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Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents
CLAY-02888; No of Pages 8 Applied Clay Science xxx (2014) xxx–xxx
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Research paper
Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents R. Duarte-Silva a, M.A. Villa-García b,⁎, M. Rendueles a, M. Díaz a a b
Department of Chemical Engineering and Environmental Technology, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain Department of Organic and Inorganic Chemistry, University of Oviedo, Julian Clavería 8, 33006 Oviedo, Spain
a r t i c l e
i n f o
Article history: Received 27 June 2011 Received in revised form 12 December 2012 Accepted 22 December 2013 Available online xxxx Keywords: Kaolinite Metakaolinite Organo–kaolinite hybrid material Structural properties Textural characteristics Whey proteins adsorption
a b s t r a c t The structural, textural and protein adsorption properties of kaolinite from clay sedimentary deposits, metakaolinite obtained by thermal dehydroxylation of kaolinite, and the organic derivative prepared by reacting kaolinite with the silane coupling agent tert-butyldimethylchlorosilane, were studied. The retention capacities for the proteins α-lactalbumin (A-LA), bovine serum albumin (BSA) and β-lactoglobulin (B-LG) and the nature of the interactions responsible for protein binding were studied by adsorption experiments, performed at room temperature and pH 5.0. The protein adsorption capacity and the selectivity show a clear dependence on the chemical nature of the adsorbents surface and on the textural properties. Kaolinite behaves as a strong adsorbent for A-LA and BSA, and exhibits a very high affinity for B-LG. Metakaolinite shows good retention capacity for A-LA and B-LG, but does not retain significant amounts of BSA. The adsorption capacity of the organo–kaolinite hybrid considerably increases for BSA and A-LA. FTIR results indicate the absence of hydrogen bonding between the adsorbents surface and the polypeptides. The interactions responsible for protein binding are closely related to the hydrophilic or hydrophobic character of the adsorbent surface and the amino acid composition of the proteins, steric effects also should be considered for the adsorption patterns. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The interactions of peptides and proteins with clay surfaces have been studied extensively in the past (Bujdák and Rode, 1997; Bujdák et al., 1996; Causserand et al., 1997; Ding and Henrichs, 2002; Fusi et al., 1989; Gupta et al., 1983; Quiquampoix et al., 1993, 1995; Rigou et al., 2006; Violante et al., 1995; Yu et al., 2000). Since clays are very abundant, their use as inorganic hosts is important not only from an economic perspective, but also for their unique physical and chemical properties, and the ease with which these materials can be modified and adjusted to new uses. Kaolinite has a wide variety of applications in industry (Bergaya and Lagaly, 2001; Bergaya et al., 2000; Braggs et al., 2006). This mineral has two different basal cleavage faces. One face consists of a tetrahedral siloxane surface with inert –Si–O–Si– links; the other basal surface consists of an octahedral gibbsite (Al(OH)3) sheet (Frost et al., 2002a, 2002b). Kaolinite has different surface structures between base planes (001) and edge planes (110) and (010). The charge on the edges is due to the protonation/deprotonation of hydroxyl groups and depends on the pH of the solution. The hydroxyl groups located at the edge planes are considered the major reactive sites of kaolinite surfaces (Rausell-Colom and Serratosa, 1987).
⁎ Corresponding author. Tel.: +34 985102976; fax: +34 985103446. E-mail address: [email protected] (M.A. Villa-García).
Kaolinite surface is hydrophilic but it can be rendered hydrophobic by reaction with organo–functional molecules (Dai and Huang, 1999). Organic derivatives of clays are generally obtained by using silane coupling agents (Dai and Huang, 1999; Ishida and Miller, 1985; Waddell et al., 1981). After surface modification, the organic groups can be attached to the clay by chemical bonding, adsorption and coating. Kaolinite surface can also be modified by thermal treatment. At temperatures higher than 450 °C dehydroxylation occurs to form metakaolinite (Al2O3 · 2SiO2), and at 650 °C dehydroxylation is by ca 90% complete (Grim, 1968). The dairy industry generates many by-products with high protein contents. These residues can cause severe environmental contamination when they are not properly disposed, and it is necessary to find solutions to prevent this pollution problem. Furthermore, the recovered proteins can be used to obtain high quality protein rich food products. Protein recovery by adsorption on various types of supports is a commonly used technique; however, the use of clay minerals as adsorbents of protein molecules has received considerably less attention. The adsorption/desorption of proteins on clay surfaces is a complex process controlled by different factors, such as the surface properties of the adsorbent, the structural stability of the proteins, the ionic strength and the pH of the adsorption/desorption experiments (Haynes and Norde, 1994). In a recent work we found that kaolinite showed a high adsorption capacity at the isoelectric point (IEP) of each protein (Barral et al., 2008). Moreover, there was a clear correlation between the adsorption patterns and the presence of hydrophobic or hydrophilic
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Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027
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groups in the polypeptide surface. In order to achieve a deeper insight on the nature of the interactions responsible for protein adsorption on clay surfaces and explain the retention patterns, adsorption experiments were run using as adsorbents kaolinite, metakaolinite obtained by thermal dehydroxylation of kaolinite, and the organic derivative obtained by reacting kaolinite with tert-butyldimethylchlorosilane. Three whey proteins were chosen for this study: BSA, B-LG and A-LA. Physico-chemical characterization of the adsorbents was carried out using different techniques: elemental chemical analysis, FTIR spectroscopy, X-ray diffraction (XRD), thermal gravimetric (TG) and differential thermal analysis (DTA), N2 adsorption–desorption at 77 K, mercury intrusion porosimetry and scanning electronic microscopy (SEM). Evidence of protein intercalation in kaolinite has not been reported yet. There was no significant expansion of the clay after protein binding, as shown by X-ray diffraction analysis. Kaolinite, a non swelling clay does not expand upon wetting, which prevents the exposure of internal surface, and adsorption of proteins is restricted to the external crystal surfaces and the edges of the clay (Fiorito et al., 2008; Venkateswerlu and Stotzky, 1992). As will be shown, the protein retention patterns are controlled by the textural properties and the chemical nature of the adsorbent surface. Steric effects should be also considered, since experimental data show a correlation between the extent of adsorption and protein sizes.
XRF spectrometer. Powder X-ray diffraction (XRD) patterns were obtained with a Philips X'Pert MPD Pro instrument-ray diffractometer, operating at 45 kV, 40 mA, using Cu Kα radiation. FTIR spectra of all the materials were obtained at room temperature with a Perkin-Elmer PARAGON 1000 spectrometer, by the KBr pellet technique. Thermal analysis, TG and DTA, were carried out in Metller TA-4000 TG-50 type thermobalance under dynamic heating conditions (10 °C/min heating rate) in a flowing nitrogen atmosphere of 99.999% purity, 50 mL/min. The samples were heated in alumina crucibles up to 1000 °C. Nitrogen adsorption–desorption isotherms at 77 °K were obtained with a Micromeritics ASAP 2020 instrument, using static adsorption procedures. Macroporosity and total pore volume were measured by mercury intrusion porosimetry with a Micromeritics Autopore IV instrument. The microscopic morphology of the samples was studied using a Jeol 6100 scanning electron microscope operating at 0.3–30 kV. Samples were prepared by sonicating the powdered sample in ethanol and then evaporating two droplets on carbonated copper grids. Protein concentrations in the initial solutions and the final supernatant obtained after the adsorption, were measured with the UV/VIS spectrophotometer (Heλios UV/VIS Thermo Electron Corporation) at 750 nm, according to the Folin–Ciocalteu colorimetric method (Lowry et al., 1951). A previous filtration through 0.45 μm polyvinylidene difluoride membrane syringe filters (Acrodisc) is required to retain the fine particles. 2.3. Adsorption experiments
2. Materials and methods 2.1. Materials The kaolinite mineral used in the experiments (sample K) was provided by Arcichamotas Mines in the North of Spain (Asturias). The clay was treated with H2O2 (Panreac) to eliminate organic matter present in the mineral, and then was washed thoroughly with distilled water. The solid was filtered, dried at 120 °C, ground and sieved. All the experiments were performed with kaolinite particles 100–250 μm in size. Tert-butyldimethylchlorosilane (reagent grade), imidazole (ACS reagent) and dichloromethane (ACS reagent) were purchased from Sigma. The whey proteins used were also supplied by Sigma: A-LA, type III, calcium depleted, 85% purity; B-LG, aprox. 90% PAGE purity; and BSA, fraction V, 99% purity. The sodium acetate buffer was purchased from Panreac. Metakaolinite (sample MK) was obtained from calcination of kaolinite. The clay mineral was heated in a ceramic crucible with a heating rate of 10 °C/min up to 650 °C and then was kept at that temperature for 12 h. Between 450 and 650 °C kaolinite undergoes a strong endothermic dehydration reaction, resulting in the conversion to metakaolinite. The organo–kaolinite composite (sample KR) was synthesized by reaction between tert-butyldimethylchlorosilane (TBSCl) and kaolinite in dichloromethane, in the presence of imidazole as a base catalyst. In a typical synthesis, 5 g of kaolinite was suspended in 50 mL of CH2Cl2 and 14 g of TBSCl and 6.5 g of imidazole were added, under vigorous stirring. The reaction mixture was sonicated for 48 h, the supernatant liquid was removed by decantation, the solid was thoroughly washed with dichloromethane to remove excess of organic species, and dried under vacuum to remove residual dichloromethane. The imidazolium chloride–kaolinite complex (sample KI) was obtained by bubbling hydrogen chloride, obtained by reaction of NaCl with H2SO4, through a solution containing 6.5 g of imidazole in 50 mL of CH2Cl2. Five g of kaolinite was added to the imidazolium chloride solution, and the reaction mixture was treated under the conditions described above for the preparation of the organo–clay solid. 2.2. Methods The chemical composition of the kaolinite was determined by X-ray fluorescence spectroscopy (XRF) using a Philips PW 2404
