Expanded bed adsorption (EBA) is a practical method for the separation of nanoparticulates. In order to analysis the local hydrodynamic and adsorption behavior of nanoparticle (NP)-based biological feedstock, a modified Nano Biotechnology Group EBA column with a 26-mm inner diameter was used to withdraw liquid from different axial positions of the column. Fabricated egg albumin (EA) NPs with an average size of 70 nm were employed as a model system and viral size/charge mimic to assess the relationship between hydrodynamic and adsorption performance of NPs at the different column regions. The effects of influential factors, including flow velocity and initial concentration of NPs, on NP hydrodynamic behavior and adsorption kinetics along the bed height were investigated. NP hydrodynamic studies confirmed that non-uniform behavior dominated the system and a decreasing trend of liquid mixing/dispersion with increase of bed height was observed in this column. The results demonstrated an increase in the mixing/dispersion at certain bed heights with the increase in both the velocity and feed initial concentration. Breakthrough curves were measured at various column points to determine the adsorption performance [dynamic binding capacity (DBC) and yield] in different bed positions/zones. Yield and DBC of NPs were improved along the bed height, whereas liquid velocity had the opposite effect. Increasing the initial concentration of NPs enhanced only the DBC. Separation of EA NPs under optimal conditions was 87 %, which is an excellent result for a one-pass frontal chromatography method. KeywordsEBA chromatography Á Modified NBG column Á EA NPs Á Axial hydrodynamic behavior Á Axial adsorption performance List of symbols B o Bodenstein number C Exit concentration of NPs in bed effluent (kg m -3 ) C o Initial concentration of NPs (kg m -3 ) C 9Exit concentration that has left the 9-cm sample port and is measured at this port (kg m -3 ) C 12Exit concentration that has left the 12-cm sample port and is measured at this port (kg m -3 )Volume of feed loaded onto the column until C 9 reaches a certain degree of NP breakthrough (m 3 ) V f,12Volume of feed loaded onto the column until C 12 reaches a certain degree of NP breakthrough (m 3 ) V l Liquid volume above the particles in the sampling syringe (m 3 ) V L,0-9 Liquid volume present in the bed voidage for the 0-to 9-cm column zone (m 3 ) V L,0-12 Liquid volume present in the bed voidage for the 0-to 12-cm column zone (m 3 ) V s,0-9 Settled volume of adsorbent in the 0-to 9-cm column zone (m 3 ) V s,9-12 Settled volume of adsorbent in the 9-to 12-cm column zone (m 3 ) V o Volume of the particles in the sampling syringe (m 3 )Greek letters e Bed voidage e i Local bed voidage at ith layer of bed d Variance (s)