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Flux decline in protein ultrafiltration
Journal of Membrane Science, 21 (1984) 269-283 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
269
FLUX DECLINE IN PROTEIN ULTRAFILTRATION*
k SUKI, A.G. FANE and C.J.D. FELL School of Chemical Engineering Kensington, N.S. W. (Australia)
and Industrial Chemistry,
University of New South Wales,
(Received February 8, 1984; accepted in revised form June 1, 1984)
Summary This paper examines the link between flux decline and protein which becomes deposited (bound) onto the membrane in protein ultrafiltration. For the conditions studied deposition kinetics were relatively slow, with the rate dependent on feed concentration but the “plateau” (steady-state) amount insensitive to this parameter. The amount of deposition was dependent on system hydrodynamics, membrane type and solution environment. Specific resistances of the deposited layer and the labile boundary layer were measured by analysis of unstirred and stirred permeation rates. A semi-empirical relationship, including the deposition kinetics and the deposited layer resistance, gives reasonable prediction of the observed flux decline.
Introduction It is well known that the ultrafiltration (UF) process can suffer from flux decline which may extend for several hours or days before a steady state is achieved. In protein UF the major cause of flux decline has been linked to the irreversible binding of protein to the membrane. Lee and Merson [ 11 and Cheryan and Merin [Z], using electronmicroscopy, observed protein layers of 0.5-1.0 pm thickness following UF. Ingham et al. [3] measured protein binding and referred to it as multilayer adsorption. We have also described bound protein as multilayer adsorption in studies of the UF of proteins in different solution environments with retentive [ 41 and partially permeable membranes [ 51. However, the term adsorption strictly implies an equilibrium process with partitioning of solute between a solution and a surface, and although this is probable in UF there may be the additional influence of convectioninduced deposition. We have therefore adopted the term deposition to describe the combined effects of adsorption and irreversible accumulation of solute on the membrane. Ultrafiltration flux can be considered to be controlled by several hydraulic resistances in series. This filtration analogy has been argued elsewhere [6,7] *Paper presented at IMTEC ‘83, November S-10,
0376-7388/84/$03.00
o 1984
1983,
Sydney, Australia.
Elsevier Science Publishers B.V.
270
and it provides
J = (l/A,)dV/dt
a useful basis for our analysis. = AP/v(R,
+ Rd + Rb)
Thus, (1)
where R, = membrane resistance, R,j = deposited solute resistance, and Rb = (labile) boundary layer resistance. Assuming that the solute resistances can be characterised by mean “specific resistances” we can write Rd = adi&
(2)
and Rb = CY),i&,
(3)
where Md is the mass of deposited solute per unit area of membrane, and we assume deposition to be a kinetic process, Md = Md [t] . Mb is the mass of solute held in the labile boundary layer. Thus eqn. (1) becomes
J = AP/q(R,
+ “dMd [t]
+ &bMb)
(4)
Equation (4) accounts for flux decline by incorporating the deposition kinetorder to test eqn. (4) it is necessary to have eStimk?S of Cld, CYband Mb. These parameters have been measured independently using modified filtration theory. In separate deposition studies the kinetics and nature of the deposited solute have been observed. This paper evaluates and discusses the parameters in eqn. (4) and compares measured and predicted fluxes. iCS. In
Experimental Experiments were performed in batch cell and cross-flow ultrafiltration systems. The batch cell had a capacity of 110 ml and a membrane area of 15 cm’. The cross-flow system had two flat plate modules in parallel, each of area 30 cm2 (10 X 3 cm) and 0.4 cm channel height. Operating conditions of 100 kPa and 25°C were used in both systems. For stirred ultrafiltration in the batch cell, the stirring speed was 400 rpm and for the cross-flow system the flow rates were 2 and 8 l-min-l (equivalent to Reynolds numbers of 2200 and 8800, respectively). The macrosolute used was bovine serum albumin (BSA), obtained from Calbiochem. Grade “A” (>99% electrophoretically pure) was used in the batch cell experiments, and fraction V BSA (>97% pure) was used in the cross-flow experiments. Comparative tests in the stirred batch cell produced similar results for the two grades of BSA. Solutions of BSA were prepared over a range of pH conditions, with and without 0.2 M NaCl. Most experiments used 0.1 wt. % BSA, but some were at 1.0 and 2.0%. Amicon PM30 membranes, which are polysulphone with a nominal molecular weight cut-off of 30,000 D, were used for the majority of experiments. Comparative deposition tests were also made on other membranes, the Amicon XMlOO (Dynel)
271
and YM30 (regenerated cellulose), DDS GR61P (polysulphone) and Millipore PTGC (polysulphone). Ultrafiltration runs in the cross-flow system were all 5 hours duration. Flux histories and protein deposition were measured. At the end of each run the membrane was carefully removed and then rinsed with distilled water prior to protein determination. Rinsing with saline solutjon instead of distilled water did not produce any difference in the results. Protein was removed from the membrane with sodium dodecyl sulphate and then measured by the modified Lowry method [8] with the spectrophotometer set at 750 nm. Details of the technique may be found elsewhere [ 91. Long-term runs were also performed in the stirred batch cell, in which case the solvent was continuously replenished from a feed reservoir connected via an in-line filter (Millipore 0.2 pm). Protein deposition was measured at the end of each run. A series of runs, of duration 0.3 to 8 hr for solutions of 0.1, 1.0 and 2.0 wt. % BSA at pH 5 provided a direct measure of deposition kinetics. The batch cell was also used to estimate the specific resistance of the deposited solute by unstirred experiments. Specific resistances were obtained by analysis of the time, t, and permeate volume, V, data. Equation (1) may be rewritten as
&‘A,)dV/dt
= AP/q(R,
+ RP)
(5)
where R, is the “polarised” solute resistance. For unstirred conditions over the relatively short duration (<0.2 hr) we neglect solute diffusion and apply conventional filtration theory, where R,
= ~,(VCB/&)
Combining equation
eqns. (5) and (6) and integrating
(6)
gives the well-known
filtration
(7) A linear t/V vs. V plot therefore gives ayp from the slope. This specific resistance represents the mean effect of gradually accumulating layers of solute in the absence of stirring. We have assumed that ap gives an approximation to the specific resistance of the bound (deposited) protein obtained in stirred ultrafiltration, i.e., ad s? ap. Unstirred tests were performed at pH 2 (or 3), 5 and 10 with and without 0.2 M NaCl on BSA “A” and “V”. The solute in the labile boundary layer in stirred ultrafiltration will have a concentration gradient and its effective specific resistance will be less than lyp The values for Qb have therefore been taken from the averaged slopes of t/V vs. V plots for the initial polarisation period under stirred conditions in the batch cell, as explained below. For these measurements the permeate was recorded at 1.0~second intervals by an automatic balance.
