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Influence of protein adsorption on flow resistance of microfiltration membrane
Colloids and Surfaces A: Physicochemical and Engineering Aspects 89 (1994) 15-22
ELSEVIER
zkLomS SURFACESA
Influence of protein adsorption on flow resistance of microfiltration membrane E. Iritani*, S. Tachi, T. Murase Department of Chemical Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-01, Japan
Received 3 August 1993; accepted 1 March 1994
Abstract It is well known that the flow resistance and sieving property of a membrane change when protein adsorption, which is a specific interaction between the protein and membrane, occurs within the pores. The influence of the protein adsorption on the membrane hydraulic permeability was examined using bovine serum albumin (BSA) as the model protein and a nitrocellulose microfiltration membrane. The Kozeny-Carman equation, which has been applied to the laminar flow in a granular bed, was employed for the description of the flow property of the tortuous pore membranes. The flow resistance of the membrane after BSA adsorption was well evaluated from the reduced porosity due to the protein adsorption in the pores. Also, the equivalent diameter of the pore space of both the clean and preadsorbed membranes was evaluated by introducing the expression for the hydraulic mean diameter. Furthermore, it was found that a more significant protein adsorption at around the isoelectric point brings about a larger increase of the flow resistance of the preadsorbed membrane. Keywords: Fouling; Kozeny-Carman
equation; Membrane filtration; Microfiltration;
1. Introduction Membrane considerable
filtration processes have attracted a amount of attention in recent years
in such widely diversified fields as biotechnology, biomedicine, food and beverage processing, and water treatment. The most serious operational constraint to the widespread use of membrane filtration is the flux decline over time due to membrane fouling. Also, the membrane fouling per se may alter the sieving properties significantly. There exist many factors influencing the fouling behavior. Adsorption of macromolecular solutes, especially of proteins, is one of the causes of such a fouling phenomenon. Therefore fundamental *Corresponding author. 0927-7757/94/$07.000 1994Elsevier Science B.V. All rights reserved SSD10927-7757(94)02854-L
Protein adsorption
studies [l-l l] of adsorption of proteins both in and on ultrafiltration and microfiltration membranes have received considerable attention in recent years. Although it is well known that protein adsorption within the pores of the membrane can dramatically alter the flow resistance of the membrane, the quantitative relation between protein adsorption and flow resistance of the membrane is quite complex and as yet not fully understood. This study aims to clarify the role of the membrane-solute interaction (adsorption) in membrane hydraulic permeability. We measured the amount of bovine serum albumin (BSA) adsorption within microfiltration membranes. A method has been developed for obtaining the flow resistance of the membrane in which BSA is adsorbed. In addition, the effects of solution pH on the amount of BSA
16
E. Zritani et aL/Colloids Surfaces A: Physicochem. Eng. Aspects 89 (1994) 15-22
adsorbed, and hence the flow resistance associated with protein adsorption, are investigated.
2. Experimental 2.1. Materials
In all the experiments the protein employed is bovine serum albumin (BSA; Fraction V) with a molecular weight of about 67000 Da provided by Katayama Chemical Industries Corp. This is one of the more abundant blood proteins. The solutions were prepared by carefully dissolving the powdered BSA in either 10 mM acetate or 10 mM phosphate buffer solutions to obtain the desired pH. The isoionic/isoelectric point of the solution is about 5.1. All water used was prepared by an ultrapure water system for laboratory use (Puric-R, Olgano Corp.). The other chemicals were of analytical grade. The BSA concentration ranged from 5 x low4 to 5 x 10W2by weight. Nitrocellulose microfiltration membranes with a nominal pore size of 0.1 pm, supplied by Advantec Toyo Corp., were used for all experiments. The membrane pores are substantially larger than the _,size of a BSA molecule (approximately 140 A x 40 A in diameter) [ 121. For each run, a fresh membrane was used. The membranes were pretreated by soaking them in ultrapure water for lh to remove the glycerin. The internal surface area of the membranes was determined by the BET method with nitrogen as the adsorbing gas. The thickness of the membranes was determined by the magnifying projector, and the true density of the membranes was measured with a pycnometer. The pore size of the membranes was measured by mercury intrusion porosimetry.
brane had been placed. The experiments were carried out under constant pressure by applying compressed nitrogen gas. Transmembrane pressure for all runs was set at 49 kPa. The buffer solution was continuously introduced from a feed solution reservoir connected to the filtration cell. The permeate weight was measured every 10 s with an electronic balance (Shimadzu Corp.) connected to an electronic timer and a printer. The weights were converted to volumes using density correlations. After the hydraulic permeability test, the membrane was carefully removed from the filtration cell and used in the protein adsorption test. BSA adsorption within the porous membranes was evaluated as follows. The membrane was submerged in a BSA solution of known concentration. After the solution was stirred at 80 rev min-’ for 12 h (except for the case of Fig. 1, see below), the membrane was removed. This adsorption time was sufficient to achieve a quasi-steady state. All experiments were conducted at ambient temperature, 22 f 2” C. The residual protein concentrations were evaluated spectrophotometrically by reading the absorbance of the solutions at 280 nm. From the difference between the protein concentration of both the bulk solution and the solution after protein adsorption, the amount of protein adsorbed could be determined, taking into account the volume excluded by the membrane. The protein concentration changed considerably by submerging the membrane in a small amount of solution. Immediately after the adsorption test, the membrane was returned to the filtration cell so that the permeability could be re-evaluated as described previously. The differences in the permeabilities of the clean and preadsorbed membranes were investigated.
