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Modification of polysulfone ultrafiltration membranes with UV irradiation and hydrophilicity increasing agents
Journal of Membrane Science, 60 (1991) Elsevier
Science
Publishers
275
275-296 B.V., Amsterdam
Modification of polysulfone ultrafiltration membranes with UV irradiation and hydrophilicity increasing agents Marianne Nystriim and Pia Jarvinen Laboratory of Technical Polymer Chemistry, Department of Chemical Technology, Lappeenranta University of Technology, P.O. Box 20, SF-53851 Lappeenranta (Finland) (Received
August 28,1990,
accepted
in revised form April 8,199l)
Abstract Hydrophobic
polysulfone
UF membranes
were modified with UV irradiation
and hydrophilicity
increasing agents. The modifications were tested with 0.5% whey-protein solution and 0.05% lysozyme solution at pH 6 and with 0.05% bovine serum albumin solution at various pH values. UV irradiation increased flux and the hydrophilicity of the membranes. The flux increases obtained varied with pH and modification agents used and could be more than 400% compared to unmodified conditions without any loss in retention. The best retentions were obtained at pH values, where both the protein and the membrane had the same charge, and a strong electrostatic repulsion was obtained. The pores enlarged to fixed sizes, which depended on the sizes of the proteins and the range of double layer forces between proteins and membranes at different states of charge density.
Keywords: ultrafiltration;
polysulfone
crease of; bovine serum albumin;
membranes;
UV irradiation;
fouling;
hydrophilicity,
in-
lysozyme; whey
Introduction
In ultrafiltration (UF ) of proteins one of the greatest problems is the fouling of the membranes, which causes a considerable flux decline. Fouling often results when small divalent ions like calcium ions are present [ 1 ] or if the protein solution contains small amounts of lipids [ 21. Also the proteins themselves seem to adsorb tightly onto the membranes, especially if the membranes are hydrophobic in nature [ 3,4]. The resulting fouling can be either reversible or irreversible. In fouling and adsorption of proteins on membranes an important factor determining adsorption seems to be the strength of the hydrophobic interaction between the proteins and the membranes. The proteins consist of both hydrophilic and hydrophobic parts, the ratio being different for different proteins. The importance of hydrophilicity for the prevention of adsorption has been shown by Gijlander and Kiss [ 51 and has been explained by Lundstrom
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[61 to depend on the fact that the hydrophilic surface attracts so much water that adsorption is prevented. When a protein adsorbs on a hydrophobic surface also conformational changes can take place [3]. The adsorption can be both entropically or enthalpically driven [ 71. Multilayer adsorption in static conditions usually consists of one layer which is more strongly bound and the rest of the layers can more easily be washed off. This is e.g. the case with BSA adsorption shown by Lee and Ruckenstein [ 81. In ultrafiltration both adsorption forces and the dynamic experimental conditions determine the amounts of fouling and flux reduction. In UF, even when the proteins seem to be hydrophilic and charged and no fouling is expected because of electrostatic repulsion between molecules and membrane, there still seems to be fouling taking place. When changes of the charge of the protein and the membrane induced by pH control do not improve the ultrafiltration results, modification of commercial membranes or the manufacture of new types of membranes seems to be the means to improve the conditions for UF of proteins. As both increased hydrophilicity and charge density of the membranes seem to be important, membrane manufacturers have tried to graft polymers of different kinds either on the ready-made membrane [5,9-111 or on the polymer from which the membrane is prepared [ 121. This grafting has mostly been initiated with a glow discharge apparatus [ 51 or by UV irradiation [lo]. Modification of the membranes has also been accomplished by chemical binding of macromolecules to the membrane [ 131 or by a partly reversible, chemical adsorption of the modification agent onto the membrane from solution [ 14,151 or by the application of a Langmuir-Blodgett film on the membrane [ 161. The problem with modification by chemical treatment of readymade commercial membranes is that the modification agent partly blocks the pores of the membranes, so that even if the membranes after modification are less apt to foul, the total flux after modification is smaller than before modification. One successful modification method seems to be that by Vigo and Uliana [ 12 1, where the polymer material is modified before the membrane is cast and in this way a good flux is obtained. In this study commercial polysulfone UF membranes were modified using UV irradiation alone or together with different modification agents in liquid environment. The criteria were to get a membrane that was less apt to foul and also a membrane with a better flux than normally without any decrease in retention of the proteins. The modification results were tested by UF experiments with different protein solutions. In some cases the hydrophilicities of the modifications were tested with contact angle measurements on a cast polymer surface treated in the same way as the membrane. Experimental
Materials The polysulfone (PSU) membranes used were of type GR 81 PP delivered from De Danske Sukkerfabrikker (DDS) with a nominal cut-off of 6000 g/
mol. These membranes have a relatively wide pore size distribution [ 171 and the mean pore diameter is approximately 2 nm for the GR 81 membrane [ 181. Smooth PSU model surfaces were prepared from PSU of the GR type obtained from DDS. The GR type polysulfone is mainly the same as UDEL polysulfone [ 191, which means that it is a polymer prepared of bisphenol A and 4,4-dichlorodiphenylsulfone, n = 50-80(Fig. 1) . The GR polysulfone differs from the UDEL polysulfone in that it contains more bisphenol A according to Fontyn et al. [ 201. The UDEL polysulfone is resistant to most chemicals but partly swells in alcohols [ 191. The reactions taking place in the chain, when irradiated with UV [21,22] are bond scissions, indicated in Fig. 1, and dissociations of the methyl side groups, either by the formation of a methyl or a hydrogen radical. As end products sulfonic acid groups can be formed. Also keto or ethenyl groups can be formed. The block copolymer (PEOB) used was synthesized according to Napper [ 231 of polyethylene oxide (PEO ) (M= 35 000 g/mol) and vinyl acetate monomer to a copolymer containing two blocks. All the dextrans used for modification were polydisperse with a W of 500 000 g/mol. They were delivered by Pharmacia, Sweden. Dextran T 500 (DEX) is non-ionic in nature. Dextran sulfate (DEXSU) is a polyanionic derivative of dextran produced by esterification with chlorosulfonic acid. The pK, of DEXSU is below 2. The relative hydrophilicity of DEXSU is lower than for DEX [ 241. Diethylaminoethyl dextran (DEAE) is a polycationic derivative of dextran containing diethylaminoethyl groups linked to the glucose residues by ether linkages. The pK, values for the amine groups of DEAE are 5.5 (tertiary), 9.2 (tertiary) and 14 (quaternary) [ 251. The plain proteins used as model macromolecules were all supplied by Sigma Chemical Co. The bovine serum albumin (BSA), grade V, essentially fatty acid free, containing 96-99% albumin, was No. A-6003, and the lysozyme (LYS) was of grade I, No. L-6876. The polypeptide used was poly (L-tyr, L-glu) -poly-
CH,
0
Fig. 1. Chain scissions that can be induced by UV irradiation of polysulfone [ 291.
278
DL-ala-poly-L-lys, No. M-3649 (240 000 g/mol; molar ratio; lys: ala: glu : tyr = 1.0: 18.3 : 3.3 : 2.1). The amino acid was D-lysine monohydrate No. L-0640. In Table 1 are given the isoelectric points (IP), the diffusion coefficients (D) and the mass transfer coefficients (k) for some of the proteins used. The values of k were calculated according to Jonsson [ 261. The whey protein used was WHEY PRO-90 supplied by Denmark Proteins A/S. Viby, Denmark. This is an undenatured whey protein concentrate produced from whey by ultrafiltration and spray-drying. The protein fraction consisted of 78-85% protein, 5-7% water, 3% ash, 4-7% fat and 4% lactose. In the preparation of the solutions p.a grade electrolytes, absolute ethanol (99.5 vol.% ) and RO-filtered water (Millipore) with a conductivity less than 1 $S/cm were used. Properties
of the membrane
surfaces and the proteins used
The GR membrane prepared of PSU is hydrophobic and non-ionic in nature. However, zeta potential data, which have been reported earlier [ 291, show that the membrane is increasingly negatively charged above pH 3.5, probably due to adsorption of either OH- ions or Cl- ions. Below this point of zero charge the charge of the membrane is either zero or slightly positive. The modified membranes can be expected to have properties arising both from irradiation and from the polymer present in the modifying solution. From TABLE 1 Isoelectric
points
serum albumin
BSA LYS Whey
TABLE
(IP), diffusion coefficients
(BSA),
Iysozyme
(LYS)
(D) and mass transfer coefficients
and whey protein
(h
1 for bovine
(Whey)
k (m/set)
IP
D (m’/sec)
5.0 [27] 11 [27] 4.2-5.5
6.8~106” 11.9x10-”
[28] [26]
intermediate
between BSA and LYS
[14]
1.86x lo-” 2.70x lo-”
2
Approximate charge conditions at different pH values for bovine serum albumin (BSA), (LYS), whey protein (Whey), a polypeptide (PPl) and the amino acid D-lysine
lysozyme
pH3
pH5
pH7
pH9
BSA LYS Whey PPI
++ ++ ++ +
i ++
-
--
++ _ -
+
D-Lysine
++
++
+
+ I ++
279
irradiation in the presence of oxygen hydrophilic carboxylic acid and sulfonic acid groups can be formed which are dissociated to anionic groups in the pH range of interest in this study (pH 3-9). Membranes modified with DEX can be expected to be neutral at low pH values and slightly negatively charged at high pH values [ 131. The positively charged DEAE attaches well to the negatively charged membrane and it gives the membrane a positive charge which increases with decreasing pH [ 131. The DEXSU modified membranes are negatively charged in the whole pH interval of interest. The PEOB is non-ionic because it is a block copolymer of uncharged monomers. The PEOB modified membranes have been presumed to be neutral or slightly negatively charged, depending on the degree of coverage with PEOB. The proteins are neutral at their IP’s although neutrality does not mean lack of charged groups, since they have as many positively as negatively charged groups at their IP’s. The assumed approximate charge conditions for the proteins, for the polypeptide used (PPl) and for the amino acid D-lySine are indicated in Table 2. Methods Preparation of the surfaces For the UF experiments the PSU membranes were modified by UV irradiation in aqueous solution containing different modification agents, with or without stirring. The device used as UV source was a Heraeus, Hanau UV immersion lamp, TNN 15/32. Most of the light has a wavelength of 254 nm and can be considered monochromatic according to the manufacturer. The radiation flux can be calculated to 6 W or 0.046 mol quanta/hr. Taking into account the transmissivity of the quartz tube (about 93% transmissivity) and the reduction in intensity due to the light passing water (2.8 cm) before hitting the membrane (the attenuation coefficient being 1 m-l for water), and considering cylindrical geometry (length of cylinder being 16 cm), the light intensity at the membrane can be calculated to 114 W/m2, not taking the rounded ends of the cylindrical lamp into account. The time of UV irradiation was chosen to give enlarged pores without loss in retention of the proteins used. The concentrations of the modification agents were selected so that a clear effect could be detected without too much pore blockage. The selection experiments were carried out at pH 5 when BSA and LYS were used and at pH 6 when Whey was used. The achieved best modification process was then used in the experiments at other pH values, too. For contact-angle measurements polysulfone surfaces were cast on a glass rod by dip-coating. Cyclohexanone solutions containing 5% PSU were used. Before use the polysulfone films on the rods were carefully dried first in air and then in vacuum for one hour so that no traces of solvent were left. The
280
absence of solvent was verified by contact-angle measurements until reproducible results were obtained. The cast surfaces were modified in a similar way as described above for the membranes. Contact-angle
measurements
The contact angles between pure water, air and the model surfaces were measured by a method similar to the Wilhelmy plate method except that a rod with a radius of 10 mm was used instead of a plate. The set-up for the measurements is pictured in Fig. 2. The contact angles were recorded with a video camera provided with magnifying optics. The advancing (0,) and receding (0,) contact angles were measured for the rod descending into the solution and then ascending from the solution, respectively. The movement of the rod was controlled to give a constant speed of 1 mm/min. Both contact angles were measured from a television screen by visual inspection. Readings were taken during constant movement after a stabilization period of 3 min. No correction in the values of the contact angles were needed for the rod because its radius was large compared with the radius of the meniscus [ 301. Ultrafiltration
experiments
The UF experiments were carried out at 25” C in a flat plate module described in detail elsewhere [31]. The membrane area was 19.3 cm2. The experiment is schematically pictured in Fig. 3. The membranes were stabilized for 2.5 hr by filtration of pure water at a pressure of 3 bar. After this period the flux was essentially constant and the pressure was dropped to 1.5 bar for 1 hr and the pure water flux (J,) was measured gravimetrically. The water flux calculated per bar did not change much after the pressure drop. Then the mod-
Fig. 2. Set-up for contact-angle measurements according to the Wilhelmy plate method using a rod instead of a plate. (1) Television screen, (2) video recorder, (3) video camera, (4) cast rod dipped into water, (5) motor attached to lifting shrew, (6) control board for lifting speed regulation, (7) light source.
