Bio-Rad Media Sampler Pack - Manual (Page 11)

Section 8
Operation and Maintenance
Macro-Prep ion exchange columns may be operated in two different modes. In bind-
elute mode, the protein of interest (target molecule) binds to the medium during loading. It
is subsequently eluted by a change of conductivity and/or pH. This allows removal of
contaminants that do not bind to the medium and contaminants that may bind more
weakly or more strongly than the protein of interest. In flow-through mode, the protein of
interest does not bind during loading, while contaminants that may bind to the medium
are removed. This approach can simplify chromatography conditions, conserve buffer,
and increase recovery of the target molecule.
For flow-through mode, a linear flow rate of 300 cm/hr in a 20 cm bed is practical.
If operating in a bind-elute mode, flow rates of 100–200 cm/hr are recommended. Lower
flow rates may support higher capacity and better resolution.
All buffers commonly used for anion or cation exchange chromatography can be
used with the appropriate ion exchange supports (see Table 3). The chemical
stability and broad operating pH range of these ion exchangers allow the use of a
variety of buffers. It is best to use buffering ions that have the same charge as the
functional group on the ion exchanger, e.g., phosphate (–) with a cation exchanger,
and Tris (+) with an anion exchanger. Buffers should be chosen so operating pH is
within 0.5 pH unit from pK
a
of buffer substance. The purification may be
optimized by changing buffers, salts, pH, additives, and gradient.
Table 3. Common Buffers for Ion Exchange Chromatography. 1, 2, 3
Type of Ion Exchanger Buffer Buffering Range
Cation Acetic acid 4.8–5.2
Citric acid 4.2–5.2
HEPES 6.8–8.2
Lactic acid
3.6–4.3
MES 5.5–6.7
MOPS 6.5–7.9
Phosphate 6.7–7.6
PIPES 6.1–7.5
TES 6.8–8.2
Tricine 7.8–8.9
Anion Bicine 7.6–9.0
Bis-Tris 5.8–7.2
Diethanolamine 8.4–8.8
Diethylamine 9.5–11.5
L-Histidine 5.5–6.0
Imidazole 6.6–7.1
Pyridine 4.9–5.6
Tricine 7.4–8.8
Triethanolamine 7.3–8.3
Tris 7.5–8.0
Note: Buffer solution of 50 mM is recommended.
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