Preparation of organo-modified kaolinite sorbents: The effect of surface functionalization on protein adsorption performance

Accepted Manuscript Title: Preparation of organo-modified kaolinite sorbents: The effect of surface functionalization on protein adsorption performance Authors: Mario D´ıaz, Mar´ıa A. Villa-Garc´ıa, Renata Duarte-Silva, Manuel Rendueles PII: DOI: Reference:

S0927-7757(17)30725-2 http://dx.doi.org/doi:10.1016/j.colsurfa.2017.07.067 COLSUA 21841

To appear in:

Colloids and Surfaces A: Physicochem. Eng. Aspects

Received date: Revised date: Accepted date:

26-5-2017 20-7-2017 21-7-2017

Please cite this article as: Mario D´ıaz, Mar´ıa A.Villa-Garc´ıa, Renata Duarte-Silva, Manuel Rendueles, Preparation of organo-modified kaolinite sorbents: The effect of surface functionalization on protein adsorption performance, Colloids and Surfaces A: Physicochemical and Engineering Aspectshttp://dx.doi.org/10.1016/j.colsurfa.2017.07.067 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Preparation of organo-modified kaolinite sorbents: The effect of surface functionalization on protein adsorption performance Mario Díaz,a

María A. Villa-García,b* Renata Duarte-Silva,a Manuel

Renduelesa a

Department of Chemical Engineering and Environmental Technology, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain b Department of Organic and Inorganic Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain ⁎ Corresponding author. Tel.: +34 985102976; fax: +34 985103446. E-mail:[email protected]

Abstract

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Kaolinite organic derivatives were obtained by functionalization of kaolinite (kaol), in order to improve its removal capacity with respect to polypeptides present in dairy industry effluents. Functionalization was carried out by grafting two organosilanes, namely tert-butyldimethylchlorosilane (TBSCl) and 3-aminopropyltriethoxysilane (APTES), onto the kaol surface, and by intercalation of the small polar molecules dimethyl sulfoxide (DMSO) and potassium acetate. FTIR spectroscopy provided qualitative evidence about the presence of organosilane molecules grafted on the kaol outer surface, as well as information about the organo-kaol intercalates. Quantitative data about the amount of grafted or intercalated organic molecules was also obtained by means of elemental and thermogravimetric analysis. Textural characterization of the samples was carried out by N2 adsorption-desorption at 77 K and by mercury intrusion porosimetry. SEM microscopy was used to study the microscopic topography. Protein adsorption experiments were carried out in batch mode, using bovine serum albumin (BSA) as a model protein to test the removal capacity of the materials obtained. The Langmuir and Freundlich adsorption models were applied to describe the equilibrium isotherms for kaol and its organic-derivatives, respectively. The results obtained showed that surface functionalization of kaol considerably improved its protein removal capacity.

Keywords: Kaolinite; functionalization; intercalation; proteins; adsorption; bovine serum albumin.

1. Introduction The dairy industry generates whey-containing effluents as by-products. The high protein content of whey-containing wastewater is one of the causes of eutrophication, which negatively affects natural water and, eventually, can promote the collapse of the

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ecosystem where these residues are being dumped. It is desirable that water treatment technologies remove proteins from the wastewater before it is returned to the environment. In most countries nowadays, thanks to restrictive legislation and greater environmental concern, a considerable effort is being made to develop techniques to allow the removal and recycling of these residues. In the particular case of whey, the total removal of the proteins dumped by the dairy industry, or at least a substantial reduction, must be achieved by using the adequate wastewater treatment. Moreover, the application of polypeptide removal technologies allows the recovery and recycling of valuable proteins. The adsorption process has been found to be one of the best water treatment technologies for removal of toxic metals and organic species from an aqueous medium, especially in terms of simplicity of design and operation and low cost [1]. In addition, this technology does not add undesirable by-products, is applicable at very low concentrations and permits the regeneration and reuse of the adsorbents. Clay minerals are probably the most promising alternatives to high cost adsorbents. Clays are abundant and widely available materials, with good chemical and mechanical stability and unique adsorption and ion-exchange properties. In addition, due to their local availability, technical feasibility and cost effectiveness, a number of studies have been carried out using clay minerals for wastewater treatment applications [2-5]. Due to their surface properties, clays behave as natural cleaning agents and take up organic and inorganic contaminants from water, both by ion exchange and adsorption mechanisms [6, 7]. Surface modification of clays has allowed the creation of new materials that exhibit many practical applications in several areas of materials science, such as adsorbents of organic and inorganic pollutants, rheological control agents, paints, cosmetics, personal care products, catalysts, soil remediation, electrodes

