Particle-loaded hollow-fiber membrane adsorbers for lysozyme separation

The separation of lysozyme (LZ), a valuable enzyme naturally present in chicken egg white, was carried out using a new type of ion exchange hollow-fiber membranes. Functionalities were incorporated into the polymeric membranes by dispersing ion-exchange resins (IERs) in a microporous structure formed by phase inversion. The obtained hollow-fibers were composed of ion-exchange particles surrounded by a polymeric matrix and possessed both high static and dynamic adsorption capacities of more than 60 mg/ml membrane. The hollow-fiber membrane adsorbers were connected in series with different numbers of fibers thereby increasing the effective thickness and the protein residence time within the module. By choosing appropriate operation conditions, the membranes adsorbed solely LZ from fresh chicken egg white (eventually also the minor component avidin), whereas the adsorption of ovalbumin, ovotransferrin, and other low isoelectric point proteins was negligible. An average separation factor for LZ of about 150 was calculated by numerical integration of the protein concentrations in the elution curve during the filtration run. The effect of the filtration flow rate, protein concentration and ionic strength on the membrane's performance was investigated to determine the optimum operation parameters.     

Dextran-grafted cation exchanger based on superporous agarose gel: Adsorption isotherms,uptake kinetics and dynamic protein adsorption performance

A novel chromatographic medium for high-capacity protein adsorption was fabricated by grafting dextran (40 kDa) onto the pore surfaces of superporous agarose (SA) beads. The bead was denoted as D-SA. D-SA, SA and homogeneous agarose (HA) beads were modified with sulfopropyl (SP) group to prepare cation exchangers, and the adsorption and uptake of lysozyme on all three cation-exchange chromatographic beads (SP-HA, SP-SA and SP-D-SA) were investigated at salt concentrations of 6–50 mmol/L. Static adsorption experiments showed that the adsorption capacity of SP-D-SA (2.24 mmol/g) was 78% higher than that of SP-SA (1.26 mmol/g) and 54% higher than that of SP-HA (1.45 mmol/g) at a salt concentration of 6 mmol/L. Moreover, salt concentration had less influence on the adsorption capacity and dissociation constant of SP-D-SA than it did on SP-HA, suggesting that dextran-grafted superporous bead is a more potent architecture for chromatographic beads. In the dynamic uptake of lysozyme to the three cation-exchange beads, the D_e/D₀ (the ratio of effective pore diffusivity to free solution diffusivity) values of 1.6–2.0 were obtained in SA-D-SA, indicating that effective pore diffusivities of SP-D-SA were about two times higher than free solution diffusivity for lysozyme. At 6 mmol/L NaCl, the D_e value in SA-D-SA (22.0 × 10⁻¹¹ m²/s) was 14.4-fold greater than that in SP-HA. Due to the superior uptake kinetics in SA-D-SA, the highest dynamic binding capacity (DBC) and adsorption efficiency (the ratio of DBC to static adsorption capacity) was likewise found in SP-D-SA. It is thus confirmed that SP-D-SA has combined the advantages of superporous matrix structure and drafted ligand chemistry in mass transport and offers a new opportunity for the development of high-performance protein chromatography.     

Functionalized anodic aluminum oxide (AAO) membranes for affinity protein separation

An ideal affinity membrane should own well uniformities. However, most existing microporous membranes used as affinity matrices generally have wide pore size distribution and some thickness variation. In this paper, chitosan (CS)–anodic aluminum oxide (AAO) composite membrane with excellent uniformities, such as narrow pore size and porosity distribution, as well as uniform membrane thickness, was fabricated, for the first time. Cu²⁺-attached affinity membrane was obtained by immobilizing Cu²⁺ on the CS–AAO membrane. The contents of CS and Cu²⁺ of affinity membranes were ∼49.7 and 27.15 mg/g membrane, respectively. The Cu²⁺-attached affinity membranes were used to recover a model protein, hemoglobin, from hemoglobin–phosphate solution (batch manner) and from the hemolysate (dynamic manner). The protein adsorption indicated that the adsorption capacity of hemoglobin was ∼17.5 mg/g membrane, and the adsorption isotherm fitted the Freundlich model well. Elution of protein showed desorption ratio was up to 91.2% using 0.5 M imidazole aqueous solution as the desorption agent. The adsorption capacities of all the tested affinity membranes did not significantly change during the repeated adsorption–desorption operations. The result of dynamic experiment showed Cu²⁺-attached affinity membranes can well purify the hemoglobin from the red cell lysate.     

