MMM Sandra 2013

mrriorS/N,

Adsorption isotherm

theerehesiffrults

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 solgel route.

2013 Elsevier Inc. All rights reserved.

iscove1,2]. Ou-41, ocid me

such as high performance liquid chromatography (HPLC) [1517].

derly 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 engineeringof materials [23]. Although it is reported in the literature the appli-cation of SBA-16 in elds such as catalysis [2426], functionaliza-tion [27], metals incorporation [28] and templating [29,30], thereare a few records [31] of the use of such materials for adsorp-tion/chromatographic purposes.

separations, this difference in structure may favor or not the ef-] found that theSBA-16 isenhancing

oxidation. Data on application of SBA-16 in the adsorptionmolecules are scarce, hence it is yet to be investigated if thisture model could positively inuence the separation ofbulking biomolecules.

Some authors have recently reported the use of synthetichydrotalcites known as Layered Double Hydroxides [3537] andmesostructured cellular foams (MCF) [38] as new potential adsor-bents for biomolecules. These materials have shown high adsorp-tion capacity for standard proteins, for example bovine serumalbumin (BSA), human serum albumin (HSA), lysozyme (LYS) andmyoglobin (Mb).

Corresponding author. Tel.: +55 8533669611; fax: +55 8533669610.

Microporous and Mesoporous Materials 180 (2013) 284292

Contents lists availab

e

seE-mail address: [email protected] (D.C.S. de Azevedo).Another mesoporous adsorbent that has received attention isthe SBA-16 whose formation mechanism is similar to that ofSBA-15 [1822]. The easiness of the method of preparation, the or-

ciency of the process. For example, Li et al. [34encapsulation of Ru complexes in nanocages ofcient method to achieve high catalytic activity by1387-1811/$ - see front matter 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.micromeso.2013.06.043an ef-waterof bio-struc-theseSBA-15 (Santa Barbara Amorphous), developed in 1998 by Zhaoet al. [3], has attracted intense interest due to their large surfaceareas, well-dened 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[810], chemical sensing [11], immobilization [12], drug deliverysystems [13,14] and separation by chromatographic techniques

Fig. 1a). It has the benets of combined micro and mesoporosityand relatively thick silica walls. The micropores are created bythe penetration of the hydrophobic ethylene oxide chain in the sil-ica walls [32]. On the other hand, SBA-16 is a mesoporous silicahaving a structure of spherical body-centered nanocages with cu-bic arrangement, wherein each sphere is connected to eight neigh-boring spheres [33] (see Fig. 1b). For operations such as catalysis orProteinEnzymeSilica

1. Introduction

In 1992, it was rst reported the drials belonging to the M41S family [als have been highlighted, the MCMroute and SBA-15, and obtained in ary of mesoporous mate-t of these, two materi-btained in basic mediadia.

Both SBA-15 as SBA-16 are synthesized in acidic media in thepresence of triblock copolymer surfactants Pluronic P123 (PEO20P-PO70PEO20) [3] and Pluronic F127 (PEO106PPO70PEO106) [18],respectively. However, the resulting materials have very distinctstructures, as shown in Fig. 1. SBA-15 is a mesoporous silica withparallel pores and highly ordered hexagonal arrangement (seeKeywords:Separation

distinct degrees of mesoporous ordering. The inuence of pH on the adsorption of BSA, LYS and CEL aswell the kinetics and adsorption isotherms were evaluated in stirred tanks. Among the materials studied,Synthesis and characterization of orderedSBA-16) for adsorption of biomolecules

Sandra Maria Lopes dos Santos, Karina Alexandre BaJeann Diniz Ferreira Lima, Ivanildo Jos da Silva JnDepartamento de Engenharia Qumica, Universidade Federal do Cear, Campus do Pici,

a r t i c l e i n f o

Article history:Received 21 May 2013Received in revised form 28 June 2013Accepted 29 June 2013Available online 12 July 2013

a b s t r a c t

The present work describesand cellulase (CEL)) on ordfrom buffered solutions. Tcharacterized by X-ray dmicroscopy (TEM). The res

Microporous and M

journal homepage: www.elesoporous silica (SBA-15 and

os Nogueira, Marlon de Souza Gama,, Diana Cristina Silva de Azevedo Bloco 710, CEP: 60455-760 Fortaleza, CE, Brazil

adsorption of biomolecules (bovine serum albumin (BSA), lysozyme (LYS)d mesoporous silicas with different pore diameters (SBA-15 and SBA-16)e adsorbents were synthesized by solgel and hydrothermal routes andaction, N2 adsorption/desorption isotherms and transmission electronby X-ray diffraction and TEM show that the synthesized materials have

le at ScienceDirect

soporous Materials

vier .com/locate /micromeso

silicas of SBA-15 and SBA-16 type and investigates their potential

wards, the reaction mixture was kept still and aged for 24 h at the

SBA

Mein the adsorption of bovine serum albumin (BSA), lysozyme (LYS)and cellulase (CEL).

2. Experimental part

2.1. Reactants

The triblock copolymers Pluronic P123 (PEO20PPO70PEO20), Plu-ronic F127 (PEO106PPO70PEO106), tetraethoxysilane (TEOS, 98%),hydrochloric acid (36%), CEL from Aspergillus niger and LYS werepurchased from Sigma Aldrich (USA). BSA was supplied by INLAB(Brazil); butyl alcohol from Aldrich (99.4%); ethanol, octane andammonium uoride from Vetec (Brazil). All chemicals were usedas received without any further purication.In this work, SBA-15 was synthesized by two distinct routes,solgel and hydrothermal. The solgel route has, as main advan-tage, the possibility of being performed under mild conditions oftemperature and pressure. However, the time required for thehydrolysis and condensation steps to occur is generally well abovethat required by hydrothermal synthesis. In turn, the latter methodoften results in materials with surface areas and pore diametersgreater than those obtained by the solgel route. Hence, it is inter-esting to compare the ability of SBA-type materials obtained bythese two routes to adsorb the biomolecules under study.