272
Results Deposited (bound) protein Deposition kinetics under ultrafiltration conditions with a PM30 membrane are shown in Fig. 1. The deposition process was rather protracted but a plateau or equilibrium value was approached. These data can be expressed by a simple relationship if it is assumed that the rate of deposition is governed by a deposition potential, i.e., dM,-J/dt =
K(d!@-
(8)
it&j)
where M$ is the plateau i&
= M$[l
deposition
2%
(9) 0
BSA .
0.1%
;: I 11 -t1
flH
BS/
__-.
-2 .
/
1/ /
: I 9
’
He
/’
/ ‘*
/I 0
-,--.A--_--
--======z-_--
/
‘0 _ ‘1 I I 11
I
So,
- eXp(-Kt)]
_BSA ,_,-2 ’ /’ // /“I, 1%
:
and K is the rate constant.
0
1 2
I 4 T
I M E
I 6
I 8
(hl
Fig. 1. Deposition 400 rpm).
kinetics during BSA ultrafiltration (stirred cell, PM30, 100 kPa,
The deposition
curves were fitted
by the following
0.1% BSA:
M$ = 65 I.lg-cmU2,
K = 0.3 hr-l
1.0% BSA:
M$ = 87 pg-cm-2,
K = 1.2 hr-l
2.0% BSA:
&I$ = 87 pg-cmb2,
K = 1.8 hr-’
values of M$ and K:
The effect of pH and membrane type on the amount of deposited protein is given in Fig. 2, which shows the enhanced deposition around the isoelectric point of the BSA (pH z 4.7). Maximum deposition was obtained with the par-
273
tially permeable XMlOO for which the measured protein may have included a component of internally bound, but removable, solute. Figure 3 also shows the effect of pH, as well as the influence of NaCl concentration and crossflow velocity. The effect of salt on protein deposition is similar to that reported previously [ 41.
50 GO 30 20 10 5
01
0
I
I
I
1
I
2
L
6
8
10
PH Fig. 2. Deposition vs. pH; 3 hr UF in stirred cell (0.1% BSA; 1 - XMlOO, 2 - PM30, 3 DDS GRGlP. 4 ~ PTGC, 5 - YM30).
200 /I
.
0
0
I
2
I
4
I
6
I
8
I
10
PH Fig. 3. Deposition vs. pH, 5 hr UF in cross-flow system (0.1% BSA, PM30; H = 1.1 m-see-’ cross-flow, L = 0.3 m-se&, S = 0.2 M NaCl, NS = no salt).
274
Resistances Plots of t/V vs. V for unstirred ultrafiltration are given in Fig. 4. The specific resistance al, is directly proportional to the slopes, and Table 1 presents the measured data which show that the largest value of crp was at pH 5 (no salt) and the lowest at pH 10. The table also shows there was little difference between BSA “A” and “V”. Literature data are comparable with this work. 150
120
> .-.. + 60
5
10
PERMEATE Fig. 4. Determination PM30,lOO
of specific
VOL
15 (ml1
resistance
of BSA from
unstirred
UF data (0.1%
BSA,
kPa).
From the measured specific resistances and the measured amounts of deposited protein, eqn. (2) can be used to calculate Rd values. These values are plotted in Figs. 5 and 6 for the stirred cell and the flow system, respectively. In the absence of salt, Rd has a maximum at pH 5, which mirrors the reported flux vs. pH values [4], With salt present, the maximum is either reduced substantially (Fig. 6) or eliminated (Fig. 5). This also agrees with the loss of the minima in the flux vs. pH plot [ 41. The effective specific resistance in the labile boundary layer, olb, was estimated from the t/V vs. V plots for initial polarisation. Figure 7 shows how
275 TABLE 1 Specific resistances of ESA (0.1 wt. %, 100 kPa) BSA conditions
Specific resistance, +
(m/kg
x
10W’5)
PH
NaCl (M)
PM30 (BSA “V”)
PM30 (BSA “A”)
2 2 5 5 10 10
0.2 0.0 0.2 0.0 0.2 0.0
4.2a 1.4 = 3.2 6.4 2.9 2.0
4.4 3.0 2.9 5.2 2.0 1.7
Literature
2.7 b 2.3 c, 4-5 d
’ pH 3.0. bPM30, pH 4.7 (acetate buffer) [7]. c PM30, pH 7.4 (0.13 M NaCl + phosphate buffer) [7]. dPMIO; PH 7 (015 M phosphate buffer), 0.2- 2.0% BSA [lo].
IO Fig. 5
4
6
PH
8
IO
Fig.
6
i 8
4
6
8
10
PH
Fig. 5. Estimated resistance of deposited layer, Rd, vs. pH (0.1% BSA, stirred cell, PM30, 100 kPa, 8 hr run; S = 0.2 M NaCl, NS = no salt). Fig. 6. Estimated resistance of deposited layer, Rd, vs. pH (0.1% BSA, cross-flow system, PM30, 100 kPa, 5 hr run; H = 1.1 m-set-I, L = 0.2 m-set-’ ).
the average slope of the t/V plot was obtained. The amount of Protein Present in this boundary layer, Mb, was estimated from the MnOUllt Of prOkiIl convected to the membrane in the initial polarisation volume, vb, The bound ary layer resistances, h!b, calculated from eqn. (3), are plotted in Fig. 8. The dependence of Rb on pH and salts is similar to that found for the deposited layer resistance.
