Table 1. column characteristics, Preparation for initial use, Buffer selection – Bio-Rad UNO® Monolith Cation Exchange Columns User Manual
Page 3
2
use of inert, biocompatible (ceramic, PEEK, titanium) solvent delivery systems
for maximum column life and recovery of sample biological activity.
Table 1. Column Characteristics
Q Polishing Column
S Polishing Column
Column volume (ml)
0.16
0.16
Recommended max. protein loading (mg)
2
2
Recommended flow-rates (ml/min)
0.1 to 1
0.1 to 1
Column Dimensions (mm)
4.6 x 10
4.6 x 10
Max. operating pressure (psi/MPa/Bar)
200/1.3/14
200/1.3/14
Preparation for Initial Use
The columns are supplied in a storage buffer of 0.1 M NaCl + 20% ethanol. The
counter ion for the Q column is Cl
-
and Na
+
for the S column. Prior to initial use
and after extended storage periods, each column should be conditioned as described
below (steps 1–4). Always use HPLC grade reagents and filter and degas the buffers.
During this operation do not exceed flow rates of 0.5 ml/min.
1. Wash with 5 ml of water. Elevate backpressures may occur when washing with
deionized water. Do not exceed 300 psi
2. Wash with 5 ml of low ionic strength start buffer [e.g. 20 mM Tris-HCl (Q) or 20
mM Sodium Phosphate (S)].
3. Wash with 5 ml of high ionic strength buffer (e.g. Starting buffer + 1.0 M NaCl).
4. Wash with 5 ml of low ionic strength start buffer [e.g. 20 mM Tris-HCl (Q) or
20 mM Sodium Phosphate (S)].
The column may now be further equilibrated in start buffer at the desired flow
rate.
Buffer Selection
Table 2 and 3 lists commonly-used buffers for anion and cation-exchange chro-
matography. The choice of whether to use an anion- or cation exchanger is deter-
mined mainly by (a) the isoelectric point (pI) and, (b) the relationship between pH
and the activity/stability of the protein of interest. Once the form of ion-exchanger
is determined, the choice of buffer and pH is also determined by the pH-activity
relationship. As a general rule, the pH used should be within ± 0.5 units of the pK
a
of the chosen buffer. This permits use of the lowest possible buffer concentration
4006078A 10/1/98 10:02 AM Page 2