The protein adsorption experiments were carried out in batch mode. In all the experiments 0.5 g of the different samples was transferred to a 250 mL Erlenmeyer flask and suspended in 100 mL of protein solution buffered using a sodium acetate buffer (pH = 5.0). The suspensions were stirred at 25 °C (250 rpm) for 6 h in a New Brunswick Scientific Excella E25 incubator-shaker. Blank experiments were run in parallel under the same experimental conditions. Using a liquid/solid ratio of 200 mL g−1, which was previously established as the most convenient value, adsorption isotherms on kaolinite for each protein were obtained by changing the initial protein concentrations. Adsorption data of each protein on kaolinite and samples MK, KR and KI were obtained under the conditions above described, using protein concentration of 2 g L−1. Every protein adsorption experiment on the different adsorbents was repeated six times, being the values obtained very close, average values are shown in this work. Protein concentrations in the initial solutions and after adsorption were measured using the Folin–Ciocalteu colorimetric method (Lowry et al., 1951) with a UV/VIS spectrophotometer. 3. Results and discussion 3.1. Kaolinite chemical composition The chemical composition of the kaolinite mineral determined by X-ray fluorescence spectroscopy (XRF) was: 50.25% SiO2; 34.22% Al2O3; 0.94% TiO2; 0.97% Fe2O3; 0.03% CaO; 0.16% MgO; 0.04% Na2O; 1.14% K2O; 12.25% H2O. 3.2. Mineralogical composition The XRD powder pattern for kaolinite (Fig. 1A) shows the diffraction lines characteristic of a high crystal order mineral, diffraction lines corresponding to quartz, mica and feldspars are also present in the diffractogram indicating the presence of these minerals as impurities. The values of the (001) d-spacing corresponding to kaolinite and the conditioned one were 7.14 Å and 7.17 Å, respectively. Conditioning did not expand the kaolinite layers, but slightly increased the defects of the original low defect kaolinite. The sample KR (Fig. 1B) has a lower crystal order due to the presence of adsorbed organic species, or due to the presence of some smaller clay particles formed during the treatment with tert-butyldimethylchlorosilane. The (001) d-spacing of
Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027
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hydroxyl deformation vibrations are very sensitive to any modification of the hydroxyl surfaces. Kaolinite contains two types of hydroxyl groups: (i) hydroxyl groups located at the internal surface between adjacent layers, called inner surface hydroxyl groups; (ii) hydroxyl groups located in the inner (shared) plane of the octahedral sheet, often referred as inner hydroxyl groups. The inner surface groups are situated in the outer (unshared) plane, whereas the inner hydroxyl groups are within the unit cell and are not exposed at the surface of the kaolinite layers (Collins and Catlow, 1991; Frost et al., 2002a, 2002b; Hess and Saunders, 1992). The FTIR spectra of natural kaolinite (Fig. 2A), and the conditioned one, show bands at 3695 cm−1 and 3651 cm− 1 attributed to the hydroxyl-stretching modes of inner surface hydroxyls, the absorption at 3620 cm−1 was assigned to the hydroxyl-stretching modes of inner hydroxyl groups and the bands that appear at 940 cm− 1 and 913 cm− 1 were assigned to hydroxyldeformation modes of the inner surface and inner hydroxyls, respectively (Farmer, 1998; Frost and Van der Gaast, 1997). Modification of kaolinite surfaces either through intercalation, grafting of organic groups, or by thermal treatment, implies changes in the IR spectrum. No hydroxyls are spectroscopically evident in the spectra of metakaolinite (sample MK). The FTIR spectrum of this sample shows absorption bands at 1080 cm−1 (very strong and very broad) and 796 cm−1 attributed to the Si–O stretching vibrations of quartz (Saikia et al., 2008), and the bands that appear at 560 cm− 1 and 484 cm− 1 were assigned to Al–O stretching modes of alumina (Boumaza et al., 2009). Surface modification of clays also can be achieved by reaction with silane coupling agents. Generally, the coupling agent can be attached to the clay by chemical bonding, adsorption or coating. Molecules of the coupling agent coating on, or adsorbing to the clay surface, basically do not modify the IR vibrations, but when they are fixed on the mineral surface by chemical bonding, changes in the vibrational spectra occur. The FTIR spectrum of sample KR (Fig. 2B), shows that the band width and the peak position of the absorptions corresponding to hydroxylstretching and hydroxyl-deformation modes of the clay mineral do
Fig. 1. X-ray diffraction patterns of (A) kaolinite; (B) organo–kaolinite compound; (C) metakaolinite; (D) kaolinite–BSA complex.
this sample did not increase, indicating lack of expansion of the clay upon reaction with the silane, and hence lack of intercalation of the organic species. The diffraction pattern of metakaolinite (Fig. 1C) shows amorphous regions centered at 2θ values of approximately 23° and 36°. Characteristic major kaolinite peaks are absent at 2θ equal 12.4° and 25°, indicating a major loss of hydroxyl groups. The peaks at 20.7°, 26.5°, 50° and 59.8° correspond to quartz. Kaolinite undergoes a structural rearrangement upon heating, the calcination step causes the liberation of the hydroxyls in the form of water vapor and, with the collapse of the original triclinic cell, a more amorphous phase is formed. The diffractograms of the different adsorbents containing retained proteins (Fig. 1D) do not show a significant shift of the basal d-spacings to higher values for the protein–kaolinite complexes. This means that there was no expansion of the clay after protein binding, indicating that the protein molecules were not intercalated in the mineral structure, but were immobilized at the external surfaces and the edges of the adsorbents. 3.3. FTIR spectroscopy The modification of kaolinite surfaces can be easily studied through vibrational spectroscopy. In particular, the hydroxyl stretching and
Fig. 2. FTIR spectra of (A) kaolinite; (B) sample KR.
Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027
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not change, indicating that kaolinite hydroxyl groups did not react with the coupling agent. The spectrum of the organo–kaolinite complex does not show the silane methyl C–H stretching doublet at 2952 cm−1 and 2923 cm−1, a new band appears at 2980 cm− 1 and the band at 2853 cm−1 assigned to C–H stretching is broadened. These changes indicate grafting of organic groups on the kaolinite surface. Since the molecules of the coupling agent are bound to the clay, the strength of the Si–O bands should be increased. However, the Si–O vibrations of kaolinite, a band at 1095 cm− 1 and a doublet with maxima at 1032 and 1008 cm− 1 assigned to Si–O stretching modes (Bougeard et al., 2000), are very strong and it is difficult to obtain additional information of the changes caused by the interaction with the coupling agent. The spectrum also shows a peak at 3135 cm−1 that can be assigned to aromatic C–H stretch of imidazolium chloride formed during the reaction. The presence of this compound is confirmed by the broad band at 3420 cm−1 that can be assigned to aromatic N–H stretch, and a sharp band at 1585 cm− 1 that correspond to N–H bending vibrations. A plausible mechanism for the silylation process is depicted in Scheme 1. It involves the hydrolysis of tert-butyldimethylchlorosilane, in the presence of imidazole as a base catalyst, to form silanol species and imidazolium chloride. The silanols are attached to the kaolinite surface through hydrogen bonding. The formation of hydrogen bonding between the clay and the silanols is supported by the fact that when sample KR is washed with water or ethanol, the transmittance of the C–H vibrations decreases, whereas these bands remained unchanged after the sample was thoroughly washed with CH2Cl2. The presence of aromatic C–H and N–H stretch bands in the FTIR spectrum of sample KR suggests that some imidazolium chloride, formed during kaolinite silylation, is also attached to kaolinite surface. In order to elucidate the role of imidazolium chloride on protein adsorption behaviour of sample KR, kaolinite was reacted with imidazolium chloride (sample KI) under the same conditions used for preparation of sample KR. As will be shown the protein adsorption capacity of kaolinite is not modified when imidazolium chloride is attached to its surface. Ultrasounds were employed in this process because significant rate enhancements of chemical reactions have been described when heterogeneous reactions are carried out in the presence of sound waves (Bartoszewicz et al., 2008). The FTIR spectra of the kaolinite–protein complexes, Fig. 3(B) and (C) do not show any significant shift of the hydroxyl stretching and deformation bands corresponding to the spectrum of kaolinite, suggesting the absence of hydrogen bonding between adsorbed protein molecules and the surface OH groups of kaolinite. Moreover, the spectra of the three pure proteins show the characteristic bands of amide I at 1654 cm−1 (mainly C = O stretch) and amide II at 1542 cm−1 (C–N stretch coupled with N–H bending mode). The IR spectra of the
Fig. 3. FTIR spectra of (A) kaolinite; (B) kaolinite/BSA complex; (C) kaolinite/β-lactoglobulin complex.
