(185e) Single-Stream Free-Flow Isoelectric Focusing with pH Gradients Induced by Water Splitting in Bipolar Membranes-Integrated Microfluidic Devices

In this abstract we report a novel microfluidic technology to generate stable pH gradients, using integrated bipolar membrane (BPM) components, for free-flow isoelectric focusing in a single-stream format.  Microfluidic free-flow isoelectric focusing (FF-IEF) is a versatile and rapid preparative separation and analysis techniques in chemistry and biology. However, stable pH gradient formation is one of the practical challenges. Several techniques have been developed to produce pH gradients, including introduction of acid and base solutions and electrolysis of water. These methods, however, require either multiple input streams for specialized sheath flow or additional setup to prevent the effect of bubble generation. We have developed a simple, single-stream microfluidic pH actuation technique that directly dopes the upstream analyte flow with proton and hydroxide ions and forms a stable pH gradient in a downstream separation channel. The excess H⁺ and OH^- ions are generated by electric field-enhanced water dissociation with two sets of BPM.  Under a reverse voltage of 4.5 V at which the anode connects to the anion exchange side, the high electric field at the BPM junction directly splits water into H⁺ and OH^- ions. Without any electron-transfer reaction to generate gas, the process produces H⁺ and OH^- ions more efficiently than electrolysis. Furthermore, the injection of H⁺/OH^- ions does not alter the total analyte volume and hence allows for pH adjustment without diluting analyte concentration. The microfluidic pH actuation was designed to offer efficient ion injection while insensitive to flow rate. Separation of a mixture of fluorescent pI markers has been successfully demonstrated by using the FF-IEF device under a flow rate of 10 μl/min and a separation electric field of 180 V/cm. The simple, high-speed FF-IEF separation makes the device applicable to proteomic analysis and flexible for further integration to downstream analysis tools, such as mass spectroscopy.