Effect of ionic strength and protein concentration on the transport of proteins through chitosan/polystyrene sulfonate multilayer membrane

The influence of ionic strength and protein concentration on the transport of bovine serum albumin (BSA), ovalbumin and lysozyme through chitosan (CHI)/polystyrenesulfonate (PSS) multilayers on polyether sulfone supports are investigated under ultrafiltration conditions. The percentage transmission and flux of BSA, ovalbumin and lysozyme were found to increase with increase in salt concentration in the protein. The percentage transmission of BSA through 9 bilayer membrane was found to increase from 5.3 to 115.6 when the salt concentration was varied from 0 to 1 M. It was observed that 0.1 M NaCl in BSA solution is capable of permeating all the BSA. When the salt concentration in BSA was further increased, a negative solute rejection (solute enrichment in permeate) was found to take place. With 9 bilayer membrane, the percentage transmission of ovalbumin was found to increase from 23.3 to 125.8 when the salt concentration in protein was increased from 0 to 0.05 M. The effect of protein concentration on protein transport is studied taking BSA as a model protein. BSA was rejected by the multilayer membrane at all the studied concentrations (0.25, 0.5, 1 and 2 mg/ml). With increase in feed concentration, maximum rejection of protein occurred at higher number of CHI/PSS bilayers. BSA solution flux was found to decrease with an increase in BSA concentration. This study indicates that it is possible to fine tune the transport properties of proteins through multilayer membranes by varying the concentration and ionic strength of protein solutions.     

Enzyme capturing and concentration with mixed matrix membrane adsorbers

This study reports the use of membrane adsorbers for lysozyme (LZ) capturing and concentration: the membrane adsorbers are prepared by incorporation of ion exchange resins into an EVAL porous matrix. The mixed matrix membrane (MMM) adsorber possesses an open and interconnected porous structure with a large ion exchange surface available for enzyme adsorption. The adsorptive membrane features both a high static as well as a high dynamic LZ adsorption capacity. The measured LZ adsorption isotherm is of the Langmuir type, with a maximum adsorption capacity of 147 mg LZ/ml membrane. Dynamic LZ adsorption capacity at a flux of 25 l/h/m² was 63 mg LZ/ml membrane, which is significantly higher than the equivalent commercial membrane Sartobind C. Since the kinetics of desorption processes are faster than the kinetics of adsorption processes, the performance can be improved by exerting the desorption processes at higher fluxes than the adsorption processes. The MMM can be reused in multiple adsorption/desorption cycles maintaining the high binding capacity performance. Fluorescence spectra of the LZ after adsorption and elution were similar to native LZ. This is confirmed by activity tests showing that the activity of LZ was maintained after an adsorption and desorption cycle.     

ANTIFOULING PROPERTIES OF POLYVINYL CHLORIDE) MEMBRANES MODIFIED BY AMPHIPHILIC COPOLYMERS P(MMA-A-MAA)

Three well-defined diblock copolymers of poly(methyl methacrylate-b-methacrylic acid)(P(MMA-b-MAA))were synthesized using atom transfer radical polymerization method and varying poly(methacrylic acid)(PMAA)chain lengths. These copolymers were blended with PVC to fabricate porous membranes via phase inversion process.Membrane morphologies were observed by scanning electron microscopy(SEM),and chemical composition changes of the membrane surfaces were measured by X-ray photoelectron spectroscopy(XPS).Static and dynamic protein adsorption experiments were used to evaluate antifouling properties of the blend membranes.It was found that,the blend membranes containing longer PMAA arm length showed lower static protein adsorption,higher water permeation flux and better protein solution flux recovery.     