adsorbed proteins show that the peak position of both bands does not change, indicating that kaolinite does not interact with C = O, C–N or N–H of the polypeptide chain and no hydrogen bond is formed. 3.4. Thermal analysis Thermogravimetric analysis of kaolinite shows the absence of any significant mass loss below 420 °C. At higher temperature a continuous mass loss (10.6%) due to kaolinite dehydroxylation is observed, that is completed at 800 °C. TG and DTA curves of sample KR are shown in Fig. 4. Thermal degradation of sample KR occurs in two steps. Below 180 °C there is not significant mass loss. With increasing temperature the elimination of the silane bonded to kaolinite begins and is terminated at 320 °C. The mass loss corresponding to this step is 45.6%. 3.5. Textural characterization
Scheme 1. Plausible mechanism for the silylation of kaolinite surface.
The porous structure of the separation media is a fundamental feature required to achieve good retention capacity. The use of materials with macropores favors a rapid mass transfer of macromolecules that improves the kinetics of retention. Moreover, large pore surfaces can accommodate protein molecules, while small pores are not accessible. Specific surface areas and mesopore size distributions were calculated
Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027
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Table 1 Textural parameters of the samples obtained from the nitrogen adsorption–desorption isotherms at 77 K. Sample
SBET (m2g−1)
Dpmax (nm)
Vp (cm3g−1 STP)
K MK KR
9 10 2
20.4 18.6 33.6
0.051 0.045 0.016
of the pores radii between 10 μm and 15 μm, the low surface area of this sample and the very large pore diameters indicate the formation of large inter particle cavities. Fig. 4. Thermogravimetric (TG) and differential TG (DTG) curves of sample KR.
3.6. Scanning electron microscopy from the nitrogen adsorption–desorption isotherms at 77 K. Sample MK is characterized by a type IV isotherm (Brunauer et al., 1940) with very narrow hysteresis loop type H3, which is usually associated to porous solids consisting of particle aggregates (Fig. 5). The low nitrogen uptake at relative pressures b0.2 indicates the absence of microporosity. The adsorption limit is not well defined at high relative pressures indicating the presence of macroporosity in the materials. The mesopore size distributions were analyzed using the method of BJH (Barret et al., 1951) applied to the adsorption band of the isotherm. BET specific surface areas (SBET), values of the most frequent pore diameters (Dpmax) obtained from the pore diameters distribution curves and the cumulative pore volume of each sample (Vp) are listed in Table 1. The specific surface area of kaolinite (9 m2g− 1) remains practically unaffected after dehydroxylation at 650 °C, (the BET specific surface area of MK is 10 m2g−1). The loss of hydroxyl groups favors the formation of smaller mesopores; the most frequent pore diameter decreases from 20.4 nm to 18.6 nm and the cumulative pore volume decreases as well (Table 1). Kaolinite reaction with the silane coupling agent considerably reduced the specific surface area to 2 m2g−1 for sample KR, as well as the pore volume, favoring the formation of wider mesopores (33.6 nm) (Table 1). Macroporosity of the samples was studied by mercury intrusion porosimetry. The pore size distribution profile of sample KR is shown in Fig. 6. The total pore volume are 0.476 mL g− 1 for samples K and MK, and 0.893 mL g−1 for sample KR that exhibits the lowest specific surface area but wider pores, which contribute more to volume than to the surface. Moreover, very large pore volumes indicate that the porosity is due to inter particle spacing, rather than pores within the structure. The macropore size distribution profile of sample K is unimodal with a maximum at 6 μm that corresponds to the most frequent pore diameters. The pore size distribution profile of sample MK shows a wider range of pore diameters with two maxima at 0.5 μm and 15 μm. Kaolinite dehydroxylation favors the formation of smaller pores, both in the mesopore and macropore range, besides a secondary pore system made of inter particle voids is created. The pore size distribution curve of sample KR (Fig. 6) shows several maxima, having most
Fig. 5. Nitrogen adsorption–desorption isotherm at 77 K of sample MK.
SEM micrographs of the samples are shown in Fig. 7. Sample K (Fig. 7A) consists of laminar particle aggregates. Sample MK (Fig. 7B) shows a less homogeneous microscopic topography and the presence of wide channel-like inter-particle voids formed during dehydroxylation. Sample KR (Fig. 7C), has a smoother surface and forms considerably larger particle aggregates with wide macropores and inter particle cavities.
3.7. Protein adsorption experiments The affinity of a protein for a given surface depends on the surface properties of the adsorbent, the composition and structural stability of the protein, the pH and the ionic strength of the solution (Haynes and Norde, 1994). Experimentally, maximum protein adsorption is observed at the isoelectric point of each protein (Barral et al., 2008; Haynes and Norde, 1994) because electrostatic repulsions between identically charged adsorbed proteins are minimized. Moreover, there is a non uniform distribution of ionic patches on the surface of the proteins that leads to electrostatic interactions between the patches and the adsorbent surface (Lee et al., 2002). However, an increase in electrostatic interactions is also accompanied by a reduction in the native structure; many proteins also adsorb with high affinity to hydrophobic surfaces. Adsorption of proteins onto hydrophilic (sample K), dehydroxylated (sample MK) and hydrophobic surfaces (samples KR and KI) may contribute to understanding the nature of the interactions responsible for protein retention. Adsorption experiments were performed at pH = 5.0, because this value is very close to the IEP of the three proteins. Table 2 lists the molecular weight and isoelectric point of each protein. Kaolinite suspension in distilled water has a pH of 3.8, resulting from proton dissociation of superficial hydroxyl groups and was buffered with sodium acetate at pH 5.0. The three proteins were readily adsorbed on kaolinite and maximal adsorption was reached after 2 h.
Fig. 6. Pore size distribution profile of sample KR, measured by mercury intrusion porosimetry.
Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027
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Fig. 8. Protein adsorption isotherms for (A) BSA; (B) A-LA; (C) B-LG.
Fig. 7. SEM images, 7500×, of (A) sample K; (B) sample MK; (C) sample KR.