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and nanocomposite precursors [8, 9]. Adsorption by organoclays has been used for the removal of both organic and inorganic contaminants in waters, due to the relatively low cost of production and high adsorption capacity [10 – 18]. Kaolinite, Al2Si2O5(OH)4, a clay mineral with the 1:1-type layered structure, has so far been used less than other clays for the preparation of organic-inorganic hybrid materials. This mineral has two different interlayer surfaces. One side of the layer is gibbsite-like, with aluminium atoms coordinated octahedrally to corner oxygen atoms and hydroxyl groups. The other side of the layer is constituted by a silica-like structure, where the silicon atoms are coordinated tetrahedrally to oxygen atoms. The adjacent layers are linked by hydrogen bonds involving aluminol (Al–OH) and siloxane (Si–O) groups. Due to hydrogen-bonding between layers kaol has been classified as nonexpandable clay. Although the majority of the reactive sites of kaol are located in interlayer spaces, since this clay mineral is not easily expandable, the hydroxyl groups located at the external surface and edge planes are the major reactive sites of the kaol surface, which makes grafting and intercalation of organic compounds in the kaol internal surface more challenging. So far, Kaol organic derivatives have scarcely been used for the removal of organic and inorganic compounds from wastewater [15, 18]. Recently, the use of kaol and its derivatives as adsorbents for whey protein removal from dairy industry effluents was reported [19, 20]. It was found that proteins were adsorbed on the external surface and there was no evidence of protein intercalation [19]. The presence of an adsorbed organic layer on the kaol surface modifies its retention capacity and selectivity, because the interactions responsible for protein binding were closely related to the hydrophilic or hydrophobic character of the surface [20]. Clay modification can be achieved using several procedures such as adsorption, ion exchange, grafting of organic compounds,

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binding of inorganic and organic anions (mainly at the edges), pillaring, intraparticle and inter particle polymerization, dehydroxylation and delamination [21]. This study reports the preparation and physicochemical characterization of organo-kaol hybrid materials obtained by grafting a monofunctional and a trifunctional organosilane,

namely

tert-butyldimethylchlorosilane

(TBSCl)

and

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aminopropyltriethoxysilane (APTES) onto the kaolinite surface, and by intercalation of dimethyl sulfoxide (DMSO) and potassium acetate (AC) in the interlayer space. Both types of reactions led to the modification of kaol properties, such as texture, hydrophobicity and adsorption capacity. The materials obtained were characterized by elemental chemical analysis, X-ray diffraction (XRD), FTIR spectroscopy, thermal analysis (TG/DTA), N2 adsorption–desorption at 77 K, mercury intrusion porosimetry and scanning electron microscopy (SEM). To test the protein removal capacity of kaol and the organo-kaol derivatives, protein adsorption experiments were carried out in batch mode, using bovine serum albumin (BSA) as model protein. 2. Materials and Methods 2.1. Materials Kaol (sample K) was obtained from Arcichamotas Mines in the North of Spain (Asturias). The clay was treated with H2O2 to eliminate any organic species present in the mineral, then it was washed with distilled water, dried at 120 °C, ground and sieved. All the experiments were performed with kaol particles 100–250 μm in size. Tertbutyldimethylchlorosilane (TBSCl) (reagent grade), imidazole (ACS reagent), 3aminopropyltriethoxysilane (APTES) (99 % purity), dimethyl sulfoxide (DMSO) (ACS reagent), 1,4-dioxane (ACS reagent), dichloromethane (ACS reagent), and

BSA,

fraction V, 99% purity, were purchased from Sigma. The sodium acetate buffer was purchased from PanReac. All the chemicals were used as received.

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2.2. Kaolinite fuctionalization 2.2.1. Grafting process The grafting reactions of kaol with monofunctional and trifunctional silylating agents were carried out in dichloromethane. The reaction mixtures were treated in an ultrasound bath to enhance the reactivity. In order to convert the hydrophilic surface to an organophilic one, kaol silylation with tert-butyldimethylchlorosilane, C6H15ClSi, was carried out by reacting TBSCl and kaol in dichloromethane, in the presence of imidazole as a base catalyst, using a procedure already described [20]. Briefly: 5 g of kaol was suspended in 50 mL of CH2Cl2 in a Schlenk tube; to the suspension was added under vigorous stirring 14 g of TBSCl and 6.5 g of imidazole. The reaction mixture was sonicated for 48 h and the supernatant liquid was removed by decantation. The solid was thoroughly washed with dichloromethane to remove excess of organic species, centrifuged and dried under vacuum to remove residual dichloromethane. Kaol silylation using 3-aminopropyltriethoxysilane, NH2(CH2)3Si(OC2H5)3, was carried out using the following procedure: 5 g of kaolinite placed in a Schlenk tube were dispersed in 50 mL of dichloromethane, then 22 mL of the organosilane were added under stirring to the above-mentioned mixture. The Schlenk tube was partially submerged, to the liquid level, in a conventional liquid sonicator (ultrasound laboratory cleaner 230 V, 150W, 50Hz). The reaction mixture was sonicated for 48 hours at 60 ºC. The resulting solid, sample KAPTES, was recovered by filtration, washed with dichloromethane (3x 50 mL) to remove non-reacted APTES, centrifuged and then dried overnight at 60 ºC. 2.2.2. Intercalation reactions Organo-kaol derivatives were obtained by intercalation of the polar molecules DMSO and potassium acetate. Both molecules were intercalated to increase the basal

