Bioseparation9: 7–19, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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Bovine serum albumin-hemoglobin fractionation: significance ofultrafiltration system and feed solution characteristics

Rishi Shukla1, Malini Balakrishnan2 & Gopal P. Agarwal31Agricultural Bioprocess Laboratory, 1302 West Pennsylvania Avenue, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA;2Tata Energy Research Institute, Darbari Seth Block, India Habitat Center,Lodi Road, New Delhi 110003, India;3Department of Biochemical Engineering and Biotechnology, IndianInstitute of Technology, Delhi, Hauz Khas, New Delhi 110016, India

Received 3 February 1999; accepted in revised form 4 November 1999

Key words:bovine serum albumin, hemoglobin, protein fractionation, separation factor, ultrafiltration, vortex flowfilter

Abstract

This work investigates the fractionation of similar molecular weight proteins bovine serum albumin (69 kD) andbovine hemoglobin (67 kD) by ultrafiltration. Three different membranes,viz. regenerated cellulose, poly(sulfone)and surface modified poly(acrylonitrile), each with a nominal molecular cutoff rating of 100 kD, were examined.The experiments were conducted in dead end, crossflow and vortex flow filtration modes and the separation wasstudied as a function of feed pH and ionic strength. Under similar system hydrodynamics, the surface modifiedpoly(acrylonitrile) membrane displayed the highest resolution with minimum membrane fouling. The separationcould be improved further by operating at low applied pressure (40 kPa) and high mass transfer (> 20× 10−6

m/s) in a vortex flow module. Under these conditions, the highest separation factor of 40 was obtained at the pI ofhemoglobin.

Introduction

Downstream processing and recovery of a specificprotein or enzyme from a complex mixture usuallyinvolves membrane based unit operations such as ul-trafiltration (UF), owing to their mild operating condi-tions combined with easy process scalability. In spiteof its advantages, UF is rarely recommended for theseparation of protein mixtures unless the target speciesdisplay a large molecular weight (MW) difference often or above (Cherkasov and Polotsky, 1996). Severalstudies, however, contradict this commonly acceptednorm. Table 1 presents the salient features of se-lective protein filtrations carried out for macrosoluteswith less than 5-fold variation in MW, which effect-ively proves that MW difference is not necessarily thelimiting factor.

It is now well recognized that other than mo-lecular sieving, protein transport is influenced byprotein–protein and protein–membrane interactions,

concentration polarization and membrane fouling. Themultidisciplinary nature of the problem therefore re-quires not only an understanding of membrane surfacechemistry and feed solution characteristics, but alsofluid dynamics, mass transfer, membrane–protein andprotein–protein forces (Mazid, 1988; Koehler et al.,1997). A variety of separation strategies have thusevolved for the fractionation of protein mixtures assummarized in Table 1. These include selecting mem-branes with optimum properties of pore size and hy-drophilicity, changing the protein conformation andshape by adjusting pH and ionic strength, manipulat-ing charge density of the protein and/or the membrane.However, with few exceptions (Nakao et al., 1988;Zhang and Spencer, 1993; Ehsani and Nyström, 1995;Balakrishnan and Agarwal, 1996b; van Reis et al.,1997a) the studies on simulated binary mixtures havelargely been confined to laboratory scale stirred unitswith limited data on modules suitable for large-scaleapplications.

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Table 1. Recent developments in protein fractionation by UF

Proteins MW pI MW Separation parameters Separation factor Reference

(kD) ratio (–)

Cytochrome c 12.4 10.6 5.5 pH, membrane cutoff Over 30 Sudareva et al., (1992)

HSA 68.5 4.9

Lysozyme 14.3 11.0 4.82 pH, ionic strength, membrane – Irtani et al. (1995, 1997);

BSA 69 4.9 modification, gas sparging, protein Millesime et al. 1996;

concentration, ultrasonic irradiation Bellara et al. (1997);

Ghosh and Cui (1998a, 1998b);

Mukai et al. (1998)

Ribonuclease 13.7 9.6 4.7 pore radius, pH – Sudareva et al., (1983)

Hemoglobin 64.5 6.8

Myoglobin 17 7.0 4.05 membrane material, applied Up to 90 Nakatsuka and Michaels (1992);

BSA 69 4.8 pressure, solute ratio, pH, ionic Ehsani and Nyström (1995)

strength

Lysozyme 13.9 10.6 3.12 mass transfer, applied pressure, pH, 3 Balakrishnan & Agarwal 1996b

Ovalbumin 43.5 4.6 ionic strength, protein concentration

BSA 67 4.8 2.3 pH, protein concentration, – Higuchi et al. (1991, 1993);

