deionized water. Do not exceed 300 psi.
2. Wash with 5 ml of 2.0 M NaCl or KCl.
3. Wash with 1 ml of 2.0 M NaOH followed by 1 ml of 0.1 M NaCl.
4. Wash with 1 ml of 50% acetic acid followed by 1 ml of 0.1 M NaCl and 1 ml
of deionized water.
5. If lipid contamination is a problem, wash with 1 ml of MeOH followed by 2ml
of 0.1 M NaCl.
6. Wash with 5 ml of 2.0 M NaCl or KCl or the salt containing the desired counter-
ion.
No repair of this column is possible. If performance deteriorates or backpressures
become excessive, then we recommend purchasing a new column.
6
4006078A 10/1/98 10:02 AM Page 6