276
01
0
I
I
I
1
2
3
PERMEATE
VOL
(ml1
Fig. 7. Determination of specific resistance for boundary layer from initial polarisation in stirred cell LJF (0.1% BSA, PM30).
I I I ' 2----i
I
I
6
I
I,
I
8
10
PH Fig. 8. Boundary layer resistance, Rb, values for stirred cell.
VS.
pH. Comparison of calculated and experimental
Instead of using separate estimates of CXband Mb to calculate Rb it is possible to obtain Rb directly from the initial ultrafiltration flux, i.e. Rb = (AP/qJi) - R,
(10)
Initial flux, Ji, may be obtained from correlations or experimentally following polarisation, and before substantial deposition. The dotted curves in Fig. 8 show Rb values obtained from eqn. (10) using the experimental Ji data Equations (3) and (10) yield very similar results. This supports our use of the filtration analogy in which the components of resistance are determined from specific resistances and mass deposition.
Discussion The amount of deposited protein reported here provides further evidence that monolayer adsorption is significantly exceeded under ultrafiltration conditions. The measured deposition kinetics show that it is a relatively slow process, and this agrees with earlier work of Dejmek et al. [ll], who followed the build-up of tagged proteins. Deposition rate constants, K, increased significantly with bulk protein concentration, Cg, but the plateau values, M*,, were notably insensitive to concentration. The initial rates, (dMd/dt)i, obtained from eqn. (8) with Md = 0 and fitted values of M$ and K, were directly proportional to the initial convec tive solute flux, JiCB, as shown in Fig. 9. Attempts to correlate (d.Md/dt)i with the estimated solute concentration at the membrane surface, Cw (= CB exp (J/k,)), were not successful.
J, Cs (yg/sJ Fig, 9. Initial deposition rate vs. initial filtration rate.
From this evidence we conclude that deposition under UF conditions is not analogous to a Langmuir-type equilibrium process, as suggested by Howell et al. [ 121. Rather, the initial rate of deposition is strongly linked to the initial rate of accumulation of solute molecules at the membrane surface. However, only a small fraction of the arriving molecules become instantaneously bound, and this fraction will probably depend on the nature of the membrane surface - as discussed below - and the conformation of the macro. molecule. Subsequent deposition is presumably linked to the filtration process in the sense that it relies on the accumulation of solute in the polarised
278
layer. Indeed, if the deposition kinetics of Fig. 1 are plotted according to conventional filtration as Md versus t’/” the data at 0.1 wt. % BSA are linearised. However, this approach does not work for the data at 1.0 and 2.0 wt. % because at these conditions a deposition “plateau” is rapidly achieved. It is more appropriate to consider the deposition process as starting with the initial accumulation of polarised solute which then provides a “reservoir” of molecules which gradually become fixed due to slow aggregation and flocculation; we have developed this concept elsewhere [ 91. The above picture of the deposition process is consistent with the observation that the “plateau” deposition is relatively insensitive to feed concentration, Cg, but is dependent on system hydrodynamics (Fig. 3). This suggests the deposition of protein ceases when the yield stress of the topmost layers of the aggregated solute is less than the local shear stress due to cross-flow or stirring. The strong influence of pH shown in Fig. 2 is expected since pH affects protein charge, stability, and tendency to aggregate. Figure 2 also shows the effect of membrane type on deposition. The results cannot be related simply to the membrane material, since the three polysulphone membranes (PM30, GR61P and PTGC) gave noticeably different data. A more plausible explanation is based on differences in the surface porosity and pore size distributions of the membranes. Available information [ 7,9,13,14] summarised in Table 2, shows that the surface heterogeneity, in terms of porosity and mean pore size of the membranes may be ranked XM300 > PM30 > PTGC > GR61P > YM30. This ranking is similar to the observed tendencies shown in Fig. 2 to accumulate deposited protein. The more heterogeneous the membrane surface the higher will be the local velocities normal to the surface. Figure 9 shows how the initial deposition rate increases with initial filtration rate for one type of membrane, and if this effect applies on a local scale around individual pores it would give more rapid initial deposition for the more heterogeneous membranes. The course of the subsequent deposition would presumably depend on the extent and nature of TABLE 2 Reported data on membrane surface characteristics Membrane type
XM300 PM30 PTGC GR61P YM30
Data obtained by EM
Ref.
Ranking
13 9 14 9 9,7
heterogeneous
Pore diam. Surface porosity (nm) (%I 24.5 12 6 a =
2%
a, -50%
b
a Pores not detectable by EM. b Order of magnitude inferred by Ref. [7 1.
I homogeneous
279 this initial deposition. In other studies differences in membrane surface micro Porosity have been used to explain variations in ultrafiltration flux [13] and permeabilities of BSA layers [ 71. The variations of the specific resistances with pH and salt content given in Table1 can be explained by the effect of these conditions on the Size and charge of the BSA molecule. We have discussed these interactions in terms of flux variations elsewhere [4]. In brief, the BSA molecule tit the isoionic point of circa pH 5 (no salt) is at its most compact and carries no net charge; this should provide the least permeable BSA layers and highest crP (as observed). Away from the isoionic pH, and in the absence of salt, the molecules expand and have significant net charge and should form permeable layers with lower orP, as observed. Addition of salt at these pH extremes will shield charges, causing molecular contraction and an increase in eP. At pH 5 the effect would be different since the addition of salt causes anion binding, enlargement of the molecule and acquisition of charge, with an expected decrease in c+, as observed. The separately determined vahres of the resistance p~~eterS (Rm, old, q,, Md and Mb) have been combined via eqn. (4) to give predicted fluxes. Figure 10 compares the final flux for various pH, salt concentrations, membrane types, ultrafiltration times, stirred and cross-flow operation, with the calculated values. The agreement is reasonably good; linear regression gives a slope of 0.95, an intercept of about 5 and a regression coefficient of 0.95; Flux declines can also be estimated from eqns. (4) and (g), i.e.,
(11)
EXPERIMENTAL FLUX lllm*-h) Pig. 10. Comparison of final UF flux (O.l-2.25% PM30,
PTGC,
DDS; pH 2-10,
time 0.2-8
hr).