2.2. Measurements
3. Theory
Prior to the protein adsorption test, the measurements of the hydraulic permeability of the membrane were performed in a dead-end filtration cell (Advantec Toto Corp.) with an effective membrane area of 27.0 cm2. The filtration cell consisted of a cylindrical vessel containing the buffer solution, surmounting a porous support on which the mem-
In order to examine the possible effects of protein adsorption on the membrane permeability, it has been commonly assumed that the pore space of the membrane is equivalent to a bundle of capillaries of uniform diameter. It has also been assumed that the pore reduces in size owing to the protein adsorption in the interior structure of the mem-
E. Iritani et al.IColloids Surfaces A: Physicochem. Eng. Aspects 89 (1994) 15-22
brane. Accordingly, the Hagen-Poiseuille equation has been employed for describing the laminar flow in the circular tube, in order to evaluate the decrease of the membrane permeability due to the protein adsorption [ 8,111. However, the real pore geometry of the membrane is extremely complicated. For a membrane possessing a polymer mesh structure, the path of a streamline through the pore space would be tortuous, and the pores are interconnected. Therefore a more realistic model would be desirable for describing the flow behavior through the membrane. Clearly, the porous membrane can be treated as a kind of granular bed consisting of non-uniform sizes and/or non-regular particle shapes. Therefore, as a first approximation, assuming that the membrane is symmetric, we have analyzed the flow property of the membrane in terms of the Kozeny-Carman relationship, which has been applied to the laminar flow in a granular bed [13]. The superficial velocity 4 can be expressed in the form
17
applied for the membrane after BSA adsorption. In this case, the Kozeny-Carman equation can be represented by 3 e&J
qp=-=
L&U,
-P
kS;,( 1 - eJ2 ,uL
(3)
where qp, Rmp, cp and S,, are the values for the membrane with adsorbed BSA. The porosity cP of the membrane after BSA adsorption can be described by E*=E-
4wpSm zD2Lpp
where wp is the amount of BSA adsorbed per unit effective internal surface area of the membrane, S, is the effective internal surface area of the membrane, and pp is the density of the protein. The density of the solute, which is defined as the reciprocal of the partial specific volume, is reported to have the value of 1.364 g cmm3 for BSA [ 123. Provided the values of qp and wp are known, one can calculate S, by using Eqs. (3) and (4). If the values of wr and S, are known, qp can be calculated.
(1) where p is the permeation pressure, p is the viscosity of the permeate, R, is the initial membrane resistance to flow, E is the porosity of the membrane, So is the effective specific surface area of the membrane, L is the thickness of the membrane, and k is generally known as Kozeny’s constant. A commonly accepted value for k is 5.0. The porosity E in Eq. ( 1) can be represented by c=l-2
4w zD2Lp,
(2)
where W, is the mass of the membrane, D is the diameter of the membrane, and P,,, is the true density of the membrane. Provided the value of 4 is known, one can calculate So by using Eqs. (1) and (2), and vice versa. In general, the internal surface area of the membrane is much larger than its upper and lower surface areas. If BSA molecules are adsorbed inside the pore structure of the membrane uniformly, then the Kozeny-Carman equation can also be
4. Results and discussion 4.1. Adsorption amount of BSA The adsorption kinetics of BSA within the membrane was investigated under static immersion. Fig. 1 illustrates the mass wp of BSA adsorbed per unit internal surface area of the membrane plotted as a function of the adsorption time &. The adsorption process of BSA is only fast at the very beginning; it reaches a nearly steady state within 3 h. As also can be observed in Fig. 1, the equilibrium amount adsorbed is about 1.9 mg me2 under this condition. A typical adsorption isotherm of BSA onto the pore surface of the membrane at pH 5.1 is shown in Fig. 2. The adsorbed amount of BSA is plotted against the equilibrium mass fraction s of BSA in the solution. The adsorption isotherm rapidly attained a plateau level. Under this condition, the saturated amount of adsorbed BSA is about 2.0 mg rne2.
18
E. Iritani et al.JColloids Surfaces A: Physicochem. Eng. Aspects 89 (1994) 15-22
1.4 CELLULOSE NITRATE MEMBRANE
CELLULOSE NITRATE MEMBRANE
01 0
I
I
2
4
I
I
I
I
6
8
IO
12
I s C-l
81 Chl Fig. 1. Variation of amount of BSA adsorbed per unit internal surface area of membrane over time.
Fig. 3. Logarithmic plot of adsorbed amount against equilibrium concentration.
2.5r-----l
4F-----l
-1
1
3oooooooooo
I
1.0 0.5 0
0
/
I 2
BSA
?
pH=5.1 &=12h
!2cr’
-
d
Eq.W
I 4
P=49kPo CELLULOSE MEMBRANE
CELLULOSE NITRATE MEMBRANE
I
h&*hA*A~
1
0 A I -
6x10-*
s C-l
-
01
Fig. 2. Adsorption isotherm for BSA within membrane.
I
0
I
I
100
NITRATE
CLEAN ADSORBED BSA,pH=5.1 s=4,35x10-3 8,=12h PREDICTED
I
200
-
I
I 300
.9 Cd
Fig. 3 shows the logarithmic plot of the adsorbed amount wP against the equilibrium concentration s. A reasonably linear relationship is obtained in accordance with the Freundlich equation represented by wP = Ksll”
(5)
where K and n are empirical constants. The solid line shown in Fig. 2 was approximated by Eq. (5). 4.2. Permeation rate of membranes In Fig. 4, typical results of the hydraulic permeability test at the equilibrium concentration s of 4.35 x 10m3are shown in the form of the permeation rate vs. the permeation time 8. The results for both the clean and preadsorbed membranes are shown. The permeation rate of the preadsorbed
Fig. 4. Dependence of permeation rate on permeation time.