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spacing of the clay, thus facilitating the adsorption of proteins in the interlayer region. Besides, the intercalation converts the hydrophilic inter-layer surface to an organophilic one. The kaol–DMSO complex, sample KDMSO, was obtained using a procedure previously described [22]. 10 g of kaol was suspended in 50 mL of DMSO, and the mixture was stirred at 80 ºC in a glass flask for 7 days. The clay–DMSO complex obtained was washed with 1,4-dioxane to remove excess DMSO, centrifuged and dried at room temperature for 3 days. The kaol-potassium acetate intercalate, sample KAC, was synthesized following the classical procedure described by Smith et al. [23]. Briefly, 1 g of kaolinite was dispersed in 25 mL of a 4 M solution of potassium acetate, the mixture was stirred in a glass flask for 30 m. The resulting material was centrifuged at 5,000 rpm and dried at 60 ºC for 3 days. 2.3. Methods Kaol chemical composition was determined by X-ray fluorescence spectroscopy (XRF) with a Philips PW 2404 XRF spectrometer. A Perkin Elmer 2400 analyser with a Perkin Elmer AD-2Z microbalance was used to determine the carbon and nitrogen content of the bare and functionalized kaol. The amount of silane grafted was determined from the difference of carbon and nitrogen content after and before grafting. X-ray powder diffraction patterns were obtained using a Philips X'Pert MPD Pro X-ray diffractometer, operating at 45 kV and 40 mA, using Cu Kα radiation. The d001 basal spacings were calculated from the 2 values. Fourier transform infrared (FTIR) spectra using the KBr pressed disk technique were performed on a Perkin-Elmer PARAGON 1000 spectrometer. The spectra were collected for each measurement over the spectral range 450–4000 cm−1, with a resolution of 4 cm−1. Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) were performed on a Mettler TA-4000 TG-50

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type thermobalance using dynamic heating conditions (10 °C/min heating rate) under a nitrogen flow (50 mL/min). The samples were heated in alumina crucibles up to 900 °C. Nitrogen adsorption–desorption isotherms at 77K were obtained with a Micromeritics ASAP 2020 instrument, using static adsorption procedures. The samples were degassed at 120 °C for 12 h in a vacuum furnace before analysis. 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. 2.4. Adsorption experiments BSA 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 BSA solution buffered with sodium acetate (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. BSA adsorption isotherms on kaol and its organic derivatives were obtained by changing the initial protein concentrations. A liquid/solid ratio of 200 mL g−1, which was previously established as the most convenient value, was used in all the experiments. Every protein adsorption experiment on the different adsorbents was repeated three times, the values obtained being very close, and the average values are shown in this study. Protein concentrations in the initial solutions, and in the final supernatant obtained after adsorption, were measured with the UV/VIS spectrophotometer Heλios UV/VIS Thermo Electron Corporation at 750 nm, using the Folin–Ciocalteu colorimetric method [24]. A previous filtration through 0.45 μm

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polyvinylidene difluoride membrane syringe filters (Acrodisc) was required to retain the fine particles. 3. Results and discussion 3.1. Chemical composition Analysis of kaol, Al2Si2O5(OH)4, using X-ray fluorescence spectroscopy (XRF) showed that the composition was: SiO2, 50.25%; Al2O3, 34.22%; TiO2, 0.94%; Fe2O3, 0.97%; CaO, 0.03%; MgO, 0.16%; Na2O, 0.04%; K2O, 1.14%; H2O, 12.25%. The chemical analysis data reveals the presence of impurities in the sample. The X-ray diffraction pattern showed that quartz and mica phases were also present at trace levels. Values of C and N content of the bare and functionalized clay obtained by elemental analysis are shown in Table 1. The high nitrogen content of sample KTBS is due to the adsorption onto the kaol surface of the imidazolium ion formed during the silylation reaction [20]. This observation was confirmed by FTIR spectroscopy. Using CN analysis data and taking into account the molecular weight of kaol, and the molecular weights of the organic species attached to the external surface or intercalated, it is possible to calculate the total amount of organic molecules loaded on 1 unit structure of kaol, the values obtained being shown in Table 1. The following formulas for the kaol hybrids were determined from C and N elemental analysis

data:

KTBS,

Al2Si2O5(OH)4(C6H16SiO)0,29(C3H5N2)2,37;

Al2Si2O5(OH)4(C3H11NSiO3)0.65;

KDMSO,

KAPTES,

Al2Si2O5(OH)4(C2H6SO)0.66;

KAC,

Al2Si2O5(OH)4(C2H3KO2)0.83. As will be shown, the small amount of TBS grafted onto the kaol external surface played an important role in the protein adsorption performance of the hybrid. 3.2. X-Ray Diffraction

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The XRD pattern of kaol, Fig. 1A, showed the diffraction lines characteristic of a well-ordered solid, with a basal value d001 of 0.715 nm. Very low intensity diffraction lines corresponding to impurities of quartz and mica are also present in the diffractogram. The diffraction patterns of samples KTBS and KAPTES, which were prepared by kaol silylation with the silane coupling agents TBSCl and APTES, respectively, are similar to that of kaol but showed a lower crystal order, probably due to the presence of adsorbed organic species, or to the presence of some smaller clay particles formed during the treatment with the silanes. In Fig. 1 is only shown the diffractogram of sample KTBS (Fig. 1A). The (001) d-value of KTBS and KAPTES did not increase, indicating lack of expansion of the clay upon reaction with the silanes and, thus, lack of intercalation of the organic species. So far, kaol silylation was reported to occur on the external surfaces [22, 25, 26]. FTIR spectroscopy provided qualitative evidence of silane grafting onto the kaol external surface. (Colour should be used for this Figure) The diffractogram of the kaol-DMSO intercalate, Fig. 1B, showed a shift at lower angle of the (001) reflection; the value d001 of kaol expanded from 0.715 to 1.12 nm, the increment of 0.405 nm in d-value of kaol indicated the intercalation of KDMSO in the interlamellar space. When kaol was intercalated with KAc, Fig. 1C, the value d001 of kaol expanded from 0.715 to 1.42 nm, which represents an expansion of 0.705 nm. According to Frost et al. [27, 28] the kaol-potassium acetate intercalate was formed from the expansion of kaol by intercalation of potassium acetate and molecular water. The extent of the intercalation for both complexes was determined from the X-ray diffractograms; the intercalation percentage was calculated as the quotient between the intensity of the first basal reflection of the new phase and the sum of this signal plus the