BSA, stirred cell and cross-flow
system;
280
The most testing comparison is between the experimental data for the crossflow system and the calculated values based on the kinetic constants obtained using the stirred cell. Figures lla, b and c, for pH 3, 5 and 10, respectively, show that the model reasonably predicts the observed trends. Typically, eqn. (11) predicts within ?15%. In many cases the discrepancy lies more in the prediction of initial flux than in the estimation of flux decline. Apart from confirming the link between protein deposition and flux decline these results suggest that eqn. (11) can be used as a semi-empirical model of the fouling process. The equation requires as input the resistance parameters and the kinetic constants. These can be obtained relatively simply in stirred batch cell apparatus and applied to larger scale cross-flow systems. In addition, attempts to reduce fouling by lowering M$ and/or K can be evaluated in the stirred batch cell with good prospects of translation to larger scale. This is the current thrust of our studies. 300 PHI
Salt -.-
No - --
0 0
a
I
I
I
I
1
2
3
4
TIME
model
(hl PH5
Salt -*0
TIME
Salt
-o- expt
(hl
No Salt -oexp ----model
5
281
pH
Salt
-.-.-
n
c
“0
I 1
I 2 TIME
10 NO
Salt
-Expt ----
I 4
I 3
Model
I 5
(h1
Fig. 11. Predicted and experimental flux declines in the cross-flow system (0.1% BSA, PM30, 0.3 m-set-‘); (a) pH 3, (b) pH 5, (c) pH 10.
Conclusions Deposition or binding of protein to membranes under ultrafiltration conditions is multilayer. The bulk concentration influences the rate of deposition, but has little influence on the ultimate “plateau” value of mass deposited, which is more dependent on system hydrodynamics, membrane type and solu. tion pH and salt content. Ultrafiltration flux can be predicted by a semi-empirical model based on membrane, deposited layer and boundary layer resistances in series. Flux decline is accounted for by a time-dependent deposited layer resistance obtained from the deposition kinetics and the measured specific resistance of the solute. Acknowledgements The authors would like to thank the Australian Research Grants Committee for financial assistance_ One of us (A.S.) would also like to thank the Agricultural University of Malaysia for support. fist of symbols membrane area (m’) f&~,$7 solute concentration in bulk, at wall (-, or kg-mm3) Am
282
J K k,
M AP
R t V a rl
flux (1-m’ -hr- ’ or m-set- ’ ) deposition rate constant (hr-’ ) solute mass transfer coefficient (m-set-’ ) deposited mass per unit membrane area (pg-cmA2) pressure (kPa) hydraulic resistance (m-l ) time (set or hr) volume (ml, m”) specific resistance (m-kg-’ ) viscosity (N-set-m-2)
Subscripts b boundary layer d deposited layer polarised layer P References 1
D.N. Lee and R.L. Merson, Examination of cottage cheese whey protein by scanning electron microscopy: Relationship to membrane fouling during ultrafiltration, J. Dairy Sci., 58 (10) (1975) 1423. 2 M. Cheryan and U. Merin, A study of the fouling phenomena during ultrafiltration of cottage cheese whey, in: A.R. Cooper(Ed.), Ultrafiltration Membranes and Applications, Plenum Press, New York, 1980, p, 619. 3 K.C. Ingham, T.F. Busby, Y. Sahlestrom and F. Castino, Separation of macro molecules by ultrafiltration: Influence of protein adsorption, protein-protein interaction and concentration polarisation, in: A.R. Cooper (Ed.). Ultrafiltration Membranes and Applications, Plenum Press, New York, 1980, p. 141. 4 A.G. Fane, C.J.D. Fell and A. Suki, The effect of pH and ionic environment on the ultrafiltration of protein solutions with retentive membranes, J. Membrane Sci., 16 (1983) 195-210. 5 A.G. Fane, C.J.D. Fell and A.G. Waters, Ultrafiltration of protein solutions through partially permeable membranes - The effect of adsorption and solution environment, J. Membrane Sci., 16 (1983) 211-224. 6 A.G. Fane, C.J.D. Fell and M.T. Nor, Ultrafiltration/activated sludge systems Development of a predictive model, in: A.R. Cooper (Ed.), Ultrafiltration Membranes and Applications, Plenum Press, New York, 1980, pp. 631-658. 7 H. Reihanian, CR. Robertson and A.S. Michaels, Mechanism of polarisation and fouling of ultrafiltration membrane by proteins, J. Membrane Sci., 16 (1983) 237-258. assay for both water8 H.H. Hess, M.B. Lees and J.E. Derr, A linear Lowry-Folin soluble and sodium dodecyl sulfate-solubilized proteins, Anal. Biochem., 85 (1978) 295-300. 9 A. Suki, Factors affecting flux in protein ultrafiltration, Ph.D. Thesis, University of New South Wales, 1984. 10 M.W. Chudacek and A.G. Fane, The dynamics of polarisation of unstirred and stirred ultrafiltration, J. Membrane Sci., 21 (1984) 145-160. 11 P. Dejmek, B. Hallstrom, A. Kilma and L. Winge, A method for measuring membrane fouling in ultrafiltration, Lebensm.-Wiss. Technol., 6 (1) (1973) 26-29. 12 J.A. Howell, D. Velicangil, M.S. Le and A.L. Herrera Zeppelin, Ultrafiltration of
283
13
14
protein solution: A theoretical model, Proc. Symp. on Membrane Processes, Manchester, 12 Nov. 1980, Inst. Chem. Eng., North Western Branch, 1980, Papers No. 4, p. 5.1. A.G. Fane, C.J.D. Fell and A.G. Waters, The relationship between membrane surface pore characteristics and flux for ultrafiltration membranes, J. Membrane Sci., 9 (1981) 245-262. U. Merin and M. Cheryan, Ultrastructure of the surface of a polysulphone ultrafiltration membrane, J. Appl. Polym. Sci., 25 (1980) 2139-2142.
