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Resolution in reversed phase chromatography
For a detailed discussion on zone broadening and resolution in general, see Basic Principles in Gel Filtration: 5 Resoution in gelfiltration
In RPC, selectivity (distance between and order of eluted peaks) depends on the hydrophobic properties of the individual sample components.
With proteins and peptides the hydrophobic properties are influenced by the running pH.
Ion pairing agents influence the hydrophobic properties by "blocking" either positive or negative charges and thus selectivity.
Gradients will influence peak-spacing but not elution order.
Efficiency (counteraction of zone broadening) depends on bead size, quality of the packed bed and flow rate in isocratic and gradient modes.
Best resolution is theoretically obtained in isocratic mode.
For practical reasons, however, gradient mode is the most frequently used elution technique.
With large volumes of complex samples, the first purification step often aims at concentrating the sample and removing the bulk of the contaminants by a capture step. Here the step elution mode is to be preferred because of its high loading capacity and that high flow rates can be applied.
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