Step SMBC Mode
In contrast to Isocratic Mode, the Step Mode uses multiple solvents and establishes four independent zones for binding, washing, elution, and regeneration steps analogous to conventional batch chromatography protocols, but operated in a continuous cycle.
The figure shows a schematic animation of an 8-column Step SMBC configuration running at equilibrium. The main advantage of this method is the ability to achieve high recovery and concentration of target proteins, and it is especially useful when working with dilute samples.
As in Isocratic Mode, “column switching” is actually performed by simultaneously switching all fluid streams one column forward at defined intervals, which has the effect of “moving” the solid phase in the opposite direction of the fluid flow.
Fluid streams, represented by colored lines and arrows, indicate the direction of fluid flow. The columns are fixed in place and connected in series to form a continuous loop. In Step Mode, four independent zones having different buffer conditions are established by closing connections between them.
In the Wash zone, the inlet stream provides a slightly higher imidazole concentration to remove untagged proteins and low affinity contaminants. In the Elute zone, the Desorbent stream provides a high imidazole concentration to elute the target protein as Extract.
In the Regeneration zone, a low imidazole concentration re-equilibrates the columns to prepare them for the next binding cycle.