281
WASHING 300
x_x-x-x /
IJPZ 200
r \ 0 *
0
YOOIFICATION
.
100 54 h
H20, 3l.m 0
,
100
1/".l.5;
$0,1.5bar 200
3b0
protein 15bar
H20,1.5bar . 400 Time [mini
UF experiment. Symbols explained in the test. All permeate fluxes given as relative values compared to the water flux before modification. The example experiments are made with 0.5 g/dm” BSA at pH 9, one without modification and the other with a membrane modified with 10 ppm PEOB during 10 min of UV irradiation.
Fig.
3. Schematized
ification, if any, was carried out. After that the permeate flux of water was measured, as above, after one hour (J,). Then a protein solution of fixed concentration, pH and ionic strength was adjusted and the flux was measured for one hour, where the flux at the start (J,, ) and at the end (J,,) were registered. In the experiments made with LYS the protein was added in portions. Instantaneous flux reduction (FR, ), flux reduction from protein addition at the end of the experiment (FR, ) , and total flux reduction (FR ) were calculated from eqn. (1) , where the second terms describe the respective flux losses.
FR, =I-J,,/J,,, FR, = I- J,,z/J,,l
(1)
FR = I- Jp2/J,,, After this the protein concentration in the permeate was calculated from measured UV absorbances at 280 nm. Corrections for concentration polarization were made according to eqn. (2))
282
(2)
cm=cb ew(Jp21h)
where c, is the membrane surface concentration and c,, the bulk concentration of protein. The observed (R, ) and true (R,) retentions of the membranes were calculated according to
R, = 1 - cP/c,,
R, = 1 - c,/c,
and
(3)
Results and discussion Influence of modification conditions on water flux In Fig. 4 can be seen the influence of UV irradiation time on water flux (J,). In one case the modification was made without stirring and in the other one with stirring in plain water. A series of experiments where 45 vol.% ethanol was present in the irradiation bath is also included. No macromolecules were present in the irradiation bath. Figure 5 shows the change in hydrophilicity as described by the contact angles of the GR material for the case without stirring. The results indicate that there is a rapid increase in hydrophilicity during the first minutes of UV exposure. At the same time a decrease in flux is registered. This phenomenon can possibly be explained by crosslinking of the membrane material when radicals and chain scissions are induced by the UV irradiation. This is in analogy * Jm/Jo* 100% 600.
500-
400-
300o-0 without stirring 0-0 with stirring x--x with stirring + 45%
200-
‘“&f_~-~/~ 0
5
10
15
ethanol
)
20 time Lminl
Fig. 4. Influenceof UV irradiation time on water flux (J,,,). J,, is the water flux before modification. The experiments were made in water with or without stirring. One series of experiments was with 45 vol.% ethanol.
283
B[degl 100
60
0-l 0
l
10
30
20
Irradiation time Iminl Fig. 5. Change in hydrophilicity
during UV irradiation
irradiation times pictured by the advancing fied polysulfone surface against water.
(S,)
in water without stirring after different
and receding
(0,) contact angles of the modi-
with experiments reported by Shimomura et al. for polyacrylonitrile (PAN) RO membranes modified with plasma [ 321. ESCA analysis of our samples, made at the Institute of Surface Chemistry in Stockholm, verify an increase in the oxygen/carbon ratio. The primary increase in hydrophilicity of the membrane is induced by direct irradiation and not transmitted by small molecules or macromolecules in the irradiation bath. This was also tested by measuring the contact angles of the “dark” sides of the rods, which had been exposed to the solutions in question but not irradiated. These contact angles decreased very little, or not at all, compared with the values obtained by direct adsorption of the macromolecule without irradiation. When stirring has been used a flux decrease is noticed only during the first five minutes of irradiation, and after that the flux increases with irradiation time (Fig. 4 ) . This effect of stirring could be explained by an increased amount of available oxygen, as stirring enhances material transport to the membrane. The oxygen probably also favors the formation of carboxylic and sulfonic acid groups on the membranes. These are dissociated and negatively charged at neutral pH values of the water and a state of internal repulsion in the pores is developed. This repulsion causes an enlargement of the pores, which results in an increase in water flux. This effect, as well as the destructive effect of oxygen, thus contributes to the flux increase. The destructive effect of oxygen could be detected as deformations of the membrane surface with a microscope attached to FTIR equipment (Enso-Gutzeit, Imatra). Some chain scissions could be
284 TABLE
3
Water flux one hour after modification (J,,) compared to flux before modification different irradiation times (IT), with ( + ) or without ( - ) stirring Stirring
_
_
_
35
-
_
38 88
10 15 “0 5 10 15 20 5 10 15 20
+ + + + + + + +
agent
J,lJ,,
IT (min)
5
Modification
(J,,) after
100 23
89 225 432
-
616
45 vol.% ethanol
50 315 488 627
45 vol.% ethanol 45 vol.% ethanol 45 vol.% ethanol
572 1500
10 10
+
45 vol.% ethanol + 5 ppm DEAE
-c
45 vol.% ethanol + 5 ppm PEOB
15 15
+ + + +
BSA BSA BSA
2 mm 5 mm 10 wm
423
BSA
5 ppm
485
1.5 20 15 15 15 15 15
176 33
Whey Whey PPl PPl D-lysine
10 15 5 10 5
ppm ppm ppm ppm ppm
420 292 392 210 130
15 15 15
+ + +
DEAE DEX DEXSU
5 ppm 10 ppm 10 ppm
388 525 306
10
+ + +
PEOB PEOB PEOB
10ppm 10ppm
293 1120
15 10
(s 1
5 ppm
392
observed from the FTIR spectra as changes in phenyl substitution. The spectra also showed an increase in oxygenated groups, verifying the results above obtained with ESCA. When the modification is made in aqueous solutions of different hydrophilic
285 a
b
Fig. 6. Pores with hydrophilic chains protruding in solution in different types of solvents. (a) Hydrophilic chains in “good” solvent, (b) hydrophilic chains in “poor” solvent i.e. wrong pH, high ionic strength or hydrophobic solvent, (c) hydrophilic negatively charged chains in “good” solvent, forming internal electrostatic repulsion and enlargement of the pore without loss in retention.