269
FLUX DECLINE IN PROTEIN ULTRAFILTRATION*
k SUKI, A.G. FANE and C.J.D. FELL School of Chemical Engineering Kensington, N.S. W. (Australia)
and Industrial Chemistry,
University of New South Wales,
(Received February 8, 1984; accepted in revised form June 1, 1984)
Summary This paper examines the link between flux decline and protein which becomes deposited (bound) onto the membrane in protein ultrafiltration. For the conditions studied deposition kinetics were relatively slow, with the rate dependent on feed concentration but the “plateau” (steady-state) amount insensitive to this parameter. The amount of deposition was dependent on system hydrodynamics, membrane type and solution environment. Specific resistances of the deposited layer and the labile boundary layer were measured by analysis of unstirred and stirred permeation rates. A semi-empirical relationship, including the deposition kinetics and the deposited layer resistance, gives reasonable prediction of the observed flux decline.
Introduction It is well known that the ultrafiltration (UF) process can suffer from flux decline which may extend for several hours or days before a steady state is achieved. In protein UF the major cause of flux decline has been linked to the irreversible binding of protein to the membrane. Lee and Merson [ 11 and Cheryan and Merin [Z], using electronmicroscopy, observed protein layers of 0.5-1.0 pm thickness following UF. Ingham et al. [3] measured protein binding and referred to it as multilayer adsorption. We have also described bound protein as multilayer adsorption in studies of the UF of proteins in different solution environments with retentive [ 41 and partially permeable membranes [ 51. However, the term adsorption strictly implies an equilibrium process with partitioning of solute between a solution and a surface, and although this is probable in UF there may be the additional influence of convectioninduced deposition. We have therefore adopted the term deposition to describe the combined effects of adsorption and irreversible accumulation of solute on the membrane. Ultrafiltration flux can be considered to be controlled by several hydraulic resistances in series. This filtration analogy has been argued elsewhere [6,7] *Paper presented at IMTEC ‘83, November S-10,
0376-7388/84/$03.00
o 1984
1983,
Sydney, Australia.
Elsevier Science Publishers B.V.
270
and it provides
J = (l/A,)dV/dt
a useful basis for our analysis. = AP/v(R,
+ Rd + Rb)
Thus, (1)
where R, = membrane resistance, R,j = deposited solute resistance, and Rb = (labile) boundary layer resistance. Assuming that the solute resistances can be characterised by mean “specific resistances” we can write Rd = adi&
(2)
and Rb = CY),i&,
(3)
where Md is the mass of deposited solute per unit area of membrane, and we assume deposition to be a kinetic process, Md = Md [t] . Mb is the mass of solute held in the labile boundary layer. Thus eqn. (1) becomes
J = AP/q(R,
+ “dMd [t]
+ &bMb)
(4)
Equation (4) accounts for flux decline by incorporating the deposition kinetorder to test eqn. (4) it is necessary to have eStimk?S of Cld, CYband Mb. These parameters have been measured independently using modified filtration theory. In separate deposition studies the kinetics and nature of the deposited solute have been observed. This paper evaluates and discusses the parameters in eqn. (4) and compares measured and predicted fluxes. iCS. In
Experimental Experiments were performed in batch cell and cross-flow ultrafiltration systems. The batch cell had a capacity of 110 ml and a membrane area of 15 cm’. The cross-flow system had two flat plate modules in parallel, each of area 30 cm2 (10 X 3 cm) and 0.4 cm channel height. Operating conditions of 100 kPa and 25°C were used in both systems. For stirred ultrafiltration in the batch cell, the stirring speed was 400 rpm and for the cross-flow system the flow rates were 2 and 8 l-min-l (equivalent to Reynolds numbers of 2200 and 8800, respectively). The macrosolute used was bovine serum albumin (BSA), obtained from Calbiochem. Grade “A” (>99% electrophoretically pure) was used in the batch cell experiments, and fraction V BSA (>97% pure) was used in the cross-flow experiments. Comparative tests in the stirred batch cell produced similar results for the two grades of BSA. Solutions of BSA were prepared over a range of pH conditions, with and without 0.2 M NaCl. Most experiments used 0.1 wt. % BSA, but some were at 1.0 and 2.0%. Amicon PM30 membranes, which are polysulphone with a nominal molecular weight cut-off of 30,000 D, were used for the majority of experiments. Comparative deposition tests were also made on other membranes, the Amicon XMlOO (Dynel)
271
and YM30 (regenerated cellulose), DDS GR61P (polysulphone) and Millipore PTGC (polysulphone). Ultrafiltration runs in the cross-flow system were all 5 hours duration. Flux histories and protein deposition were measured. At the end of each run the membrane was carefully removed and then rinsed with distilled water prior to protein determination. Rinsing with saline solutjon instead of distilled water did not produce any difference in the results. Protein was removed from the membrane with sodium dodecyl sulphate and then measured by the modified Lowry method [8] with the spectrophotometer set at 750 nm. Details of the technique may be found elsewhere [ 91. Long-term runs were also performed in the stirred batch cell, in which case the solvent was continuously replenished from a feed reservoir connected via an in-line filter (Millipore 0.2 pm). Protein deposition was measured at the end of each run. A series of runs, of duration 0.3 to 8 hr for solutions of 0.1, 1.0 and 2.0 wt. % BSA at pH 5 provided a direct measure of deposition kinetics. The batch cell was also used to estimate the specific resistance of the deposited solute by unstirred experiments. Specific resistances were obtained by analysis of the time, t, and permeate volume, V, data. Equation (1) may be rewritten as
&‘A,)dV/dt
= AP/q(R,
+ RP)
(5)
where R, is the “polarised” solute resistance. For unstirred conditions over the relatively short duration (<0.2 hr) we neglect solute diffusion and apply conventional filtration theory, where R,
= ~,(VCB/&)
Combining equation
eqns. (5) and (6) and integrating
(6)
gives the well-known
filtration
(7) A linear t/V vs. V plot therefore gives ayp from the slope. This specific resistance represents the mean effect of gradually accumulating layers of solute in the absence of stirring. We have assumed that ap gives an approximation to the specific resistance of the bound (deposited) protein obtained in stirred ultrafiltration, i.e., ad s? ap. Unstirred tests were performed at pH 2 (or 3), 5 and 10 with and without 0.2 M NaCl on BSA “A” and “V”. The solute in the labile boundary layer in stirred ultrafiltration will have a concentration gradient and its effective specific resistance will be less than lyp The values for Qb have therefore been taken from the averaged slopes of t/V vs. V plots for the initial polarisation period under stirred conditions in the batch cell, as explained below. For these measurements the permeate was recorded at 1.0~second intervals by an automatic balance.
