The present invention generally relates to devices and methods for the separation of species, including biological species. Some embodiments involve the use of free flow isoelectric focusing (FF-IEF) devices in the separation of a mixture of species. Various device configurations and/or features may enhance the performance of the devices, providing faster and more efficient devices and methods for separating mixtures of species with high resolution. In some embodiments, separation of a mixture of species may be achieved with high resolution and with short sample residence times. Such devices may provide simplified, inexpensive, and optionally disposable devices for the separation of chemical and biological species and may optionally be integrated with orthogonal separation techniques, such as SDS-PAGE or capillary electrophoresis.