Whey protein fouling of large pore-size ceramic microfiltration membranes at small cross-flow velocity

Microfiltration of whey protein solutions by tubular ceramic membranes, under constant cross-flow and trans-membrane pressure, with periodic backwashing, is investigated using a fully instrumented pilot unit. Relatively large nominal membrane pore size (0.8 μm) insures very high protein transmission, which is desirable in applications such as microbial load reduction. In the first of a sequence of three filtration-backwashing cycles, irreversible and reversible fouling are identified, over the tested pressure range of 5–17.5 psi. Early in the first cycle, especially at the higher pressures, a pore constriction/blocking mechanism appears to be responsible for the irreversible fouling. In the other two cycles only the reversible fouling is significant, possibly due to some kind of protein layer formation on the membrane surface. The permeate flux level tends to increase by increasing trans-membrane pressure up to a near-optimum value of 10 psi, beyond which pressure has a negative effect. This interesting trend is attributed to the interplay of cross-flow velocity, which tends to reduce fouling by promoting re-suspension and breakage of colloidal protein agglomerates, with the trans-membrane pressure (and related flux) which leads to protein layer formation on the membrane and may impose compressive stresses, thereby increasing its resistance to permeation.     

Resistance-in-series analysis in cross-flow ultrafiltration of fermentation broths of Bacillus subtilis culture

Quantitative analysis of various resistances that lead to flux decline during cross-flow ultrafiltration (UF) of the fermentation broth of Bacillus subtilis ATCC (American Type Culture Collection) 21332 culture was studied. Polyethersulfone membrane with a molecular weight cut-off (MWCO) of 100 kDa was used. Prior to cross-flow UF, the broth was treated by acid precipitation (pH 4.0) and centrifugation, and the precipitate was re-dissolved in NaOH solution. Experiments were performed at a feed pH of 7.0, a feed surfactin concentration of 1.48 g L⁻¹, and a cross-flow velocity of 0.32 m s⁻¹ but at different transmembrane pressures (ΔP, 20–100 kPa). The resistance-in-series model was used to analyze the flux behavior, which involves the resistances of membrane itself and cake as well as those due to adsorption and solute concentration polarization. It was shown that the resistance due to solute concentration polarization and of membrane dominated under the conditions examined. The resistances due to cake formation and solute adsorption were comparable, and their sum contributed below 20% of the overall resistance.     

Surface properties of Reactive Yellow 2 immobilised pHEMA and HEMA/chitosan membranes: characterisation of their selectivity to different proteins

Poly(hydroxyethyl methacrylate), pHEMA, and a composite pHEMA/chitosan networks were synthesized in the membrane form via UV initiated photo-polymerisation in the presence of an initiator ,′-azoisobutyronitrile. Reactive Yellow 2 (RY-2) was covalently immobilised as a dye–ligand onto both membranes. The polarity and surface energy of the investigated membranes were determined by contact angle measurement. The incorporation of chitosan in the pHEMA networks produced more hydrophilic surface, as indicated by contact angle analysis. The binding characteristics of lysozyme, γ-globulins, human serum albumin (HSA) and bovine serum albumin (BSA) to pHEMA-RY-2 and pHEMA/chitosan-RY-2 affinity membranes have been investigated from aqueous solution and their dye–ligand free forms were used as control systems. When chitosan was incorporated in the pHEMA network as a cationic polymer led to higher adsorption capacity for the lysozyme. Selective adsorption behaviour was also observed in the case of pHEMA/chitosan-RY-2 membrane for the lysozyme. The non-specific adsorptions of the lysozyme on the pHEMA and pHEMA/chitosan membranes were about 1.9 and 7.2 mg/ml, respectively. These were negligible for all others investigated proteins. The lysozyme adsorption data was analysed using the first-order and the second-order models. The first-order equation in both affinity membrane systems is the most appropriate equation to predict the adsorption capacities of the adsorbents. The adsorption isotherms well fitted the combined Langmuir–Freundlich model. A theoretical analysis has been conducted to estimate the thermodynamic contributions (changes in enthalpy, entropy and Gibbs free energy) for the adsorption of lysozyme to both dye–ligand immobilised membranes. The adsorption capacities of both dye–ligand immobilised membranes increased with increasing the temperature while decreased with increasing the NaCl concentration. Both affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated separation–elution cycles.     

