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The possibility in fast separation of intact proteins was investigated by sub-2μm nonporous silica (NPS)-based packing materials.Chromatographic cake (or named caky column) was a novel fast separation tool having diameter to be much greater than its thickness or column length.630nm and lμm NPS microspheres were synthesized respectively, further modified with PEG600.The NPS-based HIC packings were laterally packed into different sizes of chromatographic cakes (1~10 mm ×10mmI.D.), then applied for the fast separation of intact proteins on conventional HPLC equipment in 2 minutes, even seconds.The column capacity, permeability, and efficiency were evaluated through dynamic experiments, it was concluded that chromatographic cake packing with sub-2μm NPS particles had shown high efficiency and good permeability.The efficiencies of columns packed with sub-2μm NPS particles and commercially 2.5μm butyl-NPR particles were compared in hydrophobic interaction chromatography using intact proteins as analytes.Under high flow rate(10ml/min), the mixture of seven intact proteins was separated completely in 48s and 12s for buffer exchange, system re-equilibrium, and column re-life with an acceptable backpressure(<20MPa).Thus continuous five separations of seven intact proteins could be carried out in five minutes.