doi:10.1016/j.bej.2004.09.014

Biochemical Engineering Journal 22 (2005) 221228

A low cost porous polyvinylbutyral membrane for BSA adsorption

Deniz Tanyolac, Hakan So nmezsk, Ahmet R. O zdural

Chemical Engineering Department, Faculty of Engineering, Hacettepe University, Beytepe Campus, 06532 Ankara, Turkey

Received 24 September 2004; accepted 27 September 2004

Abstract

In this work, a feasible membrane was prepared in uniform thickness for the range 120145 m from a commercial resin, Mowital B30HH (polyvinylbutyral) using phase inversion technique. For the production of the membrane, the most appropriate solvent, polymer concentration, temperature and the composition of casting solution were investigated. Macroporous membranes were produced with 620% (w/v) polymerconcentration using N,N-dimethylacedamid and water as the solvent and casting solution, respectively, at 20 C, determined to be the optimumchemicals and conditions. It was found that pore size, pore density, water permeation rate, water content, and elongation of the membranes decreased while breakpoint stress increased with the increase of polymer concentration. FT-IR studies proved the abundance of hydroxyl groups on the membrane surface, which were activated later by glutaraldehyde for bovine serum albumin (BSA) separation. Preliminary adsorption runs were conducted at pH 5.0, determined to be the optimum for BSA adsorption, with the membrane prepared at 9% polymer concentration in a batch reactor. For 10 mg/ml initial BSA concentration, the adsorbed BSA was calculated as 427 g/cm2 (35.44 mg/ml membrane) denoting a remarkable capacity for BSA adsorption compare to those of other membranes in literature. 2004 Elsevier B.V. All rights reserved.

Keywords: Membrane; Phase inversion; Polyvinylbutyral; Bovine serum albumin (BSA); Adsorption

1. Introduction

There are many successful applications of membrane sep- aration processes in areas such as seawater desalination, food processing, effluent treatment, and downstream separation. Membrane based processes are gradually becoming impor- tant in pharmaceuticals, petrochemicals and other respective industries which have impact on the environment. Neverthe- less, economic considerations with respect to membrane life- time, pre/post-treatment steps, and flux decline are the major reasons why most membrane applications on the industrial scale have lagged behind their expected growth [1]. The cost of a membrane system is highly dependent on the surface area required, which is determined by the flux of the membrane. Therefore, the role and influence of the membrane material on flux reduction as well as aspects of thermal and chemical resistance of the membrane material during separation and

Corresponding author. Tel.: +90 312 2976162; fax: +90 312 2992124.E-mail address: deniztan@hacettepe.edu.tr (D. Tanyolac). regeneration has been the subject of considerable research attention [24].Recently, membrane affinity chromatography has become advantageous to conventional bead-packed column chro- matography due to significant disadvantages such as high- pressure drops, internal diffusion limitation, compressibil- ity of the soft beads and clogging experienced in the latter [57]. In contrast to the column chromatography, the mem- brane chromatography brings the solute into the proximity of the affinity ligand groups on the membrane by convec- tion, thus reducing the resistance to mass transfer and allow- ing lower pressure drops and higher flow rates. Membrane chromatography is operated either as a stack of macroporous membranes or as a bundle of hollow fibre membranes en- abling excessive surface area. Various ligand groups have been coupled to porous membranes based on nylon, regen- erated cellulose, perfluoropolymer, poly(glycidyl methacry- late), polyethylene, chitosan, poly(vinyl)alcohol, polysul- fone, polypropylene and polymer latex, dextran, agorose and silica [814]. Many applications of these membranes for se- lective protein separation and purification have been reported

1369-703X/$ see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2004.09.014

D. Tanyolac et al. / Biochemical Engineering Journal 22 (2005) 221228

D. Tanyolac et al. / Biochemical Engineering Journal 22 (2005) 221228

in the literature [1520]. In some cases, proteins have been adsorbed onto the support surface with cross-linking by glu- taraldehyde [21]. As a low cost material, polyvinylbutyral has been used in the form of microbeads for the removal of metal ions from aqueous solutions and BSA adsorption [22,23]. However, there are few studies in the literature made use of polyvinylbutyral resin as a membrane [2426].In this work a commercial resin, namely Mowital wasused as the polymer to produce a new and feasible mem- brane with excellent characteristics for BSA adsorption. The work elaborated the method of phase inversion for mem- brane preparation and facilitated hydroxyl and acetate groups on the membrane surface for selective binding. Production conditions were optimised to yield highly functional mem- brane samples. Finally, glutaraldehyde was used to activate the membrane for preliminary adsorption experiments of a model protein, bovine serum albumin (BSA), in a batch re- actor.

