Synthesis and characterization of ordered mesoporous silica (SBA-15 and SBA-16) for adsorption of biomolecules

Synthesis and characterization of ordered mesoporous silica (SBA-15 and SBA-16) for adsorption of biomolecules

Microporous and Mesoporous Materials 180 (2013) 284–292 Contents lists available at ScienceDirect Microporous and Mesoporous Materials journal homep...

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Microporous and Mesoporous Materials 180 (2013) 284–292

Contents lists available at ScienceDirect

Microporous and Mesoporous Materials journal homepage: www.elsevier.com/locate/micromeso

Synthesis and characterization of ordered mesoporous silica (SBA-15 and SBA-16) for adsorption of biomolecules Sandra Maria Lopes dos Santos, Karina Alexandre Barros Nogueira, Marlon de Souza Gama, Jeann Diniz Ferreira Lima, Ivanildo José da Silva Júnior, Diana Cristina Silva de Azevedo ⇑ Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, S/N, Bloco 710, CEP: 60455-760 Fortaleza, CE, Brazil

a r t i c l e

i n f o

Article history: Received 21 May 2013 Received in revised form 28 June 2013 Accepted 29 June 2013 Available online 12 July 2013 Keywords: Separation Adsorption isotherm Protein Enzyme Silica

a b s t r a c t The present work describes the adsorption of biomolecules (bovine serum albumin (BSA), lysozyme (LYS) and cellulase (CEL)) on ordered mesoporous silicas with different pore diameters (SBA-15 and SBA-16) from buffered solutions. These adsorbents were synthesized by sol–gel and hydrothermal routes and characterized by X-ray diffraction, N2 adsorption/desorption isotherms and transmission electron microscopy (TEM). The results by X-ray diffraction and TEM show that the synthesized materials have distinct degrees of mesoporous ordering. The influence of pH on the adsorption of BSA, LYS and CEL as well the kinetics and adsorption isotherms were evaluated in stirred tanks. Among the materials studied, hydrothermally synthesized SBA-15 showed the highest maximum adsorption capacity of BSA and LYS (329 and 636 mg/g, respectively). CEL was best adsorbed in SBA-15 synthesized by the sol–gel route. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction In 1992, it was first reported the discovery of mesoporous materials belonging to the M41S family [1,2]. Out of these, two materials have been highlighted, the MCM-41, obtained in basic media route and SBA-15, and obtained in acid media. SBA-15 (Santa Barbara Amorphous), developed in 1998 by Zhao et al. [3], has attracted intense interest due to their large surface areas, well-defined pore structure, inert framework, nontoxicity, high biocompatibility [4] and thermal and hydrothermal stability [5], which allows them to be used in catalysis [6,7], adsorption [8–10], chemical sensing [11], immobilization [12], drug delivery systems [13,14] and separation by chromatographic techniques such as high performance liquid chromatography (HPLC) [15–17]. Another mesoporous adsorbent that has received attention is the SBA-16 whose formation mechanism is similar to that of SBA-15 [18–22]. The easiness of the method of preparation, the orderly structure, and control over the size and shape of their pores, above all of these factors, makes SBA-16 a versatile material, potentially applicable in many areas of science and engineering of materials [23]. Although it is reported in the literature the application of SBA-16 in fields such as catalysis [24–26], functionalization [27], metals incorporation [28] and templating [29,30], there are a few records [31] of the use of such materials for adsorption/chromatographic purposes. ⇑ Corresponding author. Tel.: +55 8533669611; fax: +55 8533669610. E-mail address: [email protected] (D.C.S. de Azevedo). 1387-1811/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.micromeso.2013.06.043

