A microfluidic free-flow isoelectric focusing glass chip for separation of proteins is described. Free-flow isoelectric focusing is demonstrated with a set of fluorescent standards covering a wide range of isoelectric points from pH 3 to 10 as well as the protein HSA. With respect to an earlier developed device, an improved microfluidic FFE chip was developed. The improvements included the usage of multiple sheath flows and the introduction of preseparated ampholytes. Preseparated ampholytes are commonly used in large-scale conventional free-flow isoelectric focusing instruments but have not been used in micromachined devices yet. Furthermore, the channel depth was further decreased. These adaptations led to a higher separation resolution and peak capacity, which were not achieved with previously published free-flow isoelectric focusing chips. An almost linear pH gradient ranging from pH 2.5 to 11.5 between 1.2 and 2 mm wide was generated. Seven isoelectric focusing markers were successfully and clearly separated within a residence time of 2.5 s and an electrical field of 20 V mm-1. Experiments with pI markers proved that the device is fully capable of separating analytes with a minimum difference in isoelectric point of ∆(pI) = 0.4. Furthermore, the results indicate that even a better resolution can be achieved. The theoretical minimum difference in isoelectric point is ∆(pI) = 0.23 resulting in a peak capacity of 29 peaks within 1.8 mm. This is an 8-fold increase in peak capacity to previously published results. The focusing of pI markers led to an increase in concentration by factor 20 and higher. Further improvement in terms of resolution seems possible, for which we envisage that the influence of electroosmotic flow has to be further reduced. The performance of the microfluidic free-flow isoelectric focusing device will enable new applications, as this device might be used in clinical analysis where often low sample volumes are available and fast separation times are essential.