substances (Table 3) it can be observed that one can control the resulting permeability by changing the amount of macromolecule present as modification agent or by changing the irradiation time. When the amount of macromolecule in solution is increased the pores get blocked. This blocking effect is larger for the macromolecules containing positively charged groups, as they preferentially attach to the surface making ionic bonds with the negatively charged groups formed by irradiation of the polysulfone membrane. This also is an indication of the formation of negatively charged groups by irradiation. When 45 vol.% ethanol is present the alcohol can swell the membrane; since ethanol as a solvent [ 191 is not a “good” solvent for the formed hydrophilic groups [33] they adhere to the membrane pore walls leaving the pores open resulting in a permeability increase (Fig. 6b ), which seems to persist even after changing the solvent back to water. With the optical microscope also large destructions of the skin layer were observed. The situation with enlargement of pores due to a “poor” solvent can also occur at high ionic strength or at unfavorable pH, when the groups lingering into solution are charged [ 33,341. The contact angles for the cases with stirring with or without ethanol did not actually change from the case without stirring. The maximal effect is reached very fast and this effect is not changed by moving the rod a little closer or a little further away from the irradiation source. This means that the increases in permeability after 10 min of irradiation mainly result from the etching effect of oxygen and the resulting enlargement of the pores. Improvement
of flux during UF of protein solutions
0.5% Whey Table 4 shows the results from ultrafiltration experiments of Whey with different types of modified polysulfone GR 81 membranes. It can be seen that if only UV irradiation is used, improvement of permeate flux starts after irradiation times longer than 10 min. At 20 min of irradiation permeate flux is
286 TABLE
4
Effect of different kinds of modifications of the polysulfone CR 81 membrane on permeate flux and the observed retention (RI)during ultrafiltration of 0.5% Whey solution at 25°C without pH adjustment approximately at pH 6. IT=irradiation time Modification
agent
IT (min)
JplJ,, (so)
Retention
_
-
38
99.4
_
10 15 20
36 83 144
99.4 99.2 99.0
20 min 45% ethanol
-
19
99.3
45% ethanol 45% ethanol
10 20
83 219
99.3 91.9
5ppm 10ppm 5 ppm
PEOB DEX BSA
10 15 15
95 178 91
99.5 99.1 99.3
10ppm 15 ppm
Whey Whey
15 15
164 111
99.4 99.4
(% )
more than 300% of the permeate flux for the unmodified membrane with only a slight loss in retention. At pH 6 the whey proteins are slightly negatively charged and the irradiated membrane should be more negatively charged than the unmodified membrane, therefore the state of electrostatic repulsion between the membrane and the molecules in solution keeps retention high even when flux is increased. If ethanol is present the flux is further improved, which seems to be a combined effect of irradiation and alcohol treatment, as alcohol treatment of the membranes without irradiation decreases flux. With the largest flux increases a loss in retention results. Even better results are obtained with modifications where non-ionic polymers (PEOB, DEX) or proteins are present. The best result was obtained using a suitable concentration of whey protein as modification agent. 0.05% BSA In Figs. 7 and 8 the influence can be seen of modification on permeate flux during the ultrafiltration of a 0.05% BSA solution at different pH values after one hour. Figure 7 shows that modification with BSA and UV irradiation gives smaller increases in flux at neutral pH values than in acidic or alkaline solutions. When the modification conditions were adjusted they were chosen to give such an increase in flux, at the IP of BSA, that retention was not considerably decreased; this same modification was then used at different pH values.
287 Jp2/Jor100$
800
700
Modification
I
Cont.
KCI
o--o
0.015
M
x--x UV 15 min ; BSA 2 ppm 0-0 UV 15 min ; BSA 2 ppm
0.015
M
0.15 M
400-
300-
200-
loo-
0, 0
. 1
2
3
4
5
6
7
8
9
10
pH
Fig. 7. Influence on permeate flux (J,,) of modification and salt concentration at different pH values in the ultrafiltration of 0.05% BSA solution after one hour compared to flux before modification (J,). Modification of membrane with 2 ppm BSA and 15 min of UV irradiation. T= 25 o C, p= 1.5 bar.
The surface modified with BSA can be considered a self-rejecting surface as its charge should be approximately the same as the charge of the BSA molecules in solution. The results show that the modifications with BSA, which cause electrostatic repulsion between the membrane surface and the protein molecules in solution at pH 3 and 9, cause a considerably increased flux. Increase in salt concentration decreases this effect as can be expected due to shielding of charges. The effects of other types of modifications on flux are shown in Fig. 8. These effects are much smaller at pH values near the IP of BSA than at pH values further away from it. It can also be noted that similarity of charge is not as important as charge density of any sign or the hydrophilicity; this was also shown earlier to be the case with ovalbumin [ 351. The explanation of the positive effect in the case of attracting charges can be that first, due to electrostatic attraction, one monolayer of BSA is formed and then a state of repulsion is attained, which actually gives better flux than only an increase in hydrophilicity. At the IP of BSA hydrophilicity of the membrane is probably more important than charge density.
288 Modification
I
Jp2/Jo*100%
800
700-
0-n
~--a UV 15 min O-O UV 15 min e-0
uv
10 min
; DEAE ; DEXSU ; PEOB
5 ppm 10 ppm 10 ppm
600-
500-
400-
300-
200-
loo-
04
0
.
1
2
3
4
5
6
7
8
9
10
PH
Fig. 8. Influence on permeate flux (J,,,) of modification at different pH values in the ultrafiltration of 0.05% BSA solution after one hour compared to flux before modification (J,,). 2’~ 25 ‘C, p = 1.5 bar.
Flux reduction in experiments
with modified membranes
Pore blocking The modified membranes show a higher flux reduction than the unmodified membranes, when protein is applied to the solution. This seems to be a result of the increased water permeability of the modified membranes. With the modifications made in this study using UV irradiation, the conventional flux reduction (FR) concept can not be meaningfully used to describe the situation of adsorption and fouling. In order to investigate what happened in our modification experiments, flux reduction is divided into two parts; FR1, which describes the plugging effect and most of the concentration polarization effect, and FR2, which probably has more to do with adsorption and rearrangement of the molecular layers at the surface. FR, and/or FR, were sometimes negative especially at high pH values, as e.g. in one of the experiments reported in Fig. n
The flux loss (J,,/.J,) did not correlate with the original flux (J,,) of the membrane, nor with flux increase (JJJ,) during modification. It seemed, however, that if a higher flux had been achieved after modification the flux loss increased. Flux loss is plotted against flux after modification (J,,,) in Fig.
289 A J4 J,
*‘0°%
-. x-x O-O A-A
A
l
150
pH3 pH5 pH 7 pH9
100
50
0
I
0
100
200
.
300
J,W(m2h)l Fig. 9. Instantaneous flux loss (J,,/J,) in the beginning of ultrafiltration of 0.05% BSA solution at different pH values as a function of flux after modification (J,). T= 25 “C, p = 1.5 bar.
9. It can be seen that an almost linear relationship is obtained at pH 5, the IP of BSA. At this pH value no large electrostatic effects are expected. This means that the greater the flux, the higher flux reduction, probably due to pore plugging. It means that in ideal conditions equal size pores are formed. A relative estimate of this pore size can then be made from the flux. One can also assume that since BSA is rather neutral at pH 5, it does not favorably plug any special kind of modified membrane. At pH values near 7, the flux loss is almost the same as at pH 5 but the points are more scattered. However, at pH z 3 and even more at pH z 9 the flux loss is very small and even flux increase is observed at pH FZ9. This can be explained by an increase in pore size due to pH change and the presence of a slight salinity to stabilize the charges. Effect of concentration
on pore blocking
When the protein solution is applied, the pore blocking effect seems to be rather instantaneous even at small concentrations. Increasing the concentra-
290 Modification
I
.-.
x--x UV 10 min o--O UV 15 min;
A-a
UV 15 min
Fig. 10. Flux reduction
PEOB 5 ppm
; DEAE 5 ppm
in ultrafiltration
after different modifications.
of lysozyme
as a function
of membrane
concentration
T= 25’ C, p = 1.5 bar.
tion then increases flux reduction rather slowly as seen in Fig. 10 in ultrafiltration of LYS at different concentrations. One can see that pore plugging is almost complete at a concentration of 50 ppm. In the cases with membranes modified with DEAE and D-lysine pore blocking is slower. This may result from the fact that in these cases electrostatic repulsion conditions prevail and this slows down the process at low concentrations. Flux loss after one hour The change in flux with time as described by FR, (FR, = 1- J,,,/J,,,) for 0.5% Whey at pH 6 was studied to see if it somehow correlated to the type of modification made. Some results are shown in Table 5, which are summarized from the experiments reported in Table 4. It can be seen that there is little difference between the unmodified membrane and the UV irradiated membrane. When Whey is used as modifying substance there is very little flux reduction with time. The flux loss after one hour did not relate to the measured initial flux (J,,,) neither to the flux after modification (J,) nor to any special, calculated relative flux. It could only be noted that flux was either gained or lost so as to
291 TABLE
5
Flux reduction F&
(FR, = 1 -J,,/J,,
) after one hour of ultrafiltration
Unmodified membrane Only UV irradiated membrane UV UV UV UV
irradiation irradiation irradiation irradiation non-ionic
+ + + +
of Whey at pH 6, T= 25’ C
0.193 0.194 0.248
45% ethanol Whey BSA hydrophilic
0.031 0.102 0.253
substance
Abs. I
+ 25
50
Jpp I I/(m’h) Fig. 11. Absorbance a function
100
75 1
of the permeate at 280 nm in ultrafiltration
of 0.5% whey protein solution as
of permeate flux (Jr,,) after one hour.