Preparation of high-capacity,weak anion-exchange membranes for protein separations using surface-initiated atom transfer radical polymerization

This contribution describes a method to prepare high-capacity anion-exchange membranes for chromatographic bioseparations. Surface-initiated atom transfer radical polymerization was used to graft poly(2-dimethylaminoethyl methacrylate) (poly(DMAEMA)) nanolayers from the pore surfaces of commercially available regenerated cellulose membranes. Initial measurements were made to determine the thickness evolution of the poly(DMAEMA) nanolayers, using a model flat substrate designed to mimic the three-dimensional nature of initiator incorporation into the membrane. Thereafter, polymerization time was used as the independent variable to control the mass of polymer grafted from the membrane surfaces and, thus, the protein binding capacity. ATR-FTIR, AFM, and SEM were used to characterize changes in the chemical functionality, surface topography, and pore morphology of membranes as a result of modification. Bovine serum albumin was used to evaluate the static protein binding capacity of poly(DMAEMA)-modified membranes. Maximum static binding capacities increased with increasing polymerization time in a linear fashion for short polymerization times (<6 h). For longer polymerization times, capacity increased non-linearly, eventually reaching a plateau value of 66.3 mg/mL.     

Chitosan/poly(tetrafluoroethylene) composite membranes using in pervaporation dehydration processes

Chitosan/PTFE composite membranes were prepared from casting a γ-(glycidyloxypropyl)trimethoxysilane (GPTMS)-containing chitosan solution on poly(styrene sulfuric acid) grafted expended poly(tetrafluoroethylene) film surface. The adhesion between the chitosan skin layer and the PTFE substrate was pretty good to warrant the high performance of chitosan/PTFE composite membranes using in pervaporation dehydration processes on isopropanol. The chitosan/PTFE membrane exhibited a permeation flux of 1730 g/m² h and a separation factor of 775 at 70 °C on pervaporation dehydration of a 70 wt% isopropanol aqueous solution. The membrane also survived after a long-term operation test in 45 days.     

Anti-trade-off in dehydration of ethanol by novel PVA/APTEOS hybrid membranes

Novel organic–inorganic hybrid membranes were prepared through sol–gel reaction of poly(vinyl alcohol) (PVA) with γ-aminopropyl-triethoxysilane (APTEOS) for pervaporation (PV) separation of ethanol/water mixtures. The membranes were characterized by FTIR, EDX, WXRD and PALS. The amorphous region of the hybrid membranes increased with increasing APTEOS content, and both the free volume and the hydrophilicity of the hybrid membranes increased when APTEOS content was less than 5 wt%. The swelling degree of the hybrid membranes has been restrained in an aqueous solution owing to the formation of hydrogen and covalent bonds in the membrane matrix. Permeation flux increased remarkably with APTEOS content increasing, and water permselectivity increased at the same time, the trade-off between the permeation flux and water permselectivity of the hybrid membranes was broken. The sorption selectivity increased with increasing temperature, and decreased with increasing water content. In addition, the diffusion selectivity and diffusion coefficient of the permeants through the hybrid membranes were investigated. The hybrid membrane containing 5 wt% APTEOS has highest separation factor of 536.7 at 50 °C and permeation flux of 0.0355 kg m⁻² h⁻¹ in PV separation of 5 wt% water in the feed.     

Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive hydration forces in this hydrotrophic salt.     

