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Optimisation of reversed phase chromatography experiments
Optimizing running pH is perhaps the most effective way of obtaining satisfactory results with protein or peptide samples. The choice of RPC medium (especially silica- or polymer-based ) and ion pairing agent also influences selectivity. Optimizing 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 RPC medium with a suitable bead size (5 - 12 mm for high resolution experiments) is employed, the flow rate is less important in gradient-eluted experiments.
Based on the above, the following optimisation measures are recommended in order of priority:
- Select the type of RPC medium that provides best resolution under standard conditions ( e.g. ACN, TFA).
- Scout for the running pH that provides best resolution.
- If necessary scout for a suitable ion pairing agent.
- Select the steepest gradient that provides acceptable results.
- Scout for the flow rate that provides the narrowest peaks.
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