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signal of the non-intercalated kaol. The intercalation ratio was 67.3 % for the kaolDMSO complex and 83.7% for the kaol-potassium acetate intercalate. The diffraction patterns of the clay and its organic derivatives after BSA adsorption did not show a significant shift from the basal d-value of kaol (0.715 nm) to higher values for the protein/kaol and protein/grafted-kaol complexes. In Fig. 2A is depicted the (001) reflection corresponding to kaol and the kaol-BSA complex. BSA adsorption on the kaol-intercalates led to the complete disappearance of the 1.12 nm d001 reflection characteristic of the kaol-DMSO intercalation complex and the 1.42 nm d001 reflection characteristic of the potassium acetate intercalate. In Fig. 2B is shown the disappearance of the 1.42 nm d001 reflection characteristic of the kaol- acetate complex after BSA adsorption. Therefore, both polar molecules were totally displaced from the interlamellar spaces during the adsorption of the protein.

It was reported that when the interaction between kaol and the organic guests was due mainly to weak interactions, the kaol-intercalates are unstable in the presence of water [29]. Under the protein adsorption conditions in an aqueous medium, the guests are displaced from the interlayer spaces, resulting in the formation of kaol hydrates [30]. This means that there was no protein intercalation in the previously expanded kaolDMSO and kaol-potassium acetate complexes during the BSA adsorption process, the adsorbed protein molecules being immobilized at the external surfaces and edges of the organic clay derivatives. 3.3. FTIR Spectra of Samples When functionalization of clays is 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

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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 spectrum occur. In particular, the hydroxyl stretching region of kaolinite is sensitive to the effects of interlayer modification. Therefore, intercalation of organic species and grafting of organosilanes on the aluminol internal surface should have a major influence on the OH stretching pattern of kaol. FTIR transmission spectra of the samples, in the range 40002500 cm-1, are displayed in Figure 3. The IR spectrum of kaol, Fig. 3A, showed bands at 3696 and 3653 cm−1, assigned to stretching vibrations of the inner-surface hydroxyl groups; the peak at 3620 cm−1 corresponds to stretching vibrations of the inner-sheet hydroxyl groups [31 - 33], the broad band centred at 3434 cm−1 was assigned to the hydroxyl stretching vibrations of water. The FTIR spectra of samples KTBS and KAPTES, Fig. 3B and Fig. 3C, showed that the absorption bands corresponding to hydroxyl stretching modes of the clay do not change after silylation, confirming that the reaction with the organosilanes did not modify the 1:1 layer structure of kaol. The stretching vibration bands of the organosilanes C–H groups are observed at 2975 cm−1, 2926 cm−1 and 2853 cm−1, indicating the grafting of organic groups onto the kaol surface [17, 22].

The spectrum of sample KTBS also showed a peak at 3135 cm−1 that can be assigned to aromatic C–H stretch of imidazolium, which was used as a catalyst during the functionalization reaction. Attachment of imidazolium to the kaol surface is confirmed by the strong, broad band centred at 3420 cm−1 that can be assigned to aromatic N–H stretch overlapping with water OH stretch, and a sharp band at 1585 cm−1 that corresponds to N–H bending vibrations. Intercalation of DMSO into the kaol interlayer resulted in an important decrease in the intensity of the band at 3696 cm−1 and the disappearance of the peak at 3653

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cm−1, Fig. 3D. The spectrum showed the presence of three new bands at 3662, 3540 and 3504 cm−1 attributed to the formation of hydrogen bonds between the intercalated DMSO species and the inner-surface hydroxyl groups [34-36]. The vibration frequency of the band at 3620 cm−1, assigned to stretching vibrations of inner-layer hydroxyls, was not modified by the presence of DMSO, but the intensity of this band increased when the kaol-DMSO complex was formed. Therefore, intercalated DMSO species interact with the inner-surface hydroxyl groups of kaol and new hydrogen bonds are built in a three-dimensional structure [34, 36]. The bands at 3025 and 2937 cm−1 correspond to antisymmetric and symmetric C-H stretching peaks of intercalated DMSO species [34, 37]. The FTIR spectrum of the acetate-kaol intercalate is displayed in Fig. 3E; it showed bands at 3696 and 3653 cm−1 assigned to stretching vibrations of the innersurface hydroxyl groups of kaol, the peak at 3620 cm−1 was assigned to the inner hydroxyls. The low-intensity band at 3599 cm-1 was attributed to the hydroxyl stretching vibration of the inner surface hydroxyls, which are hydrogen bonded to the acetate [27, 28]. The broad band centred at 3450 cm-1 was attributed to the hydroxyl stretching frequency of interlayer water coordinated to acetate [28]. The FTIR spectra of the kaol–BSA and organokaol-BSA complexes do not show any significant shift of the hydroxyl stretching and deformation bands corresponding to the spectra of the different adsorbents, suggesting the absence of hydrogen bonding between adsorbed protein molecules and the OH groups of the adsorbents. Moreover, the FTIR spectra of the complexes BSA-kaol and BSA-organokaol hybrids showed that the peak position of the bands characteristic of the protein did not change, 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), indicating that the

C = O, C–N and

N–H groups of the

polypeptide chain did not interact with the hydroxyl groups of the adsorbents, as is

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shown in Fig. 4B for the kaol-BSAcomplex and in Fig. 4C for the KDMSO-BSA complex.