2. Materials and method

2.1. Materials

Mowital B30HH (polyvinylbutyral, Hoechst) is the poly- meric material for the preparation of the membrane. The sol- ubility and film formation properties, combination capabili- ties, and reactivity of the polymer are determined largely by acetalization, hydroxyl group content, and degree of poly- merisation. Type B30HH has a better solubility in aromatics than the other forms of Mowital [27]. Chloroform, ace- tone, methanol, ethanol, butanol, ethyl acetate, dimethyl- sulfooxide and N,N-dimethylacedamid were tested as the solvent for phase inversion technique to produce the mem- brane. All solvents were of highest purity and purchased from Merck.For keeping the medium at constant pH, universal pH buffers were used in the amounts as specified in manufac- turers data sheet. For preliminary adsorption experiments, bovine serum albumin (BSA) (fraction V, lyophilised) and acetate buffer were purchased from Sigma and Merck, re- spectively. Glutaraldehyde for surface activation was also purchased from Merck.

2.2. Preparation of polyvinylbutyral membranes

Polyvinylbutyral membranes were prepared by the con- ventional phase inversion method [28]. The appropriate amount of Mowital (to form 620% (w/v) solutions) was dissolved in a predetermined solvent of polyvinylbutyral [28] (chloroform, acetone, methanol, ethanol, butanol, ethyl acetate, dimethylsulfooxide, N,N-dimethylacedamid and a special solution made of 10% (v/v) methanol, 40% (v/v) ethanol and 50% (v/v) acetone) and the solution was trans- ferred into a membrane template having the dimensions13 cm 18 cm 200 m placed on a glass. With a rolling bar the solution was evenly distributed within the template with an initial thickness of 200 m. After 2 min of evapo-ration at optimum temperature (20 C), the membrane filmover the glass was immersed in precipitating solution (water)at 20 C and kept there for 15 min for precipitation. The so-lidified membrane was washed with distilled and de-ionised water three times to remove impurities physically adsorbed onto the surface of the membrane and preserved in de-ionised water until use.

2.3. Activation of the membrane with glutaraldehyde

A coupling agent, glutaraldehyde, was used to activate the membranes prepared with 9% (w/v) initial polyvinylbu- tyral concentration for BSA adsorption. The 1 cm2 square cut membrane samples (60 in total) were kept in double distilled water for about 24 h and washed on a glass filter with 0.1 M HCl solution and then again placed in distilled and de-ionised water to remove impurities. Prior to activation, samples were again washed with double distilled water three times. Aque- ous glutaraldehyde solution (100 ml) with an initial concen- tration of 4% (v/v) was prepared and pH of the solution was fixed at 7.4 with saline phosphate buffer [29]. All samples were then added to this solution while it was magneticallystirred at 4 C and 200 rpm in dark in order to prevent thepolymerisation of glutaraldehyde. After 24 h of activation, the membranes samples were removed and washed with dis- tilled water five times to remove the excess activation agent and impurities, then preserved in distilled and de-ionised wa- ter until use.

2.4. Preliminary BSAadsorption experiments

Adsorption experiments were conducted batchwise for up to 2 h at 20 C with a stirring rate of 100 rpm in a 50 cm3Pyrex reactor containing 25 cm3 medium. In a typical adsorp- tion experiment, appropriate amount of BSA was dissolved in a 25 ml of buffer solution at specified pH and 60 square membrane pieces (1 cm2 each) were added to start the ad- sorption reaction. At appropriate time intervals, liquid sam- ples were taken to analyse the solution BSA concentration. The concentration of BSA in the medium was determined spectrophotometrically at 730 nm with Lowry method [30] using a calibration curve prepared previously. The amount of BSA adsorbed from membranes was calculated by measur- ing the initial and final concentrations of BSA in the medium. In the runs, initial BSA concentration and medium pH were changed in the ranges 110 mg/ml and 37 using acetate and phosphate buffers, respectively. In adsorption experiments, only the glutaraldehyde-activated membranes prepared with9% (w/v) initial polyvinylbutyral concentration were used.

