The effects of electrolyte concentration and pH on protein aggregation and deposition: critical flux...

Journal of Membrane Science 185 (2001) 177–192

The effects of electrolyte concentration and pH onprotein aggregation and deposition:

critical flux and constant flux membrane filtration

R. Chan, V. Chen∗UNESCO Centre for Membrane Science and Technology, School of Chemical Engineering and Industrial Chemistry,

The University of New South Wales, Sydney, NSW 2052, Australia

Received 4 May 2000; received in revised form 23 October 2000; accepted 25 October 2000

Abstract

The performance of membranes used in the separation of solution mixtures containing proteins can be substantially limitedby aggregation and deposition inside membrane pores and on their surfaces. In this study flux, stepping and constant fluxoperation have been used in conjunction with varying electrolyte concentrations and pHs in the microfiltration of aqueoussolutions of bovine serum albumin. Experiments were performed to investigate the hydrodynamic and solution conditionsduring the incipient fouling stage which lead up to eventual cake formation. Results from flux stepping showed that an increasein wall concentration coincides with the onset of an apparent critical flux. This is followed by a time lag before an increasein observed rejection is exhibited. Sub-critical constant flux operation showed that there exists an aggregation and depositiontime lag after which the membrane suddenly experiences a rapid increase in hydraulic resistance due to protein aggregatesblocking a majority of membrane pores. These incipient fouling conditions were shown to be dependent on pH and ionicstrength and were concluded to be the product of a balance between electrostatic, solubility and hydrophobic effects whichwere manifested in protein–protein and protein–membrane interactions. © 2001 Elsevier Science B.V. All rights reserved.

Keywords:Protein fouling; Microfiltration; Critical flux

1. Introduction

In the membrane filtration of solutions consistingof proteins as at least one component, fouling phe-nomena such as pore narrowing and pore blockingcan substantially hinder the desired transmission ofone or more components. Transmission is impor-tant for many microfiltration processes, includingthe separation of plasma proteins from blood cellsand the removal of bacteria or yeast cells from

∗ Corresponding author. Tel.:+61-2-9385-4813;fax: +61-2-9385-5966.E-mail address:[email protected] (V. Chen).

broth mixtures containing proteins [1]. Mechanismsthat have been suggested for protein fouling dur-ing microfiltration include pore narrowing by nativeprotein molecules or aggregates adsorbing to porewalls and pore blocking by protein aggregates, ora sequential combination of these [2,3]. The for-mer suggests an internal fouling phenomena whilstthe latter is more suggestive to surface fouling. Ag-gregate formation is affected by pumping, temper-ature, crossflow (shear), stirring and pressure [4–8]as well as by electrostatic forces and hydrophobicinteractions [9]. Kelly and Zydney [5] suggested afouling mechanism that involved initial aggregatedeposition onto membrane surfaces followed by the

0376-7388/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0376-7388(00)00645-1

178 R. Chan, V. Chen / Journal of Membrane Science 185 (2001) 177–192

chemical attachment of native protein molecules ontothese aggregates via a thiol–disulfide interchangereaction.

Certain solution conditions, such as the pH andionic strength can either minimise or exacerbate pro-tein aggregation and fouling in membrane filtration[10–14]. Kelly and Zydney found that the rate ofBSA aggregation during protein storage increasedwith increasing pH [9]. They suggested that increasedionisation of the free thiol group at higher pH wasresponsible for the aggregation (thiol–disulfide in-terchange) reaction. However, the presence of saltscan alter electrostatic protein–protein interactions andproduce the effect of shielding charge, and hence, adampening out of intermolecular protein interactions.At pHs above and below the IEP of BSA, it has beenshown that this shielding effect can promote proteinagglomeration and produce thick, compact proteindeposits that result in decreased membrane permeabil-ities and lower fluxes [10–14]. Fane et al. [10] showedthat a transient increase in flux occurred at the IEPwhen salt was added in the ultrafiltration of BSA dueto intramolecular charge repulsion caused by anionbinding. Further addition of salt dampened out thesecharge effects and eventually reduced permeability.Although the presence of salts can lead to the damp-ening of electrostatic charge, it also increases the sol-ubility of the protein [15,16]. Ions are known to bindelectrostatically to charged groups on a protein macro-molecule at low salt concentrations, carrying hydra-tion water with them into the vicinity of the protein[17], causing a “salting-in” effect. Such an increasein solubility may have the effect of initially reducingprotein fouling.