3.4. Thermal Analysis Thermal analysis techniques provide qualitative and quantitative information on the thermal behaviour of the materials. Furthermore, thermal analysis makes it possible to gain more insight into the grafting and intercalation process, since thermal analysis data can be used to determine the amount of organic molecules intercalated, or anchored on the clay surface and edges. In Fig. 5 are displayed the TG and DTA curves of all the samples obtained under a nitrogen flow. Thermal analysis of kaol, Fig. 5A, showed a small mass loss (0.2%) below 100 ºC, corresponding to the removal of small amounts of physically adsorbed water. The mass loss associated in the DTA with an endothermic peak centred at 520 ºC, is due to the loss of structural water by kaol during its transformation to metakaolinite (Al2Si2O5(OH)4 → Al2Si2O7 + 2H2O); its value (12.98 %) is in good agreement with the value of 13.95% calculated from the theoretical formula of pure kaol. The thermal degradation of sample KTBS took place in two steps, as is shown in Fig. 5B. Below 180 °C there is no significant mass loss. The removal of the silane and the imidazole attached to kaol occurred between 180 and 330 °C, with a DTA endothermic peak at 320 ºC; the mass loss corresponding to this step is 45.36%. After the removal of the organic species, the thermal behaviour of the remaining structure is essentially identical to what was observed in the case of pure kaol, and the second endothermic peak at 520ºC is due to dehydroxylation of the clay. The TG curve of sample KAPTES, Fig. 5C, showed a continuous multistep mass loss from approximately 50 to 800 ºC. The endothermic peak at 65 ºC was attributed to the elimination of physically adsorbed water molecules [38]. The endothermic DTA peak at 125ºC was attributed to organic fragments resulting from the decomposition of the

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grafted organosilane [22]. The mass loss between 100 and 400ºC was 24.35 %, which corresponded to the elimination of the organosilane. Kaol dehydroxylation took place above 400º C, with a DTA endothermic peak at 510 ºC.

The TG curve of sample KDMSO showed three mass losses, Fig. 5D. The first mass loss with DTA endothermic peaks at 95 ºC, it is due to the removal of adsorbed water; a second mass loss of 15.91%, with DTA peak at 190ºC, was attributed to the removal of intercalated DMSO [29, 34]. The third mass loss, DTA peak at 520 ºC, is due to dehydroxylation of the kaol layers. The TG curve of kaol-acetate intercalation complex shows a mass loss of 1.36 %, DTA peak at 95 ºC, which is due to the removal of adsorbed water. The second mass loss of 20.93%, with DTA peak at 380 ºC, was attributed to the removal and decomposition of acetate together with the removal of intercalated water coordinated with acetate [39]. The mass loss with DTA peak at 490 ºC is due to dehydroxylation of the kaol–potassium acetate intercalation complex and the non-intercalated kaol (since full intercalation was not achieved) [39-41]. The dehydroxylation temperature of the kaol-acetate intercalate was lower than that of the pure clay, which is probably caused by the degradation of the crystal structure of kaol during the intercalation and deintercalation processes. The region of the thermograms between 100 and 400 °C, which corresponds to the removal of the organic molecules during thermal treatment, can be considered for quantitative determination of both silane coverage and the ratio of DMSO intercalation. For the kaol-acetate intercalate TG analysis data could not provide the adequate information, because within the temperature range of decomposition of intercalated acetate, the removal of intercalated water also takes place. Therefore, it is difficult to distinguish the water loss from the acetate loss process [39]. As seen in Table 2, the

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mass loss of the hybrids corresponding to the removal of the loaded organic species determined by TG analysis, is in good agreement with the values of the mass loss calculated from the formula proposed for the hybrids, which was obtained from C and N analysis data (Table 1).

3.5. Textural characterization It is known that the adsorption behaviour depends on various factors, which include adsorbate-adsorbent interactions controlled by the nature of the functional groups present on their surface, pH, batch or column conditions, specific surface area, and pore size distribution of the adsorbents. The porous structure of the separation media is a fundamental feature required to achieve good retention capacity. The use of macroporous adsorbents favours a rapid mass transfer of macromolecules that improves the kinetics of retention. Moreover, large pore surfaces can accommodate large protein molecules, while small pores are not accessible. Therefore, a detailed study of the surface area and the type of porosity, micro/meso/macro, as well as the pore size distribution are needed to understand the protein adsorption behaviour of kaol and the organokaol hybrids. Specific surface areas and mesopore size distributions were calculated from the nitrogen adsorption–desorption isotherms at 77 K. The profile of the isotherms is typical of type IV of the BDDT classification [42], with very narrow hysteresis loops, type H3, which are usually associated with slit-shaped pores. In Fig. 6A is depicted the isotherm corresponding to sample KTBS. All the isotherms show a low nitrogen uptake at relative pressures lower than 0.2, which indicates the absence of microporosity. The adsorption limit is not well-defined at relative pressures close to one, indicating the presence of macroporosity in the solids.

