Isoelectric focusing using non-amphoteric buffers in free solution: I. Determination of stable concentration profiles

For large-scale separations of proteins, the use of simple non-amphoteric buffers in free solution and in multicompartment electrolyzers seems promising for industrial applications. The stabilization of a pH profile with this type of buffer requires the strict observation of two conditions: choice of an adequate buffer; stationary profiles of concentrations. During electrolysis in free solution, the ions of the buffer are displaced across the compartments by migration and by diffusion. To keep a stationary composition, the inflow and outflow of all individual ionic species through each compartment must be identical. At high current, diffusion may be neglected against migration and the ionic flows will be identical if the transport number of each ion is constant at each location within the cell. In these conditions, stationary compositions will be independent of the electric current. This condition of constant transport numbers implies the use of profiles of buffer concentrations different from those published up to now. The new equations for these profiles of concentrations are given in the present paper. The constant migration of the ions must be compensated in the end compartments of the isoelectric focusing cell to provide a stable steady state. Two methods are proposed in the literature: the buffer renewal method and the external recycling method (rheoelectrolysis). Here modified buffer renewal method is proposed. Using stationary mass balances, analytical equations are given to calculate the flows and the composition of the solutions to be recycled or added. Using these equations and the profiles of concentrations to keep constant transport numbers, it is demonstrated that only a renewal of the buffers in the end compartments may lead to stable pH profiles and thus to valid conditions of separation.