Bio-Rad UNO® Monolith Cation Exchange Columns - Manual (Page 6)

Gradient Volumes & Salt Concentrations
As a starting point for developing a separation, we recommend using the UNO
Q Polishing column with a simple gradient profile over 2–6 ml.
Protocol: Use a flow-rate of 0.5 ml/min. Following sample application, wash
unbound proteins from the column with 2 ml of Start Buffer A. For elution, use a gra-
dient volume of 6 ml to a Cl
- concentration of 0.5 M (50% B). Follow this segment
of the gradient by raising the salt concentration to 1.0 M (100% B) over 2 ml and then
hold at 1.0 M for 2 ml before re-equilibrating the column with 6 ml of start buffer
A. This gradient is shown schematically in Figure 1. Once an initial separation has
been performed and the elution position of the protein of interest determined, the
gradient composition and volume is adjusted to achieve maximum resolution.
Normally, a gradient volume of 2 to 6 is sufficient. The slope of the gradient will
affect resolution. A steep gradient will result in relatively small peak volumes but
short peak-to-peak distances. A shallower gradient normally gives greater resolu-
tion but peak volumes are larger.
Fig. 1 Schematic gradient for separation on UNO Polishing column.
Use of Detergents
Cationic or non-ionic detergents may be used with the UNO Q support. Anionic
or non-ionic detergents may be used with the UNO S support. We recommend the
use of the reduced form of Triton X-100 to minimize UV absorption artifacts. It is
essential to thoroughly equilibrate the column with the detergent-containing buffer
prior to sample application. Pay particular attention to pH, which influences the sol-
ubility of the various classes of detergents. Problems may arise when using salt gra-
dient elution if the starting conditions include detergent below its critical micelle
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8 10 12 14 16
Load Sam ple
5
[Buffer B] M
Load sample
Volume (ml)
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