The mesopore size

distributions were analysed using the method of Barret, Joyner and Halenda (BJH) [43]

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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 diameter distribution curves and the cumulative pore volume of each sample (Vp) are listed in Table 3.

Kaol functionalization with TBSCl drastically reduced the specific surface area from 9 to 2.1 m2g−1; the pore volume was also considerably reduced (Table 3), which may be accounted for by the silane blockage of the narrower pores. Kaol silylation with APTES, and DMSO intercalation in the kaol interlayer, resulted in a slight increase of the BET area to 10.7 m2g-1 for sample KAPTES and 10.2 m2g-1 for KDMSO; the pore volume was also increased (Table 3). Intercalation of potassium acetate into the clay significantly decreased the specific surface area to 2.7 m2g−1 and the pore volume (Table 3), which is probably due to the modification of the kaol microstructure during the intercalation process. Macroporosity of the samples was studied by mercury intrusion porosimetry. The pore size distribution profile of sample KTBS is shown in Fig. 6B. Values of the total pore volume (Vp) and most frequent pore diameters (Dpfreq) are listed in Table 4.

Sample K shows a narrow pore size distribution profile, with a maximum at 6 μm that corresponds to the most frequent pore diameter. The total pore volume and the pore size of the organokaol hybrids were modified during the functionalization process. The higher values of the pore volume, even for samples KTBS and KAC that exhibit very low specific surface areas (Table 3), is due to the formation of a secondary pore system made of wide inter-particle voids, which contribute more to volume than to the surface. The pore size distribution curve of sample KTBS, Figure 6B, showed two maxima, most of the pores having radii between 10 and 17 μm. The low surface area of this

18

sample and the very large pore diameters are an indication that porosity may be essentially formed by inter particle cavities. The pore size distribution profile of samples KAPTES and KDMSO showed three maxima (Table 4); kaol functionalization with APTES and DMSO favoured the formation of smaller macropores, and also larger inter particle cavities. Intercalation of potassium acetate resulted in the modification of the kaol microstructure; acetate intercalation favoured particle aggregation and, as a result, a substantial decline in the specific surface area and mesopore volume. However, total pore volume was increased due to the formation of wide interparticle voids (Table 4).

3.6. Microstructural characterization Microstructure changes induced by functionalization are clearly observed in Fig. 7, in which SEM micrographs of kaol and those of the functionalized solids are depicted. Kaol particles show a flake aspect, and are grouped forming plates and stacks, Fig. 7A. The functionalized solids maintain the crystalline habit. However, the interaction of kaol with TBSCl favoured the formation of considerably larger particle aggregates and wider pores, Fig. 7B. Kaol functionalization with APTES caused a partial disintegration of the particles with formation of a spongier texture, Fig. 7C. SEM images of kaol-DMSO and kaol-acetate intercalates are shown in Fig.7D and 7E, respectively. Both intercalation complexes consist of flake type particle aggregates, being considerably larger those corresponding to sample KAC, which exhibited lower surface area.

3.7. Protein adsorption experiments

19

The affinity of a protein for a given adsorbent depends on several factors, such as the adsorbate-adsorbent interactions, the pH and the ionic strength of the solution [44, 45]. Maximum protein adsorption was observed at the isoelectric point of each protein (IEP) [20, 45, 46], because electrostatic repulsions between identically charged adsorbed proteins are minimized. Therefore, adsorption experiments were performed at pH = 5.0 because this value is very close to the IEP (4.9) of BSA [47, 48]. To investigate the influence of the adsorbent surface properties on protein retention, adsorption experiments were run using kaol and the organo-kaol hybrids synthesized. Kaol suspension in distilled water has a pH of 3.8, resulting from proton dissociation of superficial hydroxyl groups [49], which is responsible for the acid character of the mineral suspension. The suspension was buffered with sodium acetate at a pH of 5.0. Aqueous suspensions of the organo-kaol hybrids were also buffered with sodium acetate at pH of 5.0. The ability of modified kaol to adsorb BSA from aqueous solution was evaluated by measuring the adsorption isotherm for each organo-kaol hybrid. Under equilibrium conditions, the adsorption processes between adsorbent and adsorbate can be characterized by the amount of adsorbed protein per gram of the modified kaol (q). This value was calculated from the initial concentration of BSA added (c0) and those at the equilibrium point (c). Profiles of the adsorption isotherms are shown in Fig. 8, representing the amount of adsorbed BSA versus the concentration of the solution under equilibrium conditions. Protein adsorption on bare kaol followed the Langmuir equation for a liquid state sorption system (Fig. 8). 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:

20

q

qm  c kd  c

Where q is the equilibrium protein concentration on the adsorbent (mg g-1), c is the equilibrium liquid phase concentration (g L-1), qm is a constant related to the area occupied by a monolayer 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), obtaining the Langmuir parameters qm and kd that appear in Table 5.