Both SBA-15 as SBA-16 are synthesized in acidic media in the presence of triblock copolymer surfactants Pluronic P123 (PEO20PPO70PEO20) [3] and Pluronic F127 (PEO106PPO70PEO106) [18], respectively. However, the resulting materials have very distinct structures, as shown in Fig. 1. SBA-15 is a mesoporous silica with parallel pores and highly ordered hexagonal arrangement (see Fig. 1a). It has the benefits of combined micro and mesoporosity and relatively thick silica walls. The micropores are created by the penetration of the hydrophobic ethylene oxide chain in the silica walls [32]. On the other hand, SBA-16 is a mesoporous silica having a structure of spherical body-centered nanocages with cubic arrangement, wherein each sphere is connected to eight neighboring spheres [33] (see Fig. 1b). For operations such as catalysis or separations, this difference in structure may favor or not the efficiency of the process. For example, Li et al. [34] found that the encapsulation of Ru complexes in nanocages of SBA-16 is an efficient method to achieve high catalytic activity by enhancing water oxidation. Data on application of SBA-16 in the adsorption of biomolecules are scarce, hence it is yet to be investigated if this structure model could positively influence the separation of these bulking biomolecules. Some authors have recently reported the use of synthetic hydrotalcites known as Layered Double Hydroxides [35–37] and mesostructured cellular foams (MCF) [38] as new potential adsorbents for biomolecules. These materials have shown high adsorption capacity for standard proteins, for example bovine serum albumin (BSA), human serum albumin (HSA), lysozyme (LYS) and myoglobin (Mb).

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Fig. 1. Schematic illustration of the channels of SBA-15 (a) and nanocages of SBA-16 (b). Adapted from Zhao et al. [3] and Flodström et al. [21], respectively.

In this work, SBA-15 was synthesized by two distinct routes, sol–gel and hydrothermal. The sol–gel route has, as main advantage, the possibility of being performed under mild conditions of temperature and pressure. However, the time required for the hydrolysis and condensation steps to occur is generally well above that required by hydrothermal synthesis. In turn, the latter method often results in materials with surface areas and pore diameters greater than those obtained by the sol–gel route. Hence, it is interesting to compare the ability of SBA-type materials obtained by these two routes to adsorb the biomolecules under study. Thereby, this work aimed at synthesizing ordered mesoporous silicas of SBA-15 and SBA-16 type and investigates their potential in the adsorption of bovine serum albumin (BSA), lysozyme (LYS) and cellulase (CEL). 2. Experimental part 2.1. Reactants The triblock copolymers Pluronic P123 (PEO20PPO70PEO20), Pluronic F127 (PEO106PPO70PEO106), tetraethoxysilane (TEOS, 98%), hydrochloric acid (36%), CEL from Aspergillus niger and LYS were purchased from Sigma Aldrich (USA). BSA was supplied by INLAB (Brazil); butyl alcohol from Aldrich (99.4%); ethanol, octane and ammonium fluoride from Vetec (Brazil). All chemicals were used as received without any further purification.

wards, the reaction mixture was kept still and aged for 24 h at the same temperature without stirring. Subsequently, the temperature was raised to 353 K and kept at this value for 48 h. The solids were filtered, washed with abundant deionized water to remove excess surfactant. Finally, the samples were dried at 333 K for 12 h and calcined at 823 K for 6 h. The molar composition of the synthesis gel was 1.00 TEOS:0.017 P123:6 HCl:160 H2O. Hydrotermal synthesis of SBA-16 followed the synthesis method reported by Kleitz et al. [41]. The mesostructured SBA-16 silica materials were prepared using a mixture of Pluronic F127 and butyl alcohol as structure-directing agents. The silica source was TEOS. In a typical synthesis 4.0 g F127 were dissolved in a 0.4 M HCl (200 mL) aqueous solution. The synthesis was carried out in a closed polypropylene bottle. After complete dissolution, 13.75 g of butyl alcohol was added at once at 318 K. After 1 h under stirring, 19.3 g of TEOS was quickly added to this mixture. The molar composition of the synthesis gel was 1.00 TEOS:0.0035 F127:1.78 BuOH:0.88 HCl:119 H2O. The mixture was further stirred vigorously at 318 K for 24 h to allow for the formation of the mesostructured product. Subsequently, the reaction mixture was heated in an autoclave at 373 K for 24 h under static conditions. The white precipitate was then filtered without washing and dried at 373 K for 24 h in air. To remove the copolymer template, the solid was briefly rinsed at room temperature with an ethanol/HCl mixture for 20 min, filtered, dried, and then calcined at 823 K for 2 h. 2.3. Characterization