compensate for the effects caused by instantaneous flux loss. The results were the same for BSA at different pH values and for Whey at pH 6. It could be noted that FR, actually smoothed out the differences in pore size between the membranes. This could be seen as a decrease in the standard deviation in the values of the fluxes for Jpl and JP2 for different modifications. Retention
of proteins
with the modified membranes
One objective of the modification experiments was to get membranes with a retention, that was at least as good as with the membranes without modifications. Therefore the testing of the modification conditions was done rather near the IP’s of the proteins in order that the electrostatic repulsive effects favorable for retention would be as small as possible. For BSA the modifica-
292 TABLE
6
Permeate fluxes (Jr,) in l/ (m’/hr), observed retentions (R,) and true retentions (R,) in % after one hour of ultrafiltration of BSA at different pH values with unmodified and modified polysulfone membranes. IT= irradiation time Modification
IT
pH3
pH7
pH5
pH9
(min)
J P2 _
_
BSA2ppm PEOB 10 ppm PPI 5 ppm DEAE 5 ppm DEXSU 10 ppm
15 10 15 15 15
TABLE
R,
R,
Rz
96.8
98.2
15
98.9 99.0
17
98.5 98.9
98 99.7 91 99.7 109 99.4 104 >99.7 76 98.8
99.9 99.9 99.9 >99.9 99.6
44 41 61 41 35
98.6 99.1 97.1 99.1 98.9
44 28 53 40 37
98.6 99.4 97.0 98.8 99.4
40
Jp2
Rz Jp2 R,
R,
99.3 99.5 98.9 99.5 99.3
99.2 99.6 99.9 99.3 99.7
Jp2
R,
R,
35
99.0
99.4
154 100 120 107 130
99.4 99.7 99.7
99.9 99.9 >99.9 z99.9 >99.9
>99.7 99.7
7
Observed retention (R,) and true retention (R2) in the end of the ultrafiltration made with LYS in Fig. 9. IT= irradiation time Modification
IT
R,
R2
99.6 98.8 84.6 98.5 97.8
99.7 98.8 88.9 98.7 97.9
experiments
(min) PEOB 5 ppm DEAE 5 ppm D-Lysine 5 ppm
_ 10 15 15 15
tions were also tested at different pH values. Table 4 gives the retentions of Whey for differently modified membranes. It can be noted that the observed retention is very high in all cases. If flux is increased considerably a slight loss in retention occurs. When modifications are made with Whey there seems to be both a good flux and a good retention. At the rather neutral pH of the experiment and near the IP of Whey no large differences in retention depending on electrostatic forces are expected, since the whey proteins are either slightly charged or not charged at all. The correlation of flux with retention was tested further by plotting absorbance at 280 nm versus flux (Fig. 11) . Up to fluxes of ca. 25 l/m’-hr the absorbance does not change, which indicates almost total retention of the proteins (the measured background absorbance probably emanates from impurities present in the Whey used). At higher fluxes there is an increase in absorbance, and the increase is faster at higher fluxes. As the whey proteins are of different sizes and have different IP’s it is natural that the increase in absorbance along with flux is not linear. The point where absor-
293
bance starts to increase should correspond to the size of the smallest of the whey protein molecules. Besides on molar mass this size depends also on the conformational state and the charge of the protein molecule. For BSA, differences in retention at different pH values are shown in Table 6. At intermediate pH values retention is slightly lower than at pH 3 and pH 9, probably because BSA is near its IP and therefore not highly charged. The best retentions are achieved at high pH. At pH 3 the best retentions are achieved with membranes modified with positively charged polymers, as was expected, and the lowest retention is obtained for DEXSU, which is negatively charged (not taking into account the unmodified hydrophobic PSU membrane). The hydrophilicities of the modified membranes were, according to the contactangle measurements, almost equal, and also equal to the hydrophilicity obtained by irradiation only. For the experiments made with LYS depicted in Fig. 10 the retentions are given in Table 7. It can be seen that retention is higher when electrostatic repulsion is achieved as in the cases with DEAE and D-lysine, and nearly as good as without modification even if the fluxes are higher. For the non-ionic PEOB retention is not so good. The true retentions show the same trends as the observed retentions, even if they are somewhat higher. Scope of modification: flux results
estimation
of protein sizes and double layer forces from
When commercial hydrophobic polysulfone membranes are modified with UV irradiation and hydrophilicity increasing substances, it is possible to enlarge the pores more than without irradiation. Normally, the membrane pores are somewhat flexible and open up to some extent depending on the modification agent used and the pH of the experiment. But without the destructive influence of irradiation there is a limit to this opening effect. With irradiation the pores can be enlarged and permeability increased to an extent which is proportional to irradiation time. When a particular protein solution is ultrafiltrated through the modified membrane, the pores stabilize to a size characteristic for the protein at the specific pH of the solution. This fact can be deduced from flux measurements and an assumed correlation between pore size and flux. This pore size seems to give an observed retention of more than 99% and a true retention near 100% for BSA and Whey. This means that the by modification obtained leaky membrane almost instantaneously gets its larger pores plugged by the protein in question, until the membrane stabilizes and retention is almost ideally 100%. If the opening of the pores is too large so that even a monolayer of the protein in the pores still cannot retain the protein, then the modification has not been successful if high retention was the goal. In the very first minutes after the beginning of the ultrafiltration experiment with the protein solution in question, pore plugging takes place; it depends only
294
on the flux after modification (J,) and is actually not at all proportional to the original flux of the membrane (J,). With time flux increases or decreases so as to stabilize the pore sizes to the pH and protein in question. When comparing the final fluxes (J,,) at the same pH for different hydrophilic modification agents, the best flux is obtained if electrostatic repulsion is obtained. This repulsion can be obtained either as repulsion between the protein and the modification layer or by a monolayer of the protein (self-rejection) formed on the membrane by the attractive forces. In this study the monolayer is formed when the pore size is modelled by modification and therefore it does not reduce the final flux of the membrane. If the pore size is fixed from the beginning, as in the modifications without irradiation, the adsorbed layer will possibly cause a reduction in pore size (in the case of a leaky membrane), but since stabilization of pore size takes place when the protein solution is present, the final pore size will become as large as with a surface originally equally charged as the protein. The influence of pH on pore size and flux is determined by the way the pH affects the charges of the proteins, and the solution properties of the hydrophilic modification agents. At suitable salinities and pH values the modification agents protrude in the solution in the pores blocking the passage (Fig. 6 ). The protein has a larger volume if highly charged, and if electrostatic repulsion exists the range of the repulsive forces determine the final pore size. This was also the case for BSA as the flux ratios between the different pH values pH5 : pH7 : pH9 : pH3 were 1: 0.9 : 2.8: 2.2, respectively. The pore size depends on electric double layer forces, is sensitive to salinity and should decrease with increasing salt concentration (Fig. 6b ). This could be observed in the experiments made at higher salt concentration (0.15 M KCl, Fig. 7) with BSA as the modification agent at pH 3 and 9. If several proteins are present as in the experiments with Whey, the flux stabilizes so that the pore size correlates to the smallest protein molecule in solution, the size depending on its state of charge. For Whey this protein is the a-lactalbumin (M”= 14200 g/mol, [27] ), the size of which is much smaller than the size of BSA (M”=68000 g/mol, [27] ). Comparing the fluxes after modification for Whey and BSA near their IP’s, when retention is almost lOO%, one can see that the flux for Whey (Fig. 11) is much smaller than for BSA (Table 6)) which is in accordance with the assumptions. If a correlation of flux and protein size can be found it should be possible to estimate both maximal fluxes and electrostatic repulsive forces from the modification results. It has to be taken into account that the experiments above have been performed only with one type of membrane (GR 81) at specific conditions (fixed temperature, flow rate and pressure). If e.g. the pressure is increased, the protein molecules deform more easily and plug the pores to a higher extent and the optimizations above might not apply in the same way.
295
Conclusions
In the present study hydrophobic polysulfone UF membranes were modified with UV irradiation and hydrophilicity increasing agents. The modifications were tested with protein solutions. UV irradiation increased flux and the hydrophilicity of the membranes. Most of the flux increase came from the etching effect of oxygen during stirring conditions. The flux increases obtained varied with pH and modification agents used, and could be more than 400% compared to unmodified conditions without any loss in retention. The best retentions were obtained at pH values, where a strong electrostatic repulsion was obtained. The pores enlarged to a fixed size that can be taken as an estimate of the protein size, which due to the range of double layer forces acting between proteins and membranes cannot penetrate the pore. Acknowledgements
The authors want to thank Techn. Lit. Markku Laatikainen for prereading and commenting the manuscript, and miss Jaana Tuisku for her excellent technical assistance.
References 1
2 3
4 5 6 7 8
9 10 11
J.H. Hanemaaijer, T. Robbertsen, Th. van den Boomgaard and J.W. Gunnink, Fouling of ultrafiltration membranes. The role of protein adsorption and salt precipitation, J. Membrane Sci., 40 (1989) 199. C. Taddei, G. Daufin, P. Aimar and V. Sanchez, Role of some whey components on mass transfer in ultrafiltration, Biotechn. Bioeng., 34 (1989) 171. I. Lundstrom, Protein conformation at surfaces, in: P.-O. Glantz, S.A. Leach and T. Ericson (Eds.), Oral Interfacial Reactions of Bone, Soft Tissue and Saliva, IRL Press, Oxford, 1985, pp. 9-23. E. Matthiasson, Macromolecular adsorption and fouling in ultrafiltration and their relationships to concentration polarization, Ph.D. Thesis, Lund University, 1984. C.-G. Gijlander and E. Kiss, Protein adsorption on functionalized and ESCA-characterized polymer films studied by ellipsometry, J. Colloid Interface Sci., 121 (1988) 240. I. Lundstrom, Surface physics and biological phenomena, Physica Scripta, T4 (1983) 5. W. Norde, Adsorption of proteins from solution at the solid-liquid interface, Adv. Colloid Interface Sci., 25 (1986) 267. S.H. Lee and E. Ruckenstein, Adsorption of proteins onto polymeric surfaces of different hydrophilicities: A case study with bovine serum albumin, J. Colloid Interface Sci., 125 (1988) 365. F. Vigo, M. Nicchia and C. Uliana, Poly(viny1 chloride) ultrafiltration membranes modified by high frequency discharge treatment, J. Membrane Sci., 36 (1988) 187. W. Yan, P. Yang and Y. Wang, UV-radiation grafting of acrylamide onto cellulose acetate reverse osmosis membrane, (In Chinese), Shuichuli Jishu, 14(4) (1988) 213. F. Vigo, C. Uliana and G. Dondero, Ultrafiltration membranes obtained by poly(acrylonitrile) grafted onto poly(viny1 chloride), Desalination, 70 (1988) 277.
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F. Vigo and C. Uliana, Ultrafiltration
13
onto poly(viny1 chloride), J. Appl. Polym. Sci., 38 (1989) 1197. M. Wahlgren, B. Sivik and M. Nystrom, Dextran modifications branes:
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obtained
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fouling characteristics,
monomers
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Stand.
