Using channel depth to isolate and control flow in a micro free-flow electrophoresis device.

A multiple-depth micro free-flow electrophoresis chip (mu-FFE) has been fabricated with a 20-microm-deep separation channel and 78-microm-deep electrode channels. Due to the difference in channel heights, the linear velocity of buffer in the electrode channels is approximately 15 times that of the buffer in the separation channel. Previous mu-FFE devices have been limited by electrolysis product formation at the electrodes. These electrolysis products, manifested as bubbles, decreased the electric field and disrupted the buffer flow profile, limiting performance and preventing continuous operation. Using channel depth to control buffer flow over the electrodes and in the separation channel effectively removes electrolysis products, allowing continuous operation. The linear velocities in the channels were confirmed using particle velocimetry and compared well with values predicted using lubrication theory. A separation potential of 645 V could be applied before significant Joule heating was observed. This corresponded to an electric field of 586 V/cm in the separation channel, a 4-fold increase over our previous design. A separation of fluorescent standards was demonstrated using the new mu-FFE device. Resolution increased by a factor of 1.3 over our previous design, even when operated under similar conditions, suggesting that effective removal of electrolysis products is more important than originally thought.