Microchip free flow isoelectric focusing for protein prefractionation using monolith with immobilized pH gradient

Microchip free flow IEF (μFFIEF) with monolithic IPG was proposed for protein prefractionation. The monolithic materials were first prepared by UV irradiation in a microchamber of 43 mm length, 23 mm width, and 38 μm depth. Carrier ampholytes (CAs) were further immobilized on the monolith by chemical bonding, to form a stable pH gradient. By such a technique, the continuous introduction of CAs in traditional μFFIEF could be avoided, not only to decrease the operation cost, but also to avoid the interference of CAs for the further protein identification by MS/MS. With a fluorescence microscope as the detector, under the optimal conditions, the separation of FITC labeled β-lactoglobulin and carbonic anhydrase, with 0.9 unit difference on pI, was achieved with good reproducibility. The experimental results demonstrated that under the experimental conditions we applied, the separation mechanism of μFFIEF with M-IPG materials might be the cooperative effects of IEF and CZE, and the former one plays a predominant role.