Field gradient focusing: A novel method for protein separation

Equilibrium gradient techniques constitute a class of separation methods that combine the steps of separation and concentration by using a gradient in one or more counteracting forces to create a stable equilibrium point at which a protein can focus. Different proteins focus at different equilibrium points, creating a steady-state distribution of isolated proteins. Equilibrium gradient techniques can be adapted to a specific separation by choosing appropriate counteracting forces based on differences in the physical properties of the proteins involved. Zone electric field gradient focusing (FGF) is a new addition to this class of separation techniques with the unique property of using a gradient in the electric field to establish an equilibrium point instead of using a gradient in the velocity or pH. This paper presents two mathematical models which can be used to predict the steady-state concentration profiles obtained by zone electric field gradient focusing. The first model applies only at very low protein concentrations where nonlinear effects can be ignored, e.g., less than 1 mg/mL, but it can be solved analytically and is useful in understanding the basic principles engendered in the method. The second model applies at all concentrations and allows for variations in the electric field strength where the protein focuses, but requires numerical solution. The design of an experimental device is also reported, as well as the results of two experiments: (1) the focusing of the protein hemoglobin from a dilute solution and (2) the separation of different oxidation states of the protein myoglobin.