γ -globulin 160 6.3–8.4 membrane chemistry Najarian and Bellhouse (1996)

HSA 68.5 4.9 2.26 Gas sparging, pH, protein Almost Li and Pepper (1997)

IgG 155 7.0 concentration, ionic strength complete

BSA 69 4.7 2.25 pH, ionic strength, protein 50 Zhang and Spencer (1993);

IgG 155 7.0 concentration Nel et al. (1994)

Saksena and Zydney (1994);

BSA monomer 69 – 2.0 Mass transfer , concentration 32 van Reis et. al (1997a)

BSA dimer 138 polarization, pressure

Cytochrome C 12.4 10 1.3 pH 40 Yang and Tong (1997)

Myoglobin 17 7.0

Lysozyme 13.9 10.6 1.21 Protein concentration 3 Balakrishnan and Agarwal (1996b)

Myoglobin 16.9 6.8

BSA 69 4.8 ∼1.0 Membrane modification 140 Musale and Kulkarni (1997)

Hemoglobin 67 6.8 pH, ionic strength van Eijndhoven et al. (1995)

The present work explores UF for the fractionationof similar MW proteins. Bovine serum albumin (BSA)with a MW of 69,000 daltons and isoelectric point(pI) of 4.8, and bovine hemoglobin (Hb) with a MWof 67,000 daltons and pI of 6.8 were used as modelproteins. This particular separation problem has beenaddressed recently by other groups as well. van Eijnd-hoven et al. (1995) examined BSA-Hb fractionationusing 100 kD commercial modified poly(ethersulfone)membranes (Omega, Pall-Filtron). By adjusting thesolution pH and ionic strength, they claimed sep-

aration factors of more than 70 at pH 7.0 and lowionic strength (0.0023 M). In contrast, Musale andKulkarni, (1997) employed indigenously developedpoly(acrylonitrile-co-acrylamide) membranes with amean pore diameter of 20 nm. Their studies were car-ried out with varying membrane hydrophilicity andsolution pH. The best separation was obtained withthe poly(acrylonitrile) membrane at pH 4 when boththe BSA and Hb are positively charged.

The objective of this work was to understand therelative importance of the major operating paramet-

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ers influencing the fractionation of close MW mac-rosolutes. The specific variables investigated weremembrane material, module hydrodynamics and feedsolution properties. Factors like the feed pH and ionicstrength have been the subject of detailed analysis andtheir role in protein selectivity is well documented.The effect of module hydrodynamics and mass trans-fer is, however, not evident since the reported datafor a particular separation is often limited to a singlemodule type. Nevertheless, this aspect is importantto obtaining optimal separation, especially when at-tempting large scale processing. In this study, we haveexamined the effect of mass transfer by comparingthe separation in three different module configurationsviz, dead end, annular crossflow and vortex flow. Fur-ther, varying the solution pH and ionic strength wouldprovide insight into the comparative significance ofthese variables on BSA-Hb fractionation.

Materials and methods

Materials

ChemicalsThe proteins, BSA (A-7070, Lot 120H0218, 98%purity) and Hb (80% purity, single band on SDS-PAGE) were procured respectively from Sigma Chem-ical Company, St. Louis, MO, USA and the Cen-ter for Biochemical Technology, New Delhi, India.Table 2 lists the important physico-chemical char-acteristics of these proteins. The buffers were citricacid-Na2HPO4 (pH 2.6–7.6) and borate (pH 8.5), pre-pared with ultrapure water (resistivity 18.2 Mohm-cm)obtained from a Milli-Q unit (Millipore Corp., Bed-ford, MA, USA). The solution ionic strength wascontrolled by the addition of appropriate amounts ofsodium chloride. All reagents were analytical grade.The protein solutions (0.1% BSA and 0.01% Hb) wereprepared fresh for each experiment, which was con-ducted within 24 h. All solutions were filtered througha 0.45µm microfilter prior to each run to remove anyparticulate or colloidal matter.

Membranes and modulesExperiments were performed using three membranes,viz. m-PAN (modified poly(acrylonitrile)), RC (regen-erated cellulose) and m-PS(modified poly(sulfone)),all with a nominal molecular weight cut off rating of100 kD. The salient features of these membranes arelisted in Table 3.