2.5. Analysis of the structure

FT-IR spectra of the activated and non-activated mem- branes and Mowital in powder form were obtained by using

a DRS-FT-IR spectrophotometer (FT-IR 8110 Series, Shi- madzu). For the FT-IR of polyvinylbutyral in powder form,0.1 g dry Mowital was completely mixed with 0.1 g KBr(IR grade, Merck), and pressed into a form of tablet, and the spectrum was then recorded. FT-IR spectra of the membranes were directly taken with the ATR attachment. To observe the surface topography of the membrane, scanning electron mi- crographs of gold-coated membrane samples were taken with a SEM device (Model: Raster Electronen Microscopy, Leitz- AMR-1000).

2.6. Analysis of membrane properties

The swelling behaviour of polyvinylbutyral membranes was determined in distilled and de-ionised water. Dry mem- brane pieces (60 1 cm2 square cut) were placed in distilled andde-ionised water kept at a constant temperature, 20 0.5 C.Swollen membranes were periodically removed and weighedby an electronic balance (GEC-Avery Model VA304, UK,0.1 mg). The water content of the swollen membranes wascalculated using the following expression:Ws W0 dimethylacetamid since the rest of the solvents yielded non- porous and non-permeable membranes. Therefore, all mem- branes were prepared with N,N-dimethylacetamid as the sol- vent. For precipitation medium distilled and de-ionised water was determined as the most appropriate. Process tempera- ture had a profound effect on the uniformity and quality ofthe membranes produced and 20 C was experienced as theoptimum temperature for the membrane production in tem- perature range 1535 C. The polymer concentration effecthas been elucidated in the available range 620% (w/v). Uni- form membrane formation has not been achieved with less than 6% (w/v) polymer concentration while jell formation and air entrapment were experienced within the membrane along with solubility difficulties for polymer concentrations higher than 20% (w/v).Fig. 1a and b shows the SEM micrographs of Mowitalmembranes for surface and cross-section views, respectively, prepared from a solution of 9% (w/v) polyvinylbutyral. As seen in Fig. 1a and b, the membrane possesses evenly dis- tributed almost mono-size pores while a highly porous struc- ture was observed inside of the membrane. Fig. 2a and b denotes again the SEM micrographs of the membrane forWswelling ratio (%) = 100 (1)0 surface and cross-section views respectively, prepared froma solution of 12% (w/v). In contrast to Fig. 1a, the densitywhere W0 and Ws are the weights of the membranes before and after swelling, respectively.Membrane thickness was determined by measuring the overall thickness of 50 uniform membrane samples with a compass having 0.1 mm accuracy. Each measurement was repeated with three different sets of the membrane samples prepared under the same conditions.Membrane permeation tests were performed using dou- ble distilled and de-ionised water under 370 mmHg vacuum and repeated five times for each membrane sample succes- sively. During the measurements, the more porous side of the membrane was placed at the top where the water entered.The physical strength of the membranes was tested with a KarlFrank (Germany) shear stress apparatus. Membranesamples were prepared in 100 mm 15 mm dimensions, in-serted in the apparatus and shear was applied in the directionof the membrane casting until the breakpoint of the sample. The shear stress and membrane elongation were determined at the breakpoint. The strength measurement was performedat 20 C.

3. Results and discussion

In this study, polyvinylbutyral membranes with uni- form thickness were prepared in the range 120145 m. These polyvinylbutyral-based membranes possess rather hy- drophilic structure. The polyvinylbutyral resin consists of vinylbutyral, vinylalcohol and vinylacetate co-monomers in the approximate ratio 75:22:3, respectively [27].With preliminary experiments the most appropriate sol- vent for producing porous membrane was determined as N,N- of the pores are lower and uneven, however void structure (Fig. 2b) is more orderly and less tortuous than that of Fig. 1b. Fig. 3 presents again the cross-section view of the membrane

Fig. 1. SEM photographs of the membrane prepared from 9% (w/v)polyvinylbutyral solution: (a) surface view and (b) cross-section view.

