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) polymer
concentration using N,N-dimethylacedamid and water as the solvent and casting solution, respectively, at 20 C, determined to be the
optimum
chemicals 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
1369-703X/$ see front matter 2004 Elsevier B.V. All rights
reserved. doi:10.1016/j.bej.2004.09.014

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
methacrylate),
polyethylene, chitosan, poly(vinyl)alcohol, polysul- fone,
polypropylene and polymer latex, dextran, agorose and
silica [814]. Many applications of these membranes for selective protein separation and purification have been
reported

D. Tanyolac et al. / Biochemical Engineering Journal 22 (2005) 221

228

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 was
used as the polymer to produce a new and feasible membrane 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,Ndimethylacedamid 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 manufacturers data sheet. For preliminary adsorption experiments,
bovine serum albumin (BSA) (fraction V, lyophilised) and
acetate buffer were purchased from Sigma and Merck, respectively. Glutaraldehyde for surface activation was also
purchased from Merck.
2.2.
Preparation
membranes

of

polyvinylbutyral

Polyvinylbutyral membranes were prepared by the conventional 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 transferred into a membrane template having the dimensions
13 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 evaporation at optimum temperature (20 C), the membrane film
over the glass was immersed in precipitating solution
(water)
at 20 C and kept there for 15 min for precipitation. The solidified 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 deionised water until use.
2.3.
Activation
glutaraldehyde

of

the

membrane

with

A coupling agent, glutaraldehyde, was used to activate

the membranes prepared with 9% (w/v) initial polyvinylbutyral 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 magnetically
stirred at 4 C and 200 rpm in dark in order to prevent the
polymerisation 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 deionised wa- ter until use.
2.4. Preliminary BSAadsorption experiments
Adsorption experiments
were conducted batchwise for

up
cm3to 2 h at 20 C with a stirring3rate of 100 rpm in a 50
Pyrex reactor containing 25 cm 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 with
9% (w/v) initial polyvinylbutyral concentration were used.
2.5. Analysis of the structure
FT-IR spectra of the activated and non-activated membranes and Mowital in powder form were obtained by

using

D. Tanyolac et al. / Biochemical Engineering Journal 22 (2005) 221

228

a DRS-FT-IR spectrophotometer (FT-IR 8110 Series, Shimadzu). 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
properties

of

membrane

The swelling behaviour of polyvinylbutyral membranes

was determined in distilled and de-ionised water. Dry membrane pieces (60 1 cm2 square cut) were placed in distilled
and
de-ionised water kept at a constant temperature, 20 0.5
C.
Swollen membranes were periodically removed and
weighed
by an electronic balance (GEC-Avery Model VA304, UK,
0.1 mg). The water content of the swollen membranes was
calculated using the following expression:
swelling ratio (%) =

Ws W0
100
W0

(1)

where 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 double distilled and de-ionised water under 370 mmHg vacuum
and repeated five times for each membrane sample successively. 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. Membrane
samples were prepared in 100 mm 15 mm dimensions,
inserted in the apparatus and shear was applied in the
direction
of the membrane casting until the breakpoint of the sample.
The shear stress and membrane elongation were determined
at the breakpoint. The strength measurement was performed
at 20 C.
3. Results and discussion
In this study, polyvinylbutyral membranes with uniform 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

dimethylacetamid since the rest of the solvents yielded nonporous and non-permeable membranes. Therefore, all membranes 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 of
the membranes produced and 20 C was experienced as the
optimum
temperature
the membrane
production ineffect
temperature range
1535 for
C. The
polymer concentration
has 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 Mowital
membranes 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 for

surface and cross-section views respectively, prepared from

a solution of 12% (w/v). In contrast to Fig. 1a, the density
in the approximate ratio 75:22:3, respectively [27].
With preliminary experiments the most appropriate solvent 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 prepared 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 decreased while C O band at 1740 cm1 increased compared
to
inactivated 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 glutaraldehyde activation at 1740 cm1 , respectively. These bands
confirmed 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 examination 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,

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. 5. Swelling percent and water penetration rate of membranes as a

func- tion of polyvinylbutyral content.

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 pH
5.0 as 97 g BSA/cm2 membrane which was the isoelectric
point 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.
The
time
BSA
onto 9% (w/v)
polyvinylbutyral
at pH8.5.0
and
20 profile
C withofanadsorbed
initial BSA
concentration
of 10
mg/ml.

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 optimum pH 5.0 as a function of initial BSA concentration was
presented in Fig. 10. Up to 10 mg/ml initial BSA concentration, the amount adsorbed onto activated membranes increased 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. 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 as
12.95 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 (thickness
0.1 m) and realized as high as 64.6 mg/ml BSA adsorption 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 adsorption. 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 activated 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 as
427 g protein/cm2 (35.44 mg/ml), significantly higher than
many of literature adsorption studies with membranes.

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