The adsorption isotherms for the three proteins are depicted in Fig. 8. They show Langmuir characteristics; the protein uptake increases with protein concentration and finally reaches a plateau. The Langmuir isotherm assumes monolayer coverage of adsorbate over a homogeneous adsorbent surface; at equilibrium a saturation point is reached where no further adsorption/desorption occurs. The Langmuir equation is expressed as: q¼
qm c kd þ c
Where q is the equilibrium protein concentration on the adsorbent (mg g− 1), c is the concentration of the liquid phase at equilibrium (g L−1), qm is a constant related to the area occupied by a monolayer Table 2 Molecular weight and IEP of the proteins (Hambling et al., 1992). Protein
Molecular weight (kDa)
IEP
A-LA B-LG BSA
28.4 (dimer) 18.4 69
4.7 – 5.1 5.2 – 5.6 4.9
of adsorbate, reflecting the adsorption capacity and kd is a direct measure for the intensity of the adsorption process. Experimental data were fitted to the Langmuir equation, using the program Scientist (Micromath), to obtain the Langmuir parameters qm and kd (Table 3). Kaolinite adsorption capacity was different for each protein: 53 mg for BSA/g kaolinite, 108 mg for A-LA/g kaolinite and 540 mg for B-LG/g kaolinite. Proteins are made up of different amino acids with lateral chains containing hydrophobic or hydrophilic organic groups that can be negatively, neutrally or positively charged at pH 5.0. The chemical nature of the protein lateral chains and the electric surface charge of kaolinite will condition the nature of the interactions responsible for protein binding. To explain the different retention patterns a number of factors must be considered, such as the textural properties and the electric surface properties of the adsorbents, the amino acid composition, the IEP of each protein and the molecular sizes. BSA, which is the least retained protein (Table 3), contains a larger number of non-polar groups (253) that reach external positions, shielding the contact between the polar groups and the hydrophilic centres of the adsorbent, and thus minimizing the strength of the electrostatic interactions. Moreover, due to its large molecular size, steric effects should also be considered. The adsorption isotherms showed typical Langmuir characteristics, which involve monolayer coverage of the adsorbate over the adsorbent surface; this implies a clear correlation between the extent of the adsorption and protein sizes, being the largest protein (BSA, 69 kDa) the least adsorbed. The retention capacity for A-LA increases to 108 mg/g kaolinite. This protein is smaller (28.4 kDa) and contains only 44 non-polar groups. Finally, B-LG which is the smallest protein (18.4 kDa) shows the highest retention capacity, 540 mg/g kaolinite. This protein contains more non-polar groups (150 for the dimer) than A-LA, but under the adsorption conditions (pH 5.0, slightly below its IEP) there is a non uniform distribution of positive ionic patches on the surface of the protein that leads to an increase in the electrostatic attraction between the hydrophilic centres of the clay and the protein. To investigate the influence of the adsorbent surface properties on protein retention, adsorption experiments were run on kaolinite, dehydroxylated kaolinite (sample MK) and on the organo derivatives (samples KR and KI), using a liquid/solid ratio of 200 mLg−1 and protein concentrations of 2 g L−1. The results obtained are given in Table 4. Sample MK did not retain significant amounts of BSA (10 mg/g). Dehydroxylation involves elimination of the ionizable OH groups and
Table 3 Parameters of Langmuir isotherms. Protein
BSA
A-LA
B-LG
qm(mg protein/g kaolinite) Kd (g protein/L)
53 4.66
108 5.52
540 19.1
Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027
R. Duarte-Silva et al. / Applied Clay Science xxx (2014) xxx–xxx Table 4 Protein retention capacity (average values) of the different samples (mg/gsample), obtained using a liquid/solid ratio of 200 mL g−1 and protein concentrations of 2 g L−1. Protein
Kaolinite
Metakaolinite
Kaolinite-tertbutyl
A-LA B-LG BSA
98 370 43
176 135 10
159 316 125
decrease of the strength of the electrostatic interactions, which for BSA are minimized by the presence of non-polar groups. The adsorption of BSA on sample KR (125 mg/g) was increased by a factor of three with respect to the adsorption capacity shown by natural kaolinite (43 mg/ g sample). Treatment with the silane coupling agent considerably reduced the BET surface area, but created pores in the micrometer range that are accessible to large bio-molecules. Furthermore, the presence of hydrophobic groups in the sorbent surface leads to strong hydrophobic interactions with the protein, yielding high affinity for BSA for this sample. A-LA adsorption on sample MK (176 mg/g) is 56% higher than on sample K. Thermal treatment of kaolinite increased the specific surface area by 1 m2 g−1, but the total volume per unit mass did not change. However, a secondary pore system made of inter particle voids was created and A-LA is small enough to easily access the pores in the micrometer range, which leads to an increased retention capacity. A-LA adsorption on sample KR (159 mg/g) is smaller than on sample MK, probably due to the low specific surface area of this material. Adsorption of B-LG on sample KR is very high (316 mg/g sample), being 15% lower than on sample K (370 mg/g). This can be explained considering the low BET surface area of sample KR. Adsorption of B-LG on metakaolinite (135 mg/g sample) is considerably lower, probably due to a decrease in the electrostatic attraction forces between the metakaolinite surface and the protein. Protein adsorption experiments were also performed on the imidazolium chloride–kaolinite complex. Imidazolium chloride adsorbed on the kaolinite surface is readily dissolved in the buffer solution containing the proteins, and does not modify the clay retention capacity.
4. Conclusions Kaolinite obtained from clay sedimentary deposits was a strong adsorbent for α-lactalbumin and bovine serum albumin, at pH 5.0 and 25 °C, showing a very high affinity for β-lactoglobulin (540 mg/g kaolinite). The high retention capacity and different selectivity of this mineral for the three proteins, along with its low cost and absence of toxicity, especially when compared with standard polymeric resins used for protein adsorption, render kaolinite a very suitable adsorbent for the retention of proteins contained on dairy industry by-products. By thermal dehydroxylation, the electrical charge of the kaolinite surface was modified, and a secondary pore system made of inter particle voids was created, which led to drastic changes on the adsorbent selectivity, decreasing considerably the BSA retention capacity BSA (10 mg/g sample) and increasing the adsorption of A-LA (176 mg/g sample). Kaolinite reaction with the silane coupling agent TBSCl, allowed modifying the hydrophilic character of the surface to hydrophobic. It also caused important changes in the textural parameters with a significant reduction of the BET surface area, the formation of wider mesopores and a secondary pore system made of inter particle voids. As a result, the retention of BSA considerably increased. By surface modification of low cost, non toxic clay minerals obtained from sedimentary deposits, protein adsorbents with high retention capacity and desired selectivity patterns can be obtained. Additional research in this area will lead to the development of improved adsorbents for the recovery of bio-molecules present in food industry effluents,
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in order to avoid environmental contamination. Furthermore, the recovered proteins can be used to obtain protein rich food products. Acknowledgments We thank Ministerio de Educación y Ciencia of Spain for financial support (Project CTQ2007-62182/PPQ) and Dr. Humberto RodríguezSolla for helpful suggestions. References Barral, S., Villa-García, M.A., Rendueles, M., Díaz, M., 2008. Interactions between whey proteins and kaolinite surfaces. Acta Mater. 56, 2784–2790. Barret, E.P., Joyner, L.G., Halenda, P.P., 1951. The determination of pore volume and area distributions in porous substances. I. Computations from nitrogen isotherms. J. Am. Chem. Soc. 73, 373–380. Bartoszewicz, A., Kalek, M., Stawinski, J., 2008. Iodine-promoted silylation of alcohols with silyl chlorides. Synthetic and mechanistic studies. Tetrahedron 64, 8843–8850. Bergaya, F., Lagaly, G., 2001. Surface modification of clay minerals. Appl. Clay Sci. 19, 1–3. Bergaya, F., Theng, B.K.G., Lagaly, G., 2006. Handbook of Clay Science. vol. 1. Elsevier, Amsterdam. Bougeard, D., Smirnov, K.S., Geidel, E., 2000. Vibrational spectra and structure of kaolinite: a computer simulation study. J. Phys. Chem. B 104, 9210–9217. Boumaza, A., Favaro, L., Lédion, J., Sattonnay, G., Brubach, J.B., Berthet, P., Huntz, A.M., Roy, P., Tétot, R., 2009. Transition alumina phases induced by heat treatment of boehmite: an X-ray diffraction and infrared spectroscopy study. J. Solid State Chem. 182, 1171–1176. Braggs, B., Ralston, J., St Clair, R., 2000. Surface modification of kaolinite. US Patent 6 (071), 335. Brunauer, S., Deming, L.S., Deming, W.S., Teller, E., 1940. On a theory of the Van der Waals adsorption of gases. J. Am. Chem. Soc. 62, 1723–1732. Bujdák, J., Rode, B.M., 1997. Silica, alumina, and clay-catalyzed alanine peptide bond formation. J. Mol. Evol. 45, 457–466. Bujdák, J., LeSon, H., Rode, B.M., 1996. Montmorillonite catalyzed peptide bond formation: the effect of exchangeable cation. J. Inorg. Biochem. 63, 119–124. Causserand, C., Jover, K., Aimar, P., Meireles, M., 1997. Modification of clay cake permeability by adsorption of protein. J. Membr. Sci. 137, 31–44. Collins, D.R., Catlow, C.R.A., 1991. Energy-minimized hydrogen-atom positions of kaolinite. Acta Crystallogr. B47, 678–682. Dai, J.C., Huang, J.T., 1999. Surface modification of clays and clay-rubber composite. Appl. Clay Sci. 15, 51–65. Ding, X., Henrichs, S., 2002. Adsorption and desorption of proteins and polyamino acids by clay minerals and marine sediments. Mar. Chem. 77, 225–237. Farmer, V.C., 1998. Differing effects of particle size and shape in the infrared and Raman spectra of kaolinite. Clay Miner. 33, 601–604. Fiorito, T.M., Icoz, I., Stotzky, G., 2008. Adsorption and binding of the transgenic plant proteins, human serum albumin, β-glucuronidase, and Cry3Bb1, on montmorillonite and kaolinite: microbial utilization and enzymatic activity of free and clay-bound proteins. Appl. Clay Sci. 39, 142–150. Frost, R.L., Van der Gaast, S.J., 1997. Kaolinite hydroxyls; a Raman microscopy study. Clay Miner. 32, 471–484. Frost, R.L., Mako, E., Kristof, J., Kloprogge, J.T., 2002a. Modification of kaolinite surfaces through mechanochemical treatment — a mid-IR and near-IR spectroscopic study. Spectrochim. Acta A58, 2849–2859. Frost, R.L., Kloprogge, J.T., Kristof, J., 2002b. Raman and infrared spectroscopic study of modification of kaolinite surfaces by intercalation organic molecules. In: Somasundaran, P., Hubbard, A. (Eds.), Encyclopedia of Surface and Colloid Science. Marcel Dekker, New York, pp. 4438–4452. Fusi, P., Ristori, G.G., Calamai, L., Stotzky, G., 1989. Adsorption and binding of protein on “clean“(homoionic) and “dirty“(coated with Fe oxyhydroxides) montmorillonite, illite and kaolinite. Soil Biol. Biochem. 21, 911–920. Grim, R.G., 1968. Clay Mineralogy.McGraw-Hill, New York 185–233. Gupta, A., Loew, G.H., Lawless, J., 1983. Interaction of metal ions and amino acids: possible mechanisms for the adsorption of amino acids on homoionic smectite clays. Inorg. Chem. 22, 111–120. Hambling, S.G., McAlpine, A.S., Sawyer, L., 1992. Beta-lactoglobulin. Advanced dairy chemistry. Proteins, vol. 1. Elsevier, London, pp. 141–190. Haynes, C.A., Norde, W., 1994. Globular proteins at solid/liquid interfaces. Colloids Surf. B: Biointerfaces 2, 517–566. Hess, C.A., Saunders, V.R., 1992. Periodic ab initio Hartree-Fock calculations of the low-symmetry mineral kaolinite. J. Phys. Chem. 96, 4367–4374. Ishida, H., Miller, J.D., 1985. Cyclization of methacrylate-functional silane on particulate clay. J. Polymer Sci. Polym. Phys. 23, 2227–2242. Lee, W.K., Ko, J.S., Kim, H.M., 2002. Effect of electrostatic interaction on the adsorption of globular proteins on octacalcium phosphate crystal film. J. Colloid Interface Sci. 246, 70–77. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin Phenol Reagent. J. Biol. Chem. 193, 265–275. Quiquampoix, H., Staunton, S., Baron, M.H., Ratcliffe, R.G., 1993. Interpretation of the pH dependence of protein adsorption on clay mineral surfaces and its relevance to the understanding of extracellular enzyme activity in soil. Colloids Surf. A: Physicochem. Eng. Aspect 75, 85–93.