In addition to that induced by the presence of salts,aggregation can also be promoted via hydrophobiceffects. At the IEP of a protein when electrostatic in-teraction is minimal, aggregation is likely to occur byhydrophobic attraction between exposed hydrophobicpatches on protein surfaces. Shear denaturation canexpose such hydrophobic groups, further enhancingaggregation [4]. Salt ions are also known to competewith protein species for hydration water, effectively“pulling” water molecules away from the protein andexposing additional hydrophobic groups [15,18].

Apart from solution conditions, controlling hy-drodynamic conditions can have a strong bearingon the severity of protein fouling. In constant pres-

sure mode filtration, flux, the dependent variable,tends to produce constantly changing conditions inthe boundary layer due to the uncontrolled convec-tion of solute toward the membrane surface [19].When constant flux operation is employed solutetransport is better controlled so that fouling can bemonitored more accurately through transmembranepressure (TMP). Constant flux operation also al-lows for filtration to take place initially at a lowflux followed by slow and controlled flux increasein increments of constant flux. Such operation al-lows for the identification and gradual approach tothe fouling threshold often referred to as the “criti-cal flux”, or the flux below which there is negligibledeposition on the membrane [20]. Below the criticalflux the associated TMP is low, independent of timeand increases linearly with the imposed flux. Abovethe critical flux, the TMP rises rapidly with the im-posed flux and is dependent of time. In previouswork involving the membrane filtration of proteinssuch as BSA, flux steps have typically been keptconstant for periods of 30 min or less [21–23]. Whileit is known that factors such as crossflow velocity,particle size, pore size, pH, bulk concentration andionic strength can affect the location of the criticalflux [21–25], it is not known how long an imposedsub-critical flux can maintain a stable TMP. If negli-gible fouling takes place below the critical flux thenthe TMP should be stable and independent of timefor an indefinite duration. This facilitates the needto examine constant flux operation for longer timeperiods.

The primary objective of this investigation wasto study the solution and hydrodynamic conditionswhich precede cake-layer fouling in the membranefiltration of proteins. Flux-stepping was employedfor the identification of the critical flux in the micro-filtration of aqueous BSA solutions at different pHsand ionic strengths. This was complemented by con-ducting sub-critical constant flux experiments in or-der to verify the critical flux. Previous studies haveconcentrated on observing flux and rejection charac-teristics under constant pressure operation and underrelatively heavy fouling conditions [10,11,13,14].Hence, not much is known about the precursorsto cake fouling, especially the combined effectsof pH, added salts and the imposed hydrodynamicconditions.

R. Chan, V. Chen / Journal of Membrane Science 185 (2001) 177–192 179

2. Methods and materials

2.1. Materials

The microfiltration membranes used were 0.2mmpolycarbonate track-etched membranes from Poret-ics and 0.22mm PVDF membranes (GVWP fromMillipore, Australia). A new membrane strip wasused for each experiment. Bovine serum albu-min (Fraction V) was purchased from CalbiochemAustralia, weighed out, and dissolved in Milli-QTM

deionised water and made up to solutions of 0.1 wt.%(1.0 g/l). The pH was adjusted by dropwise addi-tion of 0.1 M solutions of HCl and/or NaOH, whilethe desired ionic strength was obtained by weighingout appropriate amounts of laboratory grade solidsodium chloride crystals and dissolving in the BSAsolution. All solutions were prepared less than 2 hprior to the commencement of each experimentalrun.

Fig. 1. Diagram of the crossflow rig.