Table 2. Protein physico-chemical properties (Nakatsuka and Michaels,1992; Musale and Kulkarni, 1997)

Property BSA Hb

Molecular weight 69,000 67,000

Sedimentation coefficient (S) 4.31×10−13 4.22×10−13

Diffusion coefficient, D20 (m2s−1) 5.9×10−11 6.4×10−11

Partial specific volume (m3kg−1) 7.34×10−4 7.5×10−4

Dimension (Å) 140×40×40 70×55×55

Stoke–Einstein radius (Å) 36 32.75

Isoelectric pH 4.8 6.8

Net charge

pH 7.4 −22 −7.2

pH 6.8 −17.5 0

Amino acids content 44.07:56.16 54.16:43.43

(g/100 g mixture)

hydrophobic: hydrophilic

The experiments in the dead end mode were per-formed in a stirred unit (Model 52, Amicon Corp.,MA, USA) with a membrane area of 14.5 cm2. Theprocessing volume was 55 ml and the system was pres-surized using N2 gas. The crossflow and vortex flowruns were conducted on a Benchmark Gx vortex flowfilter (Membrex Inc., NJ, USA). Details of the moduleare described elsewhere (Balakrishnan and Agarwal,1996a).

Methods

UltrafiltrationThe dead end filtration studies were performed in theconcentration mode with a 9-fold volume reduction inthe feed solution. Prior to each run, the membrane wasmaintained in contact with 55 ml of the correspondingprotein solution for 30 min. Both the initial equilibra-tion as well as the UF were carried out at an ambienttemperature of 24±2 ◦C. The volumetric flux wasmeasured by the time taken to collect a known volumeof the permeate.

The UF runs in the crossflow and the vortex flowmodes were also carried in a concentration mode. Oneliter of the initial feed solution was reduced to ap-proximately 170 ml to give a six-fold concentration.Operating temperature was maintained constant at 21±1 ◦C using a Haake circulator. The flux measure-ment was similar to that in the dead end experiment. Amore detailed description of the vortex flow filter andthe corresponding experimental setup can be obtainedfrom an earlier paper (Balakrishnan and Agarwal,

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Table 3. Membrane characteristics

Membrane Manufacturer NMWCO Material Contact angle Pure water permeability1

(kD) (◦) (m3m−2 s−1kPa−1)

MX100 Membrex 100 Surface modified 42 1.46×10−6

polyacrylonitrile

YM100 Amicon 100 Regenerated 313 2.15×10−6

cellulose

PTHK Millipore 100 Modified 331 1.38×10−6

polysulfone

1 Manufacturer Catalogs (1995).2 Hodgins and Samuelson (1990).3 Jucker and Clark (1994).

1996a). Crossflow mode was achieved by zero rotationin the vortex flow module while experiments in vortexflow mode were conducted at 4000 RPM. All exper-iments were carried out at constant transmembranepressure (TMP) of 40 kPa. In case of vortex flow, thepressure was adjusted to account for centrifugal forcedue to rotation.

Membrane fouling (Equation 1) was estimated bypure water permeability (PWP) measurements with ul-trapure water before and after protein UF experiments.Membranes were flushed with several volumes of ul-trapure water before measurements were taken. PWPreduction was an indication of irreversible fouling dueto protein adsorption on the membrane surface or poreplugging. The membranes were cleaned by soakingthem in 0.1-0.5 N NaOH for up to 12 h before flushingthoroughly with Milli-Q water. The cleaning proced-ure was repeated, if necessary, until PWP was within95% of the initial value. The cleaned membranes werestored between runs in 0.1% sodium azide at roomtemperature.

Fouling(%)= PWPfinal− PWPinitial

PWPinitial× 100 (1)

Protein recovery at the end of the run was estimatedby the procedure of van Eijndhoven et al. (1995).

Recovery= 1−(

1− Permeate Volume

Initial Volume

)τ(2)

whereτ = observed transmission as defined in Equa-tion (3).

Protein analysisProtein concentration was determined directly fromabsorbance measurements at 280 nm for BSA and416 nm for Hb solutions. The standard protein con-centrationvs. absorbance curves for each of these

two proteins was prepared at both the wavelengths.The analysis was performed using quartz cuvettes ona Shimadzu Graphicord UV 240 spectrophotometerequipped with a PR-1 graphic printer and an OPI-4computer interface (Shimadzu Corp., Kyoto, Japan).An initial wavelength scan indicated that Hb absorbsstrongly at 416 nm. In contrast, BSA does not showany measurable absorbance at this wavelength. How-ever, at 280 nm, BSA displays a higher absorbance ascompared to Hb. Thus, the BSA concentration in theBSA–Hb mixture was calculated by first measuringthe Hb concentration at 416 nm and then subtractingthe computed Hb contribution at 280 nm from the totalabsorbance at this wavelength. The observed proteintransmission and observed separation factor (SF) aredefined as:

Observed protein transmission (τ ),%= Cp

Cb× 100 (3)

Observed separation factor (SF)= τHb

τBSA(4)

where Cp = protein concentration in the permeate andCb = protein concentration in the feed.