Ch-se-
14
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by modification of ultrafiltration membranes, Progr. Colloid Polym. Sci., 82 (1990) 321. K.J. Kim, A.G. Fane and C.J.D. Fell, The performance of ultrafiltration membranes pre-
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treated by polymers, Desalination, 70 (1988) 229. K.J. Kim, A.G. Fane and C.J.D. Fell, The effect of Langmuir-Blodgett
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on the performance of ultrafiltration membranes, J. Membrane Sci., 43 (1989) 187. J.-L. Nilsson, A study of ultrafiltration membrane fouling, Ph.D. Thesis, Lund University,
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1989. G. Jonsson, Personal communication, 1986. Udel polysulfone, A temperature resistant transparent
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Carbide. M. Fontyn,
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membranes by spectroscopic B. RHnby and J.F. Rabek,
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Polymers. Principles and Applications, John Wiley & Sons, London, 1975, pp. 223-227. J.F. McKellar and N.S. Allen, Photochemistry of Man-made Polymers, Applied Science Pub-
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lishers Ltd, London, 1979, pp. 156-160. D.H. Napper, Flocculation studies of sterically
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Sci., 32 (1970) 106. A. Carlson, Factors influencing
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tion, Sep. Sci. Techn., 23(8,9) (1988) Dextran fractions, dextran sulphate,
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search, Pharmacia Fine Chemicals. G. Jonsson, Boundary layer phenomena
K. Turku and P. Jlrvinen,
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to fouling accomplished
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(In Swedish
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1, Union
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stabilized
dispersions,
the use of aqueous two phase partition 785. DEAE-dextran.
Defined
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of
J. Colloid Interface for protein
polymers of dextran
purifica-
for biological
re-
and whey protein
27
solutions, Desalination, 51 (1984) 61. W.G. Burton, K.D. Nugent, T.K. Slattery, B.R. Summers and L.R. Snyder, Separation of proteins by reversed-phase high-performance liquid chromatography. I. Optimizing the col-
28
umn, J. Chromatogr., 443 (1988) 363. C. Tanford, Physical Chemistry of Macromolecules, pp. 357-358.
29
M. Nystriim,
30 31 32
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35
and E. Matthiasson,
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acterization of ultrafiltration membranes, Colloid Surf., 36 (1989) 297. J.F. Padday, Surface Tension. Part I. The theory of surface tension, in: E. Matijevic’ (Ed.), Surface and Colloid Science, Vol. 1, Wiley-Interscience, New York, NY, 1969, pp. 39-150. M. Lindstriim, M. Nystriim and M. Laatikainen, Interactions between chlorolignin and polysulfone ultrafiltration membranes, Sep. Sci. Technol., 23 (1988) 703. T. Shimomura, M. Hirakawa, I. Murase, M. Sasaki and T. Sano, Preparation of polyacrylonitrile reverse osmosis membrane by plasma treatment, J. Appl. Polym. Sci., Appl. Polym. Symp., 38 (1984) 173. H. Iwata and T. Matsuda, Preparation and properties of novel environment-sensitive membranes (1988)
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Y. Osada, K. Honda and M. Ohta, Control of water permeability by mechanochemical contraction of poly (methacrylic acid)-grafted membranes, J. Membrane Sci., 27 (1986) 327. M. Nystrom, Fouling of unmodified and modified polysulfone ultrafiltration membranes by ovalbumin, J. Membrane Sci., 44 (1989) 183.
Science
Publishers
275
275-296 B.V., Amsterdam
Modification of polysulfone ultrafiltration membranes with UV irradiation and hydrophilicity increasing agents Marianne Nystriim and Pia Jarvinen Laboratory of Technical Polymer Chemistry, Department of Chemical Technology, Lappeenranta University of Technology, P.O. Box 20, SF-53851 Lappeenranta (Finland) (Received
August 28,1990,
accepted
in revised form April 8,199l)
Abstract Hydrophobic
polysulfone
UF membranes
were modified with UV irradiation
and hydrophilicity
increasing agents. The modifications were tested with 0.5% whey-protein solution and 0.05% lysozyme solution at pH 6 and with 0.05% bovine serum albumin solution at various pH values. UV irradiation increased flux and the hydrophilicity of the membranes. The flux increases obtained varied with pH and modification agents used and could be more than 400% compared to unmodified conditions without any loss in retention. The best retentions were obtained at pH values, where both the protein and the membrane had the same charge, and a strong electrostatic repulsion was obtained. The pores enlarged to fixed sizes, which depended on the sizes of the proteins and the range of double layer forces between proteins and membranes at different states of charge density.
Keywords: ultrafiltration;
polysulfone
crease of; bovine serum albumin;
membranes;
UV irradiation;
fouling;
hydrophilicity,
in-
lysozyme; whey
Introduction
In ultrafiltration (UF ) of proteins one of the greatest problems is the fouling of the membranes, which causes a considerable flux decline. Fouling often results when small divalent ions like calcium ions are present [ 1 ] or if the protein solution contains small amounts of lipids [ 21. Also the proteins themselves seem to adsorb tightly onto the membranes, especially if the membranes are hydrophobic in nature [ 3,4]. The resulting fouling can be either reversible or irreversible. In fouling and adsorption of proteins on membranes an important factor determining adsorption seems to be the strength of the hydrophobic interaction between the proteins and the membranes. The proteins consist of both hydrophilic and hydrophobic parts, the ratio being different for different proteins. The importance of hydrophilicity for the prevention of adsorption has been shown by Gijlander and Kiss [ 51 and has been explained by Lundstrom
0376-7388/91/$03.50
0 1991 Elsevier
Science
Publishers
B.V.
All rights reserved.
[61 to depend on the fact that the hydrophilic surface attracts so much water that adsorption is prevented. When a protein adsorbs on a hydrophobic surface also conformational changes can take place [3]. The adsorption can be both entropically or enthalpically driven [ 71. Multilayer adsorption in static conditions usually consists of one layer which is more strongly bound and the rest of the layers can more easily be washed off. This is e.g. the case with BSA adsorption shown by Lee and Ruckenstein [ 81. In ultrafiltration both adsorption forces and the dynamic experimental conditions determine the amounts of fouling and flux reduction. In UF, even when the proteins seem to be hydrophilic and charged and no fouling is expected because of electrostatic repulsion between molecules and membrane, there still seems to be fouling taking place. When changes of the charge of the protein and the membrane induced by pH control do not improve the ultrafiltration results, modification of commercial membranes or the manufacture of new types of membranes seems to be the means to improve the conditions for UF of proteins. As both increased hydrophilicity and charge density of the membranes seem to be important, membrane manufacturers have tried to graft polymers of different kinds either on the ready-made membrane [5,9-111 or on the polymer from which the membrane is prepared [ 121. This grafting has mostly been initiated with a glow discharge apparatus [ 51 or by UV irradiation [lo]. Modification of the membranes has also been accomplished by chemical binding of macromolecules to the membrane [ 131 or by a partly reversible, chemical adsorption of the modification agent onto the membrane from solution [ 14,151 or by the application of a Langmuir-Blodgett film on the membrane [ 161. The problem with modification by chemical treatment of readymade commercial membranes is that the modification agent partly blocks the pores of the membranes, so that even if the membranes after modification are less apt to foul, the total flux after modification is smaller than before modification. One successful modification method seems to be that by Vigo and Uliana [ 12 1, where the polymer material is modified before the membrane is cast and in this way a good flux is obtained. In this study commercial polysulfone UF membranes were modified using UV irradiation alone or together with different modification agents in liquid environment. The criteria were to get a membrane that was less apt to foul and also a membrane with a better flux than normally without any decrease in retention of the proteins. The modification results were tested by UF experiments with different protein solutions. In some cases the hydrophilicities of the modifications were tested with contact angle measurements on a cast polymer surface treated in the same way as the membrane. Experimental
Materials The polysulfone (PSU) membranes used were of type GR 81 PP delivered from De Danske Sukkerfabrikker (DDS) with a nominal cut-off of 6000 g/
mol. These membranes have a relatively wide pore size distribution [ 171 and the mean pore diameter is approximately 2 nm for the GR 81 membrane [ 181. Smooth PSU model surfaces were prepared from PSU of the GR type obtained from DDS. The GR type polysulfone is mainly the same as UDEL polysulfone [ 191, which means that it is a polymer prepared of bisphenol A and 4,4-dichlorodiphenylsulfone, n = 50-80(Fig. 1) . The GR polysulfone differs from the UDEL polysulfone in that it contains more bisphenol A according to Fontyn et al. [ 201. The UDEL polysulfone is resistant to most chemicals but partly swells in alcohols [ 191. The reactions taking place in the chain, when irradiated with UV [21,22] are bond scissions, indicated in Fig. 1, and dissociations of the methyl side groups, either by the formation of a methyl or a hydrogen radical. As end products sulfonic acid groups can be formed. Also keto or ethenyl groups can be formed. The block copolymer (PEOB) used was synthesized according to Napper [ 231 of polyethylene oxide (PEO ) (M= 35 000 g/mol) and vinyl acetate monomer to a copolymer containing two blocks. All the dextrans used for modification were polydisperse with a W of 500 000 g/mol. They were delivered by Pharmacia, Sweden. Dextran T 500 (DEX) is non-ionic in nature. Dextran sulfate (DEXSU) is a polyanionic derivative of dextran produced by esterification with chlorosulfonic acid. The pK, of DEXSU is below 2. The relative hydrophilicity of DEXSU is lower than for DEX [ 241. Diethylaminoethyl dextran (DEAE) is a polycationic derivative of dextran containing diethylaminoethyl groups linked to the glucose residues by ether linkages. The pK, values for the amine groups of DEAE are 5.5 (tertiary), 9.2 (tertiary) and 14 (quaternary) [ 251. The plain proteins used as model macromolecules were all supplied by Sigma Chemical Co. The bovine serum albumin (BSA), grade V, essentially fatty acid free, containing 96-99% albumin, was No. A-6003, and the lysozyme (LYS) was of grade I, No. L-6876. The polypeptide used was poly (L-tyr, L-glu) -poly-
CH,
0
Fig. 1. Chain scissions that can be induced by UV irradiation of polysulfone [ 291.
