 |
Optimisation of ion exchange chromatography experiments
Optimizing running pH is by far the most effective way of obtaining satisfactory results with protein or peptide samples. The choice of ion exchanger type (anion or cation exchanger) may also influence selectivity quite considerably.
Optimising the gradient slope only influences distances between the peaks and will not change their elution order.
Using an optimal flow rate is important in isocratic experiments in order to keep zone broadening at a minimum. Provided an IEX medium with a suitable bead size (10 - 30 mm for high resolution experiments) is employed, the flow rate is less important in gradient-eluted experiments.
Based on this, the following optimization measures are recommended in order of priority:
- Select the type of ion exchanger that provides best resolution under standard conditions ( e.g. pH 5,0 for cation exchangers and pH 8 for anion exchangers).
- Scout for the running pH that provides best resolution.
- Select the steepest gradient that provides acceptable results.
- Scout for the flow rate that provides the narrowest peaks.
|
|
