Micro free-flow IEF enhanced by active cooling and functionalized gels

Rapid free-flow IEF is achieved in a microfluidic device by separating the electrodes from the focusing region with porous buffer regions. Moving the electrodes outside enables the use of large electric fields without the detrimental effects of bubble formation in the active region. The anode and cathode porous buffer regions, which are formed by acrylamide functionalized with immobilized pH groups, allow ion transport while providing buffering capacity. Thermoelectric cooling mitigates the effects of Joule heating on sample focusing at high field strengths (similar to 500 V/cm). This localized cooling was observed to increase device performance. Rapid focusing of low-molecular-weight pI markers and Protein G-mouse IgG complexes demonstrate the versatility of the technique. Simulations provide insight into and predict device performance based on a well-defined sample composition.