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Reduced bovine serum albumin adsorption by prephosphatation of powdered zirconium oxide
COLLOIDS AND ELSEVIER
Colloids and Surfaces A: ehysicochemicaland Engineering Aspects 121 (1997) 81-88
A
SURFACES
Reduced bovine serum albumin adsorption by prephosphatation of powdered zirconium oxide Bruno Putman, Paul Van der Meeren *, Danny Thierens University of Gent, Faculty of Agricultural and Applied Biological Sciences, Laboratory of Applied Physical Chemistry, Coupure Links 653, B-9000 Gent, Belgium Received 4 July 1996; accepted 23 November 1996
Abstract
The adsorption of phosphate anions to ZrO2 and its influence on protein adsorption have been studied. Phosphate adsorption experiments revealed that phosphate anions had a high affinity for zirconia: in appropriate conditions, a monomolecular layer was adsorbed. Using the electrophoretic light scattering technique, it was shown that the isoelectric point of the zirconium oxide was shifted from pH 6.4 down to pH 3.8. From this shift, it was concluded that specific interactions were responsible for the adsorption behaviour. In addition, the adsorption of bovine serum albumin (which was selected as a model protein) to both bare and phosphated zirconia was investigated: prephosphatation reduced the BSA adsorption by over 50%. Comparing the adsorption behaviour at pH 4.15 and 5.35, it was concluded that electrostatic interactions were of minor importance. From contact angle measurements, the different adsorption behaviour was shown to be due to the increased hydrophilicity of the phosphated adsorbent. On the basis of the present results, the observed effect of phosphate ions in ultrafiltration of proteins on ZrO2 ceramic ultrafiltration membranes may be explained.
Keywords: Adsorption; Contact angle; Electrophoretic mobility; Fouling
1. Introduction
Protein adsorption to solid surfaces is an intensively studied phenomenon [1-5]. Several applications have been designed to apply this adsorption behaviour. The immobilization of enzymes [6], uses in biosensor applications [7] and the use of protein-coated particles in immunological agglutination tests [8] are only a few examples to illustrate the widely different applications of the protein adsorption phenomenon. In membrane-filtration technology, however, protein adsorption is giving rise to serious prob* Corresponding author. Tel.: +32 9 2646001; Fax: +32 9 2646242. 0927-7757/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S0927-7757 (96) 03978-7
lems [9-12]. The fouling of the filter membranes is often due to this adsorption, thus affecting the hydraulic permeability and the rejection properties of the membrane. Trying to prevent protein deposition on these membranes is an important goal for researchers in this area. An important tool in achieving this is the modification of the filtration membrane. Chemical modification of polymer membranes has been proved to be a powerful method for improving the membrane's properties in terms of hydraulic and rejection properties [13-16]. However, very little information is available about the modification of ceramic membranes, used for the ultra-filtration of protein solutions. D u m o n and Barnier [12] observed that a distinctively better permeate flux
82
Bruno Putman et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 121 (1997) 81 88
was obtained by adding a phosphate buffer to an ovalbumin solution before filtering it through a zirconium oxide membrane. They proved by IR spectroscopy that the surface of the zirconium oxide filter was modified due to phosphate adsorption. In the present study, phosphate adsorption to zirconium oxide is investigated in more detail. The surface modifications of the phosphated powder obtained were detected by ~-potential measurements. Also, the adsorption of bovine serum albumin to bare and to phosphated zirconia powders was compared. The influence of the zirconium oxide and protein surface-charge characteristics on the adsorption behaviour of BSA was investigated.
2. Materials and methods
phosphate solution was adjusted by adding small volumes of either a 0 . 2 N or a 0 . 5 N N a O H solution. Following 48 h of equilibration, the dispersion was filtered on a 0.45 lam mixed cellulose esters filter membrane (Millipore). Subsequently, the filter cake was washed five times with 50 ml of distilled water in order to remove the free phosphate ions from the interstitial pores of the oxide. In the last washing liquid, no phosphate could be detected in the eluate. After vacuum-drying the powder for 2 h at 26 mbar, the adsorbed phosphate was desorbed by boiling 100 mg of the dry powder for 20 min in 20 ml 20% H z S O 4. Subsequently, 20ml of water was added, the mixture was boiled again for 20 min and then transferred to a 100ml volumetric flask. The amount of phosphate adsorbed was calculated from the phosphate content of the clear supernatant.
2.1. Materials
2.3. Protein adsorption experiments
Powdered zirconium oxide (ZrO2) was supplied by Magneson Electron. The specific area of this adsorbent as determined by BET Nz-adsorption amounted to 21 m 2 g - 1. The bovine serum albumin (BSA) was purchased from Sigma (St. Louis, USA). Potassium dihydrogen phosphate (KHzPO4) was from Merck.
Experiments on the adsorption of the BSA protein to zirconium oxide were performed by adding increasing amounts of the protein in 10ml of distilled water or 10mM acetate buffer (pH 4.14 and 5.35) to 50 mg ZrO2 powder. After shaking the suspension for 2 h at 20°C, it was centrifuged for 10 min at 5000g. The protein content of the supernatant was determined by the method of Schaterle and Pollack [18].