2.2. Synthesis SBA-15 was synthesized by sol–gel and hydrothermal routes. SBA-16 was only synthesized hydrothermally. Hydrothermal synthesis of SBA-15 (SBA-15 HD) was performed according to the procedure reported by Zhang et al. [39] with adaptations. As synthesis procedure, 4.6 g of P123 was dissolved in 160 mL HCl solution (1.3 M), followed by the addition of 0.052 g of NH4F. After a few hours under stirring at 298 K, a clear solution was obtained and then 21.72 g of octane and 9.76 g of TEOS were added. The resulting mixture was stirred for 24 h and then transferred to an autoclave for further reaction at 373 K for 48 h. The solid was filtered, dried in air and calcined at 823 K for 5 h to remove the organic templates. The molar composition of the synthesis gel was 1.00 TEOS:0.018 P123:6.36 HCl:201.8 H2O:0.033 NH4F:4.7 octane. Sol–gel synthesis of SBA-15 (SBA-15 SG) was carried out as reported by Esparza et al. [40] with adaptations. 1.7 g P123 was dissolved in 50.4 mL aqueous HCl solution (2 M) and kept under stirring at 323 K until a transparent solution was obtained. 3.75 mL TEOS was added drop-wise under vigorous stirring. After-

X-ray diffraction (XRD) was used to identify the crystal phases of the synthesized solids. These experiments were performed with a Siemens D5000 power X-ray diffractometer equipped with a CuKa radiation source (wavelength 1.5418 Å). Measurements were obtained for 2h ranging from 1° to 10°. The transmission electron micrographs (TEM) were obtained with the aid of a high resolution microscope Philips CCCM 200 Supertwin-DX4. Measurements of N2 adsorption/desorption at 77 K were carried out using a volumetric adsorption equipment (AUTOSORB-1MP, Quantachrome Instruments). The specific surface area (SBET) of the samples was estimated with the Brunauer, Emmet and Teller (BET) method [42], using the adsorption data in the range of relative pressures from 0.05 to 0.18 and 0.05 to 0.23 for the samples SBA-15 and SBA-16 respectively, where conditions of linearity and considerations regarding the method were fulfilled [43]. The pore size distribution was calculated from the desorption branch of the isotherms by using the nonlocal density functional theory (NLDFT) [44]. The total pore volume was taken as the adsorbed volume at p/ p0 = 0.95.

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2.4. Protein adsorption experiments Batch adsorption experiments were carried out in an orbital shaker (Tecnal, Brazil). For this aim, 15 mg adsorbent were put in contact with 3.0 mL buffer solution containing the target biomolecule (BSA, LYS or CEL). Initially, the effect of pH on the biomolecule uptake was evaluated. For BSA, an initial concentration of 1.0 mg/ mL in acetate buffer (50 mmol/L) was used with pH ranging from 3.6 to 5.6 by adding either HCl or NaOH. For the other biomolecules, the initial concentration was 3.0 mg/mL, using sodium bicarbonate buffer (25 mmol/L) with pH ranging from 6.5 to 12.0 for LYS and acetate buffer (50 mmol/L) with pH ranging from 3.0 to 5.4 for CEL. For kinetic experiments, the initial concentrations used were the same as those of pH tests. The samples were collected from the experimental tubes at pre-determined time intervals. For the measurement of the adsorption isotherms, different initial concentrations of BSA (1.0–15.0 mg/mL), LYS (1.0–15.0 mg/mL) and CEL (1.0–7.0 mg/mL) at fixed pH and ionic strength were shaken for enough time to ensure equilibrium. The solid/liquid ratio used in the determination of adsorption isotherms was 5.0 mg/mL. In all experiments, after a given time, the samples were collected and centrifuged for 10 min at 10,000g (refrigerated microcentrifuge Cientec CT-15000R, USA). The concentration in the supernatant was analyzed with a UV spectrophotometer at 280 nm (UV–Vis spectrophotometer Biomate 3, Thermo Scientific, USA). The adsorbed concentration of each biomolecule was calculated using a simple mass balance, according to Eq. (1):



V sol ðC 0  C eq Þ mads

ð1Þ

where Vsol represents the volume of biomolecule solution at equilibrium (typically 3 mL); C0 the initial concentration of the biomolecule (mg/mL); Ceq is the equilibrium concentration (mg/mL) and mads is the mass of adsorbent (typically 15 mg). 3. Results and discussion 3.1. Characterization of SBA-15 and SBA-16