2.2. Apparatus

All experiments were performed using a non-commercial experimental prototype perspex crossflowmodule of channel dimensions 2 mm high, 25 mmwide and 210 mm long. A feed pressure of 50 kPa wasobtained via the use of a peristaltic feed pump (Mas-ter Flex Model 7529-00 by Cole Parmer) and adjustedby using a ball valve situated at the module outlet. Asmaller peristaltic pump (Master Flex by Cole ParmerModel 7518-00) situated on the permeate line wasused to control the flux and to perform flux-steppingwhen required. A personal computer was used to logdata from the following: two pressure transducers(PDCR 800 by Druck) — one at the roof of the cross-flow module and the other on the permeate line, a flowtransducer, a type-T thermocouple located upstreamof the module, and a digital balance which measuredflux by timed collection of the permeate. Fig. 1 givesan illustration of the crossflow rig. A UV–VIS spec-


trophotometer set at a wavelength of 280 nm was usedto measure the absorbance of permeate and retentatesamples. Electron micrograph images of membranesamples were obtained from a field emission scanningelectron microscope (S-900 by Hitachi). The presenceof protein aggregates was determined from laser lightscattering analysis via a Zeta Plus — Zeta PotentialAnalyser by Brookhaven Instruments Corporation.

2.3. Experimental protocol

A membrane strip of the appropriate dimensionswas cut out from flat a sheet and inserted into thecrossflow module. A pure water flux was obtainedby running the apparatus under constant pressureconditions (without permeate pump operation) at50 kPa feed pressure using Milli-QTM water. Solu-tions were prepared during this time to the desiredBSA concentration, ionic strength and pH. Thiswas followed by a membrane pre-adsorption pe-riod which involved removing the membrane stripfrom its module and immersing it in the preparedsolution for a period of 1 h. Flux-stepping experi-ments were performed by setting the permeate pump

Table 1Summary of results from flux-stepping experiments using 0.1 wt.% solutions of BSA at varying salt (NaCl) concentrations

Membrane pH Salt concentration(mol/l NaCl)

Apparent criticalflux (l/m2 h)

Remarks

0.2mm track-etched 3.0 0 220 Rate of TMP increase similar for no NaCl and0.017 M, but greater than that of 0.1 M NaCl0.02 220

0.1 220

4.8 0 170 Rate of TMP greater at lowersalt concentration0.02 170

0.1 170

9.0 0 220 Rate of TMP increase greatest at 0.017 M, lowerat 0.1 M and lowest in the absence of salt0.02 220

0.1 220

0.22 mm GVWP (PVDF) 3.7 0 500 Rate of TMP increase decreased with saltconcentration0.05 500

0.15 500

4.8 0 470 Rate of TMP increase decreased with saltconcentration. At 0.15 M NaCl TMP increaseremained very low throughout experiment

0.05 4700.15 470

8.4 0 470 Rate of TMP increase decreased with saltconcentration0.05 470

0.15 470

to deliver a slow flowrate initially, and manuallyincreasing this flow in constant flux incrementseach of 10 or 15 min duration. Constant flux ex-periments were conducted by setting the permeatepump at a constant flowrate for the entire durationof the experiment. Samples from the permeate andretentate were withdrawn manually for analysis by theUV spectrophotometer.

3. Results and discussion

3.1. Effect of ionic strength and pH on the locationof the apparent critical flux

With the 0.2mm track-etched membrane, the crit-ical flux was determined for pH values 3.0, 4.8 (cor-responding to the IEP of BSA) and 9.0. At each pH,three ionic strength conditions were examined: noNaCl, 0.02 M NaCl and 0.1 M NaCl. Flux steppingwas used to determine the apparent critical flux ineach case. Table 1 summarises these results. At pH3.0, the apparent critical flux was shown to be approx-imately 220 l/m2 h for all three ionic strengths. The


Fig. 2. Flux stepping using the 0.2mm track-etched membrane at pH 3.0.

