Bio-Rad ENrich™ High-Resolution Ion Exchange Columns - Manual (Page 10)

6 ENrich Instruction Manual
Gradient Volumes and Salt Concentrations
As a starting point for developing a separation, we recommend using
the ENrich column with a simple linear gradient over 15 column volumes
(15 ml).
Suggested protocol for ENrich 5 x 50 ion exchange columns:
• Use a flow rate of 1.0 ml/min. Equilibrate the column with 5 column
volumes (5 ml) of equilibration buffer. Then apply the sample.
Following sample application, wash unbound proteins from the
column with 3 column volumes (3 ml) of equilibration buffer A. For
elution, use a gradient volume of 15 column volumes (15 ml) to an
NaCl concentration of 0.5 M.
• Follow this segment of the gradient by stepping the salt
concentration to 1.0 M (100% B) and then hold at 1.0 M for 3 ml
before
re-equilibrating the column with 3 ml of start buffer A. This gradient
is shown schematically in Figure 1. When an initial separation has
been performed and the elution position of the protein of interest
determined, the gradient composition and volume are adjusted to
achieve maximum resolution. Normally, a gradient volume of 10 to
20 ml per ml of column bed volume 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 resolution
but peak volumes are larger.
Fig. 1. Schematic gradient for separation on an ENrich IEX column. Volume,
column volume.