2.2. Phosphate adsorption experiments
In the first experiment, phosphate adsorption was accomplished by adding increasing amounts of KHzPO 4 in 30 ml of distilled water to 200 mg ZrO 2 powder. This mixture was shaken at 20°C for at least 48 h at 160 rpm. The amount adsorbed was calculated by difference from the concentration of the added phosphate solution and the equilibrium phosphate concentration of the clear supernatant, obtained by centrifugation of the dispersed zirconia for 10min at 5000g. Quantitative phosphate determination was made according to the method proposed by Scheel [17]. The pH of the equilibrium solutions was 5.9_+ 0.2. Alternatively, 500 mg of powdered zirconia was dispersed in 30ml of a 0.1 M phosphoric acid solution. Hence, the phosphate-to-ZrO2 ratio was kept constant in these experiments. The pH of the
2.4. Electrophoretic light scattering measurements
The ~ potentials were determined by a Malvern Zetasizer IIc. The samples were prepared by dispersing some mg of zirconia in 50 ml of a 0.01 N potassium chloride solution using a bath sonicator. The pH was adjusted by addition of either diluted HC1 or NaOH. All measurements were carried out at 20°C. 2.5. Contact angle determinations
The advancing contact angle measurements were performed with a DCA 322 Dynamic Contact Angle Analyser (Cahn) operating at 264 ~tm s 1 Sample plates were prepared by coating a micro-
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Bruno Putman et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 121 (1997) 81-88
scope cover glass with the powdered zirconia using adhesive Spray Mount (3M).
3. Results and discussion 3.1. Adsorption o f phosphate on zirconium oxide
The adsorption of phosphate ions to powdered zirconium oxide was investigated at 20°C. Fig. 1 reveals that the experimental adsorption data were satisfactorily described by the Langmuir equation describing monolayer adsorption. Although the conditions which form the basis of the theoretical Langmuir model, such as the independency of the energy of adsorption from the surface coverage, are actually not fully fulfilled in the case of the adsorption of ions, but this model is still widely used to estimate the adsorption maximum [19]. Linear regression of the ratio of the equilibrium phosphate concentration to the adsorbed amount as a function of the equilibrium phosphate concentration revealed that the phosphate adsorption maximum /"max amounted to
66.8 ~tmol P g - 1 ZrO2; the correlation coefficient was 0.991. As Vissers [20] demonstrated, in the conditions used in our experiments, a phosphate molecule covered a zirconium-oxide surface of about 40 A2. It follows that about 16m 2 g-1 of the zirconium oxide is coated by the phosphate groups at the estimated value of the adsorption maximum. As the specific area of the powder was about 21 m 2 g - l , it follows that the surface of the powder is almost completely covered. From this first experiment, it follows that zirconium oxide has a high affinity for phosphate ions. For the sake of completeness, it should be mentioned that the pH of all equilibrium solutions was within the range 5.9_+0.2. According to Bowden et al. [21] and Barrow [22], this behaviour is due to the combined effect of the release of one surface hydroxyl group per HzPO 4 ion adsorbed, followed by the release of one proton from the adsorbed phosphate group due to the decrease of its pK values upon adsorption [23,24]. Subsequently, the phosphate equilibrium solution was decanted and replaced by 30 ml of distilled water. From the phosphate concentration of the
O~
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8
B
4o 30
i 20JII l
~
= adsorpl~on -
Langmuir fit
0 0
0.5
1
1.5
2
EQUILIBRIUM CONCENTRATION (mmol/I) Fig. 1. Adsorption isotherm of phosphate to powdered ZrO2 at 20°C. The amount of phosphate adsorbed was determined before ( • ) and after (~) the phosphated powder was washed with distilled water. Each experimentwas performedin duplicate. The smooth curve corresponds to the best-fitting Langmuir adsorption isotherm.
Bruno Putman et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 121 (1997) 81-88
84
supernatant after 48h of equilibration, the desorbed amount of phosphate was calculated. Here, a correction was introduced to take account of the free phosphate ions which were retained within the interstitial pores of the powder upon decantation. Fig. 1 reveals that the phosphate adsorption was highly irreversible, especially at lower phosphate loads. In a second experiment, we tried to increase the adsorbed amount further by changing the experimental conditions. First, the concentration of the phosphate solution was increased significantly (up to 0.1 M). Also, the pH of the phosphate solutions was controlled by addition of NaOH, because it is well known that the specific adsorption of anions is promoted at low pH [21,23]. From the results of these experiments, summarised in Table 1, it can be seen that similar values of the adsorbed amount were found using acidic phosphate solutions. As predicted by the four-layer phosphate adsorption model of Bowden et al. [21,22], the adsorption was largely reduced when the pH was larger than the pK2 value of phosphoric acid. According to Bowden et al. [21], this reduced adsorption is due to the combined effect of a decreased amount of HzPO4-, which is considered to be the most reactive phosphate species, and an increased electrostatic repulsion between the surface of the oxide and the phosphate anions. Table 1 reveals that the estimation of the adsorption maximum at a pH of about 5.9, as obtained from the Langmuir adsorption isotherm fitting to the data of the above-mentioned adsorption experiment, was slightly underestimated. Adding an excess of phosphate anions, about 80 ~tmol of phosphate was adsorbed per gram of Z r O 2 , whereas an adsorption maximum of only 68 ~tmol g 1 was estimated by extrapolation of the data obtained at low phosphate concentrations. From the projected Table 1 Phosphate adsorption to powdered zirconium oxide. The a m o u n t of phosphate adsorbed to zirconium oxide (X) was determined as a function of the pH; 30 ml of a 0.1 M phosphoric acid solution, the p H of which was adjusted by addition of N a O H , was added to 500 mg of powdered ZrO 2 pH X(gmol g-l)
1.54 89
2.09 83
2.47 91
3.73 86
6.12 79
8.08 61
area of the phosphate ions, it follows that 19 m 2 g-1 is phosphated. On the basis of the results of the phosphate adsorption experiments, all phosphated zirconia samples which were used for further research were prepared by equilibrating in 0.1 M phosphoric acid for at least two days, followed by the removal of the phosphate excess by washing with distilled water.
