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In this research, we present a chip based capillary electrochromatography (CEC) carrying out a highly efficient separation of biomolecules through gradient hydrophobic stationary phase (GHSP) provided by multiwalled carbon nanotubes (MWCNTs) nanocolumns. 4-azidoaniline was employed for the surface modification of the MWCNTs to form a gradient stationary phase by a photochemical reaction through a gradient light filter. This device was used for separating proteins (20 Â¿M FITC-BSA and FITC-Cytochrome C) with different hydrophobic characteristics, and MALDI-TOF-MS detection was employed to verify the separation efficiency in this MWCNTs nanocolumn. The best separation resolution of the FITC-BSA column efficiency was 2.5 Ã 104 (plats/m).