Bio-Rad ENrich™ High-Resolution Ion Exchange Columns - Manual (Page 7)

ENrich Instruction Manual 3
Section 2 : Use of the ENrich
Columns
2.1 Preparation for Initial Use
The columns are supplied in a storage solvent of 20% ethanol
in water. Prior to initial use and after extended storage periods,
each column should be conditioned as described in steps 1–4.
Always use HPLC-grade reagents and be sure to filter and degas
solvents. During these four steps do not exceed more than 50%
of the recommended flow rates (see Table 1).
1. Wash with 5 column volumes of water.
2. Wash with 5 column volumes of low ionic strength
equilibration buffer (such as 20 mM buffer salt).
3. Wash with 5 column volumes of high ionic strength limit
buffer (such as 1.0 M NaCl).
4. Wash with 5 column volumes of low ionic strength
equilibration buffer.
The column may now be further equilibrated in the start buffer at
the desired flow rate.
2.2 Buffer Selection
Table 2 lists commonly used buffers for ion exchange
chromatography. The buffers are specific to the type of ion
exchange. Therefore, it is important not to use anionic buffers
with the ENrich Q, which would interact with the anionic
exchange group on the support.
The choice of whether to use an anion or cation exchanger is
determined mainly by the isoelectric point (pI) and the relationship
between pH and the activity/stability of the protein(s) of interest.
When the type of ion exchanger is determined, the pH-activity
relationship also determines the choice of buffer. As a general
rule, the chosen buffer should be used within ±1.0 pH unit of
its pKa value. This permits use of the lowest possible buffer
concentration while maintaining maximum buffering capacity. We
recommend a minimum buffer concentration of 20 mM.