membrane is smaller than that of the clean membrane, owing to the BSA adsorption onto the pore surface. The most interesting feature is that the difference in the permeation rate of both membranes is not as large as expected. In general, the flux decline during micro filtration of protein solutions may be much greater than can be explained by protein adsorption. Such a substantial decrease may be interpreted in terms of protein deposition resulting from shear induced distortion of the molecules on passage through the micropores [14]. Some interesting work associated with this behavior is to be found elsewhere [ 15,161. In each case in Fig.A, the permeation rate is constant throughout the run. Fig. 4 includes the prediction
E. Iritani et al./Colloids Surfaces A: Physicochem. Eng. Aspects 89 (1994) 15-22
of the permeation rate for the preadsorbed membrane. It can be estimated as follows. The porosity e of each clean membrane was calculated from Eq. (2). Therefore the effective specific surface area S,, of the clean membrane can be obtained from the measurements of the permeation rate 4 and the porosity E, using Eq. (1). For example, in the data used for Fig. 4, the value thus obtained was 2.86 x 10’ m-l. This was in good agreement with the value of 3.05 x 10’ m-l obtained by the BET method. The porosity of the preadsorbed membrane decreases slightly, but significantly, compared with that of the clean membrane, owing to the BSA adsorption onto the pore surface. It can be calculated from Eq. (4). Consequently the effective specific surface area S, of the preadsorbed membrane can be computed from the measurements of the permeation rate qp and the porosity Ed, using Eq. (3). Surprisingly, the specific surface area SOP of the preadsorbed membrane was almost the same as that of the clean membrane. However, the BSA molecule is regarded as a prolate (rod-shaped) ellipsoid of revolution with major axis 2a = 14 nm and minor axis 2b=4 nm [ 121. The surface area S and the volume V of the prolate ellipsoid of revolution are, respectively, described by sin-’ E
S = 2nb2 + 2nab 7 V= i nab’
where E (= (a2 - b2)‘/2/a) is the eccentricity. As the specific surface area is defined as S/V, the sum of the specific surface area of the adsorbed BSA molecules onto the pore surface of the membrane can be calculated from the data of the amount of adsorbed BSA using Eqs. (6) and (7). For example, in the data used for Fig. 4, the value was found to be 7.52 x 10’ m-l, significantly larger than the measurement of the specific surface area of the preadsorbed membrane based on the permeation test. If the liquid flows through the interstices of the preadsorbed BSA molecules, the permeation rate of the preadsorbed membrane would be, of course, considerably reduced compared with that
19
of the clean membrane. The experimental results suggest that the specific surface area of the adsorbed BSA does not act as the flow resistance of the permeation effectively. The predicted line shown in Fig. 4 was obtained from the following relation using the values of porosity of the preadsorbed membrane and the specific surface area of the clean membrane: 3
P
EP
-P
kS;( 1 - ‘p)2 pL
qp=-= dLlp
(8)
The predicted line is in very good agreement with the experimental data, indicating that Eq. (8) accurately describes the change in the flux associated with protein adsorption. A comparison between Eqs. (1 ) and (8) leads to 9p q
E;f( 1- El2 .?(
1 - E&J2
(9)
It is instructive to compare the pore size of the preadsorbed membrane with that of the clean membrane, in order to evaluate the sieving property of the membrane. The equivalent diameter of the pore space can be evaluated by introducing the hydraulic mean diameter [ 171. The hydraulic mean diameter d, for a flow passage of the membrane is given by d, =
4E Ml-E)
(10)
Given a knowledge of the porosity E and the specific surface area So of the membrane, the hydraulic mean diameter de can be evaluated quantitatively from Eq. (10). For instance, in the data used for Fig. 4, the pore size of the clean membrane calculated from Eqs. (l), (2) and (10) on the basis of the data from the hydraulic permeability test is 0.291 urn. The median pore diameter of the membrane determined by porosimetry is 0.203 urn. This value is smaller than the calculated one. It is considered that the pore size of the membrane depends on the method of experimental measurement. The pore size of the preadsorbed membrane can be obtained by substituting ep for E in Eq. (10). The porosity ep of the preadsorbed membrane can be obtained from Eq. (4) on the basis of the data from the adsorption
E. Iritani et al./Colloid Surfaces A: Physicochem. Eng. Aspects 89 (1994) 15-22
20
tests. The pore size thus obtained is 0.274 pm, and hence the average value of the thickness of the adsorbed BSA layer is 8.5 nm. Whether the BSA molecules are adsorbed side-on onto the internal surface of the membrane or are adsorbed end-on is not clear from these data alone. The porosity ep of the preadsorbed membrane can, however, also be obtained from Eq. (9), using the data of the decrease of the permeation rate due to BSA adsorption. The pore size thus obtained is 0.275 pm. It is interesting to note that this value is in excellent agreement with that obtained on the basis of the measurement of the adsorbed amount. This good agreement indicates that Eq. (10) provides a reasonable description of the mean pore size of the membrane. In Fig. 5, the cumulative permeation volume u collected per unit area of active membrane is plotted against the permeation time 0. The solid line indicates the value calculated from the following relation using the predicted value of the permeation rate qP of the preadsorbed membrane, and it is in good agreement with the experimental values: (11)
up = qpe
where vP is the cumulative permeation volume collected per unit area of active membrane in the preadsorbed membrane. In Fig. 6, the resistance Rmp of the preadsorbed
854, CELLULOSE NITRATE MEMBRANE
2.0
t
(Rm,h.
Ccm-‘I
Fig. 6. Resistance of preadsorbed membrane.
membrane calculated mental value of qp is values. The predicted Eq. (8) using the adsorbed amount.
from Eq. (8) using the expericompared with the predicted values can be obtained from value of ep based on the
4.3. Efect ofpH The effect of changing the solution pH was also studied. Fig. 7 shows the pH dependence of the plateau adsorption of BSA. It is obvious that adsorption was quite sensitive to the pH, indicating the electrical nature of this adsorption process. Over the pH range examined, the amount of BSA adsorbed was largest at around its isoelectric point. As the BSA molecule carries no net charge at that
8 3
I
,
/
I
,
I
BSA si=5.ox10-3
F 6 :: p
I
-
PREDICTED
4
0
0
100
200
300
8 cs1
Fig. 5. Relation between cumulative permeation volume collected per unit active membrane area and permeation time at pH 5.1.
Oh
2
4
6
8
IO
PH
Fig. 7. Dependence of plateau adsorption of BSA on pH.