Intensity

The X-ray diffraction patterns were used to identify structural ordering of mesoporous materials under study. The results are shown in Fig. 2. In the case of SBA-15 SG, three main diffraction peaks are present referring to the crystal planes corresponding to Miller indices (1 0 0), (1 1 0) and (2 0 0). These first three peaks are characteristic of a two-dimensional hexagonal pore arrangement, commonly found in materials like SBA-15 [45,46], indicating a well

(a) (b) (c) 0

defined mesostructure. On the other hand SBA-15 HD is far less orderly and practically shows only a well resolved peak at 2h  1°. After 2h = 1° there are unresolved peaks which may indicate that there is some degree of mesoporous arrangement to be confirmed by TEM. SBA-16 also only shows one strong peak at low angle. Its position corresponds to the strong peak observed with polyhedral particles and indexed by plane (1 1 0) [47]. Such arrangement of materials may also be observed through TEM images illustrated in Fig. 3. The hexagonally arranged pore arrays of the pure-silica SBA-15 samples can be clearly observed in both synthesis routes (Fig. 3A1, B1, A2 and B2). As expected, the TEM images of SBA-16 do not show the hexagonal ordering as in SBA-15, but rather interconnected cage-like pores. Figs. 4 and 5 shows the N2 adsorption/desorption isotherm for SBA-15 and SBA-16 samples, which can be classified as type IV isotherms. This is characteristic of mesoporous materials with a hysteresis loop typical of parallel cylindrical pores in the case of Fig. 4 (SBA-15 samples). SBA-16 shows a notable hysteresis loop type H2 [48], typical of cage-like mesoporous materials [49]. In Figs. 4b and 5b, the pore size distributions of the three samples reveal that pore sizes are practically all beyond 2 nm (mesopores according to IUPAC classification). Considerably larger pores sizes were obtained for SBA-15 HD and this may be one of the reasons for the rather less ordered material. It is interesting to note that SBA-16 exhibits a bimodal pore size distribution, which corresponds to the size of the cages (approximately 11 nm) and the pores interconnecting them (approximately 3 nm). The textural characteristics of the adsorbent were obtained from N2 adsorption/desorption isotherm at 77 K and are summarized in Table 1. It is possible to observe that, despite having the lowest specific surface area, SBA-15 HD shows the highest pore volume and pore diameter, interesting features for adsorption of biomolecules. SBA-15 SG and SBA-15 HD show very distinct textural and structural properties due to the different synthesis conditions, with regards to stirring time and temperature as well as the addition of pore expanding agents. SBA-15 HD was synthesized hydrothermally with the aid of octane as a pore expanding agent and ammonium fluoride as a solubility enhancer. According to Zhang et al. [50], the addition of such reactants promotes the formation of organized structures at low temperatures, which is beneficial, because PPO (propylene oxide) blocks tend to become more hydrophilic at low temperatures and to be easily hydrated, which impairs micelle formation and aggregation. In fact, even though hydrothermal synthesis usually leads quite ordered SBA-15 with well resolved XRD (1 0 0) reflection peaks, SBA-15 HD is fairly disordered, probably due to the relatively low temperature (298 K) in the condensation step. On the other hand, the presence of alkanes, such as octane, tends to suppress the hydration of hydrophobic PPO, whereas the solubilization of such alkane with the aid of ammonium fluoride expands the hydrophobic nuclei of micelles [51]. The combination of all these factors (low condensation temperature and addition of pore expanding agents) results in mesoporous silicas with larger pore diameters (more than 10 nm); nevertheless, they tend to be more disordered than those synthesized following a conventional procedure, such as SBA-15 SG. The latter was prepared without the addition of pore expanders or solubility enhancers and the condensation steps occurred at 323 K, which led to a more effective micelle aggregation and condensation and hence more ordered materials, but with a narrower pore size. 3.2. Biomolecules adsorption

2

4

6

8

10

2 theta Fig. 2. X-ray diffraction patterns of SBA-15 SG (a), SBA-15 HD (b) and SBA-16 (c) samples.

3.2.1. pH effect on the adsorption of BSA, LYS and CEL Adsorption of BSA, LYS and CEL onto SBA-15 and SBA-16 samples was investigated at different pH values and the results are shown in Fig. 6a–c, respectively.

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Fig. 3. TEM images of samples SBA-15 SG (A1 and A2), SBA-15 HD (B1 and B2) and SBA-16 (C1 and C2).

