Protein separation using free‐flow electrophoresis microchip etched in a single step

A one‐step etching method was developed to fabricate glass free‐flow electrophoresis microchips with a rectangle separation microchamber (42 mm‐long, 23 mm‐wide and 28 μm‐deep), in which two glass bridges (0.5 mm‐wide) were made simultaneously to prevent bubbles formed by electrolysis near the Pt electrode from entering the separation chamber. By microchip free‐flow zone electrophoresis, with 200 V voltage applied, the baseline separation of three FITC labeled proteins, ribonuclease B, myoglobin and β‐lactoglobulin, was achieved, with resolution over 1.78. Furthermore, with 2.5 mM Na₂SO₄ added into the electrode buffer to form higher electrical field strength across separation microchamber than electrode compartments, similar resolution of samples was achieved with the applied voltage decreased to 75 V, which could obviously decrease Joule heat during continuous separation. All these results demonstrate that the free‐flow electrophoresis microchip fabricated by one‐step etching method is suitable for the continuous separation of proteins, which might become an effective pre‐fractionation method for proteome study.