Zeta-potential measurementThe membrane zeta-potential was measured followingthe procedure described by Bowen and Clark (1984).The membrane sample, having an effective area of6.6 cm2, was positioned with the active layer facingthe bottom electrode. The electrodes were platinumwires in the form of spiral coils. The membrane wascompletely immersed in the 0.01 M KCl electrolytemedium (conductivity 1.18×10−3 Scm−1, dielectricconstant 78). The solution pH was adjusted usingappropriate amounts of HCl or NaOH and the electro-osmotic flux was measured over the initial 10 min

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by monitoring the weight gain on a balance accur-ate to 10−4 g. Each run was repeated with a freshmedium and the average value (reproducible within6%) was used to calculate the zeta potential by theSmoluchowski equation.

Results and discussion

All experiments were conducted in duplicate with aclean membrane. The permeate flux was reproduciblewithin±10% and the BSA and Hb transmission valueswere reproducible within±10% and±5% respect-ively. The deviation in the BSA transmission valueswas higher as the samples had to be diluted 10- fold toensure reliable BSA concentration measurement. Onthe other hand, the analysis for Hb was conductedwithout diluting the sample.

Preliminary studies indicated that the differencesin BSA and Hb transmission were greater in the pres-sure dependent regime (Shukla, 1996). Further, aTMP of 40 kPa was found to result in a near op-timum in the separation factor. Ideally, the membranecomparisons should be conducted at constant wallconcentration (van Reis et al., 1997b) since variationsin the membrane material characteristics would affectthe wall concentration, thus resulting in a flux vari-ation. However, our experimental setup did not permitsuch a mode of operation. Consequently, instead ofthe constant flux mode, the constant pressure mode ofoperation was adopted in all our experiments.

Membrane evaluation

The UF characteristics of BSA-Hb mixture for threedifferent membranes are shown in Figure 1. The highinitial ratio of BSA to Hb concentration was usedto increase the accuracy of the concentration meas-urements for the very low BSA concentration in thepermeate. Experiments were performed in an unstirredbatch cell and the volumetric flux and protein trans-mission monitored up to 2 h. Dead end mode wasspecifically chosen to highlight the influence of themembrane material without contributions from masstransfer and related phenomena. All the three mem-branes, viz. m-PAN, RC and m-PS, displayed practic-ally complete transmission (τobs ∼80–100%) of both0.1% BSA and 0.01% Hb when the proteins wereultrafiltered alone (Table 4). Transmission remainedvirtually unchanged during the UF of the BSA-Hbmixture for the m-PS membrane. The RC membrane,

Table 4. Effect of membrane mater-ial on single protein transmission indead end UF (40 kPa TMP, pH 6.8,0.15 M ionic strength)

Membrane Transmission (%)

BSA Hb

MX100 98 90

YM100 97 95

PTHK 87 75

however, displayed a significant reduction in the trans-mission of both the components. In contrast, m-PANrestricted BSA transmission to 55% while permittingrelatively unhindered passage of Hb.

Fouling was strongly influenced by the nature ofthe membrane material. The m-PAN membrane exhib-ited relatively low fouling ( 10%) as compared to RCand m-PS, which displayed 40% and 60% loss in purewater permeability respectively. This agrees with theirrelative hydrophilicity (m-PAN> RC > m-PS) asdeduced by their contact angles in Table 3. Our obser-vations conform to reported findings that hydrophilicmembranes, in general, foul less severely (Sheldon etal., 1991; Ko and Pellegrino, 1992; Koehler et al.,1997).

From the results of the single protein UF, it isnoticed that the m-PS membrane displays relativelylower transmission (87% for BSA and 75% for Hb)as compared to the more hydrophilic RC and m-PANmembranes. The latter exhibit almost complete trans-mission (>90%) for both the proteins. However, thisbehavior changes during the UF of the protein mix-ture. The m-PS membrane was an exception with theUF profiles for the mixture closely matching the datafor the corresponding single proteins. An analogousbehavior has been observed previously for other pro-tein mixtures also, under well-defined experimentalconditions. It was noticed that at low transmembranepressure using dilute protein solutions, the transmis-sion patterns of BSA and myoglobin, both singlyand in mixture, were essentially identical (Nakatsukaand Michaels, 1992). In another study, lysozyme–ovalbumin mixtures demonstrated a similar behaviorup to 100 kPa at feed concentrations below 0.21%(Balakrishnan and Agarwal, 1996b).