rates of TMP increase in the absence of NaCl and at0.02 M NaCl were both similar, but much greater thanat 0.1 M NaCl (Figs. 2 and 3). This seems to reflect thehigher solubility of the protein at higher salt concen-tration. It is evident that the salt concentration affectsthe TMP but not the location of the apparent criticalflux at pH 3.0. The apparent critical flux was foundto be 170 l/m2 h at pH 4.8 for all salt concentrations,considerably lower than at pH 3.0. This is reasonable,as it is known that at the IEP minimal electrostatic


charge interaction can cause hydrophobic patches onprotein surfaces to attract, causing protein associationwith the increased possibility of protein aggregation[16]. Such an effect can serve to faster reduce perme-ability. The rate of TMP increase was again found tobe greater, although not as pronounced, when less saltwas present. At pH 9.0, the apparent critical flux at allionic strength conditions was found to be 220 l/m2 h.However, in contrast to the other two pHs, the rateof TMP increase was shown to be greater in the



presence of salt, being greatest at 0.02 M NaCl, lowerat 0.1 M NaCl and lowest in the absence of salt(Fig. 4). It appears that a concentration of 0.02 MNaCl is sufficient to just dampen out any electrostaticinteraction while a higher concentration of 0.1 M NaClresults in solubility effects, causing a lower TMP. Inthe absence of salt, both BSA and the membrane sur-face is negatively charged and electrostatic repulsionwill ensue, resulting in the lowest TMP. Therefore,under alkaline conditions charge interaction effectedby the pH seems to be responsible for the locationof the apparent critical flux, but solubility considera-tions seem to be responsible for the rapidity of TMPincrease.

The increase in solubility due to the presence ofa significant sodium chloride concentration can beattributed to salt ions electrostatically binding to theprotein molecules. As mentioned in the literature,macromolecules at low salt concentrations preferablybind ions [17]. Given that the high polarisability ofwater molecules results in strong interactions withions [26], at concentrations such as 0.1 M NaCl, ionsmust carry a significant amount of hydration waterinto the vicinity of the protein thus increasing itssolubility. This is known as “salting-in” of the protein.

Although only applicable to high salt concentration(∼1.0 M), the presence of salt can act as a stabilis-ing species. Arakawa and Timasheff [27] determined

the apparent binding,ν, of salt to protein for BSAat a salt concentration of 1 M NaCl and pH 5.6 tobe−16.8 mol/mol protein. The binding parameter,ν,describes preferential binding, or the excess bindingof salt ions over water relative to the bulk solventcomposition [28]. The negative number thus indicatesthat there is excess water in the immediate vicinity ofthe protein and that the salt is preferentially excludedfrom this zone. In addition to increasing protein solu-bility, the stabilising property of the salt species makesit thermodynamically unfavourable for the protein toassume a denatured form, therefore keeping it in thenative form and preventing denaturation and possibleaggregation. In the present work, this phenomenonmay contribute to less protein association and hencelower TMPs.

It is also possible that the low TMPs exhibited inthe presence of salts is due to ions dampening out theelectro-viscous effect. Such an effect results when acharged protein deposit unequally partitions cationsand anions creating a potential difference across thedeposit [14]. A greater convective flow of ions oppo-sitely charged to the deposit occurs, but at the sametime producing an electrophoretic flow of counter-ionsin a direction opposite to that of the bulk fluid. Theelectrical stresses produced as a result decrease thenet solvent flow through the deposit. Dampening outof this effect causes an increase in flux under constant


Fig. 5. Flux stepping using the 0.22mm PVDF (GVWP) membrane at pH 4.8.

pressure operation, or alternatively, a decrease in TMPwhere flux is the controlled variable.