BSA adsorption on the organokaol hybrids followed the Freundlich isotherm, Fig. 8. The Freundlich isotherm was developed to describe multi-site adsorption on heterogeneous surfaces. In this case, there is a continuously varying energy of adsorption as the most actively energetic sites are occupied first, then the surface is continually occupied until the lowest energy sites are filled at the end of adsorption process. The Freundlich equation is expressed as: ln 𝑞 = 𝑙𝑛 𝐾 + 𝑚 ln 𝑐 Where 𝐾 and 𝑚 are the Freundlich constants related to adsorption capacity and adsorption intensity, respectively. So, the plot of ln 𝑞 against ln 𝑐 of the Freundlich equation should give a linear relationship, from which m and 𝐾 can be determined from the slope and the intercept, respectively. Experimental data were fitted to the Freundlich equation using the program Scientist (Micromath), obtaining the parameters 𝑚 and 𝐾 that are presented in Table 5. The correlation coefficients of each isotherm equations are also shown in Table 5.

21

As was shown in Fig 8, the adsorption behaviour of the initial raw kaol is different from that of the organic derivatives. BSA adsorption on kaol followed the Langmuir isotherm, which assumes monolayer adsorption of adsorbate over a homogeneous adsorbent surface, the total adsorption capacity being 53mg BSA/ g kaol. The isotherms of the organokaol hybrids followed the Freundlich equation, clearly indicating that multilayer adsorption took place. Additional information about the influence of the adsorbent surface on protein retention was obtained by running BSA adsorption experiments, using a liquid/solid ratio of 200 mLg−1 and protein concentrations of 2 g L−1. The results obtained are given in Table 6. To explain the different retention capacity a number of factors must be

considered. The affinity of a protein for a given surface depends on its composition and structural stability, the surface properties of the adsorbent and the pH [45]. Steric effects should also be considered; therefore, the textural properties of the adsorbents also have an influence in the adsorption behaviour [20]. Kaol behaved as a good adsorbent for the whey protein BSA at pH 5 (44 mgBSA/g kaolinite); X-ray diffraction data showed that protein adsorption took place on the external surface. FTIR spectrum of the kaol/BSA complex showed that no hydrogen bonding occurred between the adsorbed protein molecules and the surface hydroxyl groups of kaol. BSA adsorption on sample KTBS was considerably increased, 36% higher (Table 6); the formula proposed for this hybrid, Al2Si2O5(OH)4(C6H16SiO)0,29(C3H5N2)2,37,

showed

that

the

amount

of

tert-

butyl(dimethyl)silanol grafted on the kaol external surface was low, thus, only a partial hydrophobisation of the kaol surface was achieved. The amount of imidazolium

22

attached to the kaol surface was considerably larger; however, it was found in a previous study [20] that the BSA adsorption capacity of kaol was not modified when imidazolium chloride is adsorbed on its surface, since it is readily dissolved in the buffer solution containing the protein during the adsorption process. Even though kaol grafting with TBS considerably reduced the BET surface area, it favoured the formation of wider pores, in the micrometer range, which are easily accessible to large biomolecules, such as BSA. The increased adsorption capacity of sample KTBS can be explained by taking into account the presence of hydrophobic patches on the adsorbent surface, which favoured the formation of strong hydrophobic interactions with the large number of non-polar groups (253) that reach the external positions of the protein [48, 50]. BSA adsorption on KAPTES (100 mg/g) was increased by a factor of 2.3 with respect to that of kaol. The presence of electrophilic amino groups on the surface of the silanized adsorbent was responsible for the formation of electrostatic interactions between the cationic centres of the modified clay and the polar centres of the protein, which would explain the increased adsorption shown by this hybrid. Sample KDMSO also showed a higher BSA adsorption capacity (66 mg/g) than kaol. Both samples had similar textural properties (Table 3). XRD data showed that BSA adsorption on the kaol-DMSO intercalate led to the disappearance of the 1.12 nm d001 peak characteristic of the DMSO-kaol intercalation complex, indicating that DMSO was displaced from the interlamellar spaces during the adsorption of the protein. However, the FTIR spectrum of the KDMSO/BSA complex, Fig. 4C, showed absorption bands at 3540, 3503, 3025 and 2937 cm−1, characteristic of DMSO, which indicates that during the BSA adsorption process deintercalated DMSO is adsorbed on the kaolinite external surface. The attachment of this polar molecule to the kaol surface considerably increased the adsorption capacity, 66 mg/g, probably due to electrostatic interactions between the

23

polar groups attached to the adsorbent surface and the ionic patches existing at the interface of the protein [46, 51]. Finally, BSA adsorption on sample KAC was very low, 15 mgBSA/g. Potassium acetate intercalation and deintercalation in kaol drastically modified its structural and textural properties, lowering its adsorption capacity.

Conclusions Kaolinite is a good adsorbent for the whey protein BSA at pH 5.0 and 25ºC. The low cost and absence of toxicity of this clay, especially when compared with standard polymeric resins, render kaol a very appropriate adsorbent for the retention of proteins contained in dairy industry effluents. Kaolinite functionalization by grafting the organosilanes TBSCl and APTES, and by intercalation of DMSO and potassium acetate, caused important changes in the texture and microstructure. In addition, it allowed modification of the hydrophilic character of the surface to hydrophobic or electrophilic, which resulted in increased BSA retention capacity. Partial hydrophobisation of the kaol surface increased BSA adsorption capacity by 36%. The presence of electrophilic cationic centres on the surface increased its protein adsorption capacity by a factor of 2.3. Intercalation of small polar molecules to expand the interlayer spacing, in order to favour adsorption into the inner kaolinite surface, it is not appropriate for enhancing the adsorption capacity. On the contrary, it reduces the adsorption capacity. By surface functionalization of low cost non-toxic clay minerals, protein adsorbents with improved retention capacity can be obtained. Additional research in this area will allow the development of improved adsorbents for the recovery of bio-molecules present in food industry effluents.

