Microfluidic and lab-on-a-chip devices have gained widespread attendance in separation sciences since they are characterized by advantageous properties like a gain in throughput, time, cost, performance and analyte amount. Beyond these advantages, microfluidic devices open access to completely new separation or particle sorting phenomena far from thermal equilibrium based on Brownian motion and non linear dynamics induced through microstructuring. Here, we demonstrate the migration phenomena of absolute negative mobility (ANM) for colloidal particles in structured microfluidic devices and discuss its application for separation of particles and biomolecules, such as DNA.