Fig. 2. SEM photographs of the membrane prepared from 12% (w/v)polyvinylbutyral solution: (a) surface view and (b) cross-section view.

prepared from 15% (w/v) polyvinylbutyral concentration. Al- though the polymer structure is very uniform and the least tor- tuous, the surface placed on the glass during the production is not porous, disabling the penetration through the membrane. The membrane prepared from 20% (w/v) polymer concen- tration gave a completely non-penetrating membrane with no pores on both surfaces (figure not shown). Considering the membrane for a potential application in a stacked membrane reactor, the membrane of 9% (w/v) was preferred since it yielded many pores on the surface as well as larger surface

Fig. 3. The SEM photograph for cross-section view of the membrane pre- pared from 15% (w/v) polyvinylbutyral solution. area inside the structure enabling adequate penetration and adsorption of the selected compound.Fig. 4 denotes the FT-IR spectra of powder Mowital ,non-activated and glutaraldehyde activated membranes. The OH peak intensity was greater in powder form (Fig. 4a) than that of the membrane (Fig. 4b) due to possible inactivation of the OH groups on the surface during reformation of the poly- mer structure during the precipitation. After glutaraldehyde activation, the intensity of the OH band on the membrane decreased due to the reaction took place between OH and aldehyde groups (Fig. 4c). In this spectrum, OH band peak de-creased while C O band at 1740 cm1 increased compared toinactivated membrane due to reaction of glutaraldehyde with OH groups to yield C O bands. Therefore, the intensity of H C O and (CH2 )3 C O groups increased after glutaralde-hyde activation at 1740 cm1 , respectively. These bands con-firmed the reaction of glutaraldehyde with OH groups on the polyvinylbutyral membranes.The membranes have been kept in water and in ambient air at room temperature for 4 months and after the examina- tion of FT-IR spectrum and physical test results, no change in chemical and physical properties was detected. Thus, poly- meric structure of the membrane is considered resistant to degradation.The experiments done for physical and adsorption charac- teristics of membranes were repeated three times and average values of corresponding data (with a standard deviation not more than 5%) were presented in the figures.The polyvinylbutyral membranes swell rapidly, and the equilibrium is achieved in about 15 min. Fig. 5 shows the change of swelling percent and water permeation rate of membranes at different initial polymer concentrations. With the increase of polymer concentration, number and the radius of the pores on the surface decreases (as viewed in Figs. 13) and accordingly permeation rate of the membrane is dras- tically reduced from 39 to 5 cm3 water/cm2 min almost lin- early. Higher polymer content in the membrane production process resulted in a dense membrane structure with less pores and void fraction, therefore the water capacity of the membrane structure decreased from 94 to 80% with a linear trend.Fig. 6 shows the change of final membrane thickness with the polymer content. The final membrane thickness changed from 120 to 145 m within the range 620% (w/v) of polymer content with an exponential trend. This implies the exponen- tial increase of void fraction of the membrane with linear increase of polymer content within specified range.The change of physical strength of the membrane in terms of breakpoint shear stress and maximum elongation with polymer content of the solution is presented in Fig. 7. Break- point shear stress is almost directly proportional to poly- mer content while elongation decreased hyperbolically at the point of break. Undoubtedly, higher polymer content made the membrane more stiff and less flexible, but meanwhile more robust due to increased cross-bonds in the structure at high polymer concentrations. Membranes prepared at 6%

Fig. 4. FT-IR spectra of (a) powder Mowital , (b) non-activated membrane and (c) glutaraldehyde activated membrane.

(w/v) could not be tested for shear stress and elongation since the structure was very weak and non-homogeneous.For adsorption runs, the sufficient time to reach to the equilibrium was determined with a dynamic batch run carried out pH 5.0 with an initial BSA concentration of 10 mg/ml, a higher limit encountered in many literature studies. Since higher initial adsorbent concentration delays the equilibrium,

Fig. 5. Swelling percent and water penetration rate of membranes as a func- tion of polyvinylbutyral content. the time to reach equilibrium for 10 mg/ml is far sufficient for lower initial BSA concentrations. The change of BSA adsorbed by time is given in Fig. 8. As is clear from the figure, adsorbed BSA amount remains constant after 120 min, so this duration was considered a standard time to measure BSA equilibrium concentration for all runs.