3.2. Electrokinetic charge characteristics of phosphated zirconia In order to check the influence of phosphate adsorption on the properties of the zirconia, the electrophoretic mobility of bare and phosphated zirconium oxide in 0.01 N KC1 was determined as a function of the pH. From Fig. 2, it follows that the surface charge characteristics of the zirconia were significantly affected by phosphate adsorption: phosphatation decreased the value of the electrophoretic mobility of the zirconium oxide. Hence, highly negative values were obtained at neutral pH. As a further consequence, the IEP was lowered by phosphatation. From Fig. 2, it follows that the isoelectric point (IEP) of zirconium oxide was approximately 6.35, which is in close agreement with the data of Parks [25]. As the amount of phosphate adsorbed was increased, the IEP decreased to about 4.5 at the highest value under investigation. From this observation, it follows that the phosphate ions are sorbed by a specific adsorption mechanism. The experimental data can be explained by the ligand-exchange model [21-23]: upon phosphate adsorption, surface hydroxyl ions are replaced by H2PO 4 anions. Upon adsorption, the pK value of the phosphate ions is decreased [23,24], so that a proton is set free. As a consequence, phosphate adsorption renders the metal oxide surface more negatively charged, and hence the IEP is reduced. The increase of the adsorbed amount when the powdered zirconia was first equilibrated in a 0.1 M phosphate solution also became obvious from the determination of the electrokinetic properties of the phosphated zirconia samples. From Fig. 3, very similar ~-potential characteristics were observed for the three samples which had been
Bruno Putman et aL / Colloids Surfaces A." Physicochem. Eng. Aspects 121 (1997) 81-88
85
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I
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pH 8.08
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pH Fig. 3. Electrophoretic mobility spectra of phosphated zirconium oxide in 0.0l M KC1 as a function of the pH of the 0.1 M phosphatation solution.
86
Bruno Putman et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 121 (1997) 81 88
phosphated at acid pH, characterised by an isoelectric point of about 3.7. As compared to the sample with the highest phosphate content of the previous adsorption experiment, this represents a further decrease of the isoelectric point by about 0.8 pH units. The close resemblance of the i-potential spectra indicates that these samples contained a comparable amount of adsorbed phosphate. Actually, the differences of the adsorbed amount of phosphate to zirconia up to pH 3.73 (shown in Table 1) should primarily be ascribed to experimental error. The samples which were treated at pH 6.12, and especially at pH 8.08, on the other hand, had significantly different ~-potential characteristics (Fig. 3), from which it may be concluded that different amounts of phosphate had been adsorbed. Hence, it follows that the electrokinetic measurements (Fig. 3) fully support the estimated values of the adsorbed amount of phosphate (Table 1). 3.3. Adsorption o f B S A on phosphated zirconium oxide
The adsorption behaviour of proteins to both bare and phosphated zirconia was compared. Bovine serum albumin (BSA) was selected as a model protein because it is commercially available in high purity. The adsorption experiments were performed in 10mM acetate buffer solutions; pH 4.15 and 5.35 were selected because they are quite symmetrical about the isoelectric point of BSA, which is located at pH 4.8 according to Kondo and Higashitani [5]. As it was shown experimentally that the change of the pH during the adsorption experiments amounted to 0.1 at the most, it can be concluded that the buffering capacity of the buffers used was sufficiently high. From Fig. 4, the widely different adsorption behaviour of the bare and the phosphated zirconia becomes immediately obvious: much less protein is adsorbed to the phosphated zirconia surface. In addition, Fig. 4 reveals that the adsorption of the protein was pH-dependent. From the results of this experiment (Fig. 4), a very strong adsorption of the protein to the bare zirconia surface was observed: at the lowest protein concentration considered, about 70-80% of the
BSA added was adsorbed. According to Norde [1], this pronounced adsorption behaviour is typical for a soft protein, such as BSA, at low ionic strength. Also, Fig. 4 reveals that the adsorbed amount does not tend to a maximum; instead, the adsorbed amount keeps on increasing as the added protein concentration is increased. This is also known to be a feature of soft-protein adsorption [1]. Comparing the protein adsorption on bare and phosphated zirconium oxide, highly significantly differences were noticed. At the same initial protein concentration, the treated zirconia adsorbed over 50% less protein. These results are in agreement with the ultra-filtration flux observations of Dumon and Barnier [12]: following the treatment of ceramic zirconia membranes with phosphate solutions, much higher permeate fluxes were obtained during the ultra-filtration of ovalbumin. The latter should be ascribed to a decreased fouling tendency, thanks to a lower adsorption affinity. Similarly, Nishida et al. [26] found that the adsorption of BSA to aluminium hydroxide was reduced in the presence of phosphate. Comparing the adsorption behaviour of BSA onto bare and phosphated zirconia samples at pH 4.15 and 5.35, it follows that electrostatic interaction does not suffice to interpret the experimental results: from an electrostatic point of view, protein adsorption should be maximised on the bare zirconia at pH 5.35, since the protein and the adsorbent have an opposite charge, whereas phosphated zirconia should be the best adsorbent at pH 4.15. In the latter case, both the protein and the bare zirconia are positively charged, whereas the phosphated zirconia surface is negatively charged. Also, electrostatic interactions would favour BSA adsorption to phosphated zirconia at pH 4.15 as compared to pH 5.35. As these expectations have to be rejected on the basis of the experimental data of Fig. 4, it follows that protein-surface electrostatic interactions play only a minor role in the adsorption process. According to Norde [1], the maximum adsorption is usually found around the isoelectric point of the soft protein, irrespective of the IEP of the adsorbent, because of structural rearrangements in the adsorbing proteins at either side of the protein's IEP. Hence, it follows that the pH dependence of the adsorption phenomenon
87
Bruno Putman et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 121 (1997) 81-88 35 pH 4.15
g 30O
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.
