Methods, apparatus and systems are provided for efficient separation of various small and large molecules, especially biomolecules such as proteins, nucleic acids and polysaccharides. In particular, an electrophoresis apparatus is employed in a wide bore electrophoresis of samples in larger amounts than those in conventional capillary electrophoresis. The electrophoresis apparatus comprises: an electrophoresis chamber comprising a cathode, an anode and a housing; and a separation chamber positioned within the housing and comprising an inlet end, an outlet end, and one or more cooling capillaries positioned inside the separation chamber such that the longitudinal axis of at least one of the cooling capillaries is parallel to the direction of electric current flow from the anode to the cathode, wherein the end of the cooling capillary (or capillaries) is adapted to be coupled to a cooling device that allows cooling medium to pass through the cooling capillary. Electrophoresis systems incorporated with cooling mechanism can utilize wide bore separation tubes and tolerate much higher electric current than that in conventional CE systems. The apparatus can be used to separate and analyze a wide variety of molecules in an automatic, high throughput, and high performance manner. By using the apparatus, methods and systems, large amounts of biomolecules such as proteins can be separated with high resolution and in their native states. Samples separated by this system should be sufficient for many post-separation analyses by other techniques, such as mass spectrometry and crystallography. These features are especially advantageous in building and mining databases of biomolecules in functional genomics, structural genomics and proteomics in the post-genome era.