Enhanced desulfurization performance and swelling resistance of asymmetric hydrophilic pervaporation membrane prepared through surface segregation technique

The severe swelling behavior of most hydrophobic membranes has always been an obstinate problem when separating organic mixtures by pervaporation. In some cases, hydrophilic membranes may be an appropriate alternative. In this study, amphiphilic copolymer Pluronic F127 was employed as a surface modifier to fabricate polyethersulfone (PES) asymmetric pervaporation membranes via surface segregation. The scanning electron microscopy (SEM) photographs showed an asymmetric structure of PES/Pluronic F127 membranes. The Fourier transform-infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and static water contact angle measurements confirmed the hydrophilic modification of the membrane surface. Based on the distinct difference of solubility in water between thiophene and n-octane, the prepared membranes were utilized to remove thiophene from n-octane by pervaporation. The effect of Pluronic F127 content on the pervaporation performance was evaluated experimentally. It has been found that both the permeation flux and enrichment factor exhibited a peak value of approximately 60 wt% of the Pluronic F127 content. The highest enrichment factor was around 3.50 with a permeation flux of 3.10 kg/(m² h) for 500 mg/L sulfur in the feed at 30 °C. The influence of various operating parameters on the pervaporation performance was extensively investigated.     

Albumin separation with Cibacron Blue carrying macroporous chitosan and chitin affinity membranes

Cibacron Blue F3GA, Procion Red HE-3B and Procion Blue MX-R were immobilized on macroporous chitosan and chitin membranes with concentrations as high as 10–200 μmol/ml membrane. These dyed membranes were chemically and mechanically stable, could be reproducibly prepared, and operated at high flow rates. Human serum albumin (HSA) and bovine serum albumin (BSA) were selected as model proteins, and their adsorption on and desorption from the dyed chitosan membranes investigated. The Cibacron Blue F3GA membranes had a higher protein adsorption capacity, much greater for HSA than BSA, than the other dyed membranes. About 8.4 mg HSA/ml membrane were adsorbed at saturation by Cibacron Blue F3GA–chitosan membranes from a 0.05 M Tris–HCl/0.05 M NaCl, pH 8 solution. The chitin membranes had a lower dye content and hence a lower protein adsorption capacity than the chitosan membranes. The effects of important operation parameters (flow rate, protein concentration and loading) were also investigated. Cibacron Blue F3GA–chitosan membranes were employed for the separation of HSA from human plasma and high purity HSA thus obtained. This suggests that these membranes could be used for large-scale plasma fractionation.     

Separation and preconcentration of trace manganese from various samples with Amberlyst 36 column and determination by flame atomic absorption spectrometry

This work assesses the potential of a new adsorptive material, Amberlyst 36, for the separation and preconcentration of trace manganese(II) from various media. It is based on the sorption of manganese(II) ions onto a column filled with Amberlyst 36 cation exchange resin, followed by the elution with 5 mL of 3 mol/L nitric acid and determination by flame atomic absorption spectrometry (FAAS) without interference of the matrix. Different factors including pH of sample solution, sample volume, amount of resin, flow rate of sample solution, volume and concentration of eluent, and matrix effects for preconcentration were investigated. Good relative standard deviation (3%) and high recovery (>95%) at 100 μg/L and high enrichment factor (200) and low analytical detection limit (0.245 μg/L) were obtained. The adsorption equilibrium was described well by the Langmuir isotherm model with maximum adsorption capacity of 88 mg/g of manganese on the resin. The method was applied for the manganese determination by FAAS in tap water, commercial natural drinking water, commercial treated drinking water and commercial tea bag sample. The accuracy of the method is confirmed by analyzing the certified reference material (tea leaves GBW 07605). The results demonstrated good agreement with the certified values.     

Synthesis of non-porous poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application in separation of biopolymers

The monodisperse, 5.0 μm non-porous poly(glycidylmethacrylate-co-ethylenedimethacrylate) (P_GMA/EDMA) beads were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. Based on this media, a weak cation exchange (WCX) stationary phase for high performance liquid chromatography (HPLC) was synthesized by a new chemical modification method. The prepared resin has advantages of biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The measured bioactivity recovery for lysozyme was 97 ± 5%. The dynamic protein loading capacity of the synthesized WCX packings was 20.5 mg/g. Four proteins were completely separated in 3.0 min using the synthesized WCX stationary phase. The experimental results show that the obtained WCX resin has very weak hydrophobicity. The WCX resin was also used for the rapid separation and purification of lysozyme from egg white in 3.0 min with only one step. The purity and specific bioactivity of the purified lysozyme was found more than 95% and 70.264 IU/mg, respectively.     