278
DL-ala-poly-L-lys, No. M-3649 (240 000 g/mol; molar ratio; lys: ala: glu : tyr = 1.0: 18.3 : 3.3 : 2.1). The amino acid was D-lysine monohydrate No. L-0640. In Table 1 are given the isoelectric points (IP), the diffusion coefficients (D) and the mass transfer coefficients (k) for some of the proteins used. The values of k were calculated according to Jonsson [ 261. The whey protein used was WHEY PRO-90 supplied by Denmark Proteins A/S. Viby, Denmark. This is an undenatured whey protein concentrate produced from whey by ultrafiltration and spray-drying. The protein fraction consisted of 78-85% protein, 5-7% water, 3% ash, 4-7% fat and 4% lactose. In the preparation of the solutions p.a grade electrolytes, absolute ethanol (99.5 vol.% ) and RO-filtered water (Millipore) with a conductivity less than 1 $S/cm were used. Properties
of the membrane
surfaces and the proteins used
The GR membrane prepared of PSU is hydrophobic and non-ionic in nature. However, zeta potential data, which have been reported earlier [ 291, show that the membrane is increasingly negatively charged above pH 3.5, probably due to adsorption of either OH- ions or Cl- ions. Below this point of zero charge the charge of the membrane is either zero or slightly positive. The modified membranes can be expected to have properties arising both from irradiation and from the polymer present in the modifying solution. From TABLE 1 Isoelectric
points
serum albumin
BSA LYS Whey
TABLE
(IP), diffusion coefficients
(BSA),
Iysozyme
(LYS)
(D) and mass transfer coefficients
and whey protein
(h
1 for bovine
(Whey)
k (m/set)
IP
D (m’/sec)
5.0 [27] 11 [27] 4.2-5.5
6.8~106” 11.9x10-”
[28] [26]
intermediate
between BSA and LYS
[14]
1.86x lo-” 2.70x lo-”
2
Approximate charge conditions at different pH values for bovine serum albumin (BSA), (LYS), whey protein (Whey), a polypeptide (PPl) and the amino acid D-lysine
lysozyme
pH3
pH5
pH7
pH9
BSA LYS Whey PPI
++ ++ ++ +
i ++
-
--
++ _ -
+
D-Lysine
++
++
+
+ I ++
279
irradiation in the presence of oxygen hydrophilic carboxylic acid and sulfonic acid groups can be formed which are dissociated to anionic groups in the pH range of interest in this study (pH 3-9). Membranes modified with DEX can be expected to be neutral at low pH values and slightly negatively charged at high pH values [ 131. The positively charged DEAE attaches well to the negatively charged membrane and it gives the membrane a positive charge which increases with decreasing pH [ 131. The DEXSU modified membranes are negatively charged in the whole pH interval of interest. The PEOB is non-ionic because it is a block copolymer of uncharged monomers. The PEOB modified membranes have been presumed to be neutral or slightly negatively charged, depending on the degree of coverage with PEOB. The proteins are neutral at their IP’s although neutrality does not mean lack of charged groups, since they have as many positively as negatively charged groups at their IP’s. The assumed approximate charge conditions for the proteins, for the polypeptide used (PPl) and for the amino acid D-lySine are indicated in Table 2. Methods Preparation of the surfaces For the UF experiments the PSU membranes were modified by UV irradiation in aqueous solution containing different modification agents, with or without stirring. The device used as UV source was a Heraeus, Hanau UV immersion lamp, TNN 15/32. Most of the light has a wavelength of 254 nm and can be considered monochromatic according to the manufacturer. The radiation flux can be calculated to 6 W or 0.046 mol quanta/hr. Taking into account the transmissivity of the quartz tube (about 93% transmissivity) and the reduction in intensity due to the light passing water (2.8 cm) before hitting the membrane (the attenuation coefficient being 1 m-l for water), and considering cylindrical geometry (length of cylinder being 16 cm), the light intensity at the membrane can be calculated to 114 W/m2, not taking the rounded ends of the cylindrical lamp into account. The time of UV irradiation was chosen to give enlarged pores without loss in retention of the proteins used. The concentrations of the modification agents were selected so that a clear effect could be detected without too much pore blockage. The selection experiments were carried out at pH 5 when BSA and LYS were used and at pH 6 when Whey was used. The achieved best modification process was then used in the experiments at other pH values, too. For contact-angle measurements polysulfone surfaces were cast on a glass rod by dip-coating. Cyclohexanone solutions containing 5% PSU were used. Before use the polysulfone films on the rods were carefully dried first in air and then in vacuum for one hour so that no traces of solvent were left. The
280
absence of solvent was verified by contact-angle measurements until reproducible results were obtained. The cast surfaces were modified in a similar way as described above for the membranes. Contact-angle
measurements
The contact angles between pure water, air and the model surfaces were measured by a method similar to the Wilhelmy plate method except that a rod with a radius of 10 mm was used instead of a plate. The set-up for the measurements is pictured in Fig. 2. The contact angles were recorded with a video camera provided with magnifying optics. The advancing (0,) and receding (0,) contact angles were measured for the rod descending into the solution and then ascending from the solution, respectively. The movement of the rod was controlled to give a constant speed of 1 mm/min. Both contact angles were measured from a television screen by visual inspection. Readings were taken during constant movement after a stabilization period of 3 min. No correction in the values of the contact angles were needed for the rod because its radius was large compared with the radius of the meniscus [ 301. Ultrafiltration
experiments
The UF experiments were carried out at 25” C in a flat plate module described in detail elsewhere [31]. The membrane area was 19.3 cm2. The experiment is schematically pictured in Fig. 3. The membranes were stabilized for 2.5 hr by filtration of pure water at a pressure of 3 bar. After this period the flux was essentially constant and the pressure was dropped to 1.5 bar for 1 hr and the pure water flux (J,) was measured gravimetrically. The water flux calculated per bar did not change much after the pressure drop. Then the mod-
Fig. 2. Set-up for contact-angle measurements according to the Wilhelmy plate method using a rod instead of a plate. (1) Television screen, (2) video recorder, (3) video camera, (4) cast rod dipped into water, (5) motor attached to lifting shrew, (6) control board for lifting speed regulation, (7) light source.
281
WASHING 300
x_x-x-x /
IJPZ 200
r \ 0 *
0
YOOIFICATION
.
100 54 h
H20, 3l.m 0
,
100
1/".l.5;
$0,1.5bar 200
3b0
protein 15bar
H20,1.5bar . 400 Time [mini
UF experiment. Symbols explained in the test. All permeate fluxes given as relative values compared to the water flux before modification. The example experiments are made with 0.5 g/dm” BSA at pH 9, one without modification and the other with a membrane modified with 10 ppm PEOB during 10 min of UV irradiation.
Fig.
3. Schematized
ification, if any, was carried out. After that the permeate flux of water was measured, as above, after one hour (J,). Then a protein solution of fixed concentration, pH and ionic strength was adjusted and the flux was measured for one hour, where the flux at the start (J,, ) and at the end (J,,) were registered. In the experiments made with LYS the protein was added in portions. Instantaneous flux reduction (FR, ), flux reduction from protein addition at the end of the experiment (FR, ) , and total flux reduction (FR ) were calculated from eqn. (1) , where the second terms describe the respective flux losses.
FR, =I-J,,/J,,, FR, = I- J,,z/J,,l
(1)
FR = I- Jp2/J,,, After this the protein concentration in the permeate was calculated from measured UV absorbances at 280 nm. Corrections for concentration polarization were made according to eqn. (2))
282
(2)
cm=cb ew(Jp21h)
where c, is the membrane surface concentration and c,, the bulk concentration of protein. The observed (R, ) and true (R,) retentions of the membranes were calculated according to
R, = 1 - cP/c,,
R, = 1 - c,/c,
and
(3)
Results and discussion Influence of modification conditions on water flux In Fig. 4 can be seen the influence of UV irradiation time on water flux (J,). In one case the modification was made without stirring and in the other one with stirring in plain water. A series of experiments where 45 vol.% ethanol was present in the irradiation bath is also included. No macromolecules were present in the irradiation bath. Figure 5 shows the change in hydrophilicity as described by the contact angles of the GR material for the case without stirring. The results indicate that there is a rapid increase in hydrophilicity during the first minutes of UV exposure. At the same time a decrease in flux is registered. This phenomenon can possibly be explained by crosslinking of the membrane material when radicals and chain scissions are induced by the UV irradiation. This is in analogy * Jm/Jo* 100% 600.
500-
400-
300o-0 without stirring 0-0 with stirring x--x with stirring + 45%
200-
‘“&f_~-~/~ 0
5
10
15
ethanol
)
20 time Lminl
Fig. 4. Influenceof UV irradiation time on water flux (J,,,). J,, is the water flux before modification. The experiments were made in water with or without stirring. One series of experiments was with 45 vol.% ethanol.
283
B[degl 100
60
0-l 0
l
10
30
20
Irradiation time Iminl Fig. 5. Change in hydrophilicity
during UV irradiation
irradiation times pictured by the advancing fied polysulfone surface against water.