In this investigation, it was observed that the RCand m-PAN membranes deviate from the above trend.RC displayed a sharp decrease in the transmission ofboth BSA and Hb when the proteins were ultrafiltered

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Figure 1. BSA–Hb fractionation: effect of membrane material (40 kPa TMP, pH 6.8, 0.15 M ionic strength, dead end UF).

together, while m-PAN showed a noticeable decreasein the BSA transmission in the presence of Hb. Thisoutcome can be interpreted by considering the com-bined effect of protein properties, membrane charac-teristics and operating conditions on the membraneselectivity. At pH 6.8, Hb is a zwitterion with equalpopulation of positive and negative charges. BSA, onthe other hand, is strongly negatively charged, witha net charge of−17.5 (Vilker et al., 1981). Thus,the experimental conditions favor the hydrophobic in-teractions between the neutral Hb and the relativelyhydrophobic RC membrane, which leads to percept-ible fouling and reduced Hb transmission. Further,the Hb layer on the membrane surface hinders thepassage of BSA thus resulting in a drop in the BSAtransmission as well.

Earlier studies on protein transmission through m-PAN membranes indicated that the m-PAN membranepossessed a negative charge at neutral pH (Balakrish-nan and Agarwal, 1996a). This is confirmed from themembrane zeta-potential measurement as a function ofsolution pH (Figure 2). Thus, at pH 6.8, BSA is negat-ively charged and will be repelled by the membrane

surface while the neutral Hb is preferentially polar-ized. Since a higher wall concentration of the proteinleads to higher transmission by diffusion (Lentsch etal., 1993), Hb is almost completely transmitted underthese conditions.

The PS membrane is also reported to carry a slightnegative charge (−5 to –10 mV between pH 4–9) as in-dicated from zeta potential measurements (Nabe et al.,1997). However, the charge on the m-PAN is strongerwith a zeta potential of –15 to –40 mV in the pH rangeof 4–12. Thus, the charge effects are pronounced withthe m-PAN membrane.

It is pertinent to note here that the electrostaticprotein–protein and protein–membrane interactionsare partially shielded in the presence of 0.15 M NaClin the feed solution. This leads to the formation ofa relatively porous Hb layer, which shields the pas-sage of the similar size BSA molecules. Thus, BSAtransmission of around 55% is observed, yielding aseparation factor of 1.6. In single protein experiments(Table 4), the salt concentration was maintained at0.15 M as well, which leads to masking of coulombicforces between BSA and the membrane at pH 6.8 to a

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Table 5. Estimation of mass transfer coefficients

Configuration Equation Data Reference

Dead-end k =(

Dπt

)1/2t=7200 sec Zeman and Zydney, (1996)

Crossflow Sh = 1.86×Re0.33× Sc0.33×(

dhL

)0.33R=3.23× 10−2m Zeman and Zydney (1996)

d=3× 10−3m

L=15.87× 10−2m

Vortex flow Sh = 0.29×Ta0.66×Sc0.33 R, d, L same as Agarwal, (1997)

crossflow

ω=4000

Where:ν = 9.48× 10−7 m2s−1; µ = 10−3 kgm−1s−1; D values given in Table 2.

Figure 2. Variation of membrane zeta-potential with pH (m-PAN).

certain extent. Still, the repulsion is not strong enoughto overcome the convective transport due to the ap-plied pressure. Consequently, BSA alone in solutionexhibits almost complete transmission and the effectof the protein surface charge becomes apparent onlywhen both the proteins are ultrafiltered together.

Since both RC and m-PS membranes displayedcomparatively poorer resolution of BSA and Hb, thesewere not examined any further. All subsequent invest-igations were thereafter performed solely on m-PAN.

Module comparison

The effect of mass transfer coefficient on protein frac-tionation was evaluated by using three different flow

geometries, viz. dead end, crossflow and vortex flowmodes.

In dead end filtration, the transmission values ofBSA and Hb stayed constant in the 55–60% and 85–90% range respectively (Figure 3). Owing to repulsionbetween the similarly charged membrane surface andBSA molecules, the polarization layer is relatively richin Hb, thus leading to a higher Hb transmission. Aseparation factor of 1.6 was obtained in this instance.However, flux drop averaged 75% in 1 h. The dynam-ics of the concentration polarization responsible forthis behavior is explained by the constant pressure fil-tration theory (Suki et al., 1984). The concentration ofeach component on the membrane surface is governedby the net convective flow towards the membrane sur-

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face flowing against the solute diffusion back in thebulk solution. This flow is responsible for the gradualincrease in the concentration polarization layer thatcontinues to develop until the solute convective flowis balanced by the solute diffusion back into the bulksolution. In this instance, the quasi steady state ofequal flow was not reached and the layer of rejec-ted protein molecules increased in thickness over thecourse of the UF. This led to a continuous increase inthe resistance to flow and hence the flux continued todecline until the end of the experiment.