金属螯合亲和膜吸附分离与纯化溶菌酶的研究(Ⅱ)──不同金属螯合亲和膜对溶菌酶的吸附性能

采用低温氧或氨等离子体法改性聚丙烯微孔膜,基于等离子体改性膜的活化、偶联及螯合过程的机理,制备了Fe³⁺,Ni²⁺,Cu²⁺和Zn²⁺等金属离子螯合亲和膜,并用于溶菌酶的吸-脱附实验.实验结果表明,Ni²⁺和Cu²⁺离子螯合亲和膜对溶菌酶具有较高的吸附量,螯合过程中采用氯化物盐溶液制得的膜对溶菌酶吸附量比采用硫酸盐溶液制得的膜的吸附量高.两种膜的重复吸-脱附性能相近,而Fe³⁺螯合亲和膜基本上不能用于重复吸-脱附实验.采用补充金属螯合离子,能部分恢复亲和膜对溶菌酶的吸附量,是实现亲和膜重复使用的有效方法.     

Modeling of the permeate flux decline during MF and UF cross-flow filtration of soy sauce lees

Cross-flow ultrafiltration and microfiltration have been used to recover refined soy sauce from soy sauce lees for over 25 years. The precise mechanism which dominated the permeate flux during batch cross-flow filtration has not been clarified. In the present study, we proposed a modified analytical method incorporated with the concept of deadend filtration to determine the initial flux of cross-flow filtration and carried out the permeate recycle and batch cross-flow filtration experiments using soy sauce lees. We used UF and MF flat membrane (0.006 m² polysulfone) module under different transmembrane pressures (TMP) and cross-flow velocities. The modified analysis provided an accurate prediction of permeate flux during the filtration of soy sauce lees, because this model can consider the change in J₀ at initial stage of filtration which was caused by the pore constriction and plugging inside membrane, and these changes may not proceed when the cake was formed on the membrane surface. Mean specific resistance of the cake increased with TMP due to the compaction of the cake and decreased with cross-flow velocity due to the change of deposited particle size, but less depended on the membrane in the present study. These results indicate that the value of J₀ determined by modified method was relevant to exclude the effects of the initial membrane fouling by pore constriction due to protein adsorption and plugging with small particles. The modified analytical method for the cake filtration developed in the present study was considered to be capable of selecting an appropriate operating conditions for many cross-flow filtration systems with UF, MF membranes.     

Cross-linked macroporous chitosan anion-exchange membranes for protein separations

Macroporous chitosan membranes with controlled pore sizes and good mechanical properties were prepared and cross-linked with ethylene glycol diglycidyl ether to increase their chemical stability. Because of their amine groups, they can serve as anion-exchangers (with an ion-exchange capacity as high as 0.83 meq/g dry cross-linked membrane) and can be employed for protein separations in the ion-exchange mode. At pH<7, their surface is positively charged, and they can adsorb proteins with a pI<6 at appropriate pHs. Five proteins, namely ovalbumin (pI=4.6), human serum albumin (pI=4.8), soybean trypsin inhibitor (pI=4.5), lysozyme (pI=11) and cytochrome C (pI=10.6) were selected as model proteins to investigate their adsorption on the chitosan membranes. Relatively high dynamic capacities were achieved at a flow rate of 2 ml/min, namely 11.6, 19 and 20.8 mg/ml membrane for human serum albumin, ovalbumin and soybean trypsin inhibitor, respectively. These proteins could be efficiently recovered (91–98%) from the membranes using a 1 N NaCl in 0.02 N sodium phosphate solution (pH 6) as eluant. Protein separations were performed from binary mixtures (ovalbumin–lysozyme, human serum albumin–cytochrome C, and soybean trypsin inhibitor–cytochrome C), and high purity products (∼99%) obtained in a single pass. These membranes showed high stability and reproducibility.