With the 0.22mm PVDF membrane at pH 3.7, theapparent critical flux was found to be 500 l/m2 h un-der the ionic strength conditions of no NaCl, 0.05 MNaCl and 0.15 M NaCl. At both pH values of 4.8 and8.4, the apparent critical flux was also the same underall ionic strength conditions, but only slightly lowerat 470 l/m2 h. At each pH, the rate of TMP increasedropped with increasing ionic strength, being greatestfor no NaCl and lowest for 0.15 M NaCl. At pH 4.8,the rate of TMP increase with 0.15 M NaCl remainedlow and almost constant until very high fluxes wereimposed (Fig. 5). Therefore, the solubility effect is

Table 2Summary of results from constant sub-critical flux experiments

Membrane pH Salt concentration(mol/l NaCl)

Time lag for TMPincrease at sub-criticalflux of 65 l/m2 h (min)

Remarks

0.2mm track-etched 4.8 0.001 – Where TMP increase is observed, the rate is lowerfor lower salt concentration. At 0.001 M NaClTMP increase was not observed within theduration of the experiment (120 min)

0.005 900.01 700.1 70

7.0 0.1 – Negligible TMP increase observed in 120 min

9.0 0.1 – Negligible TMP increase observed in 120 min

exceptionally pronounced for this type of membrane,and especially at the protein IEP.

3.2. Effect of ionic strength and pH on constant fluxoperation

Negligible deposition and TMP increase was ex-pected when fluxes were held constant and well be-low the apparent critical flux. For the length of timeover which these experiments were conducted, con-stant flux microfiltration of aqueous BSA solutionsshowed that the TMP remained stable or exhibiteda non-discernible TMP increase in the absence ofsalts. When salt was present the TMP remained stable


Fig. 6. Effect of ionic strength on sub-critical constant flux operation at 65 l/m2 h using 0.2mm track-etched membrane at pH 4.8.

for a considerable duration before the TMP startedto rapidly rise. Table 2 summarises these results.When a constant sub-critical flux of 65 l/m2 h wasimposed using the 0.2mm track etched membrane atpH 4.8 and ionic strength 0.1 M, the time lag beforeTMP increase was observed to be about 60–70 min(Fig. 6). This increase was associated with significantaggregation of the protein as evidenced by electronmicroscope images of the membrane surface. Size-able aggregates were observed at 60 min just prior tothe TMP increase, some of which completely blockedthe membrane pores (Fig. 7a). No evidence of porenarrowing was observed, indicating that pore block-ing resulting from the deposition of large aggregateswas the initial fouling mechanism present. At 90 minmore aggregates deposited on the surface and moreevidence of pore blockage occurred (Fig. 7b). After225 min of constant flux filtration pore narrowing aswell as blockage was observed, and evidence of cakeformation was exhibited (Fig. 7c).

The origin of the aggregates observed in the ini-tial fouling is not certain. Whether they were formedaround protein nucleation sites already deposited onthe membrane, or in situ in solution is not known asresults from laser light scattering were not conclusive.This is because the laser light scattering was per-formed on the bulk solution where the population ofaggregates is much lower relative to that directly adja-cent to the membrane surface. In a previous study, cir-

culation of the protein feed solution prior to filtrationsuggested that pumping was not a major contributorto the initial aggregation in this system [15]. This ex-periment involved pumping and recirculating the feedsolution around the crossflow rig without permeatewithdrawal for 1 h prior to filtration. Results showedthere to be little difference in the TMP between runswhere pre-filtration circulation was conducted andwhere the usual static pre-adsorption was carried out.The time lag associated was the same in both cases.It is suggested that aggregation accelerates only whenfiltration occurs.

At constant subcritical flux, the rise in TMP at pH4.8 was highest at a concentration of 0.01 M NaCl,slightly lower at 0.1 M NaCl, lower again at 0.005 Mand lowest at 0.001 M NaCl where no significant TMPrise was exhibited (Fig. 6). At 0.01 M NaCl it seemsthat the amount of salt ions present is sufficient to justmask out the charged groups on the protein molecules.While the protein molecule already possesses a “net”zero charge at this pH (the IEP of BSA), the salt ionshave the effect of dampening out localised chargessuch that there are greater hydrophobic forces be-tween the proteins, causing attraction and hence en-hancing the aggregation process. The TMP is lower atthe higher salt concentration of 0.1 M NaCl becausesolubility effects become more dominant.