Figure 4 shows the UF characteristics for the cross-flow mode of operation. Flux declined steadily byover 60% in 1 h as the layer of the rejected proteinmolecules did not reach a steady state in this casetoo. This can possibly be attributed to the low masstransfer coefficient arising from low feed crossflowvelocity (0.0148 ms−1) which was not effective incontrolling the concentration polarization (Zeman andZydney, 1996). However, in this configuration, thoughthe transmission of Hb remained constant during thecourse of the experiment, the BSA transmission de-clined steadily. As explained earlier, both the m-PANmembrane and the BSA molecules carry strong negat-ive charge at pH 6.8 causing the BSA molecules to berepelled from the membrane surface. However, Hb be-ing at its isoelectric point, is electrically neutral. As theboundary layer develops, the Hb concentration riseson the membrane surface leading to a protein layerpredominantly composed of Hb molecules. Thus, theHb transmission remained high (> 80%). On thecontrary, the BSA transmission dropped continuouslysince crossflow operation affords a mechanism otherthan solute diffusion for the BSA molecules to betransported away from the membrane surface. Thisled to a steady depletion in the BSA concentration inthe polarization layer thus resulting in a correspondingdecrease in protein transmission.

In the vortex flow filter system, flux decline ismuch less as indicated by only a 25% drop in fluxin 1 h (Figure 5). The mass transfer coefficient in thevortex flow filter is high (> 22× 10−6 ms−1) resultingin the most effective polarization control among thethree modules tested. It has been reported that the highshear sweeping action due to Taylor vortices reducesthe thickness of the gel layer on the membrane surface(Cheryan, 1998). In addition, flux in the vortex flowfilter was almost twice that observed in the cross flowmode. Since the fouling as measured by the PWP losswas almost identical (∼10%) for all the three configur-ations, it can be inferred that the high fluxes observed

Table 6. BSA-Hb fractionation with m-PAN membrane: com-parison of module types (40 kPa TMP, pH 6.8, 0.15M ionicstrength)

Parameter Dead end Cross flow Vortex flow

kBSA(×106 m/s) 5.1×10−2 1.22 22.26

kHb(×106 m/s) 5.31×10−2 1.29 23.51

Average flux (×106 m/s) 2.5 6 10

Transmission (%)

BSA 55 15 < 1

Hb 88 80 40

Separation factor (–) 1.6 5.3 ∼40

Recovery (%)

BSA 70.1 23.3 1.8

Hb 85.5 75.8 50.8

for the vortex flow filter are due to the high trans-verse shear rates generated on the membrane surfacein this system. This system provides a high averageseparation factor of∼40.

It should be noted here that a high separationfactor by itself could be misleading since the value ishighly sensitive to changes in the denominator, espe-cially when the latter is less than 1% (Ghosh and Cui,1998a). This situation is observed in the experimentswith the vortex flow filter where the BSA transmissionis consistently below 1%. Thus, for the BSA-Hb frac-tionation to be effective, it is essential to obtain a highseparation factor combined with high Hb transmission.

Table 6 summarizes the performance of the differ-ent module configurations. In general, increasing themass transfer coefficient ‘k’ improves both the fluxand the separation. It is estimated that ‘k’ in the vor-tex flow filter could be up to 18 times higher thanthat in the crossflow mode (Table 5). This is reflec-ted in a 400% higher flux of about 10×10−6 ms−1

= 36 L m−2 h−1, which implies a higher systemthroughput, or lower processing time. In addition, analmost pure Hb stream is obtained with this device.However, since the Hb recovery is modest (50.8%),it needs to be improved further while maintainingthe clarity of resolution and desired separation factor.Thus, subsequent investigations were performed in thevortex flow module to examine the effect of feed solu-tion properties like the ionic strength and pH on theseparation characteristics.

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Figure 3. BSA-Hb fractionation with m-PAN membrane: dead end UF. (♦: Flux;�; BSA;N: Hb, 40 kPa TMP, pH 6.8, 0.15 M ionic strength).

