Bio-Rad ENrich™ High-Resolution Ion Exchange Columns - Manual (Page 9)

ENrich Instruction Manual 5
2.3 Sample Preparation
Proper adjustment of the sample pH and ionic strength is critical
for consistent and reproducible chromatography. For best
results, the sample should be exchanged into the start buffer or
diluted to the start buffer’s concentration. Buffer exchange can
be performed using Bio-Spin
® 6 and Micro Bio-Spin ™ 6 columns,
Econo-Pac ® 10DG desalting columns, Bio-Gel ® P-6DG gel, or
the Bio-Scale ™ Mini desalting cartridges with Bio-Gel P-6. The
choice of product depends on sample volume. Centrifuge or filter
the sample
(0.2–0.45 μm filter) to remove particulates. Application of turbid
or lipid-containing samples may reduce the column lifetime.
Sample Load
The maximum sample load for each column is shown in
Table 1. This amount may vary somewhat depending on the
actual sample composition. We do not recommend overloading
the column as both resolution and column lifetime will decrease.
For larger loads, perform several chromatographic runs
with a reduced load. Ideally, samples should be bound in a
concentrated zone at the top of the column. Higher sample
loads produce a broad application zone in which components
with less charge are displaced by more highly charged
components. This may result in a shift of certain peaks to an
earlier elution position in the gradient. The recommended sample
load is approximately 20% of the maximum.
2.4 Elution Conditions
Separations by ion exchange are typically accomplished by
increasing the salt concentration of the eluent either as a step or
as a continuous gradient. Sodium chloride (NaCl) and potassium
chloride (KCl) are the most common elution salts. For many
separations, varying the pH of the elution buffer in addition to its
salt concentration may be advantageous.