According to these results, the maximum adsorption uptake occurs at pH 4.8 for BSA, 10.6 for LYS and 4.0 for CEL. In the case of proteins (BSA and LYS), these values are close to their isoeletric point (pIBSA = 4.8 and pILYS = 11.0), at which the net protein charge is zero. At a pH below the pI, protein/enzyme is positively charged, whereas it is negatively charged at a pH above the pI. Therefore, the lateral repulsions between adsorbed proteins are minimal when pH equals pI. The charge difference can promote or not the biomolecule interaction with the adsorbent and these interactions may be governed by hydrophobic forces, electrostatic interactions or hydrogen bonding. In our case, the interactions between protein/enzyme and SBA-15 are likely to be hydrophilic interaction,

due to the presence of hydroxyl groups on the surface of SBA-15 and the functional groups of protein/enzyme [52]. Another explanation for favoring the adsorption at the isoelectric point is the change in chain conformation of the protein. In the case of BSA and LYS, it is reported in the literature that these molecules have the ability to fold or unfold their structure according to the pH of the medium. BSA, for example, exists in a compact form between pH 4.3 and 10.5 [53,54] . The decrease of pH may lead to the transition of BSA conformation from compact heart-shape (N form) to unfolded cigar-shape (F form) at pH 4.5 and the conformation changes are irreversible when pH < 4.0 [54,55], this transition always involves an expansion of the molecule. Gao et al. [54]

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640

(a) 1200

(a)

560 1000

Vads (cm3/g)

3

Vads (cm /g)

480 800 600 400

400 320 240 160

200

80 0

0

0.0

0.2

0.4

0.6

0.8

0.0

1.0

0.2

0.4

(b)

12

0.05

0.8

1.0

(b)

0.04

dV/dw (cm3/g)

3

dV/dw (cm /g)

10 8 6 4

0.03 0.02 0.01

2 0

0.6

p/p0

p/p0

0

5

10

15

20

25

30

35

0.00

40

0

5

Pore size (nm) Fig. 4. N2 adsorption isotherm (a) and NLDFT pore size distribution (b) for SBA-15 SG (j) and SBA-15 HD (s).

reported that when the pH value is decreased from 5.0 to 2.4, both hydrodynamic diameters and the molecular volume of BSA increase significantly. It implies that the observed decrease of the adsorption capacity with the decrease of pH could be considered as a result of the unfolding and increase of the molecular volume of BSA. Similar phenomenon should happens with the LYS, which also changes its structure with pH, according to the literature [56]. Literature records have not been found related to CEL with respect to this matter, but we believe that similar changes in tridimensional conformation take place at pH lower them pI. It should be taken into account that cellulase is a pool of three enzymes (cellobiohydrolase, endobeta-1,4-glucanase, and beta-glucosidase or cellobiase), each one having its own pI [57]. Therefore, this may explain the higher uptakes at pH 4 (Fig. 6c), a slightly lower pH then reported pI of CEL. 3.2.2. Kinetic and adsorption isotherm of BSA, LYS and CEL onto SBA15 and SBA-16 In Fig. 7, the uptake curves as a function of time are shown for BSA (a), LYS (b) and CEL (c) on SBA-15 HD, SBA-15 SG and SBA-16. According to these results, we can observe a rapid decrease in the biomolecules concentration in liquid phase. The equilibrium was reached in about 240 min (4 h) for BSA and LYS in both SBA-15 samples and 120 min (2 h) in SBA-16. In the case of CEL, the equilibrium was reached in 240 min for all adsorbents. This information is useful for the knowledge of the contact time required for adsorption equilibrium to be established. Figs. 8–10 show the adsorption isotherms of BSA, LYS and CEL, respectively, obtained in stirred tanks with SBA-15 SG, SBA-15 HD and SBA-16. The curves shown in all figures represent the

10

15

20

25

30

35

Pore size (nm) Fig. 5. N2 adsorption isotherm (a) and NLDFT pore size distribution (b) for SBA-16. Table 1 Textural properties of the SBA-15 HD, SBA-15 SG and SBA-16 samples. Samples

SBET (m2/g)

Dp (nm)

Vp (cm3/g)