Ionic strength has a significant effect on the com-mencement of fouling. As the salt concentration is


Fig. 7. Scanning electron microscope image of a constant flux (65 l/m2 h) filtration of 0.1 wt.% BSA+ 0.1 M NaCl, pH 4.8, using 0.2mmtrack-etched membrane taken after (a) 60 min; (b) 90 min ; and (c) 225 min.


Fig. 7. (Continued).

reduced, the aggregation/deposition time lag length-ens. In order to check the stability of the TMP at nosalt concentration, a constant flux filtration at 65 l/m2 hwas carried out in excess of 120 min (Fig. 8). It canbe seen from the results that the TMP remained stable

Fig. 8. Sub-critical constant flux (65 l/m2 h) filtration of 0.1 wt.% BSA (no salt), pH 4.8, using 0.2mm track-etched membrane.

over a filtration period of 225 min. Furthermore, scan-ning electron microscope images showed very littledeposition on the membrane surface (Fig. 9).

The effect of pH on the time lag was examinedusing solutions of ionic strength 0.1 M at higher pH


Fig. 9. Scanning electron microscope image taken after 225 min of a constant flux (65 l/m2 h) filtration of 0.1 wt.% BSA (no salt), pH 4.8,using 0.2mm track-etched membrane.

Fig. 10. Effect of pH on sub-critical constant flux operation at 65 l/m2 h with an ionic strength of 0.1 M NaCl using 0.2mm track-etchedmembrane.


values. A very slight TMP increase was observed atpH 7.0 and 9.0 (Fig. 10), indicating that pH valuesabove the IEP can suppress, or hinder the formationof aggregates. At pH 4.8, a time lag of about 65 minwas exhibited before TMP rise occurred. This is pos-sibly due to the higher pH being further away fromthe protein IEP and causing greater ionisation of neg-atively charged groups which a concentration of 0.1 MNaCl is insufficient to fully mask out. At pH 7.4 BSApossesses a large (and negative) net charge number of−20.4 [29]. Despite charge repulsion being damped,the overall surface charge of the BSA molecules re-main negative, resulting in electrostatic repulsion be-tween proteins and the negatively charged membranesurface thereby reducing aggregation and deposition.

When constant flux operation, using a flux of65 l/m2 h, was imposed using the PVDF membraneat an ionic strength of 0.1 M, pH 4.8, 50 kPa feedpressure, the TMP remained stable below 10 kPa forthe entire 135 min duration of the experiment. Theapparent sustained stability of TMP is likely to bedue to the morphology of this membrane, although itis not known how long this stability can be sustained.As suggested by Ho and Zydney [30], isotropic mem-branes such as the PVDF possess an interconnectedpore structure which allows fluid to flow around de-posited aggregates. The overall effect of this is thatpore blockage would have lesser impact in terms ofhydraulic resistance on this type of membrane thanfor a membrane with straight-through capillary-typepores such as the track-etched membrane.

Fig. 11. Wall concentration and rejection at pH 3.0 and at varying ionic strengths using 0.2mm track-etched membrane.