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.--+
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EQUILIBRIUM CONCENTRATION (rag/I) Fig. 4. The adsorption of bovine serum albumin to bare ZrO2 (dotted lines) and to phosphated ZrO2 (solid lines) in 10 mM acetate buffer at pH 4.15 and 5.35, respectively.
is primarily determined by the properties of the protein, and that protein protein interactions and structural rearrangements in the protein molecules are of utmost importance. The magnitude of the adsorption phenomenon, however, is highly affected by the nature of the adsorbent. Thus, Anzai et al. [27] observed that BSA adsorbs more efficiently to hydrophobic than to hydrophilic surfaces. According to these authors, the electrostatic force of attraction or repulsion between the BSA molecules and the surface does not play a primary role in determining the adsorption behaviour of the protein. Also, it is well known that protein adsorption to ultra-filtration membranes, with the concomitant filtrate-flux decline, is strongly reduced by rendering the surface more hydrophilic [13-16]. Based on these data, the decreased BSA adsorption to the phosphated zirconia samples most probably originates from an increased hydrophilicity of the surface of the powder.
Table 2 Contact angles of phosphated zirconia. The advancing contact angle (O,) of powdered zirconium oxide was determined as a function of the amount of phosphate adsorbed (it) X (gmol g-l) Oa (°)
0 59
10 54
38 49
68 44
Table 2 indicates that the contact angle decreased from 59 ° to 44 ° as the amount of phosphate adsorbed increased from 0 to 65 gmol g-1. F r o m these data, the above-mentioned hypothesis was corroborated, i.e. the amount of protein adsorbed at a given p H is largely determined by the surface hydrophilicity. Hence, it should be concluded that hydrophobic interactions are of major importance in controlling the adsorption of proteins to solid surfaces.
4. C o n c l u s i o n s
3.4. Contact angle determinations
From contact angle determinations of bare and phosphated zirconium oxide powders (Table 2), it was concluded that phosphate adsorption not only affected the surface charge characteristics, but also the surface hydrophilicity of the powdered oxide.
Phosphate ions have been shown to interact strongly with powdered zirconium oxide. The adsorption was shown to affect both the surface charge properties and the hydrophilicity of the surface of the oxide. Prephosphatation of the powdered oxide has been shown to significantly
88
Bruno Putman et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 121 (1997) 81-88
affect the subsequent adsorption of bovine serum albumin. Hence, protein adsorption may be controlled. In our opinion, the observed phenomena are of the utmost importance in membrane technology. The results provide a fundamental explanation of the experimentally observed improvement of the ultra-filtration flux of ovalbumin solutions on ceramic zirconium oxide membranes in the presence of phosphate ions.
Acknowledgment Professor G. Buckton, School of Pharmacy, University of London, is gratefully acknowledged for allowing us to perform the contact angle determinations in his laboratory. Thanks are also due to Dr. Ir. R. Leysen and Ing. W. Doyen for providing us with the zirconium oxide samples, and for stimulating discussions concerning their properties. P.V.d.M. is a Senior Research Associate of the Belgian Fund for Scientific Research (NFWO).
References [1] W. Norde, Adv. Colloid Interface Sci., 25 (1986) 341. [2] T. Arai and W. Norde, Colloids Surfaces, 51 (1990) 1. [3] W.R. Bowen and D.T. Hughes, J. Membrane Sci., 51 (1990) 189. [4] W. Norde, J. Dispersion Sci. Technol., 13 (1992) 363. [5] A. Kondo and K. Higashitani, J. Colloid Interface Sci., 150 (1992) 344.
[6] M. Gille and E. Staude, Biotechnol. Bioeng., 44 (1994) 557. [7] R.A. Williams and H.W. Blanch, Biosensors Bioelectron., 9 (1994) 159. [8] K. Yoshinaga, A. Kondo, K. Higashitani and T. Kito, Colloids Surfaces A: Physicochem. Eng. Aspects, 7 (1993) 101. [9] R.M. McDonogh, H. Bauser, N. Stroh and H. Chmiel, Desalination, 79 (1990) 217. [10] J.L. Nilsson, J. Membrane Sci., 52 (1990) 121. [11] W.R. Bowen and Q. Gan, Biotechnol. Bioeng., 38 (1991) 688. [12] S. Dumon and H. Barnier, J. Membrane Sci., 74 (1992) 289. [13] K.B. Hvid, P.S. Nielsen and F.F. Stengaard, J. Membrane Sci., 53 (1990) 189. [14] C.P.G.H. Schroen, M.C. Wijers, M.A. Cohen-Stuart, A. Van der Padt and K. Van't Riet, J. Membrane Sci., 80 (1993) 265. [15] R.H. Li and T.A. Barbari, J. Membrane Sci., 88 (1994) 115. [16] K.J. Kim and A.G. Fane, J. Membrane Sci., 99 (1995) 149. [17] K.C. Scheel, Z. Anal. Chem., 105 (1936) 256. [18] G.R. Schaterle and R.L. Pollack, Anal. Biochem., 51 (1973) 654. [19] W. Stumm and J.J. Morgan, Aquatic Chemistry, 2nd ed., Wiley, New York, 1981. [20] D.R. Vissers, J. Phys. Chem., 72 (1968) 3236. [21] J.W. Bowden, S. Nagarajah, N.J. Barrow, A.M. Posner and J.P. Quirk, Australian J. Soil Res., 18 (1980) 49. [22] N.J. Barrow, Adv. Agronomy, 38 (1985) 183. [23] W. Stumm, R. Kummert and L. Sigg, Croatica Chemica Acta, 53 (1980) 291. [24] M. Nanzyo and Y. Watanabe, Soil Sci. Plant Nutrition, 28 (1982) 359. [25] G.A. Parks, Chem. Rev., 65 (1965) 177. [26] M. Nishida, A. Ookubo, Y. Hashimura, A. Ikawada, Y. Yoshimura, K. Ooi, T. Suzuki, Y. Tomita and J. Kawada, J. Pharmaceut. Sci., 81 (1992) 828. [27]J.I. Anzai, B. Guo and T. Osa, Bioelectrochem. Bioenerget., 40 (1996) 35.
