A New Elution System for Isoelectric Focusing in Sucrose Gradients

Abstract

This chapter describes a new elution system for isoelectric focusing in sucrose gradients. Preparative isoelectric focusing in sucrose gradient is a separation method with a very high resolving power. This resolving power is only at a maximum so long as the electric field over the gradient is maintained as this field greatly counteracts the diffusion occurring in the focused protein and Ampholine bands. It is found that when the isoelectric focusing has finished, the sucrose gradient is eluted by allowing the pump to convey the most dense sucrose solution through the inlet, and at the same time applying suction with pump at the outlet. The pump must apply suction at higher velocity than that at which pump is pumping. In this way a very stable contact surface on a level with the edge of the outlet is obtained between dense liquid coming from the bottom, and less dense liquid that comes from above. The liquid from above will all the time in the contact surface meet a denser liquid, and from the contact surface be conveyed to the outlet and on to a fraction collector