E. Iritaniet al./ColloicisSurfaces A: Physicochem.Eng. Aspects89 (1994) 15-22
point, the molecule is in its most compact state. The BSA molecules deposit themselves rather densely onto the pore walls of the membrane to form a compact configuration with a smaller lateral electrical interaction between the molecules. As a result of this, the protein is very adsorptive at around the isoelectric point [ 18-211. This is similar to the observation concerning ultrafiltration of BSA solutions made by Fane and co-workers [l-3,22]. In Fig. 8, the permeation volume at pH 8.0 are plotted against the permeation time 8. Compared with the result for the isoelectric point in Fig. 5, the permeation volume does not decrease SQmuch because the amount of BSA adsorbed is relatively small, as can be seen in Fig. 7.
21
complex, our analysis indicates that the relation between the flow resistance of the membrane and the amount of adsorbed protein can be relatively well described using the Kozeny-Carman relationship. In addition, the importance of pH in adsorption was highlighted. It has been proven that the higher amount of protein adsorption at around the isoelectric point brings about a larger increase in the flow resistance of the preadsorbed membrane. As our current analysis postulates that the membrane is symmetric, future work must consider the effect of membrane heterogeneity. Also, future work is planned to examine the effect of protein adsorption on the flow resistance of fully and partially retentive ultrafiltration membranes.
Acknowledgments 5. Conclusions The amounts of BSA molecules adsorbed in a nitrocellulose microfiltration membrane were measured under various conditions. Subsequently the change in the flow resistance of the microfiltration membrane associated with the BSA adsorption was investigated. The flow resistances of both clean and preadsorbed membranes were analyzed in terms of developments of the Kozeny-Carman relationship. While the real mechanism of the fouling due to the protein adsorption is quite IO
/
I
This work was partly supported by a Grantin-Aid for Scientific Research from the Ministry of Education, Japan, Grant Nos. 03805089 and 04805098, and by the Asahi Glass Foundation. The authors wish to acknowledge with sincere gratitude the financial support leading to the publication of this article. The authors also wish to thank Hiroaki Sumi of Showa Chemical Industry Co., Ltd., for assistance with the measurements of the surface area of the membranes.
Appendix: List of symbols
I
P=49kPo
8
_
CELLULOSE MEMBRANE 0 A
-
NITRATE
CLEAN ADSORBED BSA,pH=B.O s=4.76~10-~ 8;=12h PREDICTED
: D
d,
E k
K L n 0
200
100
300
e CSI
Fig. 8. Relation between cumulative permeation volume collected per unit active membrane area and permeation time at pH 8.0.
P i, s %
s
half length of major axis (m) half length of minor axis (m) diameter of membrane (m) hydraulic mean diameter (m) eccentricity (a’ - b*)‘/*/a Kozeny’s constant constant in Eq. (5) (kg m-*) thickness of membrane (m) constant in Eq. (5) permeation pressure (Pa) superficial permeation velocity (m s- ‘) initial membrane resistance to flow (m-l) equilibrium concentration initial concentration surface area of a solute (m”)
E. Iritani et al.IColloids Surfaces A: Physicochem. Eng. Aspects 89 (1994) 15-22
22
effective internal surface area of membrane (m’) effective specific surface area of membrane &I (m-7 V cumulative permeation volume collected per unit active membrane area (m) V volume of a solute (m3) amount of solute adsorbed per unit effectWP ive internal surface area of membrane (kg m-*) WIZl mass of membrane (kg)
&II
Greek letters E
e 4
p Pm PP
porosity of membrane permeation time (s) adsorption time (s) viscosity of permeate (Pa s) true density of membrane (kg me3) density of solute (kg mp3)
Subscript P
preadsorbed membrane
References [l]
A.G. Fane, C.J.D. Fell and A.G. Waters, J. Membr. Sci., 16 (1983) 211. [2] A.G. Fane, C.J.D. Fell and A. Suki, J. Membr. Sci., 16 (1983) 195.
c31 A. Suki, A.G. Fane and C.J.D. Fell, J. Membr. Sci., 21 (1984) 269. 141 M.W. Chudacek and A.G. Fane, J. Membr. Sci., 21 (1984) 145. c51 J.H. Hanemaaijer, T. Robbertsen, Th. van den Boomgaard and J.W. Gunnink, J. Membr. Sci., 40 (1989) 199. C61 H. Yanagishita, T. Nakane, J. Aihara, S. Takatsu and H. Yoshitome, Membrane, 14 (1989) 64. c71 H. Nabetani, N. Nakajima, A. Watanabe, S. Nakao and S. Kimura, AIChE J., 36 (1990) 907. C81 K. Ogasawara, S. Tsuda, K. Ozawa and K. Sakai, Chem. Eng. J., 48 (1992) Bl. c91 L.E.S. Brink, S.J.G. Elbers, T. Robbertsen and P. Both, J. Membr. Sci., 76 (1993) 281. Cl01 M.K. Ko, J.J. Pellegrino, R. Nassimbene and P. Marko, J. Membr. Sci., 76 (1993) 101. Cl11 A.M. Bribes and M.N. de Pinho, J. Membr. Sci., 78 (1993) 265. Cl21 T. Peters, Jr., Serum albumin, Adv. Protein Chem., 37 (1985) 161. Cl31 PC. Carman, Trans. Inst. Chem. Eng., 15 (1937) 150. [141 W.R. Bowen and Q. Gan, J. Membr. Sci., 80 (1993) 165. Cl51 E. Iritani, Y. Itano and T. Murase, Membrane, 17 (1992) 101. Cl61 E. Iritani, Y. Itano and T. Murase, Kagaku Kogaku Ronbunshu, 19 (1993) 536. Cl71 E. Iritani, M. Iwata and T. Murase, Sep. Sci. Technol., 28 (1993) 1819. Cl81 H. Shirahama, Hyomen, 27 (1989) 29. Cl91 N. Muramatsu and T. Kondo, J. Colloid Interlace Sci., 153 (1992) 23. c201 E. Iritani, S. Nakatsuka, H. Aoki and T. Murase, J. Chem. Eng. Jpn., 24 (1991) 177. c211 E. Iritani, T. Watanabe and T. Murase, J. Membr. Sci., 69 (1992) 87. c221 A.G. Fane, Ultrafiltration: factors influencing flux and rejection, in R.J. Wakeman (Ed.), Progress in Filtration and Separation 4, Elsevier, Amsterdam, 1986, p. 134.