SBA-15 HD SBA-15 SG SBA-16

609 777 755

16.7 9.1 11.7

1.65 0.92 0.91

SBET = surface area; Dp = pore diameter, obtained from the NLDFT method desorption step; Vp = total pore.

regression of experimental data, according to the Langmuir (L), Henry (H) and Langmuir–Freundlich (LF) models, as expressed below (Eqs. (2)–(4)). The estimated parameters of these isotherm models are shown in Table 2–4:



qmax ðkL C eq Þ 1 þ kL C eq

ð2Þ

where q is the amount of protein adsorbed per unit weight (mg/g), Ceq is the concentration of protein in the liquid phase at adsorption equilibrium (mg/mL), kL is the dissociation constant of Langmuir (mL/mg) that is associated with the energy of adsorption and qmax is the saturation capacity per unit weight (mg/g):

q ¼ kH  C eq

ð3Þ

where kH is the constant of Henry’s law (mL/g) and Ceq is the concentration of the fluid phase (mg/mL).



qmax ðkLF C eq Þ

b

b

1 þ ðkLF C eq Þ

ð4Þ

where q is the amount of protein adsorbed per unit weight (mg/g), Ceq is the concentration of protein in the liquid phase at adsorption

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200

1.2

(a)

(a)

1.0

160

C/C0

q (mg/g)

0.8 120

0.6

80 0.4 40

0.2 0.0

0 3.2

3.6

4.0

4.4

4.8

5.2

5.6

0

6.0

1

2

3

4

5

6

7

8

pH 1.2

200

9

Time (h)

(b)

(b)

1.0

160

C/C0

q (mg/g)

0.8 120

0.6

80

0.4

40

0.2 0.0

0

6

7

8

9

10

11

12

0

1

2

3

13

pH 200

4

5

6

7

8

Time (h) 1.2

(c)

(c)

1.0

160

C/C0

q (mg/g)

0.8 120 80

0.4

40 0 3.2

0.6

0.2 0.0 3.6

4.0

4.4

4.8

5.2

5.6

pH

0

1

2

3

4

5

6

Time (h)

Fig. 6. Influence of pH on adsorption of BSA (a), LYS (b) and CEL (c) in SBA-15 obtained by sol–gel (j), hydrothermal (s) and SBA-16 (N).

Fig. 7. Kinetic of adsorption of BSA (a), LYS (b) and CEL (c) in SBA-15 SG (j), SBA-15 HD (s) and SBA-16 (N).

equilibrium (mg/mL), kLF (mL/mg)1/b is the Langmuir–Freundlich constant associated with the energy of adsorption, b is the Langmuir–Freundlich heterogeneity constant. LF trends to the Langmuir isotherm when the heterogeneity constant b is close to unity. The adsorption isotherms of BSA in SBA-15 SG and HD and LYS in all adsorbents show a considerable initial increase, suggesting a high affinity between these biomolecules and the surface of the adsorbent until finally the isotherm reaches a plateau (type Langmuir isotherm) [58]. It is observed that the value of qmax for both BSA and LYS is relatively high as compared to data reported in the literature [9,58–60], particularly for SBA-15 HD. This material shows the largest pore diameter and pore volume and will therefore accommodate more biomolecules per unit mass. The adsorption capacity of proteins/enzymes on the mesoporous molecular sieves is known to be strongly influenced by the specific area and

volume of pores [61]. Since both BSA and LYS have been adsorbed at their isoelectric point, it is likely that adsorption uptake is driven majorly by size exclusion effects. The solid and dash lines in Fig. 8 represent the fits of L and LF models for BSA adsorption on SBA-15 HD and SBA-15 SG. According to results presented in Table 2, BSA adsorptions in both SBAs were well fitted by LF. The maximum amount of BSA adsorbed in SBA-15 HD was 317.38 mg/g and 74.69 mg/g in SBA-15 SG. BSA is a large ellipsoid-shaped protein with molecular mass of 69 kDa and molecular size of 4  4  14 nm [61]. Its cross section is closer to the size of the average pore diameter of SBA-15 SG (9 nm) than to the pore diameter of SBA-15 HD (17 nm). Additionally, the pore volume of SBA-15 SG is about 55% of that of SBA-15 HD. Therefore, it is not surprising that the adsorption capacity of BSA in the former adsorbent is much lower than in the latter. Based on the same

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350

(a)

300

300

250

250

q (mg/g)

q (mg/g)