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Quiquampoix, H., Abadie, J., Baron, M.H., Leprince, F., Matumoto-Pintro, B.M., Ratcliffe, R.G., Staunton, S., 1995. Mechanisms and consequences of protein adsorption on soil mineral surfaces. Proteins interfaces II. Chapter 23. ACS Symp. Ser. 602, 321–333. Rausell-Colom, J., Serratosa, J., 1987. Chemistry of clays and clay minerals. In: Newman, A.C.D. (Ed.), Mineralogical Society Monograph, N° 6. Wiley Interscience, New York, p. 412. Rigou, P., Rezaei, H., Grosclaude, J., Staunton, S., Quiquampoix, H., 2006. Fate of prions in soil: adsorption and extraction by electroelution of recombinant ovine prion protein from montmorillonite and natural soils. Environ. Sci. Technol. 40, 1497–1503. Saikia, B.J., Parthasarathy, G., Sarmah, N.C., 2008. Fourier transform infrared spectroscopic estimation of crystallinity in SiO2 based rocks. Bull. Mater. Sci. 31, 775–779.
Venkateswerlu, G., Stotzky, G., 1992. Binding of the protoxin and toxin proteins of Bacillus thuringiensis subsp.kurstaki on clay minerals. Curr. Microbiol. 25, 225–233. Violante, A., De Cristofaro, A., Rao, M.A., Gianfreda, L., 1995. Physicochemical properties of protein-smectite and protein-Al(OH)x-smectite complexes. Clay Miner. 30, 325–336. Waddell, T.G., Leyden, D.E., De Bello, M.T., 1981. The nature of organosilane to silicasurface bonding. J. Am. Chem. Soc. 103, 5303–5307. Yu, C.H., Norman, M.A., Newton, S.Q., Miller, D.M., Teppen, B.J., Schäfer, L., 2000. Molecular dynamics simulations of the adsorption of proteins on clay mineral surfaces. J. Mol. Struct. 556, 95–103.
Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027
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Research paper
Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents R. Duarte-Silva a, M.A. Villa-García b,⁎, M. Rendueles a, M. Díaz a a b
Department of Chemical Engineering and Environmental Technology, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain Department of Organic and Inorganic Chemistry, University of Oviedo, Julian Clavería 8, 33006 Oviedo, Spain
a r t i c l e
i n f o
Article history: Received 27 June 2011 Received in revised form 12 December 2012 Accepted 22 December 2013 Available online xxxx Keywords: Kaolinite Metakaolinite Organo–kaolinite hybrid material Structural properties Textural characteristics Whey proteins adsorption
a b s t r a c t The structural, textural and protein adsorption properties of kaolinite from clay sedimentary deposits, metakaolinite obtained by thermal dehydroxylation of kaolinite, and the organic derivative prepared by reacting kaolinite with the silane coupling agent tert-butyldimethylchlorosilane, were studied. The retention capacities for the proteins α-lactalbumin (A-LA), bovine serum albumin (BSA) and β-lactoglobulin (B-LG) and the nature of the interactions responsible for protein binding were studied by adsorption experiments, performed at room temperature and pH 5.0. The protein adsorption capacity and the selectivity show a clear dependence on the chemical nature of the adsorbents surface and on the textural properties. Kaolinite behaves as a strong adsorbent for A-LA and BSA, and exhibits a very high affinity for B-LG. Metakaolinite shows good retention capacity for A-LA and B-LG, but does not retain significant amounts of BSA. The adsorption capacity of the organo–kaolinite hybrid considerably increases for BSA and A-LA. FTIR results indicate the absence of hydrogen bonding between the adsorbents surface and the polypeptides. The interactions responsible for protein binding are closely related to the hydrophilic or hydrophobic character of the adsorbent surface and the amino acid composition of the proteins, steric effects also should be considered for the adsorption patterns. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The interactions of peptides and proteins with clay surfaces have been studied extensively in the past (Bujdák and Rode, 1997; Bujdák et al., 1996; Causserand et al., 1997; Ding and Henrichs, 2002; Fusi et al., 1989; Gupta et al., 1983; Quiquampoix et al., 1993, 1995; Rigou et al., 2006; Violante et al., 1995; Yu et al., 2000). Since clays are very abundant, their use as inorganic hosts is important not only from an economic perspective, but also for their unique physical and chemical properties, and the ease with which these materials can be modified and adjusted to new uses. Kaolinite has a wide variety of applications in industry (Bergaya and Lagaly, 2001; Bergaya et al., 2000; Braggs et al., 2006). This mineral has two different basal cleavage faces. One face consists of a tetrahedral siloxane surface with inert –Si–O–Si– links; the other basal surface consists of an octahedral gibbsite (Al(OH)3) sheet (Frost et al., 2002a, 2002b). Kaolinite has different surface structures between base planes (001) and edge planes (110) and (010). The charge on the edges is due to the protonation/deprotonation of hydroxyl groups and depends on the pH of the solution. The hydroxyl groups located at the edge planes are considered the major reactive sites of kaolinite surfaces (Rausell-Colom and Serratosa, 1987).
⁎ Corresponding author. Tel.: +34 985102976; fax: +34 985103446. E-mail address: [email protected] (M.A. Villa-García).
Kaolinite surface is hydrophilic but it can be rendered hydrophobic by reaction with organo–functional molecules (Dai and Huang, 1999). Organic derivatives of clays are generally obtained by using silane coupling agents (Dai and Huang, 1999; Ishida and Miller, 1985; Waddell et al., 1981). After surface modification, the organic groups can be attached to the clay by chemical bonding, adsorption and coating. Kaolinite surface can also be modified by thermal treatment. At temperatures higher than 450 °C dehydroxylation occurs to form metakaolinite (Al2O3 · 2SiO2), and at 650 °C dehydroxylation is by ca 90% complete (Grim, 1968). The dairy industry generates many by-products with high protein contents. These residues can cause severe environmental contamination when they are not properly disposed, and it is necessary to find solutions to prevent this pollution problem. Furthermore, the recovered proteins can be used to obtain high quality protein rich food products. Protein recovery by adsorption on various types of supports is a commonly used technique; however, the use of clay minerals as adsorbents of protein molecules has received considerably less attention. The adsorption/desorption of proteins on clay surfaces is a complex process controlled by different factors, such as the surface properties of the adsorbent, the structural stability of the proteins, the ionic strength and the pH of the adsorption/desorption experiments (Haynes and Norde, 1994). In a recent work we found that kaolinite showed a high adsorption capacity at the isoelectric point (IEP) of each protein (Barral et al., 2008). Moreover, there was a clear correlation between the adsorption patterns and the presence of hydrophobic or hydrophilic
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Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027
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groups in the polypeptide surface. In order to achieve a deeper insight on the nature of the interactions responsible for protein adsorption on clay surfaces and explain the retention patterns, adsorption experiments were run using as adsorbents kaolinite, metakaolinite obtained by thermal dehydroxylation of kaolinite, and the organic derivative obtained by reacting kaolinite with tert-butyldimethylchlorosilane. Three whey proteins were chosen for this study: BSA, B-LG and A-LA. Physico-chemical characterization of the adsorbents was carried out using different techniques: elemental chemical analysis, FTIR spectroscopy, X-ray diffraction (XRD), thermal gravimetric (TG) and differential thermal analysis (DTA), N2 adsorption–desorption at 77 K, mercury intrusion porosimetry and scanning electronic microscopy (SEM). Evidence of protein intercalation in kaolinite has not been reported yet. There was no significant expansion of the clay after protein binding, as shown by X-ray diffraction analysis. Kaolinite, a non swelling clay does not expand upon wetting, which prevents the exposure of internal surface, and adsorption of proteins is restricted to the external crystal surfaces and the edges of the clay (Fiorito et al., 2008; Venkateswerlu and Stotzky, 1992). As will be shown, the protein retention patterns are controlled by the textural properties and the chemical nature of the adsorbent surface. Steric effects should be also considered, since experimental data show a correlation between the extent of adsorption and protein sizes.