Colloids and Surfaces A: ehysicochemicaland Engineering Aspects 121 (1997) 81-88
A
SURFACES
Reduced bovine serum albumin adsorption by prephosphatation of powdered zirconium oxide Bruno Putman, Paul Van der Meeren *, Danny Thierens University of Gent, Faculty of Agricultural and Applied Biological Sciences, Laboratory of Applied Physical Chemistry, Coupure Links 653, B-9000 Gent, Belgium Received 4 July 1996; accepted 23 November 1996
Abstract
The adsorption of phosphate anions to ZrO2 and its influence on protein adsorption have been studied. Phosphate adsorption experiments revealed that phosphate anions had a high affinity for zirconia: in appropriate conditions, a monomolecular layer was adsorbed. Using the electrophoretic light scattering technique, it was shown that the isoelectric point of the zirconium oxide was shifted from pH 6.4 down to pH 3.8. From this shift, it was concluded that specific interactions were responsible for the adsorption behaviour. In addition, the adsorption of bovine serum albumin (which was selected as a model protein) to both bare and phosphated zirconia was investigated: prephosphatation reduced the BSA adsorption by over 50%. Comparing the adsorption behaviour at pH 4.15 and 5.35, it was concluded that electrostatic interactions were of minor importance. From contact angle measurements, the different adsorption behaviour was shown to be due to the increased hydrophilicity of the phosphated adsorbent. On the basis of the present results, the observed effect of phosphate ions in ultrafiltration of proteins on ZrO2 ceramic ultrafiltration membranes may be explained.
Keywords: Adsorption; Contact angle; Electrophoretic mobility; Fouling
1. Introduction
Protein adsorption to solid surfaces is an intensively studied phenomenon [1-5]. Several applications have been designed to apply this adsorption behaviour. The immobilization of enzymes [6], uses in biosensor applications [7] and the use of protein-coated particles in immunological agglutination tests [8] are only a few examples to illustrate the widely different applications of the protein adsorption phenomenon. In membrane-filtration technology, however, protein adsorption is giving rise to serious prob* Corresponding author. Tel.: +32 9 2646001; Fax: +32 9 2646242. 0927-7757/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S0927-7757 (96) 03978-7
lems [9-12]. The fouling of the filter membranes is often due to this adsorption, thus affecting the hydraulic permeability and the rejection properties of the membrane. Trying to prevent protein deposition on these membranes is an important goal for researchers in this area. An important tool in achieving this is the modification of the filtration membrane. Chemical modification of polymer membranes has been proved to be a powerful method for improving the membrane's properties in terms of hydraulic and rejection properties [13-16]. However, very little information is available about the modification of ceramic membranes, used for the ultra-filtration of protein solutions. D u m o n and Barnier [12] observed that a distinctively better permeate flux
82
Bruno Putman et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 121 (1997) 81 88
was obtained by adding a phosphate buffer to an ovalbumin solution before filtering it through a zirconium oxide membrane. They proved by IR spectroscopy that the surface of the zirconium oxide filter was modified due to phosphate adsorption. In the present study, phosphate adsorption to zirconium oxide is investigated in more detail. The surface modifications of the phosphated powder obtained were detected by ~-potential measurements. Also, the adsorption of bovine serum albumin to bare and to phosphated zirconia powders was compared. The influence of the zirconium oxide and protein surface-charge characteristics on the adsorption behaviour of BSA was investigated.
2. Materials and methods
phosphate solution was adjusted by adding small volumes of either a 0 . 2 N or a 0 . 5 N N a O H solution. Following 48 h of equilibration, the dispersion was filtered on a 0.45 lam mixed cellulose esters filter membrane (Millipore). Subsequently, the filter cake was washed five times with 50 ml of distilled water in order to remove the free phosphate ions from the interstitial pores of the oxide. In the last washing liquid, no phosphate could be detected in the eluate. After vacuum-drying the powder for 2 h at 26 mbar, the adsorbed phosphate was desorbed by boiling 100 mg of the dry powder for 20 min in 20 ml 20% H z S O 4. Subsequently, 20ml of water was added, the mixture was boiled again for 20 min and then transferred to a 100ml volumetric flask. The amount of phosphate adsorbed was calculated from the phosphate content of the clear supernatant.
2.1. Materials
2.3. Protein adsorption experiments
Powdered zirconium oxide (ZrO2) was supplied by Magneson Electron. The specific area of this adsorbent as determined by BET Nz-adsorption amounted to 21 m 2 g - 1. The bovine serum albumin (BSA) was purchased from Sigma (St. Louis, USA). Potassium dihydrogen phosphate (KHzPO4) was from Merck.
Experiments on the adsorption of the BSA protein to zirconium oxide were performed by adding increasing amounts of the protein in 10ml of distilled water or 10mM acetate buffer (pH 4.14 and 5.35) to 50 mg ZrO2 powder. After shaking the suspension for 2 h at 20°C, it was centrifuged for 10 min at 5000g. The protein content of the supernatant was determined by the method of Schaterle and Pollack [18].
2.2. Phosphate adsorption experiments
In the first experiment, phosphate adsorption was accomplished by adding increasing amounts of KHzPO 4 in 30 ml of distilled water to 200 mg ZrO 2 powder. This mixture was shaken at 20°C for at least 48 h at 160 rpm. The amount adsorbed was calculated by difference from the concentration of the added phosphate solution and the equilibrium phosphate concentration of the clear supernatant, obtained by centrifugation of the dispersed zirconia for 10min at 5000g. Quantitative phosphate determination was made according to the method proposed by Scheel [17]. The pH of the equilibrium solutions was 5.9_+ 0.2. Alternatively, 500 mg of powdered zirconia was dispersed in 30ml of a 0.1 M phosphoric acid solution. Hence, the phosphate-to-ZrO2 ratio was kept constant in these experiments. The pH of the
2.4. Electrophoretic light scattering measurements
The ~ potentials were determined by a Malvern Zetasizer IIc. The samples were prepared by dispersing some mg of zirconia in 50 ml of a 0.01 N potassium chloride solution using a bath sonicator. The pH was adjusted by addition of either diluted HC1 or NaOH. All measurements were carried out at 20°C. 2.5. Contact angle determinations
The advancing contact angle measurements were performed with a DCA 322 Dynamic Contact Angle Analyser (Cahn) operating at 264 ~tm s 1 Sample plates were prepared by coating a micro-
83
Bruno Putman et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 121 (1997) 81-88
scope cover glass with the powdered zirconia using adhesive Spray Mount (3M).