Table 7. Effect of feed pH on BSA-Hb separation (m-PAN mem-brane in vortex flow UF, 40kPa TMP, 0.15 M ionic strength, 4000RPM)

Parameter pH

2.5 4.8 5.5 6.8 8.5

Charge

BSA + 0 (pI) – – –

Hb + + + 0 (pI) –

Flux (x106 ms−1) 7 0.65 6 11 12

Separation factor (–) 0.43 0.30 6.8 40 1.8

Membrane fouling (%) 60 90 80 10 <2

Effect of feed characteristics

The m-PAN membrane surface is characterized bystrong negative charge between pH 2-8 (Figure 2) withthe isoelectric point possibly located below pH 2. Inorder to fully utilize the electrostatic interactions dueto the negative charge on the membrane and to im-prove the BSA-Hb resolution, we carried out studiesat different pH and ionic strengths. The results arepresented in Figure 6 and Table 7 respectively.

The effect of ionic strength was studied at pH 6.8where Hb is electrically neutral while BSA and mem-brane surface are both negatively charged. At low saltconcentrations, the electrostatic shielding was min-

imized, leading to strong repulsion of the BSA mo-lecules from the membrane surface and consequentlylow transmission (∼1%). In contrast, the neutral Hbis present in higher concentration in the polarizationlayer on the membrane surface. However, at zero ionicstrength this did not translate into high Hb transmis-sion, which remains below 10%. This observationmay be explained as follows. The protein deposit onthe membrane surface provides the greatest hydraulicresistance at the isoelectric point due to increase indensity caused by reduction in intermolecular electro-static repulsion among the protein molecules (Palacekand Zydney 1994). In addition, the solubility of aprotein in solution is minimum around its isoelectricpoint; consequently, Hb forms a gel on the membranesurface. The increased protein density and proteinprecipitation account for the high membrane fouling(>70%) observed in the absence of electrolytes. Onincreasing the ionic strength to 0.01 M, the solvent fluxand protein transmission increased with a correspond-ing decrease in the membrane fouling (<40%). Thiswas a direct result of partial shielding in the long-rangecoulombic interactions between the protein moleculesand the membranes. The positively charged sodiumions form an electrical double layer on the membranesurface, which reduces the density of the protein layer.This behavior becomes more pronounced when theionic strength is increased further to 0.15 M. A high

16

Figure 4. BSA–Hb fractionation with m-PAN membrane: crossflow UF. (♦: Flux;�: BSA;N: Hb, 40 kPa TMP, pH 6.8, 0.15 M ionic strength,0.0148 ms−1 crossflow velocity).

Figure 5. BSA–Hb fractionation with m-PAN membrane: vortex flow UF. (♦: Flux; �: BSA; N: Hb, 40 kPa TMP, pH 6.8, 0.15 M ionicstrength, 4000 RPM).

17

Figure 6. BSA–Hb fractionation with m-PAN membrane in vortex flow: effect of ionic strength. (40 kPa TMP, pH 6.8, 4000 RPM).

salt concentration enhances the electrostatic shieldingof the protein–protein as well as protein–membraneinteractions. Consequently, there was an increase inthe transmission of both BSA and Hb. The membranefouling was less than 10% due to reduced protein ad-sorption on the membrane surface. The optimum saltconcentration, characterized by low fouling, high fluxand high separation factor, possibly lies between 0.01M and 0.15 M NaCl and needs to be identified bysystematic experimentation.

The effect of varying pH on protein fractiona-tion is summarized in Table 7. At pH 2.5, both theproteins are positively charged. Thus, membrane foul-ing was significant owing to the strong electrostaticinteractions with the negatively charged membrane.Also, Hb being further away from its pI, carries astronger charge as compared to BSA and thereforebonds more strongly to the membrane. This loweredthe transmission of Hb and the membrane preferen-tially transported BSA. By increasing the pH to the pIof BSA (pH 4.8), the fouling of the membrane wasincreased to 90%, which was also responsible for theorder of magnitude lower solvent fluxes. At this pH,

because of its low solubility, BSA precipitates on themembrane and fouls the membrane. This behavior isnot perceptible at the pI of Hb (pH 6.8) since the con-centration of Hb in bulk solution is ten folds smaller(0.01% Hb compared to 0.1% BSA).

At pH 5.5, the BSA and Hb molecules are op-positely charged. Thus, the positively charged Hbwould form a self-rejecting layer on top of the negat-ively charged membrane surface. This, in turn, wouldprovide a surface for the adsorption of negativelycharged BSA. This results in a low transmission of thenegative BSA molecules (<20%) through the mem-brane due to the long-range electrostatic repulsioneffect. The Hb transmission is also low (< 30%). Itshould also be noted that the conditions favor the elec-trostatic heteroaggregation of Hb and BSA. However,since this would involve equimolar participation ofthe two proteins, the effect may not be pronouncedowing to the 1:10 Hb-BSA ratio in the feed. A high de-gree of membrane fouling is also observed at this pH.Apart from the adsorption of the BSA molecules onthe membrane, the fouling is possibly aggravated dueto the low protein solubility as both the components

18

are close to their pI at pH 5.5. Thus, both the pro-teins contribute to the formation of the gel layer on themembrane leading to heavy fouling and consequentlylow flux.