(S,)
in water without stirring after different
and receding
(0,) contact angles of the modi-
with experiments reported by Shimomura et al. for polyacrylonitrile (PAN) RO membranes modified with plasma [ 321. ESCA analysis of our samples, made at the Institute of Surface Chemistry in Stockholm, verify an increase in the oxygen/carbon ratio. The primary increase in hydrophilicity of the membrane is induced by direct irradiation and not transmitted by small molecules or macromolecules in the irradiation bath. This was also tested by measuring the contact angles of the “dark” sides of the rods, which had been exposed to the solutions in question but not irradiated. These contact angles decreased very little, or not at all, compared with the values obtained by direct adsorption of the macromolecule without irradiation. When stirring has been used a flux decrease is noticed only during the first five minutes of irradiation, and after that the flux increases with irradiation time (Fig. 4 ) . This effect of stirring could be explained by an increased amount of available oxygen, as stirring enhances material transport to the membrane. The oxygen probably also favors the formation of carboxylic and sulfonic acid groups on the membranes. These are dissociated and negatively charged at neutral pH values of the water and a state of internal repulsion in the pores is developed. This repulsion causes an enlargement of the pores, which results in an increase in water flux. This effect, as well as the destructive effect of oxygen, thus contributes to the flux increase. The destructive effect of oxygen could be detected as deformations of the membrane surface with a microscope attached to FTIR equipment (Enso-Gutzeit, Imatra). Some chain scissions could be
284 TABLE
3
Water flux one hour after modification (J,,) compared to flux before modification different irradiation times (IT), with ( + ) or without ( - ) stirring Stirring
_
_
_
35
-
_
38 88
10 15 “0 5 10 15 20 5 10 15 20
+ + + + + + + +
agent
J,lJ,,
IT (min)
5
Modification
(J,,) after
100 23
89 225 432
-
616
45 vol.% ethanol
50 315 488 627
45 vol.% ethanol 45 vol.% ethanol 45 vol.% ethanol
572 1500
10 10
+
45 vol.% ethanol + 5 ppm DEAE
-c
45 vol.% ethanol + 5 ppm PEOB
15 15
+ + + +
BSA BSA BSA
2 mm 5 mm 10 wm
423
BSA
5 ppm
485
1.5 20 15 15 15 15 15
176 33
Whey Whey PPl PPl D-lysine
10 15 5 10 5
ppm ppm ppm ppm ppm
420 292 392 210 130
15 15 15
+ + +
DEAE DEX DEXSU
5 ppm 10 ppm 10 ppm
388 525 306
10
+ + +
PEOB PEOB PEOB
10ppm 10ppm
293 1120
15 10
(s 1
5 ppm
392
observed from the FTIR spectra as changes in phenyl substitution. The spectra also showed an increase in oxygenated groups, verifying the results above obtained with ESCA. When the modification is made in aqueous solutions of different hydrophilic
285 a
b
Fig. 6. Pores with hydrophilic chains protruding in solution in different types of solvents. (a) Hydrophilic chains in “good” solvent, (b) hydrophilic chains in “poor” solvent i.e. wrong pH, high ionic strength or hydrophobic solvent, (c) hydrophilic negatively charged chains in “good” solvent, forming internal electrostatic repulsion and enlargement of the pore without loss in retention.
substances (Table 3) it can be observed that one can control the resulting permeability by changing the amount of macromolecule present as modification agent or by changing the irradiation time. When the amount of macromolecule in solution is increased the pores get blocked. This blocking effect is larger for the macromolecules containing positively charged groups, as they preferentially attach to the surface making ionic bonds with the negatively charged groups formed by irradiation of the polysulfone membrane. This also is an indication of the formation of negatively charged groups by irradiation. When 45 vol.% ethanol is present the alcohol can swell the membrane; since ethanol as a solvent [ 191 is not a “good” solvent for the formed hydrophilic groups [33] they adhere to the membrane pore walls leaving the pores open resulting in a permeability increase (Fig. 6b ), which seems to persist even after changing the solvent back to water. With the optical microscope also large destructions of the skin layer were observed. The situation with enlargement of pores due to a “poor” solvent can also occur at high ionic strength or at unfavorable pH, when the groups lingering into solution are charged [ 33,341. The contact angles for the cases with stirring with or without ethanol did not actually change from the case without stirring. The maximal effect is reached very fast and this effect is not changed by moving the rod a little closer or a little further away from the irradiation source. This means that the increases in permeability after 10 min of irradiation mainly result from the etching effect of oxygen and the resulting enlargement of the pores. Improvement
of flux during UF of protein solutions
0.5% Whey Table 4 shows the results from ultrafiltration experiments of Whey with different types of modified polysulfone GR 81 membranes. It can be seen that if only UV irradiation is used, improvement of permeate flux starts after irradiation times longer than 10 min. At 20 min of irradiation permeate flux is
286 TABLE
4
Effect of different kinds of modifications of the polysulfone CR 81 membrane on permeate flux and the observed retention (RI)during ultrafiltration of 0.5% Whey solution at 25°C without pH adjustment approximately at pH 6. IT=irradiation time Modification
agent
IT (min)
JplJ,, (so)
Retention
_
-
38
99.4
_
10 15 20
36 83 144
99.4 99.2 99.0
20 min 45% ethanol
-
19
99.3
45% ethanol 45% ethanol
10 20
83 219
99.3 91.9
5ppm 10ppm 5 ppm
PEOB DEX BSA
10 15 15
95 178 91
99.5 99.1 99.3
10ppm 15 ppm
Whey Whey
15 15
164 111
99.4 99.4
(% )
more than 300% of the permeate flux for the unmodified membrane with only a slight loss in retention. At pH 6 the whey proteins are slightly negatively charged and the irradiated membrane should be more negatively charged than the unmodified membrane, therefore the state of electrostatic repulsion between the membrane and the molecules in solution keeps retention high even when flux is increased. If ethanol is present the flux is further improved, which seems to be a combined effect of irradiation and alcohol treatment, as alcohol treatment of the membranes without irradiation decreases flux. With the largest flux increases a loss in retention results. Even better results are obtained with modifications where non-ionic polymers (PEOB, DEX) or proteins are present. The best result was obtained using a suitable concentration of whey protein as modification agent. 0.05% BSA In Figs. 7 and 8 the influence can be seen of modification on permeate flux during the ultrafiltration of a 0.05% BSA solution at different pH values after one hour. Figure 7 shows that modification with BSA and UV irradiation gives smaller increases in flux at neutral pH values than in acidic or alkaline solutions. When the modification conditions were adjusted they were chosen to give such an increase in flux, at the IP of BSA, that retention was not considerably decreased; this same modification was then used at different pH values.
287 Jp2/Jor100$
800
700
Modification
I
Cont.
KCI
o--o
0.015
M
x--x UV 15 min ; BSA 2 ppm 0-0 UV 15 min ; BSA 2 ppm
0.015
M
0.15 M
400-
300-
200-
loo-
0, 0
. 1
2
3
4
5
6
7
8
9
10
pH
Fig. 7. Influence on permeate flux (J,,) of modification and salt concentration at different pH values in the ultrafiltration of 0.05% BSA solution after one hour compared to flux before modification (J,). Modification of membrane with 2 ppm BSA and 15 min of UV irradiation. T= 25 o C, p= 1.5 bar.
The surface modified with BSA can be considered a self-rejecting surface as its charge should be approximately the same as the charge of the BSA molecules in solution. The results show that the modifications with BSA, which cause electrostatic repulsion between the membrane surface and the protein molecules in solution at pH 3 and 9, cause a considerably increased flux. Increase in salt concentration decreases this effect as can be expected due to shielding of charges. The effects of other types of modifications on flux are shown in Fig. 8. These effects are much smaller at pH values near the IP of BSA than at pH values further away from it. It can also be noted that similarity of charge is not as important as charge density of any sign or the hydrophilicity; this was also shown earlier to be the case with ovalbumin [ 351. The explanation of the positive effect in the case of attracting charges can be that first, due to electrostatic attraction, one monolayer of BSA is formed and then a state of repulsion is attained, which actually gives better flux than only an increase in hydrophilicity. At the IP of BSA hydrophilicity of the membrane is probably more important than charge density.
288 Modification
I
Jp2/Jo*100%
800
700-
0-n
~--a UV 15 min O-O UV 15 min e-0
uv
10 min
; DEAE ; DEXSU ; PEOB
5 ppm 10 ppm 10 ppm
600-
500-
400-
300-
200-
loo-
04
0
.
1
2
3
4
5
6
7
8
9
10
PH
Fig. 8. Influence on permeate flux (J,,,) of modification at different pH values in the ultrafiltration of 0.05% BSA solution after one hour compared to flux before modification (J,,). 2’~ 25 ‘C, p = 1.5 bar.
Flux reduction in experiments
with modified membranes
Pore blocking The modified membranes show a higher flux reduction than the unmodified membranes, when protein is applied to the solution. This seems to be a result of the increased water permeability of the modified membranes. With the modifications made in this study using UV irradiation, the conventional flux reduction (FR) concept can not be meaningfully used to describe the situation of adsorption and fouling. In order to investigate what happened in our modification experiments, flux reduction is divided into two parts; FR1, which describes the plugging effect and most of the concentration polarization effect, and FR2, which probably has more to do with adsorption and rearrangement of the molecular layers at the surface. FR, and/or FR, were sometimes negative especially at high pH values, as e.g. in one of the experiments reported in Fig. n
The flux loss (J,,/.J,) did not correlate with the original flux (J,,) of the membrane, nor with flux increase (JJJ,) during modification. It seemed, however, that if a higher flux had been achieved after modification the flux loss increased. Flux loss is plotted against flux after modification (J,,,) in Fig.
289 A J4 J,
*‘0°%
-. x-x O-O A-A
A
l
150
pH3 pH5 pH 7 pH9
100
50
0
I
0
100
200
.
300
J,W(m2h)l Fig. 9. Instantaneous flux loss (J,,/J,) in the beginning of ultrafiltration of 0.05% BSA solution at different pH values as a function of flux after modification (J,). T= 25 “C, p = 1.5 bar.