XRF spectrometer. Powder X-ray diffraction (XRD) patterns were obtained with a Philips X'Pert MPD Pro instrument-ray diffractometer, operating at 45 kV, 40 mA, using Cu Kα radiation. FTIR spectra of all the materials were obtained at room temperature with a Perkin-Elmer PARAGON 1000 spectrometer, by the KBr pellet technique. Thermal analysis, TG and DTA, were carried out in Metller TA-4000 TG-50 type thermobalance under dynamic heating conditions (10 °C/min heating rate) in a flowing nitrogen atmosphere of 99.999% purity, 50 mL/min. The samples were heated in alumina crucibles up to 1000 °C. Nitrogen adsorption–desorption isotherms at 77 °K were obtained with a Micromeritics ASAP 2020 instrument, using static adsorption procedures. Macroporosity and total pore volume were measured by mercury intrusion porosimetry with a Micromeritics Autopore IV instrument. The microscopic morphology of the samples was studied using a Jeol 6100 scanning electron microscope operating at 0.3–30 kV. Samples were prepared by sonicating the powdered sample in ethanol and then evaporating two droplets on carbonated copper grids. Protein concentrations in the initial solutions and the final supernatant obtained after the adsorption, were measured with the UV/VIS spectrophotometer (Heλios UV/VIS Thermo Electron Corporation) at 750 nm, according to the Folin–Ciocalteu colorimetric method (Lowry et al., 1951). A previous filtration through 0.45 μm polyvinylidene difluoride membrane syringe filters (Acrodisc) is required to retain the fine particles. 2.3. Adsorption experiments
2. Materials and methods 2.1. Materials The kaolinite mineral used in the experiments (sample K) was provided by Arcichamotas Mines in the North of Spain (Asturias). The clay was treated with H2O2 (Panreac) to eliminate organic matter present in the mineral, and then was washed thoroughly with distilled water. The solid was filtered, dried at 120 °C, ground and sieved. All the experiments were performed with kaolinite particles 100–250 μm in size. Tert-butyldimethylchlorosilane (reagent grade), imidazole (ACS reagent) and dichloromethane (ACS reagent) were purchased from Sigma. The whey proteins used were also supplied by Sigma: A-LA, type III, calcium depleted, 85% purity; B-LG, aprox. 90% PAGE purity; and BSA, fraction V, 99% purity. The sodium acetate buffer was purchased from Panreac. Metakaolinite (sample MK) was obtained from calcination of kaolinite. The clay mineral was heated in a ceramic crucible with a heating rate of 10 °C/min up to 650 °C and then was kept at that temperature for 12 h. Between 450 and 650 °C kaolinite undergoes a strong endothermic dehydration reaction, resulting in the conversion to metakaolinite. The organo–kaolinite composite (sample KR) was synthesized by reaction between tert-butyldimethylchlorosilane (TBSCl) and kaolinite in dichloromethane, in the presence of imidazole as a base catalyst. In a typical synthesis, 5 g of kaolinite was suspended in 50 mL of CH2Cl2 and 14 g of TBSCl and 6.5 g of imidazole were added, under vigorous stirring. The reaction mixture was sonicated for 48 h, the supernatant liquid was removed by decantation, the solid was thoroughly washed with dichloromethane to remove excess of organic species, and dried under vacuum to remove residual dichloromethane. The imidazolium chloride–kaolinite complex (sample KI) was obtained by bubbling hydrogen chloride, obtained by reaction of NaCl with H2SO4, through a solution containing 6.5 g of imidazole in 50 mL of CH2Cl2. Five g of kaolinite was added to the imidazolium chloride solution, and the reaction mixture was treated under the conditions described above for the preparation of the organo–clay solid. 2.2. Methods The chemical composition of the kaolinite was determined by X-ray fluorescence spectroscopy (XRF) using a Philips PW 2404
The protein adsorption experiments were carried out in batch mode. In all the experiments 0.5 g of the different samples was transferred to a 250 mL Erlenmeyer flask and suspended in 100 mL of protein solution buffered using a sodium acetate buffer (pH = 5.0). The suspensions were stirred at 25 °C (250 rpm) for 6 h in a New Brunswick Scientific Excella E25 incubator-shaker. Blank experiments were run in parallel under the same experimental conditions. Using a liquid/solid ratio of 200 mL g−1, which was previously established as the most convenient value, adsorption isotherms on kaolinite for each protein were obtained by changing the initial protein concentrations. Adsorption data of each protein on kaolinite and samples MK, KR and KI were obtained under the conditions above described, using protein concentration of 2 g L−1. Every protein adsorption experiment on the different adsorbents was repeated six times, being the values obtained very close, average values are shown in this work. Protein concentrations in the initial solutions and after adsorption were measured using the Folin–Ciocalteu colorimetric method (Lowry et al., 1951) with a UV/VIS spectrophotometer. 3. Results and discussion 3.1. Kaolinite chemical composition The chemical composition of the kaolinite mineral determined by X-ray fluorescence spectroscopy (XRF) was: 50.25% SiO2; 34.22% Al2O3; 0.94% TiO2; 0.97% Fe2O3; 0.03% CaO; 0.16% MgO; 0.04% Na2O; 1.14% K2O; 12.25% H2O. 3.2. Mineralogical composition The XRD powder pattern for kaolinite (Fig. 1A) shows the diffraction lines characteristic of a high crystal order mineral, diffraction lines corresponding to quartz, mica and feldspars are also present in the diffractogram indicating the presence of these minerals as impurities. The values of the (001) d-spacing corresponding to kaolinite and the conditioned one were 7.14 Å and 7.17 Å, respectively. Conditioning did not expand the kaolinite layers, but slightly increased the defects of the original low defect kaolinite. The sample KR (Fig. 1B) has a lower crystal order due to the presence of adsorbed organic species, or due to the presence of some smaller clay particles formed during the treatment with tert-butyldimethylchlorosilane. The (001) d-spacing of
Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027
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hydroxyl deformation vibrations are very sensitive to any modification of the hydroxyl surfaces. Kaolinite contains two types of hydroxyl groups: (i) hydroxyl groups located at the internal surface between adjacent layers, called inner surface hydroxyl groups; (ii) hydroxyl groups located in the inner (shared) plane of the octahedral sheet, often referred as inner hydroxyl groups. The inner surface groups are situated in the outer (unshared) plane, whereas the inner hydroxyl groups are within the unit cell and are not exposed at the surface of the kaolinite layers (Collins and Catlow, 1991; Frost et al., 2002a, 2002b; Hess and Saunders, 1992). The FTIR spectra of natural kaolinite (Fig. 2A), and the conditioned one, show bands at 3695 cm−1 and 3651 cm− 1 attributed to the hydroxyl-stretching modes of inner surface hydroxyls, the absorption at 3620 cm−1 was assigned to the hydroxyl-stretching modes of inner hydroxyl groups and the bands that appear at 940 cm− 1 and 913 cm− 1 were assigned to hydroxyldeformation modes of the inner surface and inner hydroxyls, respectively (Farmer, 1998; Frost and Van der Gaast, 1997). Modification of kaolinite surfaces either through intercalation, grafting of organic groups, or by thermal treatment, implies changes in the IR spectrum. No hydroxyls are spectroscopically evident in the spectra of metakaolinite (sample MK). The FTIR spectrum of this sample shows absorption bands at 1080 cm−1 (very strong and very broad) and 796 cm−1 attributed to the Si–O stretching vibrations of quartz (Saikia et al., 2008), and the bands that appear at 560 cm− 1 and 484 cm− 1 were assigned to Al–O stretching modes of alumina (Boumaza et al., 2009). Surface modification of clays also can be achieved by reaction with silane coupling agents. Generally, the coupling agent can be attached to the clay by chemical bonding, adsorption or coating. Molecules of the coupling agent coating on, or adsorbing to the clay surface, basically do not modify the IR vibrations, but when they are fixed on the mineral surface by chemical bonding, changes in the vibrational spectra occur. The FTIR spectrum of sample KR (Fig. 2B), shows that the band width and the peak position of the absorptions corresponding to hydroxylstretching and hydroxyl-deformation modes of the clay mineral do
Fig. 1. X-ray diffraction patterns of (A) kaolinite; (B) organo–kaolinite compound; (C) metakaolinite; (D) kaolinite–BSA complex.