24

Acknowledgements Technical assistance from the Scientific-Technical Services of the University of Oviedo is gratefully acknowledged. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

25

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28 [49] G. Józefaciuk, Effect of acid and alkali treatment on surface-charge properties of

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29

Fig. 1. XRD patterns of: (A) Kaol (K), Kaol-TBS (KTBS); (B) Kaol-DMSO (KDMSO); (C) Kaol-acetate (KAC).

1000

800

A

─K

─KTBS

(001)

600

Intensity (a.u.)

400

200

0 10

10

20

20

30

30 30

40

40 40

B

10 1020 20 800

60

70

2Theta (°)

─ K ─KDMSO

(001)

1000

50

50 50 6060 70 70

30 50 40

C

50

─K

(001)

60

7070

─KAC

600

400

200

0 10

10

20

20

30

40

50

30 40 50 2 Theta (º)

60

60

70

70

2Theta (°)

Fig. 1. XRD patterns of: (A) Kaol (K), Kaol-TBS (KTBS); (B) Kaol-DMSO (KDMSO); (C) Kaol-acetate (KAC). (Color should be used for this Figure)

30

Fig. 2. (A) (001) reflection of kaol (K) and kaol-BSA complex (K-BSA); (B) (001) reflection of KAC and KAC-BSA complex.

Fig. 3. FTIR transmission spectra (4000–2500 cm-1) of: (A) kaol; (B) kaol –TBS; (C) kaol -APTES; (D) kaol-DMSO; (E) kaol-acetate.

31

\

Exothermic Endothermic

Mass Loss (%)

Fig. 4. FTIR transmission spectra of: (A) kaol; (B) kaol-BSA complex; (C) KDMSO-BSA complex.

Fig. 5. TG and DTA curves of: (A) kaol; (B) KTBS; (C) KAPTES; (D) KDMSO; (E) KAC.

32

Fig. 6. (A) Nitrogen adsorption–desorption isotherm at 77 K of sample KTBS. (B) Pore size distribution profile of sample KTBS, measured by mercury intrusion

33

A

B

C

D

E

Fig. 7. SEM images, 7500×, of: (A) Kaol; (B) KTBS; (C) KAPTES; (D) KDMSO; (E) KAC.

34

350

qm (mg BSA/g)

300 250 200 150 100 50 0 0

1

2

3

4

Ce (g BSA/L) Fig. 8. BSA adsorption isotherms for kaol and the organo-kaol derivatives: ( ■ ) KAC; (▲) KTBS; (x) KDMSO; (●) KAPTES; (♦) Kaol.

35

Table 1. C and N content (mass %) of kaol and its organic derivatives and organic material content of the hybrids, n (number of moles of organic species per kaolinite structural unit, Al2Si2O4(OH)4). Sample C (%) N (%) n K 0.21 0.07 ─ KTBS 0,29* 23.40 14.52 2.37** 6.94 2.67 0.65 KAPTES 5.36 0.03 0.66 KDMSO KAC 6.10 0.03 0.83 *moles of TBS per kaolinite structural unit [Al2Si2O5(OH)4)] ** moles of imidazolium per kaolinite structural unit [Al2Si2O5(OH)4)]

Table 2. Values of the mass loss (%) from TG data, and calculated using the formulas proposed for the hybrids. Sample Mass loss (%) Mass loss (%) (TG data) (calculated) K 13.95 12.93 KTBS

45.36

43.90

KAPTES 24.35

25.66

KDMSO

16.65

15.91

Table 3. Textural parameters of the samples obtained from the nitrogen adsorption–desorption isotherms at 77 K. Sample

SBET (m2g-1) Dpmax (nm) Vp (cm3 g-1 STP)

K

9

20.4

0.051

KTBS

2.1

33.6

0.016

KAPTES 10.7

24.5

0.065

KDMSO

10.2

19.4

0.053

KAC

2.7

31.3

0.023

36

Table 4. Textural parameters of the samples obtained by mercury intrusion porosimetry. Sample

Vp (mL g-1)

Dpfreq (µm)

K

0.476

6

KTBS

0.893

11 y 17

KAPTES

1.091

0.2, 0.8, 11

KDMSO

0.870

0.6 , 1.2, 60

KAC

0.633

10 , 40

Table 5. Isotherm constants for BSA adsorption on kaol and organokaol hybrids.

Sample K

Langmuir qm (mgBSA g-1) 53.00 Freundlich

Sample

K (mgBSA g-1) KTBS 18.494 KAPTES 72.554 KDMSO 11.222 KAC 0.817

R2 Kd (gBSA L-1) 0.36 0.97 m

R2

2.2017 1.1922 3.076 4.0

0.98 0.99 0.97 0.98

Table 6. Protein retention capacity (average values) of the different samples (mg BSA/g sample), obtained using a liquid/solid ratio of 200 mLg−1 and protein concentrations of 2 g L−1. Sample

K

KTBS

KAPTES

KDMSO

KAC

mg BSA/g

44

60

100

66

15