ELSEVIER
zkLomS SURFACESA
Influence of protein adsorption on flow resistance of microfiltration membrane E. Iritani*, S. Tachi, T. Murase Department of Chemical Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-01, Japan
Received 3 August 1993; accepted 1 March 1994
Abstract It is well known that the flow resistance and sieving property of a membrane change when protein adsorption, which is a specific interaction between the protein and membrane, occurs within the pores. The influence of the protein adsorption on the membrane hydraulic permeability was examined using bovine serum albumin (BSA) as the model protein and a nitrocellulose microfiltration membrane. The Kozeny-Carman equation, which has been applied to the laminar flow in a granular bed, was employed for the description of the flow property of the tortuous pore membranes. The flow resistance of the membrane after BSA adsorption was well evaluated from the reduced porosity due to the protein adsorption in the pores. Also, the equivalent diameter of the pore space of both the clean and preadsorbed membranes was evaluated by introducing the expression for the hydraulic mean diameter. Furthermore, it was found that a more significant protein adsorption at around the isoelectric point brings about a larger increase of the flow resistance of the preadsorbed membrane. Keywords: Fouling; Kozeny-Carman
equation; Membrane filtration; Microfiltration;
1. Introduction Membrane considerable
filtration processes have attracted a amount of attention in recent years
in such widely diversified fields as biotechnology, biomedicine, food and beverage processing, and water treatment. The most serious operational constraint to the widespread use of membrane filtration is the flux decline over time due to membrane fouling. Also, the membrane fouling per se may alter the sieving properties significantly. There exist many factors influencing the fouling behavior. Adsorption of macromolecular solutes, especially of proteins, is one of the causes of such a fouling phenomenon. Therefore fundamental *Corresponding author. 0927-7757/94/$07.000 1994Elsevier Science B.V. All rights reserved SSD10927-7757(94)02854-L
Protein adsorption
studies [l-l l] of adsorption of proteins both in and on ultrafiltration and microfiltration membranes have received considerable attention in recent years. Although it is well known that protein adsorption within the pores of the membrane can dramatically alter the flow resistance of the membrane, the quantitative relation between protein adsorption and flow resistance of the membrane is quite complex and as yet not fully understood. This study aims to clarify the role of the membrane-solute interaction (adsorption) in membrane hydraulic permeability. We measured the amount of bovine serum albumin (BSA) adsorption within microfiltration membranes. A method has been developed for obtaining the flow resistance of the membrane in which BSA is adsorbed. In addition, the effects of solution pH on the amount of BSA
16
E. Zritani et aL/Colloids Surfaces A: Physicochem. Eng. Aspects 89 (1994) 15-22
adsorbed, and hence the flow resistance associated with protein adsorption, are investigated.
2. Experimental 2.1. Materials
In all the experiments the protein employed is bovine serum albumin (BSA; Fraction V) with a molecular weight of about 67000 Da provided by Katayama Chemical Industries Corp. This is one of the more abundant blood proteins. The solutions were prepared by carefully dissolving the powdered BSA in either 10 mM acetate or 10 mM phosphate buffer solutions to obtain the desired pH. The isoionic/isoelectric point of the solution is about 5.1. All water used was prepared by an ultrapure water system for laboratory use (Puric-R, Olgano Corp.). The other chemicals were of analytical grade. The BSA concentration ranged from 5 x low4 to 5 x 10W2by weight. Nitrocellulose microfiltration membranes with a nominal pore size of 0.1 pm, supplied by Advantec Toyo Corp., were used for all experiments. The membrane pores are substantially larger than the _,size of a BSA molecule (approximately 140 A x 40 A in diameter) [ 121. For each run, a fresh membrane was used. The membranes were pretreated by soaking them in ultrapure water for lh to remove the glycerin. The internal surface area of the membranes was determined by the BET method with nitrogen as the adsorbing gas. The thickness of the membranes was determined by the magnifying projector, and the true density of the membranes was measured with a pycnometer. The pore size of the membranes was measured by mercury intrusion porosimetry.
brane had been placed. The experiments were carried out under constant pressure by applying compressed nitrogen gas. Transmembrane pressure for all runs was set at 49 kPa. The buffer solution was continuously introduced from a feed solution reservoir connected to the filtration cell. The permeate weight was measured every 10 s with an electronic balance (Shimadzu Corp.) connected to an electronic timer and a printer. The weights were converted to volumes using density correlations. After the hydraulic permeability test, the membrane was carefully removed from the filtration cell and used in the protein adsorption test. BSA adsorption within the porous membranes was evaluated as follows. The membrane was submerged in a BSA solution of known concentration. After the solution was stirred at 80 rev min-’ for 12 h (except for the case of Fig. 1, see below), the membrane was removed. This adsorption time was sufficient to achieve a quasi-steady state. All experiments were conducted at ambient temperature, 22 f 2” C. The residual protein concentrations were evaluated spectrophotometrically by reading the absorbance of the solutions at 280 nm. From the difference between the protein concentration of both the bulk solution and the solution after protein adsorption, the amount of protein adsorbed could be determined, taking into account the volume excluded by the membrane. The protein concentration changed considerably by submerging the membrane in a small amount of solution. Immediately after the adsorption test, the membrane was returned to the filtration cell so that the permeability could be re-evaluated as described previously. The differences in the permeabilities of the clean and preadsorbed membranes were investigated.
