For a detailed discussion on zone broadening and resolution in general, refer to Basic Principles in Gel Filtration: 5. Resolution in gel filtration.
In HIC, selectivity (distance between and order of eluted peaks) depends on the hydrophobic properties of the individual sample components and on the type of ligand chosen.
Protein and peptide hydrophobicities, however are not easily influenced by any practical means, a fact that leaves you with the choice of HIC ligand as the most useful means of influencing 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.
Although best resolution is theoretically obtained in isocratic mode, gradient mode is the most frequently used elution technique in HIC.
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. |