this sample did not increase, indicating lack of expansion of the clay upon reaction with the silane, and hence lack of intercalation of the organic species. The diffraction pattern of metakaolinite (Fig. 1C) shows amorphous regions centered at 2θ values of approximately 23° and 36°. Characteristic major kaolinite peaks are absent at 2θ equal 12.4° and 25°, indicating a major loss of hydroxyl groups. The peaks at 20.7°, 26.5°, 50° and 59.8° correspond to quartz. Kaolinite undergoes a structural rearrangement upon heating, the calcination step causes the liberation of the hydroxyls in the form of water vapor and, with the collapse of the original triclinic cell, a more amorphous phase is formed. The diffractograms of the different adsorbents containing retained proteins (Fig. 1D) do not show a significant shift of the basal d-spacings to higher values for the protein–kaolinite complexes. This means that there was no expansion of the clay after protein binding, indicating that the protein molecules were not intercalated in the mineral structure, but were immobilized at the external surfaces and the edges of the adsorbents. 3.3. FTIR spectroscopy The modification of kaolinite surfaces can be easily studied through vibrational spectroscopy. In particular, the hydroxyl stretching and
Fig. 2. FTIR spectra of (A) kaolinite; (B) sample KR.
Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027
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not change, indicating that kaolinite hydroxyl groups did not react with the coupling agent. The spectrum of the organo–kaolinite complex does not show the silane methyl C–H stretching doublet at 2952 cm−1 and 2923 cm−1, a new band appears at 2980 cm− 1 and the band at 2853 cm−1 assigned to C–H stretching is broadened. These changes indicate grafting of organic groups on the kaolinite surface. Since the molecules of the coupling agent are bound to the clay, the strength of the Si–O bands should be increased. However, the Si–O vibrations of kaolinite, a band at 1095 cm− 1 and a doublet with maxima at 1032 and 1008 cm− 1 assigned to Si–O stretching modes (Bougeard et al., 2000), are very strong and it is difficult to obtain additional information of the changes caused by the interaction with the coupling agent. The spectrum also shows a peak at 3135 cm−1 that can be assigned to aromatic C–H stretch of imidazolium chloride formed during the reaction. The presence of this compound is confirmed by the broad band at 3420 cm−1 that can be assigned to aromatic N–H stretch, and a sharp band at 1585 cm− 1 that correspond to N–H bending vibrations. A plausible mechanism for the silylation process is depicted in Scheme 1. It involves the hydrolysis of tert-butyldimethylchlorosilane, in the presence of imidazole as a base catalyst, to form silanol species and imidazolium chloride. The silanols are attached to the kaolinite surface through hydrogen bonding. The formation of hydrogen bonding between the clay and the silanols is supported by the fact that when sample KR is washed with water or ethanol, the transmittance of the C–H vibrations decreases, whereas these bands remained unchanged after the sample was thoroughly washed with CH2Cl2. The presence of aromatic C–H and N–H stretch bands in the FTIR spectrum of sample KR suggests that some imidazolium chloride, formed during kaolinite silylation, is also attached to kaolinite surface. In order to elucidate the role of imidazolium chloride on protein adsorption behaviour of sample KR, kaolinite was reacted with imidazolium chloride (sample KI) under the same conditions used for preparation of sample KR. As will be shown the protein adsorption capacity of kaolinite is not modified when imidazolium chloride is attached to its surface. Ultrasounds were employed in this process because significant rate enhancements of chemical reactions have been described when heterogeneous reactions are carried out in the presence of sound waves (Bartoszewicz et al., 2008). The FTIR spectra of the kaolinite–protein complexes, Fig. 3(B) and (C) do not show any significant shift of the hydroxyl stretching and deformation bands corresponding to the spectrum of kaolinite, suggesting the absence of hydrogen bonding between adsorbed protein molecules and the surface OH groups of kaolinite. Moreover, the spectra of the three pure proteins show the characteristic bands of amide I at 1654 cm−1 (mainly C = O stretch) and amide II at 1542 cm−1 (C–N stretch coupled with N–H bending mode). The IR spectra of the
Fig. 3. FTIR spectra of (A) kaolinite; (B) kaolinite/BSA complex; (C) kaolinite/β-lactoglobulin complex.
adsorbed proteins show that the peak position of both bands does not change, indicating that kaolinite does not interact with C = O, C–N or N–H of the polypeptide chain and no hydrogen bond is formed. 3.4. Thermal analysis Thermogravimetric analysis of kaolinite shows the absence of any significant mass loss below 420 °C. At higher temperature a continuous mass loss (10.6%) due to kaolinite dehydroxylation is observed, that is completed at 800 °C. TG and DTA curves of sample KR are shown in Fig. 4. Thermal degradation of sample KR occurs in two steps. Below 180 °C there is not significant mass loss. With increasing temperature the elimination of the silane bonded to kaolinite begins and is terminated at 320 °C. The mass loss corresponding to this step is 45.6%. 3.5. Textural characterization
Scheme 1. Plausible mechanism for the silylation of kaolinite surface.
The porous structure of the separation media is a fundamental feature required to achieve good retention capacity. The use of materials with macropores favors a rapid mass transfer of macromolecules that improves the kinetics of retention. Moreover, large pore surfaces can accommodate protein molecules, while small pores are not accessible. Specific surface areas and mesopore size distributions were calculated
Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027
R. Duarte-Silva et al. / Applied Clay Science xxx (2014) xxx–xxx
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Table 1 Textural parameters of the samples obtained from the nitrogen adsorption–desorption isotherms at 77 K. Sample
SBET (m2g−1)
Dpmax (nm)
Vp (cm3g−1 STP)
K MK KR
9 10 2
20.4 18.6 33.6
0.051 0.045 0.016
of the pores radii between 10 μm and 15 μm, the low surface area of this sample and the very large pore diameters indicate the formation of large inter particle cavities. Fig. 4. Thermogravimetric (TG) and differential TG (DTG) curves of sample KR.
3.6. Scanning electron microscopy from the nitrogen adsorption–desorption isotherms at 77 K. Sample MK is characterized by a type IV isotherm (Brunauer et al., 1940) with very narrow hysteresis loop type H3, which is usually associated to porous solids consisting of particle aggregates (Fig. 5). The low nitrogen uptake at relative pressures b0.2 indicates the absence of microporosity. The adsorption limit is not well defined at high relative pressures indicating the presence of macroporosity in the materials. The mesopore size distributions were analyzed using the method of BJH (Barret et al., 1951) applied to the adsorption band of the isotherm. BET specific surface areas (SBET), values of the most frequent pore diameters (Dpmax) obtained from the pore diameters distribution curves and the cumulative pore volume of each sample (Vp) are listed in Table 1. The specific surface area of kaolinite (9 m2g− 1) remains practically unaffected after dehydroxylation at 650 °C, (the BET specific surface area of MK is 10 m2g−1). The loss of hydroxyl groups favors the formation of smaller mesopores; the most frequent pore diameter decreases from 20.4 nm to 18.6 nm and the cumulative pore volume decreases as well (Table 1). Kaolinite reaction with the silane coupling agent considerably reduced the specific surface area to 2 m2g−1 for sample KR, as well as the pore volume, favoring the formation of wider mesopores (33.6 nm) (Table 1). Macroporosity of the samples was studied by mercury intrusion porosimetry. The pore size distribution profile of sample KR is shown in Fig. 6. The total pore volume are 0.476 mL g− 1 for samples K and MK, and 0.893 mL g−1 for sample KR that exhibits the lowest specific surface area but wider pores, which contribute more to volume than to the surface. Moreover, very large pore volumes indicate that the porosity is due to inter particle spacing, rather than pores within the structure. The macropore size distribution profile of sample K is unimodal with a maximum at 6 μm that corresponds to the most frequent pore diameters. The pore size distribution profile of sample MK shows a wider range of pore diameters with two maxima at 0.5 μm and 15 μm. Kaolinite dehydroxylation favors the formation of smaller pores, both in the mesopore and macropore range, besides a secondary pore system made of inter particle voids is created. The pore size distribution curve of sample KR (Fig. 6) shows several maxima, having most
Fig. 5. Nitrogen adsorption–desorption isotherm at 77 K of sample MK.
SEM micrographs of the samples are shown in Fig. 7. Sample K (Fig. 7A) consists of laminar particle aggregates. Sample MK (Fig. 7B) shows a less homogeneous microscopic topography and the presence of wide channel-like inter-particle voids formed during dehydroxylation. Sample KR (Fig. 7C), has a smoother surface and forms considerably larger particle aggregates with wide macropores and inter particle cavities.