2.2. Measurements
3. Theory
Prior to the protein adsorption test, the measurements of the hydraulic permeability of the membrane were performed in a dead-end filtration cell (Advantec Toto Corp.) with an effective membrane area of 27.0 cm2. The filtration cell consisted of a cylindrical vessel containing the buffer solution, surmounting a porous support on which the mem-
In order to examine the possible effects of protein adsorption on the membrane permeability, it has been commonly assumed that the pore space of the membrane is equivalent to a bundle of capillaries of uniform diameter. It has also been assumed that the pore reduces in size owing to the protein adsorption in the interior structure of the mem-
E. Iritani et al.IColloids Surfaces A: Physicochem. Eng. Aspects 89 (1994) 15-22
brane. Accordingly, the Hagen-Poiseuille equation has been employed for describing the laminar flow in the circular tube, in order to evaluate the decrease of the membrane permeability due to the protein adsorption [ 8,111. However, the real pore geometry of the membrane is extremely complicated. For a membrane possessing a polymer mesh structure, the path of a streamline through the pore space would be tortuous, and the pores are interconnected. Therefore a more realistic model would be desirable for describing the flow behavior through the membrane. Clearly, the porous membrane can be treated as a kind of granular bed consisting of non-uniform sizes and/or non-regular particle shapes. Therefore, as a first approximation, assuming that the membrane is symmetric, we have analyzed the flow property of the membrane in terms of the Kozeny-Carman relationship, which has been applied to the laminar flow in a granular bed [13]. The superficial velocity 4 can be expressed in the form
17
applied for the membrane after BSA adsorption. In this case, the Kozeny-Carman equation can be represented by 3 e&J
qp=-=
L&U,
-P
kS;,( 1 - eJ2 ,uL
(3)
where qp, Rmp, cp and S,, are the values for the membrane with adsorbed BSA. The porosity cP of the membrane after BSA adsorption can be described by E*=E-
4wpSm zD2Lpp
where wp is the amount of BSA adsorbed per unit effective internal surface area of the membrane, S, is the effective internal surface area of the membrane, and pp is the density of the protein. The density of the solute, which is defined as the reciprocal of the partial specific volume, is reported to have the value of 1.364 g cmm3 for BSA [ 123. Provided the values of qp and wp are known, one can calculate S, by using Eqs. (3) and (4). If the values of wr and S, are known, qp can be calculated.
(1) where p is the permeation pressure, p is the viscosity of the permeate, R, is the initial membrane resistance to flow, E is the porosity of the membrane, So is the effective specific surface area of the membrane, L is the thickness of the membrane, and k is generally known as Kozeny’s constant. A commonly accepted value for k is 5.0. The porosity E in Eq. ( 1) can be represented by c=l-2
4w zD2Lp,
(2)
where W, is the mass of the membrane, D is the diameter of the membrane, and P,,, is the true density of the membrane. Provided the value of 4 is known, one can calculate So by using Eqs. (1) and (2), and vice versa. In general, the internal surface area of the membrane is much larger than its upper and lower surface areas. If BSA molecules are adsorbed inside the pore structure of the membrane uniformly, then the Kozeny-Carman equation can also be
4. Results and discussion 4.1. Adsorption amount of BSA The adsorption kinetics of BSA within the membrane was investigated under static immersion. Fig. 1 illustrates the mass wp of BSA adsorbed per unit internal surface area of the membrane plotted as a function of the adsorption time &. The adsorption process of BSA is only fast at the very beginning; it reaches a nearly steady state within 3 h. As also can be observed in Fig. 1, the equilibrium amount adsorbed is about 1.9 mg me2 under this condition. A typical adsorption isotherm of BSA onto the pore surface of the membrane at pH 5.1 is shown in Fig. 2. The adsorbed amount of BSA is plotted against the equilibrium mass fraction s of BSA in the solution. The adsorption isotherm rapidly attained a plateau level. Under this condition, the saturated amount of adsorbed BSA is about 2.0 mg rne2.
18
E. Iritani et al.JColloids Surfaces A: Physicochem. Eng. Aspects 89 (1994) 15-22
1.4 CELLULOSE NITRATE MEMBRANE
CELLULOSE NITRATE MEMBRANE
01 0
I
I
2
4
I
I
I
I
6
8
IO
12
I s C-l
81 Chl Fig. 1. Variation of amount of BSA adsorbed per unit internal surface area of membrane over time.
Fig. 3. Logarithmic plot of adsorbed amount against equilibrium concentration.
2.5r-----l
4F-----l
-1
1
3oooooooooo
I
1.0 0.5 0
0
/
I 2
BSA
?
pH=5.1 &=12h
!2cr’
-
d
Eq.W
I 4
P=49kPo CELLULOSE MEMBRANE
CELLULOSE NITRATE MEMBRANE
I
h&*hA*A~
1
0 A I -
6x10-*
s C-l
-
01
Fig. 2. Adsorption isotherm for BSA within membrane.
I
0
I
I
100
NITRATE
CLEAN ADSORBED BSA,pH=5.1 s=4,35x10-3 8,=12h PREDICTED
I
200
-
I
I 300
.9 Cd
Fig. 3 shows the logarithmic plot of the adsorbed amount wP against the equilibrium concentration s. A reasonably linear relationship is obtained in accordance with the Freundlich equation represented by wP = Ksll”
(5)
where K and n are empirical constants. The solid line shown in Fig. 2 was approximated by Eq. (5). 4.2. Permeation rate of membranes In Fig. 4, typical results of the hydraulic permeability test at the equilibrium concentration s of 4.35 x 10m3are shown in the form of the permeation rate vs. the permeation time 8. The results for both the clean and preadsorbed membranes are shown. The permeation rate of the preadsorbed
Fig. 4. Dependence of permeation rate on permeation time.