350

200 150

200 150

100

100

50

50

0 0

2

4

6

8

10

12

14

0

16

0

1

2

Ceq (mg/mL) 100

(b)

40

Sample

Model

6

7

qmax (mg/g) SBA-15 SG

0

2

4

6

8

10

12

14

16

Ceq (mg/mL) Fig. 8. BSA adsorption isotherms at 295 K in (a) SBA-15 SG (j) and SBA-15 HD (s) and (b) SBA-16 (N). Solid lines (__) represents Langmuir model, dash lines (. . .) represents Langmuir–Freundlich model and dotted lines (- - -) represents Henry model.

a

R2

Parameters

20 Langmuir 65.02 ± 1.35 74.69 ± 2.22 LFa

kL (mL/mg), b kLF (mL/mg)1/b 70.52 ± 17.62 70.42 ± 12.05

– 0.9682 0.39 ± 0.05 0.9973

SBA-15 HD Langmuir 328.74 ± 14.86 23.10 ± 4.98 LFa 317.38 ± 10.54 25.17 ± 2.19

– 0.9195 2.21 ± 0.47 0.9501

SBA-16

– –

Langmuir – Henry –

– 6.20 ± 0.10

– 0.9951

Langmuir–Freundlich.

Table 3 Parameters of the Langmuir and Langmuir–Freundlich equation for adsorption of LYS in mesoporous silica.

800 700

Sample

Model

500

SBA-15 SG

400

R2

Parameters qmax (mg/g)

600

q (mg/g)

5

Table 2 Parameters of the Langmuir, Henry and Langmuir–Freundlich equation for adsorption of BSA in mesoporous silica.

60

0

4

Fig. 10. CEL adsorption isotherms at 22 °C ± 1 °C in SBA-15 SG (j), SBA-15 HD (s) and SBA-16 (N). Solid lines (__) represents Langmuir model and dash lines (. . .) represents Langmuir–Freundlich model.

80

q (mg/g)

3

Ceq (mg/mL)

Langmuir 429.10 ± 16.69 535.23 ± 75.08 LFa

kL (mL/mg), b kLF (mL/mg)1/b 15.79 ± 5.12 0.42 ± 0.24

– 0.9627 0.46 ± 0.12 0.9682

300

SBA-15 HD Langmuir 635.69 ± 29.99 52.71 ± 19.17 LFa 819.85 ± 221.15 0.40 ± 0.43

– 0.9129 0.38 ± 0.18 0.9504

200

SBA-16

– 0.9994 1.20 ± 0.38 0.9994

100 0

a

0

1

2

3

4

5

6

7

Langmuir LFa

76.07 ± 0.58 75.03 ± 1.60

9.35 ± 0.79 0.08 ± 0.04

Langmuir–Freundlich.

8

Ceq (mg/mL) Fig. 9. LYS adsorption isotherms at 295 K in SBA-15 SG (j), SBA-15 HD (s) and SBA-16 (N). Solid lines (__) represents Langmuir model and dash lines (. . .) represents Langmuir–Freundlich model.

reasoning, BSA is hardly adsorbed in SBA-16 (Fig. 8b), which has a clearly bimodal pore size distribution (see Fig. 4b). Even though the cages of such adsorbent are spacious enough to accommodate BSA (between 10 and 13 nm), the size of the interconnecting pores is smaller than 4 nm, which interposes serious steric hindrances. These data are in agreement as reported by Diao et al. [61], who investigated the effect of the pore size of mesoporous SBA-15 on adsorption of BSA and LYS. It was also observed that the equilibrium adsorption capacity of BSA on SBA-15 increased with increasing pore size.

LYS is a quite smaller biomolecule than BSA, with dimensions 3  3  4.5 nm [9]. It is adsorbed in all three silicas with higher maximum uptakes than those measured for BSA. In SBA-16, LYS is the only biomolecule to show sharp isotherms of Langmuir behavior, although the uptake is the lowest as compared to the other silicas. This may be explained again by the small size of the access pores to the cubic cages. As seen in Fig. 4b, the average size of such pores is around 3 nm, which may also limit the adsorption of LYS, despite the relatively large cubic cage. For the case of LYS adsorption on SBA-15 HD, SBA-15 SG and SBA-16 (Fig. 9), the solid lines and dashed lines represent the fits of L and LF models. As shown in Table 3, unlike BSA adsorption, L model was more suitable to represent the experimental data in all adsorbents. Although visually showed a better fit, LF model presented large standard deviations for the estimated parameters.