3.7. Protein adsorption experiments The affinity of a protein for a given surface depends on the surface properties of the adsorbent, the composition and structural stability of the protein, the pH and the ionic strength of the solution (Haynes and Norde, 1994). Experimentally, maximum protein adsorption is observed at the isoelectric point of each protein (Barral et al., 2008; Haynes and Norde, 1994) because electrostatic repulsions between identically charged adsorbed proteins are minimized. Moreover, there is a non uniform distribution of ionic patches on the surface of the proteins that leads to electrostatic interactions between the patches and the adsorbent surface (Lee et al., 2002). However, an increase in electrostatic interactions is also accompanied by a reduction in the native structure; many proteins also adsorb with high affinity to hydrophobic surfaces. Adsorption of proteins onto hydrophilic (sample K), dehydroxylated (sample MK) and hydrophobic surfaces (samples KR and KI) may contribute to understanding the nature of the interactions responsible for protein retention. Adsorption experiments were performed at pH = 5.0, because this value is very close to the IEP of the three proteins. Table 2 lists the molecular weight and isoelectric point of each protein. Kaolinite suspension in distilled water has a pH of 3.8, resulting from proton dissociation of superficial hydroxyl groups and was buffered with sodium acetate at pH 5.0. The three proteins were readily adsorbed on kaolinite and maximal adsorption was reached after 2 h.
Fig. 6. Pore size distribution profile of sample KR, measured by mercury intrusion porosimetry.
Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027
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R. Duarte-Silva et al. / Applied Clay Science xxx (2014) xxx–xxx
Fig. 8. Protein adsorption isotherms for (A) BSA; (B) A-LA; (C) B-LG.
Fig. 7. SEM images, 7500×, of (A) sample K; (B) sample MK; (C) sample KR.
The adsorption isotherms for the three proteins are depicted in Fig. 8. They show Langmuir characteristics; the protein uptake increases with protein concentration and finally reaches a plateau. The Langmuir isotherm assumes monolayer coverage of adsorbate over a homogeneous adsorbent surface; at equilibrium a saturation point is reached where no further adsorption/desorption occurs. The Langmuir equation is expressed as: q¼
qm c kd þ c
Where q is the equilibrium protein concentration on the adsorbent (mg g− 1), c is the concentration of the liquid phase at equilibrium (g L−1), qm is a constant related to the area occupied by a monolayer Table 2 Molecular weight and IEP of the proteins (Hambling et al., 1992). Protein
Molecular weight (kDa)
IEP
A-LA B-LG BSA
28.4 (dimer) 18.4 69
4.7 – 5.1 5.2 – 5.6 4.9
of adsorbate, reflecting the adsorption capacity and kd is a direct measure for the intensity of the adsorption process. Experimental data were fitted to the Langmuir equation, using the program Scientist (Micromath), to obtain the Langmuir parameters qm and kd (Table 3). Kaolinite adsorption capacity was different for each protein: 53 mg for BSA/g kaolinite, 108 mg for A-LA/g kaolinite and 540 mg for B-LG/g kaolinite. Proteins are made up of different amino acids with lateral chains containing hydrophobic or hydrophilic organic groups that can be negatively, neutrally or positively charged at pH 5.0. The chemical nature of the protein lateral chains and the electric surface charge of kaolinite will condition the nature of the interactions responsible for protein binding. To explain the different retention patterns a number of factors must be considered, such as the textural properties and the electric surface properties of the adsorbents, the amino acid composition, the IEP of each protein and the molecular sizes. BSA, which is the least retained protein (Table 3), contains a larger number of non-polar groups (253) that reach external positions, shielding the contact between the polar groups and the hydrophilic centres of the adsorbent, and thus minimizing the strength of the electrostatic interactions. Moreover, due to its large molecular size, steric effects should also be considered. The adsorption isotherms showed typical Langmuir characteristics, which involve monolayer coverage of the adsorbate over the adsorbent surface; this implies a clear correlation between the extent of the adsorption and protein sizes, being the largest protein (BSA, 69 kDa) the least adsorbed. The retention capacity for A-LA increases to 108 mg/g kaolinite. This protein is smaller (28.4 kDa) and contains only 44 non-polar groups. Finally, B-LG which is the smallest protein (18.4 kDa) shows the highest retention capacity, 540 mg/g kaolinite. This protein contains more non-polar groups (150 for the dimer) than A-LA, but under the adsorption conditions (pH 5.0, slightly below its IEP) there is a non uniform distribution of positive ionic patches on the surface of the protein that leads to an increase in the electrostatic attraction between the hydrophilic centres of the clay and the protein. To investigate the influence of the adsorbent surface properties on protein retention, adsorption experiments were run on kaolinite, dehydroxylated kaolinite (sample MK) and on the organo derivatives (samples KR and KI), using a liquid/solid ratio of 200 mLg−1 and protein concentrations of 2 g L−1. The results obtained are given in Table 4. Sample MK did not retain significant amounts of BSA (10 mg/g). Dehydroxylation involves elimination of the ionizable OH groups and
Table 3 Parameters of Langmuir isotherms. Protein
BSA
A-LA
B-LG
qm(mg protein/g kaolinite) Kd (g protein/L)
53 4.66
108 5.52
540 19.1
Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027
R. Duarte-Silva et al. / Applied Clay Science xxx (2014) xxx–xxx Table 4 Protein retention capacity (average values) of the different samples (mg/gsample), obtained using a liquid/solid ratio of 200 mL g−1 and protein concentrations of 2 g L−1. Protein
Kaolinite
Metakaolinite
Kaolinite-tertbutyl
A-LA B-LG BSA
98 370 43
176 135 10
159 316 125
decrease of the strength of the electrostatic interactions, which for BSA are minimized by the presence of non-polar groups. The adsorption of BSA on sample KR (125 mg/g) was increased by a factor of three with respect to the adsorption capacity shown by natural kaolinite (43 mg/ g sample). Treatment with the silane coupling agent considerably reduced the BET surface area, but created pores in the micrometer range that are accessible to large bio-molecules. Furthermore, the presence of hydrophobic groups in the sorbent surface leads to strong hydrophobic interactions with the protein, yielding high affinity for BSA for this sample. A-LA adsorption on sample MK (176 mg/g) is 56% higher than on sample K. Thermal treatment of kaolinite increased the specific surface area by 1 m2 g−1, but the total volume per unit mass did not change. However, a secondary pore system made of inter particle voids was created and A-LA is small enough to easily access the pores in the micrometer range, which leads to an increased retention capacity. A-LA adsorption on sample KR (159 mg/g) is smaller than on sample MK, probably due to the low specific surface area of this material. Adsorption of B-LG on sample KR is very high (316 mg/g sample), being 15% lower than on sample K (370 mg/g). This can be explained considering the low BET surface area of sample KR. Adsorption of B-LG on metakaolinite (135 mg/g sample) is considerably lower, probably due to a decrease in the electrostatic attraction forces between the metakaolinite surface and the protein. Protein adsorption experiments were also performed on the imidazolium chloride–kaolinite complex. Imidazolium chloride adsorbed on the kaolinite surface is readily dissolved in the buffer solution containing the proteins, and does not modify the clay retention capacity.
4. Conclusions Kaolinite obtained from clay sedimentary deposits was a strong adsorbent for α-lactalbumin and bovine serum albumin, at pH 5.0 and 25 °C, showing a very high affinity for β-lactoglobulin (540 mg/g kaolinite). The high retention capacity and different selectivity of this mineral for the three proteins, along with its low cost and absence of toxicity, especially when compared with standard polymeric resins used for protein adsorption, render kaolinite a very suitable adsorbent for the retention of proteins contained on dairy industry by-products. By thermal dehydroxylation, the electrical charge of the kaolinite surface was modified, and a secondary pore system made of inter particle voids was created, which led to drastic changes on the adsorbent selectivity, decreasing considerably the BSA retention capacity BSA (10 mg/g sample) and increasing the adsorption of A-LA (176 mg/g sample). Kaolinite reaction with the silane coupling agent TBSCl, allowed modifying the hydrophilic character of the surface to hydrophobic. It also caused important changes in the textural parameters with a significant reduction of the BET surface area, the formation of wider mesopores and a secondary pore system made of inter particle voids. As a result, the retention of BSA considerably increased. By surface modification of low cost, non toxic clay minerals obtained from sedimentary deposits, protein adsorbents with high retention capacity and desired selectivity patterns can be obtained. Additional research in this area will lead to the development of improved adsorbents for the recovery of bio-molecules present in food industry effluents,
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Please cite this article as: Duarte-Silva, R., et al., Structural, textural and protein adsorption properties of kaolinite and surface modified kaolinite adsorbents, Appl. Clay Sci. (2014), http://dx.doi.org/10.1016/j.clay.2013.12.027