9. It can be seen that an almost linear relationship is obtained at pH 5, the IP of BSA. At this pH value no large electrostatic effects are expected. This means that the greater the flux, the higher flux reduction, probably due to pore plugging. It means that in ideal conditions equal size pores are formed. A relative estimate of this pore size can then be made from the flux. One can also assume that since BSA is rather neutral at pH 5, it does not favorably plug any special kind of modified membrane. At pH values near 7, the flux loss is almost the same as at pH 5 but the points are more scattered. However, at pH z 3 and even more at pH z 9 the flux loss is very small and even flux increase is observed at pH FZ9. This can be explained by an increase in pore size due to pH change and the presence of a slight salinity to stabilize the charges. Effect of concentration
on pore blocking
When the protein solution is applied, the pore blocking effect seems to be rather instantaneous even at small concentrations. Increasing the concentra-
290 Modification
I
.-.
x--x UV 10 min o--O UV 15 min;
A-a
UV 15 min
Fig. 10. Flux reduction
PEOB 5 ppm
; DEAE 5 ppm
in ultrafiltration
after different modifications.
of lysozyme
as a function
of membrane
concentration
T= 25’ C, p = 1.5 bar.
tion then increases flux reduction rather slowly as seen in Fig. 10 in ultrafiltration of LYS at different concentrations. One can see that pore plugging is almost complete at a concentration of 50 ppm. In the cases with membranes modified with DEAE and D-lysine pore blocking is slower. This may result from the fact that in these cases electrostatic repulsion conditions prevail and this slows down the process at low concentrations. Flux loss after one hour The change in flux with time as described by FR, (FR, = 1- J,,,/J,,,) for 0.5% Whey at pH 6 was studied to see if it somehow correlated to the type of modification made. Some results are shown in Table 5, which are summarized from the experiments reported in Table 4. It can be seen that there is little difference between the unmodified membrane and the UV irradiated membrane. When Whey is used as modifying substance there is very little flux reduction with time. The flux loss after one hour did not relate to the measured initial flux (J,,,) neither to the flux after modification (J,) nor to any special, calculated relative flux. It could only be noted that flux was either gained or lost so as to
291 TABLE
5
Flux reduction F&
(FR, = 1 -J,,/J,,
) after one hour of ultrafiltration
Unmodified membrane Only UV irradiated membrane UV UV UV UV
irradiation irradiation irradiation irradiation non-ionic
+ + + +
of Whey at pH 6, T= 25’ C
0.193 0.194 0.248
45% ethanol Whey BSA hydrophilic
0.031 0.102 0.253
substance
Abs. I
+ 25
50
Jpp I I/(m’h) Fig. 11. Absorbance a function
100
75 1
of the permeate at 280 nm in ultrafiltration
of 0.5% whey protein solution as
of permeate flux (Jr,,) after one hour.
compensate for the effects caused by instantaneous flux loss. The results were the same for BSA at different pH values and for Whey at pH 6. It could be noted that FR, actually smoothed out the differences in pore size between the membranes. This could be seen as a decrease in the standard deviation in the values of the fluxes for Jpl and JP2 for different modifications. Retention
of proteins
with the modified membranes
One objective of the modification experiments was to get membranes with a retention, that was at least as good as with the membranes without modifications. Therefore the testing of the modification conditions was done rather near the IP’s of the proteins in order that the electrostatic repulsive effects favorable for retention would be as small as possible. For BSA the modifica-
292 TABLE
6
Permeate fluxes (Jr,) in l/ (m’/hr), observed retentions (R,) and true retentions (R,) in % after one hour of ultrafiltration of BSA at different pH values with unmodified and modified polysulfone membranes. IT= irradiation time Modification
IT
pH3
pH7
pH5
pH9
(min)
J P2 _
_
BSA2ppm PEOB 10 ppm PPI 5 ppm DEAE 5 ppm DEXSU 10 ppm
15 10 15 15 15
TABLE
R,
R,
Rz
96.8
98.2
15
98.9 99.0
17
98.5 98.9
98 99.7 91 99.7 109 99.4 104 >99.7 76 98.8
99.9 99.9 99.9 >99.9 99.6
44 41 61 41 35
98.6 99.1 97.1 99.1 98.9
44 28 53 40 37
98.6 99.4 97.0 98.8 99.4
40
Jp2
Rz Jp2 R,
R,
99.3 99.5 98.9 99.5 99.3
99.2 99.6 99.9 99.3 99.7
Jp2
R,
R,
35
99.0
99.4
154 100 120 107 130
99.4 99.7 99.7
99.9 99.9 >99.9 z99.9 >99.9
>99.7 99.7
7
Observed retention (R,) and true retention (R2) in the end of the ultrafiltration made with LYS in Fig. 9. IT= irradiation time Modification
IT
R,
R2
99.6 98.8 84.6 98.5 97.8
99.7 98.8 88.9 98.7 97.9
experiments
(min) PEOB 5 ppm DEAE 5 ppm D-Lysine 5 ppm
_ 10 15 15 15
tions were also tested at different pH values. Table 4 gives the retentions of Whey for differently modified membranes. It can be noted that the observed retention is very high in all cases. If flux is increased considerably a slight loss in retention occurs. When modifications are made with Whey there seems to be both a good flux and a good retention. At the rather neutral pH of the experiment and near the IP of Whey no large differences in retention depending on electrostatic forces are expected, since the whey proteins are either slightly charged or not charged at all. The correlation of flux with retention was tested further by plotting absorbance at 280 nm versus flux (Fig. 11) . Up to fluxes of ca. 25 l/m’-hr the absorbance does not change, which indicates almost total retention of the proteins (the measured background absorbance probably emanates from impurities present in the Whey used). At higher fluxes there is an increase in absorbance, and the increase is faster at higher fluxes. As the whey proteins are of different sizes and have different IP’s it is natural that the increase in absorbance along with flux is not linear. The point where absor-
293
bance starts to increase should correspond to the size of the smallest of the whey protein molecules. Besides on molar mass this size depends also on the conformational state and the charge of the protein molecule. For BSA, differences in retention at different pH values are shown in Table 6. At intermediate pH values retention is slightly lower than at pH 3 and pH 9, probably because BSA is near its IP and therefore not highly charged. The best retentions are achieved at high pH. At pH 3 the best retentions are achieved with membranes modified with positively charged polymers, as was expected, and the lowest retention is obtained for DEXSU, which is negatively charged (not taking into account the unmodified hydrophobic PSU membrane). The hydrophilicities of the modified membranes were, according to the contactangle measurements, almost equal, and also equal to the hydrophilicity obtained by irradiation only. For the experiments made with LYS depicted in Fig. 10 the retentions are given in Table 7. It can be seen that retention is higher when electrostatic repulsion is achieved as in the cases with DEAE and D-lysine, and nearly as good as without modification even if the fluxes are higher. For the non-ionic PEOB retention is not so good. The true retentions show the same trends as the observed retentions, even if they are somewhat higher. Scope of modification: flux results
estimation
of protein sizes and double layer forces from
When commercial hydrophobic polysulfone membranes are modified with UV irradiation and hydrophilicity increasing substances, it is possible to enlarge the pores more than without irradiation. Normally, the membrane pores are somewhat flexible and open up to some extent depending on the modification agent used and the pH of the experiment. But without the destructive influence of irradiation there is a limit to this opening effect. With irradiation the pores can be enlarged and permeability increased to an extent which is proportional to irradiation time. When a particular protein solution is ultrafiltrated through the modified membrane, the pores stabilize to a size characteristic for the protein at the specific pH of the solution. This fact can be deduced from flux measurements and an assumed correlation between pore size and flux. This pore size seems to give an observed retention of more than 99% and a true retention near 100% for BSA and Whey. This means that the by modification obtained leaky membrane almost instantaneously gets its larger pores plugged by the protein in question, until the membrane stabilizes and retention is almost ideally 100%. If the opening of the pores is too large so that even a monolayer of the protein in the pores still cannot retain the protein, then the modification has not been successful if high retention was the goal. In the very first minutes after the beginning of the ultrafiltration experiment with the protein solution in question, pore plugging takes place; it depends only
294
on the flux after modification (J,) and is actually not at all proportional to the original flux of the membrane (J,). With time flux increases or decreases so as to stabilize the pore sizes to the pH and protein in question. When comparing the final fluxes (J,,) at the same pH for different hydrophilic modification agents, the best flux is obtained if electrostatic repulsion is obtained. This repulsion can be obtained either as repulsion between the protein and the modification layer or by a monolayer of the protein (self-rejection) formed on the membrane by the attractive forces. In this study the monolayer is formed when the pore size is modelled by modification and therefore it does not reduce the final flux of the membrane. If the pore size is fixed from the beginning, as in the modifications without irradiation, the adsorbed layer will possibly cause a reduction in pore size (in the case of a leaky membrane), but since stabilization of pore size takes place when the protein solution is present, the final pore size will become as large as with a surface originally equally charged as the protein. The influence of pH on pore size and flux is determined by the way the pH affects the charges of the proteins, and the solution properties of the hydrophilic modification agents. At suitable salinities and pH values the modification agents protrude in the solution in the pores blocking the passage (Fig. 6 ). The protein has a larger volume if highly charged, and if electrostatic repulsion exists the range of the repulsive forces determine the final pore size. This was also the case for BSA as the flux ratios between the different pH values pH5 : pH7 : pH9 : pH3 were 1: 0.9 : 2.8: 2.2, respectively. The pore size depends on electric double layer forces, is sensitive to salinity and should decrease with increasing salt concentration (Fig. 6b ). This could be observed in the experiments made at higher salt concentration (0.15 M KCl, Fig. 7) with BSA as the modification agent at pH 3 and 9. If several proteins are present as in the experiments with Whey, the flux stabilizes so that the pore size correlates to the smallest protein molecule in solution, the size depending on its state of charge. For Whey this protein is the a-lactalbumin (M”= 14200 g/mol, [27] ), the size of which is much smaller than the size of BSA (M”=68000 g/mol, [27] ). Comparing the fluxes after modification for Whey and BSA near their IP’s, when retention is almost lOO%, one can see that the flux for Whey (Fig. 11) is much smaller than for BSA (Table 6)) which is in accordance with the assumptions. If a correlation of flux and protein size can be found it should be possible to estimate both maximal fluxes and electrostatic repulsive forces from the modification results. It has to be taken into account that the experiments above have been performed only with one type of membrane (GR 81) at specific conditions (fixed temperature, flow rate and pressure). If e.g. the pressure is increased, the protein molecules deform more easily and plug the pores to a higher extent and the optimizations above might not apply in the same way.
295
Conclusions
In the present study hydrophobic polysulfone UF membranes were modified with UV irradiation and hydrophilicity increasing agents. The modifications were tested with protein solutions. UV irradiation increased flux and the hydrophilicity of the membranes. Most of the flux increase came from the etching effect of oxygen during stirring conditions. The flux increases obtained varied with pH and modification agents used, and could be more than 400% compared to unmodified conditions without any loss in retention. The best retentions were obtained at pH values, where a strong electrostatic repulsion was obtained. The pores enlarged to a fixed size that can be taken as an estimate of the protein size, which due to the range of double layer forces acting between proteins and membranes cannot penetrate the pore. Acknowledgements
The authors want to thank Techn. Lit. Markku Laatikainen for prereading and commenting the manuscript, and miss Jaana Tuisku for her excellent technical assistance.
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