IN 45 100 010 • AA • 8 / 12
Optimization of washing and elution
Prolonged wash
A continuously decreasing UV signal during washing is an indication of unbound material being washed out (it
may be target substance if it is weakly bound). Washing should be continued until the UV absorbance signal has
reached 0.01-0.02 (“10-20 mAU”). A BabyBio column should be washed with at least 10 CV buffer.
Optimizing elution conditions
Elution can be carried out using a high salt concentration or by altering the pH to change the charge of the
adsorbed protein. A stronger binding may require higher salt concentration for elution.
Step elution
The optimal salt concentration is dependent on purity and recovery requirements as well as properties of the
target protein and the sample. Using a gradient elution gives increased purity than step elution, but step elution
may be necessary to obtain the highest possible concentration of the target protein. In order to optimize the
salt concentration for step elution an initial gradient test run can be carried out to identify a suitable step elution
conditions for purification of the target protein, see Figure 3.
Note: Remember to take the system dead volume into account when comparing the print out of the gradient
and the trace.
Figure 3. Optimization of step gradient elution with salt. A test run with linear gradient elution gives information about suitable salt concentrations to
be used during the step elution.
Conductivity, gradient elution
UV absorbance
Conductivity, step elution
Volume