membrane is smaller than that of the clean membrane, owing to the BSA adsorption onto the pore surface. The most interesting feature is that the difference in the permeation rate of both membranes is not as large as expected. In general, the flux decline during micro filtration of protein solutions may be much greater than can be explained by protein adsorption. Such a substantial decrease may be interpreted in terms of protein deposition resulting from shear induced distortion of the molecules on passage through the micropores [14]. Some interesting work associated with this behavior is to be found elsewhere [ 15,161. In each case in Fig.A, the permeation rate is constant throughout the run. Fig. 4 includes the prediction
E. Iritani et al./Colloids Surfaces A: Physicochem. Eng. Aspects 89 (1994) 15-22
of the permeation rate for the preadsorbed membrane. It can be estimated as follows. The porosity e of each clean membrane was calculated from Eq. (2). Therefore the effective specific surface area S,, of the clean membrane can be obtained from the measurements of the permeation rate 4 and the porosity E, using Eq. (1). For example, in the data used for Fig. 4, the value thus obtained was 2.86 x 10’ m-l. This was in good agreement with the value of 3.05 x 10’ m-l obtained by the BET method. The porosity of the preadsorbed membrane decreases slightly, but significantly, compared with that of the clean membrane, owing to the BSA adsorption onto the pore surface. It can be calculated from Eq. (4). Consequently the effective specific surface area S, of the preadsorbed membrane can be computed from the measurements of the permeation rate qp and the porosity Ed, using Eq. (3). Surprisingly, the specific surface area SOP of the preadsorbed membrane was almost the same as that of the clean membrane. However, the BSA molecule is regarded as a prolate (rod-shaped) ellipsoid of revolution with major axis 2a = 14 nm and minor axis 2b=4 nm [ 121. The surface area S and the volume V of the prolate ellipsoid of revolution are, respectively, described by sin-’ E
S = 2nb2 + 2nab 7 V= i nab’
where E (= (a2 - b2)‘/2/a) is the eccentricity. As the specific surface area is defined as S/V, the sum of the specific surface area of the adsorbed BSA molecules onto the pore surface of the membrane can be calculated from the data of the amount of adsorbed BSA using Eqs. (6) and (7). For example, in the data used for Fig. 4, the value was found to be 7.52 x 10’ m-l, significantly larger than the measurement of the specific surface area of the preadsorbed membrane based on the permeation test. If the liquid flows through the interstices of the preadsorbed BSA molecules, the permeation rate of the preadsorbed membrane would be, of course, considerably reduced compared with that
19
of the clean membrane. The experimental results suggest that the specific surface area of the adsorbed BSA does not act as the flow resistance of the permeation effectively. The predicted line shown in Fig. 4 was obtained from the following relation using the values of porosity of the preadsorbed membrane and the specific surface area of the clean membrane: 3
P
EP
-P
kS;( 1 - ‘p)2 pL
qp=-= dLlp
(8)
The predicted line is in very good agreement with the experimental data, indicating that Eq. (8) accurately describes the change in the flux associated with protein adsorption. A comparison between Eqs. (1 ) and (8) leads to 9p q
E;f( 1- El2 .?(
1 - E&J2
(9)
It is instructive to compare the pore size of the preadsorbed membrane with that of the clean membrane, in order to evaluate the sieving property of the membrane. The equivalent diameter of the pore space can be evaluated by introducing the hydraulic mean diameter [ 171. The hydraulic mean diameter d, for a flow passage of the membrane is given by d, =
4E Ml-E)
(10)
Given a knowledge of the porosity E and the specific surface area So of the membrane, the hydraulic mean diameter de can be evaluated quantitatively from Eq. (10). For instance, in the data used for Fig. 4, the pore size of the clean membrane calculated from Eqs. (l), (2) and (10) on the basis of the data from the hydraulic permeability test is 0.291 urn. The median pore diameter of the membrane determined by porosimetry is 0.203 urn. This value is smaller than the calculated one. It is considered that the pore size of the membrane depends on the method of experimental measurement. The pore size of the preadsorbed membrane can be obtained by substituting ep for E in Eq. (10). The porosity ep of the preadsorbed membrane can be obtained from Eq. (4) on the basis of the data from the adsorption
E. Iritani et al./Colloid Surfaces A: Physicochem. Eng. Aspects 89 (1994) 15-22
20
tests. The pore size thus obtained is 0.274 pm, and hence the average value of the thickness of the adsorbed BSA layer is 8.5 nm. Whether the BSA molecules are adsorbed side-on onto the internal surface of the membrane or are adsorbed end-on is not clear from these data alone. The porosity ep of the preadsorbed membrane can, however, also be obtained from Eq. (9), using the data of the decrease of the permeation rate due to BSA adsorption. The pore size thus obtained is 0.275 pm. It is interesting to note that this value is in excellent agreement with that obtained on the basis of the measurement of the adsorbed amount. This good agreement indicates that Eq. (10) provides a reasonable description of the mean pore size of the membrane. In Fig. 5, the cumulative permeation volume u collected per unit area of active membrane is plotted against the permeation time 0. The solid line indicates the value calculated from the following relation using the predicted value of the permeation rate qP of the preadsorbed membrane, and it is in good agreement with the experimental values: (11)
up = qpe
where vP is the cumulative permeation volume collected per unit area of active membrane in the preadsorbed membrane. In Fig. 6, the resistance Rmp of the preadsorbed
854, CELLULOSE NITRATE MEMBRANE
2.0
t
(Rm,h.
Ccm-‘I
Fig. 6. Resistance of preadsorbed membrane.
membrane calculated mental value of qp is values. The predicted Eq. (8) using the adsorbed amount.
from Eq. (8) using the expericompared with the predicted values can be obtained from value of ep based on the
4.3. Efect ofpH The effect of changing the solution pH was also studied. Fig. 7 shows the pH dependence of the plateau adsorption of BSA. It is obvious that adsorption was quite sensitive to the pH, indicating the electrical nature of this adsorption process. Over the pH range examined, the amount of BSA adsorbed was largest at around its isoelectric point. As the BSA molecule carries no net charge at that
8 3
I
,
/
I
,
I
BSA si=5.ox10-3
F 6 :: p
I
-
PREDICTED
4
0
0
100
200
300
8 cs1
Fig. 5. Relation between cumulative permeation volume collected per unit active membrane area and permeation time at pH 5.1.
Oh
2
4
6
8
IO
PH
Fig. 7. Dependence of plateau adsorption of BSA on pH.
E. Iritaniet al./ColloicisSurfaces A: Physicochem.Eng. Aspects89 (1994) 15-22
point, the molecule is in its most compact state. The BSA molecules deposit themselves rather densely onto the pore walls of the membrane to form a compact configuration with a smaller lateral electrical interaction between the molecules. As a result of this, the protein is very adsorptive at around the isoelectric point [ 18-211. This is similar to the observation concerning ultrafiltration of BSA solutions made by Fane and co-workers [l-3,22]. In Fig. 8, the permeation volume at pH 8.0 are plotted against the permeation time 8. Compared with the result for the isoelectric point in Fig. 5, the permeation volume does not decrease SQmuch because the amount of BSA adsorbed is relatively small, as can be seen in Fig. 7.