3. Results and discussion 3.1. Adsorption o f phosphate on zirconium oxide
The adsorption of phosphate ions to powdered zirconium oxide was investigated at 20°C. Fig. 1 reveals that the experimental adsorption data were satisfactorily described by the Langmuir equation describing monolayer adsorption. Although the conditions which form the basis of the theoretical Langmuir model, such as the independency of the energy of adsorption from the surface coverage, are actually not fully fulfilled in the case of the adsorption of ions, but this model is still widely used to estimate the adsorption maximum [19]. Linear regression of the ratio of the equilibrium phosphate concentration to the adsorbed amount as a function of the equilibrium phosphate concentration revealed that the phosphate adsorption maximum /"max amounted to
66.8 ~tmol P g - 1 ZrO2; the correlation coefficient was 0.991. As Vissers [20] demonstrated, in the conditions used in our experiments, a phosphate molecule covered a zirconium-oxide surface of about 40 A2. It follows that about 16m 2 g-1 of the zirconium oxide is coated by the phosphate groups at the estimated value of the adsorption maximum. As the specific area of the powder was about 21 m 2 g - l , it follows that the surface of the powder is almost completely covered. From this first experiment, it follows that zirconium oxide has a high affinity for phosphate ions. For the sake of completeness, it should be mentioned that the pH of all equilibrium solutions was within the range 5.9_+0.2. According to Bowden et al. [21] and Barrow [22], this behaviour is due to the combined effect of the release of one surface hydroxyl group per HzPO 4 ion adsorbed, followed by the release of one proton from the adsorbed phosphate group due to the decrease of its pK values upon adsorption [23,24]. Subsequently, the phosphate equilibrium solution was decanted and replaced by 30 ml of distilled water. From the phosphate concentration of the
O~
"""
8
B
4o 30
i 20JII l
~
= adsorpl~on -
Langmuir fit
0 0
0.5
1
1.5
2
EQUILIBRIUM CONCENTRATION (mmol/I) Fig. 1. Adsorption isotherm of phosphate to powdered ZrO2 at 20°C. The amount of phosphate adsorbed was determined before ( • ) and after (~) the phosphated powder was washed with distilled water. Each experimentwas performedin duplicate. The smooth curve corresponds to the best-fitting Langmuir adsorption isotherm.
Bruno Putman et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 121 (1997) 81-88
84
supernatant after 48h of equilibration, the desorbed amount of phosphate was calculated. Here, a correction was introduced to take account of the free phosphate ions which were retained within the interstitial pores of the powder upon decantation. Fig. 1 reveals that the phosphate adsorption was highly irreversible, especially at lower phosphate loads. In a second experiment, we tried to increase the adsorbed amount further by changing the experimental conditions. First, the concentration of the phosphate solution was increased significantly (up to 0.1 M). Also, the pH of the phosphate solutions was controlled by addition of NaOH, because it is well known that the specific adsorption of anions is promoted at low pH [21,23]. From the results of these experiments, summarised in Table 1, it can be seen that similar values of the adsorbed amount were found using acidic phosphate solutions. As predicted by the four-layer phosphate adsorption model of Bowden et al. [21,22], the adsorption was largely reduced when the pH was larger than the pK2 value of phosphoric acid. According to Bowden et al. [21], this reduced adsorption is due to the combined effect of a decreased amount of HzPO4-, which is considered to be the most reactive phosphate species, and an increased electrostatic repulsion between the surface of the oxide and the phosphate anions. Table 1 reveals that the estimation of the adsorption maximum at a pH of about 5.9, as obtained from the Langmuir adsorption isotherm fitting to the data of the above-mentioned adsorption experiment, was slightly underestimated. Adding an excess of phosphate anions, about 80 ~tmol of phosphate was adsorbed per gram of Z r O 2 , whereas an adsorption maximum of only 68 ~tmol g 1 was estimated by extrapolation of the data obtained at low phosphate concentrations. From the projected Table 1 Phosphate adsorption to powdered zirconium oxide. The a m o u n t of phosphate adsorbed to zirconium oxide (X) was determined as a function of the pH; 30 ml of a 0.1 M phosphoric acid solution, the p H of which was adjusted by addition of N a O H , was added to 500 mg of powdered ZrO 2 pH X(gmol g-l)
1.54 89
2.09 83
2.47 91
3.73 86
6.12 79
8.08 61
area of the phosphate ions, it follows that 19 m 2 g-1 is phosphated. On the basis of the results of the phosphate adsorption experiments, all phosphated zirconia samples which were used for further research were prepared by equilibrating in 0.1 M phosphoric acid for at least two days, followed by the removal of the phosphate excess by washing with distilled water.
