Bio-Rad Bio-Scale™ CHT™ Type I Columns - Manual (Page 7)

sharp, UV-absorbing peaks during the gradient, complicating subsequent analysis of the chro-
matogram.
Section 3
Care of the Bio-Scale CHT-I Columns
3.1 Column Hygiene
Careful preparation (especially filtration) of the sample and the buffers will maintain the
column performance and lifetime. Normally, washing with 0.5 M phosphate will remove
most bound components. However, if there is a significant decrease in column performance
(ie increasing backpressures or a significant drop in resolution) then a more extensive clean-
ing protocol as described below should be used. Always reverse the flow during this pro-
cedure so tightly bound substances at the top of the column are quickly removed.
During this operation, do not exceed more than 25% of the maximum recommended
flow rates (see Table 1).
1. Wash with 2 column volumes of 2.0 M NaCl or KCl followed by 3 column volumes of
water or low salt buffer.
2. Wash with 1 column volume of 2.0 M NaOH followed by 3 column volumes of water or
low salt buffer.
3. Change the top frit, readjust the bed height to eliminate any voids and wash with 3 col-
umn volumes of equilibration buffer.
If lipid contamination is a problem, wash with 1 column volume of methanol followed by
3 column volumes of water followed by at least 2 column volumes of 0.5 M phosphate.
Change the top frit, readjust the bed height to eliminate any voids and wash with 3 column vol-
umes of equilibration buffer.
3.2 Bed Height Adjustment
Under certain conditions of buffer composition, high flow rates or long-term use, the
resin bed may compress creating a void between the frit and the top of the bed. Normally,
the void can be eliminated by turning the lock nut clockwise until the frit just touches the top
of the bed. If the bed compresses at high flow rates, stop the pump and loosen the top fitting.
Then adjust the lock nut to remove the void, retighten the top fitting and resume pumping
buffer. If the bed has compressed from long-term use, then as a precaution the top frit should
be replaced.
3.3 Frit Removal
The top frit may need to be replaced after extensive column use or if increasing back-
pressures are noticed. Each column is supplied with a Frit Removal Tool, Polyethylene Frit
and Distribution Screen.
The screen assists in distributing the sample over the entire column surface and also acts
as a pre-filter for the frit. It should be replaced every time the frit is changed. Figure 2 describes
in detail how to remove and change the top frit.
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