21
complex, our analysis indicates that the relation between the flow resistance of the membrane and the amount of adsorbed protein can be relatively well described using the Kozeny-Carman relationship. In addition, the importance of pH in adsorption was highlighted. It has been proven that the higher amount of protein adsorption at around the isoelectric point brings about a larger increase in the flow resistance of the preadsorbed membrane. As our current analysis postulates that the membrane is symmetric, future work must consider the effect of membrane heterogeneity. Also, future work is planned to examine the effect of protein adsorption on the flow resistance of fully and partially retentive ultrafiltration membranes.
Acknowledgments 5. Conclusions The amounts of BSA molecules adsorbed in a nitrocellulose microfiltration membrane were measured under various conditions. Subsequently the change in the flow resistance of the microfiltration membrane associated with the BSA adsorption was investigated. The flow resistances of both clean and preadsorbed membranes were analyzed in terms of developments of the Kozeny-Carman relationship. While the real mechanism of the fouling due to the protein adsorption is quite IO
/
I
This work was partly supported by a Grantin-Aid for Scientific Research from the Ministry of Education, Japan, Grant Nos. 03805089 and 04805098, and by the Asahi Glass Foundation. The authors wish to acknowledge with sincere gratitude the financial support leading to the publication of this article. The authors also wish to thank Hiroaki Sumi of Showa Chemical Industry Co., Ltd., for assistance with the measurements of the surface area of the membranes.
Appendix: List of symbols
I
P=49kPo
8
_
CELLULOSE MEMBRANE 0 A
-
NITRATE
CLEAN ADSORBED BSA,pH=B.O s=4.76~10-~ 8;=12h PREDICTED
: D
d,
E k
K L n 0
200
100
300
e CSI
Fig. 8. Relation between cumulative permeation volume collected per unit active membrane area and permeation time at pH 8.0.
P i, s %
s
half length of major axis (m) half length of minor axis (m) diameter of membrane (m) hydraulic mean diameter (m) eccentricity (a’ - b*)‘/*/a Kozeny’s constant constant in Eq. (5) (kg m-*) thickness of membrane (m) constant in Eq. (5) permeation pressure (Pa) superficial permeation velocity (m s- ‘) initial membrane resistance to flow (m-l) equilibrium concentration initial concentration surface area of a solute (m”)
E. Iritani et al.IColloids Surfaces A: Physicochem. Eng. Aspects 89 (1994) 15-22
22
effective internal surface area of membrane (m’) effective specific surface area of membrane &I (m-7 V cumulative permeation volume collected per unit active membrane area (m) V volume of a solute (m3) amount of solute adsorbed per unit effectWP ive internal surface area of membrane (kg m-*) WIZl mass of membrane (kg)
&II
Greek letters E
e 4
p Pm PP
porosity of membrane permeation time (s) adsorption time (s) viscosity of permeate (Pa s) true density of membrane (kg me3) density of solute (kg mp3)
Subscript P
preadsorbed membrane
References [l]
A.G. Fane, C.J.D. Fell and A.G. Waters, J. Membr. Sci., 16 (1983) 211. [2] A.G. Fane, C.J.D. Fell and A. Suki, J. Membr. Sci., 16 (1983) 195.
c31 A. Suki, A.G. Fane and C.J.D. Fell, J. Membr. Sci., 21 (1984) 269. 141 M.W. Chudacek and A.G. Fane, J. Membr. Sci., 21 (1984) 145. c51 J.H. Hanemaaijer, T. Robbertsen, Th. van den Boomgaard and J.W. Gunnink, J. Membr. Sci., 40 (1989) 199. C61 H. Yanagishita, T. Nakane, J. Aihara, S. Takatsu and H. Yoshitome, Membrane, 14 (1989) 64. c71 H. Nabetani, N. Nakajima, A. Watanabe, S. Nakao and S. Kimura, AIChE J., 36 (1990) 907. C81 K. Ogasawara, S. Tsuda, K. Ozawa and K. Sakai, Chem. Eng. J., 48 (1992) Bl. c91 L.E.S. Brink, S.J.G. Elbers, T. Robbertsen and P. Both, J. Membr. Sci., 76 (1993) 281. Cl01 M.K. Ko, J.J. Pellegrino, R. Nassimbene and P. Marko, J. Membr. Sci., 76 (1993) 101. Cl11 A.M. Bribes and M.N. de Pinho, J. Membr. Sci., 78 (1993) 265. Cl21 T. Peters, Jr., Serum albumin, Adv. Protein Chem., 37 (1985) 161. Cl31 PC. Carman, Trans. Inst. Chem. Eng., 15 (1937) 150. [141 W.R. Bowen and Q. Gan, J. Membr. Sci., 80 (1993) 165. Cl51 E. Iritani, Y. Itano and T. Murase, Membrane, 17 (1992) 101. Cl61 E. Iritani, Y. Itano and T. Murase, Kagaku Kogaku Ronbunshu, 19 (1993) 536. Cl71 E. Iritani, M. Iwata and T. Murase, Sep. Sci. Technol., 28 (1993) 1819. Cl81 H. Shirahama, Hyomen, 27 (1989) 29. Cl91 N. Muramatsu and T. Kondo, J. Colloid Interlace Sci., 153 (1992) 23. c201 E. Iritani, S. Nakatsuka, H. Aoki and T. Murase, J. Chem. Eng. Jpn., 24 (1991) 177. c211 E. Iritani, T. Watanabe and T. Murase, J. Membr. Sci., 69 (1992) 87. c221 A.G. Fane, Ultrafiltration: factors influencing flux and rejection, in R.J. Wakeman (Ed.), Progress in Filtration and Separation 4, Elsevier, Amsterdam, 1986, p. 134.