S.M.L. dos Santos et al. / Microporous and Mesoporous Materials 180 (2013) 284–292 Table 4 Parameters of the Langmuir and Langmuir–Freundlich equation for adsorption of CEL in mesoporous silica. Sample

Model

qmax (mg/g) SBA-15 SG

a

R2

Parameters kL (mL/mg), b kLF (mL/mg)1/b

Langmuir 957.30 ± 71.91 0.09 ± 0.01 931.29 ± 301.47 0.09 ± 0.05 LFa

– 0.9989 1.01 ± 0.09 0.9987

SBA-15 HD Langmuir 845.84 ± 195.70 0.05 ± 0.01 LFa 605.19 ± 7.47 0.09 ± 0.00

– 0.9952 1.07 ± 0.00 0.9961

SBA-16

– 0.9922 1.00 ± 0.00 0.9930

Langmuir 473.38 ± 122.64 0.06 ± 0.02 LFa 412.81 ± 6.62 0.07 ± 0.00

291

oscopic pore arrangement (Fig. 2), but it also exhibits a narrower pore size distribution (Fig. 4b) as compared to the other adsorbents, which suggests that cellulase is likely to be more densely packed in SBA-15(SG) than in SBA-15(HD). Regarding SBA-16, lower adsorption capacities were expected for all studied biomolecules due to the nature of his own structure, since it presents a face-centered cubic structure connected by narrow pores. Nevertheless, the uptake of cellulase by this material (473 mg/g), reported for the first time in this paper, far exceeds the uptakes of other biomolecules, and this may be an interesting feature to be exploited in the design of chromatographic separation processes of this enzyme.

Langmuir–Freundlich.

Acknowledgments The authors are grateful to CNPq (process number 577363/ 2008-5) and CAPES (process number PE-055/2008) for providing financial support for this research. The authors thank too Dr. Karim Sapag and his group (Universidad Nacional de San Luis, Argentina) for fruitful discussions and Prof. Enrique Rodriguez-Castellon (Universidad de Malaga, Spain) for the availability of some of the characterization techniques used in this work.

140 120

q (mg/g)

100 80 60

References

40 20 0

0

2

4

6

8

10

12

14

16

Ceq (mg/mL) Fig. 11. Adsorption isotherms at 295 K in SBA-16: BSA (N), LYS (}) and CEL ().

In the case of CEL, all adsorbents showed appreciable adsorption capacity at 295 K and only slightly non-linear behavior. The fitting parameters of the L and LF equations are summarized in Table 4. According to results showed in Table 4, L model was also more suitable to represent the experimental adsorption of CEL in all SBAs. It is the biomolecule best adsorbed by SBA-16, possibly due to the different conformations and sizes of the individual enzymes that compose the cellulase pool, which leads to a less pronounced exclusion effect than that observed for BSA and LYS. This may be best appreciated in Fig. 11, in which the isotherms of the three studied biomolecules are shown for SBA-16. In fact, it is reported in Hartono et al. [57] that cellulase may assume either spherical (2.4–7.4 nm diameter) and ellipsoidal (1.3  7.9 nm to 4.2  25.2 nm). Taking into account that the pore mouth diameter of the synthesized SBA-16 is around 3 nm (see Fig. 5b), smaller enzymes or thin ellipsoidal ones will have free access to the cubic cages. 4. Conclusions SBA-15 and SBA-16 were successfully synthesized by sol–gel and hydrothermal routes. All three synthesized mesoporous silicas were capable of adsorbing the studied biomolecules (BSA, LYS and CEL) at pHs close to their pIs with relatively high capacities in most cases. Among the silicas with parallel cylindrical pores (SBA-15), the material synthesized hydrothermally had superior textural properties (pore size and specific pore volume) and showed the highest adsorption capacity for both BSA (317 mg/g) and LYS (636 mg/g), even though pore arrangement was poorly ordered. On the other hand, SBA-15 synthesized by a sol–gel route showed a larger adsorption capacity for cellulase (957 mg/g) as compared to the other materials. Not only it is the material with better mes-

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