Fig. 6. Change of final membrane thickness with polyvinylbutyral content.

Fig. 7. The variation of maximum elongation and breakpoint shear stress of the membrane with polyvinylbutyral content.

In adsorption experiments, first the effect of medium pH on the adsorption capacity of the glutaraldehyde activated (4%, v/v) polyvinylbutyral membranes was investigated with batch adsorption runs. Fig. 9 depicts the change of adsorbed BSA at equilibrium as a function of medium pH obtained with an initial BSA concentration of 2 mg/ml. Similar to other studies in literature, maximum adsorption was realized around pH5.0 as 97 g BSA/cm2 membrane which was the isoelectricpoint of BSA [31]. This result is not unusual since maximum adsorption of a protein can be accomplished when it has a neutral charge at the isoelectric point where protein solubility is at its minimum. However, acidic or basic medium causes the protein positively or negatively charged, increasing the solubility of the protein in the aqueous media. Therefore, lower or higher pH values than isoelectric point resulted in decreased BSA adsorption onto the membrane.

Fig. 9. The amount of BSA adsorbed at equilibrium onto 9% (w/v) polyvinylbutyral membrane as a function of medium pH with an initial BSA concentration of 2 mg/ml.

The amount of BSA adsorbed onto membranes at opti- mum pH 5.0 as a function of initial BSA concentration was presented in Fig. 10. Up to 10 mg/ml initial BSA concen- tration, the amount adsorbed onto activated membranes in- creased linearly with increasing initial BSA concentration values. At 10 mg/ml initial BSA concentration the adsorbed BSA was determined as high as 427 g/cm2 (35.44 mg/ml) and maximum adsorbed BSA would be larger than this value because saturation has not been achieved at this point. This adsorbed BSA value was higher than the value of 17.7 mg BSA/g adsorbent, the maximum adsorbed amount to mag- netic polyvinylbutyral microbeads [32], mainly due to more surface area available in membrane form. The adsorbed BSA value at 10 mg/ml initial BSA concentration in this study is

Fig. 8. The time profile of adsorbed BSA onto 9% (w/v) polyvinylbutyral at pH 5.0 and 20 C with an initial BSA concentration of 10 mg/ml. Fig. 10. The variation of BSA adsorbed at equilibrium onto 9% (w/v)polyvinylbutyral membrane as a function of initial BSA concentration.

also higher than those of other membrane studies made of different materials for BSA adsorption. Anspach and Petsch used a poly(ethyleneimine) coated Nylon 66 microporous membrane to adsorb BSA from 20 mM phosphate buffer at pH 7.0 and calculated the maximum binding capacity as12.95 mg/ml from a multilayer model [33]. Kubota et al. made use of cellulose acetate membranes modified with tannic acid for separation and purification of BSA and 9.49 mg/ml BSA adsorption was achieved at 0.15 mg/ml initial BSA concen- tration under optimum conditions [16]. Gebauer et al. em- ployed two different types of (prepared on nylon and mod- ified cellulose base) commercial Sartobind-S membranes to study the breakthrough of BSA [34]. They achieved 23.5 and 31 mg/ml maximum BSA adsorption capacities for ny- lon and modified cellulose membranes at 4.0 and 6.0 mg/ml initial BSA concentrations, respectively. Jones and OMelia elucidated the rate and extend of adsorption of BSA onto a regenerated cellulose ultrafiltration membrane (thickness0.1 m) and realized as high as 64.6 mg/ml BSA adsorp- tion at 25 mg/ml initial BSA concentration under optimum conditions [35]. This remarkable value was attributed to high initial BSA concentration and very thin structure of the membrane.

4. Conclusion

In this work, new and easy to manufacture membranes were made of a commercial resin, Mowital for protein ad- sorption. The phase inversion method was elaborated and reaction conditions as well as chemicals and quantities were optimised to yield a membrane with high BSA adsorption capacity. FT-IR studies revealed the presence of hydroxyl groups on the particle surface, which may be easily acti- vated through conventional techniques for bioaffinity separa- tions. Preliminary adsorption runs for bovine serum albumin were conducted with glutaraldehyde activated membranes which resulted in remarkable adsorption capacities as high as427 g protein/cm2 (35.44 mg/ml), significantly higher thanmany of literature adsorption studies with membranes.

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