Microchip free flow isoelectric focusing with immobilized pH gradient on monolithic materials

Microchip free flow electrophoresis (μFFE) is a significant microscale technique for the continuous pre-fractionation and the preparation of valuable biological samples. In our recent work, monolithic polyacylamide (PAM) materials were polymerized in microchamber by ultraviolet (UV) initiated polymerization. With the further immobilization of a stable pH gradient on the monolith, a novel microchip free flow isoelectric focusing (μFF-IEF) with monolithic immobilized pH gradient (M-IPG) materials was developed; by which fluorescein-5-isothio-cyanate (FITC) labeled glycin, pro line and lysine, with a minimum pI difference of 0. 33 units, were well separated with a resolution higher than that performed by traditional μFF-IEF. Our experimental results demonstrate that by μFF-IEF with M-IPG, not only the interference of mobile carrier ampholytes in buffer; usually indispensable in traditional μFF-IEF, on the further separation by other techniques and the identification by mass spectrometry (MS) could be avoided; but also the improved resolution and detection sensitivity could be obtained compared with traditional μFF-IEF. Therefore, such a novel technique might be promising in microscale consecutive separation and preparation of samples.