3.3. Observed rejection and wall concentration influx stepping

In general, for the 0.2mm track-etched membraneit was found that in all of the flux stepping experi-ments (at all pHs and ionic strengths) the observedrejection did not increase until after a considerabledelay following the onset of the apparent critical flux.Low and stable TMP at each imposed flux prior to theapparent critical flux reflected a low calculated wallconcentration based on the osmotic pressure model(see Appendix A). In terms of pH, wall concentra-tions decreased in the following order for a given saltconcentration: pH in the order 4.8 > 3.0 > 9.0. AtpH 3.0, the apparent critical flux was approximately220 l/m2 h for all ionic strengths and this occurredduring the 45–60 min flux step. However, experimen-tal data shows that for all salt concentrations rejectionremained close to 10% until after 90 min (at whichtime the corresponding flux was over 300 l/m2 h).Fig. 11 shows the rise in calculated wall concentra-tion and the stability in observed rejection at fluxesbelow 300 l/m2 h. When the observed rejection beganto increase, it did so more rapidly and at higher levelsupon lowering the salt concentration. The highest ob-served rejection was found to be 65% in the absenceof salt after the 120 min run and lowest at about 12%for 0.1 M NaCl. At ionic strength 0.1 M, the observedrejection remained close to 10% without any notice-able increase. The variation of wall concentrationwith time at pH 3.0 shows steep rises at flux of about


210 l/m2 h for all ionic strengths, corresponding to theoccurrence of the apparent critical flux (220 l/m2 h).The increase in wall concentration with the onset ofthe apparent critical flux was also observed at pH 4.8and 9.0.

Unlike the case at pH 3.0 where rejection remainedessentially constant throughout the 120 min run, atpH 4.8 and ionic strength 0.1 M NaCl, rejection roserapidly after 90 min. However, as for the case at pH3.0, the observed rejection also increased significantlyabout 45 min after the onset of the apparent criticalflux at pH 4.8, a phenomenon which was also observedat pH 9.0. The wall concentrations at pH 9.0 were at alower level compared with the other two acidic pHs.

Since there is no significant increase in rejectionuntil about 45 min after the onset of the apparent crit-ical flux for all three pH values there must exist atime lag before aggregate deposition reaches a surfacecoverage significant enough to effect an increase inrejection. This may consist of continual and increas-ing surface coverage over time in response to the fluxincrease, an increase in the size of aggregates in thecirculating retentate, an increase in the size of aggre-gates already deposited on the surface or a combina-tion of these. It has been suggested that such a time laginvolves the transition from internal pore-narrowingfouling to external cake fouling [2,3]. Thus it is plau-sible for the current study that the lag time involves atransition from a sporadic deposition of aggregates onthe membrane surface to cake layer formation in re-sponse to the flux increase above the apparent critical

Fig. 12. Wall concentration and rejection at pH 4.8 and at varying ionic strengths using 0.2mm track-etched membrane at a constant fluxof 65 l/m2 h.

flux. While pore narrowing may have already occurrednear the apparent critical flux, the balance betweenreduced sieving coefficient and high wall concentra-tion may result in the time delay before a noticeableincrease in observed rejection is observed.

3.4. Observed protein rejection and wallconcentration in constant sub-critical flux operation

When constant sub-critical flux mode wasemployed at about 65 l/m2 h with the track-etchedmembrane at pH 4.8, a salt concentration of 0.01 MNaCl resulted in the highest observed rejection after120 min filtration (Fig. 12). In general, and unlike inthe case of flux-stepping, observed rejection tendedto be higher for higher salt concentration. Examiningthe variation of wall concentration with time, it isfound that up to 65 min and for all salt concentrations,the calculated wall concentration remained close to1.0 g/l (the original feed concentration) before risingsteadily.

At a constant salt concentration of 0.1 M NaCl, ob-served rejection tended to be higher at higher pH. Thedifferences in pH showed greater differences in re-jection profiles than for differences in ionic strengthat any particular pH value. Rejection at pH 9.0 in-creased to about 55%, at pH 7.0 was constant at about34%, and at pH 4.8 increased to 25% at the end of thefiltration period of 135 min.

The observed rejection profile of the GVWP(PVDF) membrane (at pH 4.8 and salt concentration


0.1 M NaCl) remained constant throughout the dura-tion of the experimental run at about 21%. This is dueto the “interconnecting” (isotropic) pore structure ofthe membrane which seems to allow passage of bothsolute and solvent and at the same time not offeringany significant resistance to flow due to depositionor build up of the polarised layer. A previous studynoted that the rejection behaviour of this membraneis more like that of a 0.4mm pore size track-etchedpolycarbonate membrane rather than its 0.22mm rat-ing due to its heterogeneous pore size distribution[22].