As the pH is increased, the magnitude of positivecharge on Hb decreases. This reduces the intensityof bonding with the negatively charged membranesurface thus leading to higher transmission, whichreaches a maximum at the pI (pH 6.8). Correspond-ingly, BSA becomes more negatively charged as thepH is increased beyond its pI of 4.8. Thus, it willexperience increased repulsion owing to the intrinsicnegative charge on the membrane surface. The latterexhibits a negative zeta potential below –30 V up to pH9.0. Thus, the repulsion effect is expected to be morepronounced at higher pH values as the magnitude ofthe negative charge on the BSA would be higher. Thiswould explain the exclusion of the BSA molecules (<

5% transmission) at higher pH values of 6.8 and 8.5.Further, Hb is also negatively charged at pH 8.5. Thus,both BSA and Hb are repelled from the membrane sur-face leading to low transmissions for both. The foulingis almost non-existent; therefore the permeate flux isrelatively high.

Variation in ionic strength and pH of the feedsolution contribute to agglomeration of the proteins(Musale and Kulkarni, 1997) as well as changes inprotein-protein interactions which could lead to pre-cipitation and densification on the membrane surface.In case of protein agglomeration, the molecular sizeincreases thereby decreasing its transmission. How-ever, the permeate flux need not be affected. In case ofprotein precipitation followed by densification, trans-mission should increase since a more dense layertranslates to a greater wall concentration. Simultan-eously, the flux should decrease owing to the higherresistance offered by the dense wall layer. From thebehavior observed in our experiments, a precipitation-densification phenomenon on the membrane surfaceappears to be the dominant mechanism.

Conclusions

This work describes the ultrafiltration of BSA–Hbmixtures with the aim of understanding the role ofthe various operating parameters on the fractionationof similar molecular weight proteins. The significanceof three variables, viz. membrane material, mod-ule hydrodynamics and feed solution characteristicswere examined. Experiments with different 100 kDmembranes indicated that the surface modified m-

PAN membrane was best suited for this separation.Of the different module configurations tested, the vor-tex flow system demonstrated high steady state fluxcoupled with a high separation factor. The separationcould be further improved by adjusting the feed pHand ionic strength. This work proves conclusively thatapart from the feed and membrane properties, the sys-tem hydrodynamics is critical to the fractionation ofprotein mixtures.

Nomenclature

BSA: Bovine serum albumin; Cb: Protein concen-tration in feed (gl−1); Cp: Protein concentration inpermeate (gl−1); d: Annular gap width in vortex flowmodule (m); D: Effective diffusion coefficient (m2

s−1); dh: Hydraulic diameter (m); Hb: Hemoglobin;k: Mass transfer coefficient (ms−1); L: Membranelength in vortex flow module (m); MW: Molecularweight; pI: Isoelectric point (–); PWP: Pure wa-ter permeability (m3m−2 s−1kPa−1); R: Radius ofthe membrane cartridge in vortex flow module (m);Re: Reynolds number (udh/ν); Sc: Schmidt number(ν/D); SF: Observed separation factor(–); Sh: Sher-wood number (kd/D); t: Experiment duration (s); Ta:Taylor number ((ωRd/ν)(d/R)0.5); TMP: Transmem-brane Pressure (kPa); u: Cross flow velocity (ms−1);UF: Ultrafiltration.Greek Notation;ν: Kinematic viscosity (m2 s−1); τ :Observed protein transmission (%);ω: Revolutionsper minute (s−1); µ: Viscosity (kgm−1 s−1).

Acknowledgements

The vortex flow filter and membranes were fundedby the grant UNDP IND/89/103. The authors thankDr Deepak Musale, National Chemical Laboratory,Pune, India for the membrane zeta potential measure-ments. We also appreciate the donation of the bovinehemoglobin by Dr V. Gade, Center for BiochemicalTechnology, New Delhi, India. In addition, the authorsacknowledge the referees valuable comments in therevision of this manuscript.

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Address for correspondence: Rishi Shukla, Agricultural Biopro-cess Laboratory, 1302 West Pennsylvania Avenue, University ofIllinois at Urbana Champaign, Urbana, IL 61801, USA. E-mail:rshukla@uiuc.edu