272
Results Deposited (bound) protein Deposition kinetics under ultrafiltration conditions with a PM30 membrane are shown in Fig. 1. The deposition process was rather protracted but a plateau or equilibrium value was approached. These data can be expressed by a simple relationship if it is assumed that the rate of deposition is governed by a deposition potential, i.e., dM,-J/dt =
K(d!@-
(8)
it&j)
where M$ is the plateau i&
= M$[l
deposition
2%
(9) 0
BSA .
0.1%
;: I 11 -t1
flH
BS/
__-.
-2 .
/
1/ /
: I 9
’
He
/’
/ ‘*
/I 0
-,--.A--_--
--======z-_--
/
‘0 _ ‘1 I I 11
I
So,
- eXp(-Kt)]
_BSA ,_,-2 ’ /’ // /“I, 1%
:
and K is the rate constant.
0
1 2
I 4 T
I M E
I 6
I 8
(hl
Fig. 1. Deposition 400 rpm).
kinetics during BSA ultrafiltration (stirred cell, PM30, 100 kPa,
The deposition
curves were fitted
by the following
0.1% BSA:
M$ = 65 I.lg-cmU2,
K = 0.3 hr-l
1.0% BSA:
M$ = 87 pg-cm-2,
K = 1.2 hr-l
2.0% BSA:
&I$ = 87 pg-cmb2,
K = 1.8 hr-’
values of M$ and K:
The effect of pH and membrane type on the amount of deposited protein is given in Fig. 2, which shows the enhanced deposition around the isoelectric point of the BSA (pH z 4.7). Maximum deposition was obtained with the par-
273
tially permeable XMlOO for which the measured protein may have included a component of internally bound, but removable, solute. Figure 3 also shows the effect of pH, as well as the influence of NaCl concentration and crossflow velocity. The effect of salt on protein deposition is similar to that reported previously [ 41.
50 GO 30 20 10 5
01
0
I
I
I
1
I
2
L
6
8
10
PH Fig. 2. Deposition vs. pH; 3 hr UF in stirred cell (0.1% BSA; 1 - XMlOO, 2 - PM30, 3 DDS GRGlP. 4 ~ PTGC, 5 - YM30).
200 /I
.
0
0
I
2
I
4
I
6
I
8
I
10
PH Fig. 3. Deposition vs. pH, 5 hr UF in cross-flow system (0.1% BSA, PM30; H = 1.1 m-see-’ cross-flow, L = 0.3 m-se&, S = 0.2 M NaCl, NS = no salt).
274
Resistances Plots of t/V vs. V for unstirred ultrafiltration are given in Fig. 4. The specific resistance al, is directly proportional to the slopes, and Table 1 presents the measured data which show that the largest value of crp was at pH 5 (no salt) and the lowest at pH 10. The table also shows there was little difference between BSA “A” and “V”. Literature data are comparable with this work. 150
120
> .-.. + 60
5
10
PERMEATE Fig. 4. Determination PM30,lOO
of specific
VOL
15 (ml1
resistance
of BSA from
unstirred
UF data (0.1%
BSA,
kPa).
From the measured specific resistances and the measured amounts of deposited protein, eqn. (2) can be used to calculate Rd values. These values are plotted in Figs. 5 and 6 for the stirred cell and the flow system, respectively. In the absence of salt, Rd has a maximum at pH 5, which mirrors the reported flux vs. pH values [4], With salt present, the maximum is either reduced substantially (Fig. 6) or eliminated (Fig. 5). This also agrees with the loss of the minima in the flux vs. pH plot [ 41. The effective specific resistance in the labile boundary layer, olb, was estimated from the t/V vs. V plots for initial polarisation. Figure 7 shows how
275 TABLE 1 Specific resistances of ESA (0.1 wt. %, 100 kPa) BSA conditions
Specific resistance, +
(m/kg
x
10W’5)
PH
NaCl (M)
PM30 (BSA “V”)
PM30 (BSA “A”)
2 2 5 5 10 10
0.2 0.0 0.2 0.0 0.2 0.0
4.2a 1.4 = 3.2 6.4 2.9 2.0
4.4 3.0 2.9 5.2 2.0 1.7
Literature
2.7 b 2.3 c, 4-5 d
’ pH 3.0. bPM30, pH 4.7 (acetate buffer) [7]. c PM30, pH 7.4 (0.13 M NaCl + phosphate buffer) [7]. dPMIO; PH 7 (015 M phosphate buffer), 0.2- 2.0% BSA [lo].
IO Fig. 5
4
6
PH
8
IO
Fig.
6
i 8
4
6
8
10
PH
Fig. 5. Estimated resistance of deposited layer, Rd, vs. pH (0.1% BSA, stirred cell, PM30, 100 kPa, 8 hr run; S = 0.2 M NaCl, NS = no salt). Fig. 6. Estimated resistance of deposited layer, Rd, vs. pH (0.1% BSA, cross-flow system, PM30, 100 kPa, 5 hr run; H = 1.1 m-set-I, L = 0.2 m-set-’ ).
the average slope of the t/V plot was obtained. The amount of Protein Present in this boundary layer, Mb, was estimated from the MnOUllt Of prOkiIl convected to the membrane in the initial polarisation volume, vb, The bound ary layer resistances, h!b, calculated from eqn. (3), are plotted in Fig. 8. The dependence of Rb on pH and salts is similar to that found for the deposited layer resistance.
