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Optimisation of hydrophobic interaction chromatography experiments
Choosing the optimal ligand is the most effective way of obtaining satisfactory results with protein or peptide samples.
Optimizing the gradient slope only influences the 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 a HIC 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 HIC ligand that provides best resolution under standard conditions ( e.g. Buffer A: 1.2 M; pH~ 7,0; room temperature).
- Select the steepest gradient that provides acceptable results.
- Scout for the flow rate that provides the narrowest peaks.
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