3.2. Electrokinetic charge characteristics of phosphated zirconia In order to check the influence of phosphate adsorption on the properties of the zirconia, the electrophoretic mobility of bare and phosphated zirconium oxide in 0.01 N KC1 was determined as a function of the pH. From Fig. 2, it follows that the surface charge characteristics of the zirconia were significantly affected by phosphate adsorption: phosphatation decreased the value of the electrophoretic mobility of the zirconium oxide. Hence, highly negative values were obtained at neutral pH. As a further consequence, the IEP was lowered by phosphatation. From Fig. 2, it follows that the isoelectric point (IEP) of zirconium oxide was approximately 6.35, which is in close agreement with the data of Parks [25]. As the amount of phosphate adsorbed was increased, the IEP decreased to about 4.5 at the highest value under investigation. From this observation, it follows that the phosphate ions are sorbed by a specific adsorption mechanism. The experimental data can be explained by the ligand-exchange model [21-23]: upon phosphate adsorption, surface hydroxyl ions are replaced by H2PO 4 anions. Upon adsorption, the pK value of the phosphate ions is decreased [23,24], so that a proton is set free. As a consequence, phosphate adsorption renders the metal oxide surface more negatively charged, and hence the IEP is reduced. The increase of the adsorbed amount when the powdered zirconia was first equilibrated in a 0.1 M phosphate solution also became obvious from the determination of the electrokinetic properties of the phosphated zirconia samples. From Fig. 3, very similar ~-potential characteristics were observed for the three samples which had been
Bruno Putman et aL / Colloids Surfaces A." Physicochem. Eng. Aspects 121 (1997) 81-88
85
..--. 4 '7,
8
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2
:::1,,
1
-1 -2 -3 -4
|
I
2
I
|
3
T--
I
4
I
I
5
6
pH Fig. 2. Electrophoretic mobility of powdered zirconium oxide in 0.01 M KCI as a function of the amount of phosphate adsorbed.
3
fJ
0
-1
"
'
-3
-4
A
pH 6.12
x
pH 8.08
v
bare ZrO2 ,
2
,
, 4
~
, 5
, 6
7
pH Fig. 3. Electrophoretic mobility spectra of phosphated zirconium oxide in 0.0l M KC1 as a function of the pH of the 0.1 M phosphatation solution.
86
Bruno Putman et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 121 (1997) 81 88
phosphated at acid pH, characterised by an isoelectric point of about 3.7. As compared to the sample with the highest phosphate content of the previous adsorption experiment, this represents a further decrease of the isoelectric point by about 0.8 pH units. The close resemblance of the i-potential spectra indicates that these samples contained a comparable amount of adsorbed phosphate. Actually, the differences of the adsorbed amount of phosphate to zirconia up to pH 3.73 (shown in Table 1) should primarily be ascribed to experimental error. The samples which were treated at pH 6.12, and especially at pH 8.08, on the other hand, had significantly different ~-potential characteristics (Fig. 3), from which it may be concluded that different amounts of phosphate had been adsorbed. Hence, it follows that the electrokinetic measurements (Fig. 3) fully support the estimated values of the adsorbed amount of phosphate (Table 1). 3.3. Adsorption o f B S A on phosphated zirconium oxide
The adsorption behaviour of proteins to both bare and phosphated zirconia was compared. Bovine serum albumin (BSA) was selected as a model protein because it is commercially available in high purity. The adsorption experiments were performed in 10mM acetate buffer solutions; pH 4.15 and 5.35 were selected because they are quite symmetrical about the isoelectric point of BSA, which is located at pH 4.8 according to Kondo and Higashitani [5]. As it was shown experimentally that the change of the pH during the adsorption experiments amounted to 0.1 at the most, it can be concluded that the buffering capacity of the buffers used was sufficiently high. From Fig. 4, the widely different adsorption behaviour of the bare and the phosphated zirconia becomes immediately obvious: much less protein is adsorbed to the phosphated zirconia surface. In addition, Fig. 4 reveals that the adsorption of the protein was pH-dependent. From the results of this experiment (Fig. 4), a very strong adsorption of the protein to the bare zirconia surface was observed: at the lowest protein concentration considered, about 70-80% of the
BSA added was adsorbed. According to Norde [1], this pronounced adsorption behaviour is typical for a soft protein, such as BSA, at low ionic strength. Also, Fig. 4 reveals that the adsorbed amount does not tend to a maximum; instead, the adsorbed amount keeps on increasing as the added protein concentration is increased. This is also known to be a feature of soft-protein adsorption [1]. Comparing the protein adsorption on bare and phosphated zirconium oxide, highly significantly differences were noticed. At the same initial protein concentration, the treated zirconia adsorbed over 50% less protein. These results are in agreement with the ultra-filtration flux observations of Dumon and Barnier [12]: following the treatment of ceramic zirconia membranes with phosphate solutions, much higher permeate fluxes were obtained during the ultra-filtration of ovalbumin. The latter should be ascribed to a decreased fouling tendency, thanks to a lower adsorption affinity. Similarly, Nishida et al. [26] found that the adsorption of BSA to aluminium hydroxide was reduced in the presence of phosphate. Comparing the adsorption behaviour of BSA onto bare and phosphated zirconia samples at pH 4.15 and 5.35, it follows that electrostatic interaction does not suffice to interpret the experimental results: from an electrostatic point of view, protein adsorption should be maximised on the bare zirconia at pH 5.35, since the protein and the adsorbent have an opposite charge, whereas phosphated zirconia should be the best adsorbent at pH 4.15. In the latter case, both the protein and the bare zirconia are positively charged, whereas the phosphated zirconia surface is negatively charged. Also, electrostatic interactions would favour BSA adsorption to phosphated zirconia at pH 4.15 as compared to pH 5.35. As these expectations have to be rejected on the basis of the experimental data of Fig. 4, it follows that protein-surface electrostatic interactions play only a minor role in the adsorption process. According to Norde [1], the maximum adsorption is usually found around the isoelectric point of the soft protein, irrespective of the IEP of the adsorbent, because of structural rearrangements in the adsorbing proteins at either side of the protein's IEP. Hence, it follows that the pH dependence of the adsorption phenomenon
87
Bruno Putman et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 121 (1997) 81-88 35 pH 4.15
g 30O
o.o.o°-
pH 5.35
.
25 ,.]
