Bio-Rad Media Sampler Pack - Manual (Page 12)

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Operating flow rates should not exceed 80% of the maximum pressure attained
during the initial column packing in laboratory scale columns. If you do not have a
pressure indicator, do not exceed flow rates in excess of 80% of the maximum
flow rate used during packing. In process scale columns, do not exceed 70% of
the maximum pressure attained during column packing.
Buffer Preparation
When preparing buffers for ion exchange chromatography, it is important that excess
conductivity is not produced during buffer pH adjustment. For example, back-titration of
Tris-HCl with NaOH elevates the conductivity, which will lower binding capacity. To
prepare Tris buffer, begin with Tris base and titrate with Tris-HCl to the target pH. Apply
the same principle with other buffers.
Column Equilibration
To equilibrate the column, wash with 1 to 2 column volumes (CV) of buffer containing
0.5–1.0 M of the buffer salt used in the start buffer. Follow with 3 CV of the start buffer or
until pH and conductivity are stable.
Sample Preparation
Adjust salt concentration and pH as necessary to achieve desired selectivity for binding
of target or contaminants. If sample conductivity exceeds conductivity of the column
equilibration buffer, sample capacity may be reduced, or the target molecule may not
bind. Adjustment of pH and conductivity of sample can be done by dilution or buffer
exchange.
Sample Load and Adsorption
The sample load is determined empirically by loading and evaluating breakthrough of
the molecule of interest. Sample volume is not a critical factor. Large volumes of dilute
feed such as cell culture supernatant and clarified lysates may be loaded onto the
medium without prior concentration.
Wash Through
When operating in a bind-elute mode, after loading of the sample onto the column,
follow with 2 to 5 CV of the equilibration buffer. This will wash out unbound contaminants.
In flow-through mode, after sample is completely through the column, regenerate the
medium as described under "Regeneration" below.
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