In light of the findings from the use of thetrack-etched membrane, it may be deduced that only“apparent” critical fluxes were observed which aredependent on the kinetics of protein aggregation andaggregate deposition, which is in turn dependent onpH and ionic strength. While no noticeable effect isobserved in the short term, in the long term the pres-ence of salt does affect the apparent critical flux. Influx stepping, the wall concentration reaches a signif-icant level as the apparent critical flux is exceeded.This is followed by a time lag before the observed re-jection starts to increase in response to flux increase.In sub-critical constant flux operation, the wall con-centration remains low and close to the feed concen-tration until it suddenly increases due to changes insolute association, involving proteins and salt ions, asopposed to changes in hydrodynamic conditions. Thisincrease occurs simultaneously with the rapid rise inTMP which is associated with aggregate formationand their deposition on the membrane surface. Thissuggests the existence of a critical wall concentrationthat occurs with the onset the apparent critical fluxand a critical aggregation/deposition time lag whichis dictated by interparticle forces and particle aggre-gation. It can be deduced from these observations thatin the microfiltration of proteins like BSA, it may bepossible to maintain a stable TMP below the appar-ent critical flux depending on the solution conditions,but this may not last for an indefinite period of time.The slow aggregation and deposition kinetics willeventually effect rapid deposition and pore block-age. Furthermore, the abrupt changes in flow patternexperienced by protein flowing tangentially acrossthe surface of a low porosity capillary pore mem-brane (such as the track-etched membrane) results ingreater shear denaturation and increased likelihood of

hydrophobic aggregation than would be the case withisotropic membranes such as the PVDF.

4. Conclusions

While many workers such as Fane et al., and Palecekand co-workers [10,13,14] have already studied theeffects of pH and ionic strength on BSA cake filtrationand associated cake permeability, not much is knownabout the pre-cake formation interactions which occurin the incipient fouling stage. The mechanism of foul-ing and the initiation and growth of protein depositshas been studied in some detail [5], but the conditionsleading up to fouling, especially in the presence of saltsand varying pH values, are not well understood. In thecurrent study, there exists a trade-off between elec-trostatic, solubility and hydrophobic effects and theirimplications for protein aggregation and deposition.It has been shown using flux stepping experimentswith the microfiltration track-etched membrane thatthe increase in wall concentration and the onset of theapparent critical flux precede the increase in observedrejection by a finite time lag. With the constant flux ex-periments, the presence of salt exacerbated the aggre-gation process but a considerable time lag was requiredbefore this was shown through TMP increase. Athigher pH and/or less salt concentration this time lagcan be increased and aggregation and deposition canbe hindered, albeit temporarily. In addition, consid-eration must be given to the membrane morphology.While the track-etched microfiltration membraneexhibits these constant flux lag time characteris-tics, isotropic microfiltration membranes such as theGVWP (PVDF) do not seem to be affected, at least notwithin the time duration of the experiments conducted.

Acknowledgements

The authors acknowledge support from the Aus-tralian Research Council and material support fromMillipore.

Appendix A. Calculation of wall concentration

The wall concentration,Cw, was calculated usingthe osmotic pressure model (A.1) and determiningCw


by iteration.

J = 1P − 1π

RM(A.1)

where 1P is the transmembrane pressure,1π theosmotic pressure= f (Cw) (see below) andRM theresistance of the membrane, determined from usingpure water as feed.

pH Valid range ofconcentration (g/l)

Units of C A1 A2 A3

7.4 0–450 g/l 3.787× 10−1 −2.98× 10−3 1.000× 10−5

5.5 0–450 g/l 5.633× 10−2 −2.80× 10−4 2.604× 10−6

4.5 0–450 g/l 7.539× 10−2 −4.90× 10−4 1.852× 10−6

References

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3w (A.2)

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The effects of electrolyte concentration and pH on protein aggregation and deposition: critical flux...

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