276
01
0
I
I
I
1
2
3
PERMEATE
VOL
(ml1
Fig. 7. Determination of specific resistance for boundary layer from initial polarisation in stirred cell LJF (0.1% BSA, PM30).
I I I ' 2----i
I
I
6
I
I,
I
8
10
PH Fig. 8. Boundary layer resistance, Rb, values for stirred cell.
VS.
pH. Comparison of calculated and experimental
Instead of using separate estimates of CXband Mb to calculate Rb it is possible to obtain Rb directly from the initial ultrafiltration flux, i.e. Rb = (AP/qJi) - R,
(10)
Initial flux, Ji, may be obtained from correlations or experimentally following polarisation, and before substantial deposition. The dotted curves in Fig. 8 show Rb values obtained from eqn. (10) using the experimental Ji data Equations (3) and (10) yield very similar results. This supports our use of the filtration analogy in which the components of resistance are determined from specific resistances and mass deposition.
Discussion The amount of deposited protein reported here provides further evidence that monolayer adsorption is significantly exceeded under ultrafiltration conditions. The measured deposition kinetics show that it is a relatively slow process, and this agrees with earlier work of Dejmek et al. [ll], who followed the build-up of tagged proteins. Deposition rate constants, K, increased significantly with bulk protein concentration, Cg, but the plateau values, M*,, were notably insensitive to concentration. The initial rates, (dMd/dt)i, obtained from eqn. (8) with Md = 0 and fitted values of M$ and K, were directly proportional to the initial convec tive solute flux, JiCB, as shown in Fig. 9. Attempts to correlate (d.Md/dt)i with the estimated solute concentration at the membrane surface, Cw (= CB exp (J/k,)), were not successful.
J, Cs (yg/sJ Fig, 9. Initial deposition rate vs. initial filtration rate.
From this evidence we conclude that deposition under UF conditions is not analogous to a Langmuir-type equilibrium process, as suggested by Howell et al. [ 121. Rather, the initial rate of deposition is strongly linked to the initial rate of accumulation of solute molecules at the membrane surface. However, only a small fraction of the arriving molecules become instantaneously bound, and this fraction will probably depend on the nature of the membrane surface - as discussed below - and the conformation of the macro. molecule. Subsequent deposition is presumably linked to the filtration process in the sense that it relies on the accumulation of solute in the polarised
278
layer. Indeed, if the deposition kinetics of Fig. 1 are plotted according to conventional filtration as Md versus t’/” the data at 0.1 wt. % BSA are linearised. However, this approach does not work for the data at 1.0 and 2.0 wt. % because at these conditions a deposition “plateau” is rapidly achieved. It is more appropriate to consider the deposition process as starting with the initial accumulation of polarised solute which then provides a “reservoir” of molecules which gradually become fixed due to slow aggregation and flocculation; we have developed this concept elsewhere [ 91. The above picture of the deposition process is consistent with the observation that the “plateau” deposition is relatively insensitive to feed concentration, Cg, but is dependent on system hydrodynamics (Fig. 3). This suggests the deposition of protein ceases when the yield stress of the topmost layers of the aggregated solute is less than the local shear stress due to cross-flow or stirring. The strong influence of pH shown in Fig. 2 is expected since pH affects protein charge, stability, and tendency to aggregate. Figure 2 also shows the effect of membrane type on deposition. The results cannot be related simply to the membrane material, since the three polysulphone membranes (PM30, GR61P and PTGC) gave noticeably different data. A more plausible explanation is based on differences in the surface porosity and pore size distributions of the membranes. Available information [ 7,9,13,14] summarised in Table 2, shows that the surface heterogeneity, in terms of porosity and mean pore size of the membranes may be ranked XM300 > PM30 > PTGC > GR61P > YM30. This ranking is similar to the observed tendencies shown in Fig. 2 to accumulate deposited protein. The more heterogeneous the membrane surface the higher will be the local velocities normal to the surface. Figure 9 shows how the initial deposition rate increases with initial filtration rate for one type of membrane, and if this effect applies on a local scale around individual pores it would give more rapid initial deposition for the more heterogeneous membranes. The course of the subsequent deposition would presumably depend on the extent and nature of TABLE 2 Reported data on membrane surface characteristics Membrane type
XM300 PM30 PTGC GR61P YM30
Data obtained by EM
Ref.
Ranking
13 9 14 9 9,7
heterogeneous
Pore diam. Surface porosity (nm) (%I 24.5 12 6 a =
2%
a, -50%
b
a Pores not detectable by EM. b Order of magnitude inferred by Ref. [7 1.
I homogeneous
279 this initial deposition. In other studies differences in membrane surface micro Porosity have been used to explain variations in ultrafiltration flux [13] and permeabilities of BSA layers [ 71. The variations of the specific resistances with pH and salt content given in Table1 can be explained by the effect of these conditions on the Size and charge of the BSA molecule. We have discussed these interactions in terms of flux variations elsewhere [4]. In brief, the BSA molecule tit the isoionic point of circa pH 5 (no salt) is at its most compact and carries no net charge; this should provide the least permeable BSA layers and highest crP (as observed). Away from the isoionic pH, and in the absence of salt, the molecules expand and have significant net charge and should form permeable layers with lower orP, as observed. Addition of salt at these pH extremes will shield charges, causing molecular contraction and an increase in eP. At pH 5 the effect would be different since the addition of salt causes anion binding, enlargement of the molecule and acquisition of charge, with an expected decrease in c+, as observed. The separately determined vahres of the resistance p~~eterS (Rm, old, q,, Md and Mb) have been combined via eqn. (4) to give predicted fluxes. Figure 10 compares the final flux for various pH, salt concentrations, membrane types, ultrafiltration times, stirred and cross-flow operation, with the calculated values. The agreement is reasonably good; linear regression gives a slope of 0.95, an intercept of about 5 and a regression coefficient of 0.95; Flux declines can also be estimated from eqns. (4) and (g), i.e.,
(11)
EXPERIMENTAL FLUX lllm*-h) Pig. 10. Comparison of final UF flux (O.l-2.25% PM30,
PTGC,
DDS; pH 2-10,
time 0.2-8
hr).