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.--+
.4.- -°
_.. ......
a
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0
i
0
16o
i
36o
i
Zr02
phosphated
36o
ZrO2 i
400
EQUILIBRIUM CONCENTRATION (rag/I) Fig. 4. The adsorption of bovine serum albumin to bare ZrO2 (dotted lines) and to phosphated ZrO2 (solid lines) in 10 mM acetate buffer at pH 4.15 and 5.35, respectively.
is primarily determined by the properties of the protein, and that protein protein interactions and structural rearrangements in the protein molecules are of utmost importance. The magnitude of the adsorption phenomenon, however, is highly affected by the nature of the adsorbent. Thus, Anzai et al. [27] observed that BSA adsorbs more efficiently to hydrophobic than to hydrophilic surfaces. According to these authors, the electrostatic force of attraction or repulsion between the BSA molecules and the surface does not play a primary role in determining the adsorption behaviour of the protein. Also, it is well known that protein adsorption to ultra-filtration membranes, with the concomitant filtrate-flux decline, is strongly reduced by rendering the surface more hydrophilic [13-16]. Based on these data, the decreased BSA adsorption to the phosphated zirconia samples most probably originates from an increased hydrophilicity of the surface of the powder.
Table 2 Contact angles of phosphated zirconia. The advancing contact angle (O,) of powdered zirconium oxide was determined as a function of the amount of phosphate adsorbed (it) X (gmol g-l) Oa (°)
0 59
10 54
38 49
68 44
Table 2 indicates that the contact angle decreased from 59 ° to 44 ° as the amount of phosphate adsorbed increased from 0 to 65 gmol g-1. F r o m these data, the above-mentioned hypothesis was corroborated, i.e. the amount of protein adsorbed at a given p H is largely determined by the surface hydrophilicity. Hence, it should be concluded that hydrophobic interactions are of major importance in controlling the adsorption of proteins to solid surfaces.
4. C o n c l u s i o n s
3.4. Contact angle determinations
From contact angle determinations of bare and phosphated zirconium oxide powders (Table 2), it was concluded that phosphate adsorption not only affected the surface charge characteristics, but also the surface hydrophilicity of the powdered oxide.
Phosphate ions have been shown to interact strongly with powdered zirconium oxide. The adsorption was shown to affect both the surface charge properties and the hydrophilicity of the surface of the oxide. Prephosphatation of the powdered oxide has been shown to significantly
88
Bruno Putman et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 121 (1997) 81-88
affect the subsequent adsorption of bovine serum albumin. Hence, protein adsorption may be controlled. In our opinion, the observed phenomena are of the utmost importance in membrane technology. The results provide a fundamental explanation of the experimentally observed improvement of the ultra-filtration flux of ovalbumin solutions on ceramic zirconium oxide membranes in the presence of phosphate ions.
Acknowledgment Professor G. Buckton, School of Pharmacy, University of London, is gratefully acknowledged for allowing us to perform the contact angle determinations in his laboratory. Thanks are also due to Dr. Ir. R. Leysen and Ing. W. Doyen for providing us with the zirconium oxide samples, and for stimulating discussions concerning their properties. P.V.d.M. is a Senior Research Associate of the Belgian Fund for Scientific Research (NFWO).
References [1] W. Norde, Adv. Colloid Interface Sci., 25 (1986) 341. [2] T. Arai and W. Norde, Colloids Surfaces, 51 (1990) 1. [3] W.R. Bowen and D.T. Hughes, J. Membrane Sci., 51 (1990) 189. [4] W. Norde, J. Dispersion Sci. Technol., 13 (1992) 363. [5] A. Kondo and K. Higashitani, J. Colloid Interface Sci., 150 (1992) 344.
[6] M. Gille and E. Staude, Biotechnol. Bioeng., 44 (1994) 557. [7] R.A. Williams and H.W. Blanch, Biosensors Bioelectron., 9 (1994) 159. [8] K. Yoshinaga, A. Kondo, K. Higashitani and T. Kito, Colloids Surfaces A: Physicochem. Eng. Aspects, 7 (1993) 101. [9] R.M. McDonogh, H. Bauser, N. Stroh and H. Chmiel, Desalination, 79 (1990) 217. [10] J.L. Nilsson, J. Membrane Sci., 52 (1990) 121. [11] W.R. Bowen and Q. Gan, Biotechnol. Bioeng., 38 (1991) 688. [12] S. Dumon and H. Barnier, J. Membrane Sci., 74 (1992) 289. [13] K.B. Hvid, P.S. Nielsen and F.F. Stengaard, J. Membrane Sci., 53 (1990) 189. [14] C.P.G.H. Schroen, M.C. Wijers, M.A. Cohen-Stuart, A. Van der Padt and K. Van't Riet, J. Membrane Sci., 80 (1993) 265. [15] R.H. Li and T.A. Barbari, J. Membrane Sci., 88 (1994) 115. [16] K.J. Kim and A.G. Fane, J. Membrane Sci., 99 (1995) 149. [17] K.C. Scheel, Z. Anal. Chem., 105 (1936) 256. [18] G.R. Schaterle and R.L. Pollack, Anal. Biochem., 51 (1973) 654. [19] W. Stumm and J.J. Morgan, Aquatic Chemistry, 2nd ed., Wiley, New York, 1981. [20] D.R. Vissers, J. Phys. Chem., 72 (1968) 3236. [21] J.W. Bowden, S. Nagarajah, N.J. Barrow, A.M. Posner and J.P. Quirk, Australian J. Soil Res., 18 (1980) 49. [22] N.J. Barrow, Adv. Agronomy, 38 (1985) 183. [23] W. Stumm, R. Kummert and L. Sigg, Croatica Chemica Acta, 53 (1980) 291. [24] M. Nanzyo and Y. Watanabe, Soil Sci. Plant Nutrition, 28 (1982) 359. [25] G.A. Parks, Chem. Rev., 65 (1965) 177. [26] M. Nishida, A. Ookubo, Y. Hashimura, A. Ikawada, Y. Yoshimura, K. Ooi, T. Suzuki, Y. Tomita and J. Kawada, J. Pharmaceut. Sci., 81 (1992) 828. [27]J.I. Anzai, B. Guo and T. Osa, Bioelectrochem. Bioenerget., 40 (1996) 35.