BSA, stirred cell and cross-flow
system;
280
The most testing comparison is between the experimental data for the crossflow system and the calculated values based on the kinetic constants obtained using the stirred cell. Figures lla, b and c, for pH 3, 5 and 10, respectively, show that the model reasonably predicts the observed trends. Typically, eqn. (11) predicts within ?15%. In many cases the discrepancy lies more in the prediction of initial flux than in the estimation of flux decline. Apart from confirming the link between protein deposition and flux decline these results suggest that eqn. (11) can be used as a semi-empirical model of the fouling process. The equation requires as input the resistance parameters and the kinetic constants. These can be obtained relatively simply in stirred batch cell apparatus and applied to larger scale cross-flow systems. In addition, attempts to reduce fouling by lowering M$ and/or K can be evaluated in the stirred batch cell with good prospects of translation to larger scale. This is the current thrust of our studies. 300 PHI
Salt -.-
No - --
0 0
a
I
I
I
I
1
2
3
4
TIME
model
(hl PH5
Salt -*0
TIME
Salt
-o- expt
(hl
No Salt -oexp ----model
5
281
pH
Salt
-.-.-
n
c
“0
I 1
I 2 TIME
10 NO
Salt
-Expt ----
I 4
I 3
Model
I 5
(h1
Fig. 11. Predicted and experimental flux declines in the cross-flow system (0.1% BSA, PM30, 0.3 m-set-‘); (a) pH 3, (b) pH 5, (c) pH 10.
Conclusions Deposition or binding of protein to membranes under ultrafiltration conditions is multilayer. The bulk concentration influences the rate of deposition, but has little influence on the ultimate “plateau” value of mass deposited, which is more dependent on system hydrodynamics, membrane type and solu. tion pH and salt content. Ultrafiltration flux can be predicted by a semi-empirical model based on membrane, deposited layer and boundary layer resistances in series. Flux decline is accounted for by a time-dependent deposited layer resistance obtained from the deposition kinetics and the measured specific resistance of the solute. Acknowledgements The authors would like to thank the Australian Research Grants Committee for financial assistance_ One of us (A.S.) would also like to thank the Agricultural University of Malaysia for support. fist of symbols membrane area (m’) f&~,$7 solute concentration in bulk, at wall (-, or kg-mm3) Am
282
J K k,
M AP
R t V a rl
flux (1-m’ -hr- ’ or m-set- ’ ) deposition rate constant (hr-’ ) solute mass transfer coefficient (m-set-’ ) deposited mass per unit membrane area (pg-cmA2) pressure (kPa) hydraulic resistance (m-l ) time (set or hr) volume (ml, m”) specific resistance (m-kg-’ ) viscosity (N-set-m-2)
Subscripts b boundary layer d deposited layer polarised layer P References 1
D.N. Lee and R.L. Merson, Examination of cottage cheese whey protein by scanning electron microscopy: Relationship to membrane fouling during ultrafiltration, J. Dairy Sci., 58 (10) (1975) 1423. 2 M. Cheryan and U. Merin, A study of the fouling phenomena during ultrafiltration of cottage cheese whey, in: A.R. Cooper(Ed.), Ultrafiltration Membranes and Applications, Plenum Press, New York, 1980, p, 619. 3 K.C. Ingham, T.F. Busby, Y. Sahlestrom and F. Castino, Separation of macro molecules by ultrafiltration: Influence of protein adsorption, protein-protein interaction and concentration polarisation, in: A.R. Cooper (Ed.). Ultrafiltration Membranes and Applications, Plenum Press, New York, 1980, p. 141. 4 A.G. Fane, C.J.D. Fell and A. Suki, The effect of pH and ionic environment on the ultrafiltration of protein solutions with retentive membranes, J. Membrane Sci., 16 (1983) 195-210. 5 A.G. Fane, C.J.D. Fell and A.G. Waters, Ultrafiltration of protein solutions through partially permeable membranes - The effect of adsorption and solution environment, J. Membrane Sci., 16 (1983) 211-224. 6 A.G. Fane, C.J.D. Fell and M.T. Nor, Ultrafiltration/activated sludge systems Development of a predictive model, in: A.R. Cooper (Ed.), Ultrafiltration Membranes and Applications, Plenum Press, New York, 1980, pp. 631-658. 7 H. Reihanian, CR. Robertson and A.S. Michaels, Mechanism of polarisation and fouling of ultrafiltration membrane by proteins, J. Membrane Sci., 16 (1983) 237-258. assay for both water8 H.H. Hess, M.B. Lees and J.E. Derr, A linear Lowry-Folin soluble and sodium dodecyl sulfate-solubilized proteins, Anal. Biochem., 85 (1978) 295-300. 9 A. Suki, Factors affecting flux in protein ultrafiltration, Ph.D. Thesis, University of New South Wales, 1984. 10 M.W. Chudacek and A.G. Fane, The dynamics of polarisation of unstirred and stirred ultrafiltration, J. Membrane Sci., 21 (1984) 145-160. 11 P. Dejmek, B. Hallstrom, A. Kilma and L. Winge, A method for measuring membrane fouling in ultrafiltration, Lebensm.-Wiss. Technol., 6 (1) (1973) 26-29. 12 J.A. Howell, D. Velicangil, M.S. Le and A.L. Herrera Zeppelin, Ultrafiltration of
283
13
14
protein solution: A theoretical model, Proc. Symp. on Membrane Processes, Manchester, 12 Nov. 1980, Inst. Chem. Eng., North Western Branch, 1980, Papers No. 4, p. 5.1. A.G. Fane, C.J.D. Fell and A.G. Waters, The relationship between membrane surface pore characteristics and flux for ultrafiltration membranes, J. Membrane Sci., 9 (1981) 245-262. U. Merin and M. Cheryan, Ultrastructure of the surface of a polysulphone ultrafiltration membrane, J. Appl. Polym. Sci., 25 (1980) 2139-2142.