Thereby, this work aimed at synthesizing ordered mesoporous

Fig. 1. Schematic illustration of the channels of SBA-15 (a) and nanocages of

S.M.L. dos Santos et al. /Microporous and2.2. Synthesis

SBA-15 was synthesized by solgel and hydrothermal routes.SBA-16 was only synthesized hydrothermally. Hydrothermal syn-thesis of SBA-15 (SBA-15 HD) was performed according to the pro-cedure reported by Zhang et al. [39] with adaptations. As synthesisprocedure, 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 fewhours under stirring at 298 K, a clear solution was obtained andthen 21.72 g of octane and 9.76 g of TEOS were added. The result-ing mixture was stirred for 24 h and then transferred to an auto-clave for further reaction at 373 K for 48 h. The solid was ltered,dried in air and calcined at 823 K for 5 h to remove the organictemplates. The molar composition of the synthesis gel was 1.00TEOS:0.018 P123:6.36 HCl:201.8 H2O:0.033 NH4F:4.7 octane.

Solgel synthesis of SBA-15 (SBA-15 SG) was carried out as re-ported by Esparza et al. [40] with adaptations. 1.7 g P123 was dis-solved in 50.4 mL aqueous HCl solution (2 M) and kept understirring at 323 K until a transparent solution was obtained.3.75 mL TEOS was added drop-wise under vigorous stirring. After-same temperature without stirring. Subsequently, the temperaturewas raised to 353 K and kept at this value for 48 h. The solids wereltered, washed with abundant deionized water to remove excesssurfactant. Finally, the samples were dried at 333 K for 12 h andcalcined at 823 K for 6 h. The molar composition of the synthesisgel was 1.00 TEOS:0.017 P123:6 HCl:160 H2O.

Hydrotermal synthesis of SBA-16 followed the synthesis meth-od reported by Kleitz et al. [41]. The mesostructured SBA-16 silicamaterials were prepared using a mixture of Pluronic F127 and bu-tyl alcohol as structure-directing agents. The silica source wasTEOS. In a typical synthesis 4.0 g F127 were dissolved in a 0.4 MHCl (200 mL) aqueous solution. The synthesis was carried out ina closed polypropylene bottle. After complete dissolution, 13.75 gof butyl alcohol was added at once at 318 K. After 1 h under stir-ring, 19.3 g of TEOS was quickly added to this mixture. The molarcomposition of the synthesis gel was 1.00 TEOS:0.0035 F127:1.78BuOH:0.88 HCl:119 H2O. The mixture was further stirred vigor-ously at 318 K for 24 h to allow for the formation of the mesostruc-tured product. Subsequently, the reaction mixture was heated inan autoclave at 373 K for 24 h under static conditions. The whiteprecipitate was then ltered without washing and dried at 373 Kfor 24 h in air. To remove the copolymer template, the solid wasbriey rinsed at room temperature with an ethanol/HCl mixturefor 20 min, ltered, dried, and then calcined at 823 K for 2 h.-16 (b). Adapted from Zhao et al. [3] and Flodstrm et al. [21], respectively.

soporous Materials 180 (2013) 284292 2852.3. Characterization

X-ray diffraction (XRD) was used to identify the crystal phasesof the synthesized solids. These experiments were performed witha Siemens D5000 power X-ray diffractometer equipped with aCuKa radiation source (wavelength 1.5418 ). Measurements wereobtained for 2h ranging from 1 to 10. The transmission electronmicrographs (TEM) were obtained with the aid of a high resolutionmicroscope Philips CCCM 200 Supertwin-DX4. Measurements ofN2 adsorption/desorption at 77 K were carried out using a volu-metric adsorption equipment (AUTOSORB-1MP, QuantachromeInstruments). The specic surface area (SBET) of the samples wasestimated with the Brunauer, Emmet and Teller (BET) method[42], using the adsorption data in the range of relative pressuresfrom 0.05 to 0.18 and 0.05 to 0.23 for the samples SBA-15 andSBA-16 respectively, where conditions of linearity and consider-ations regarding the method were fullled [43]. The pore size dis-tribution was calculated from the desorption branch of theisotherms by using the nonlocal density functional theory (NLDFT)[44]. The total pore volume was taken as the adsorbed volume at p/p0 = 0.95.

2.4. Protein adsorption experiments

Batch adsorption experiments were carried out in an orbitalshaker (Tecnal, Brazil). For this aim, 15 mg adsorbent were put incontact with 3.0 mL buffer solution containing the target biomole-cule (BSA, LYS or CEL). Initially, the effect of pH on the biomoleculeuptake was evaluated. For BSA, an initial concentration of 1.0 mg/mL in acetate buffer (50 mmol/L) was used with pH ranging from3.6 to 5.6 by adding either HCl or NaOH. For the other biomole-cules, the initial concentration was 3.0 mg/mL, using sodium bicar-bonate buffer (25 mmol/L) with pH ranging from 6.5 to 12.0 for LYSand acetate buffer (50 mmol/L) with pH ranging from 3.0 to 5.4 forCEL. For kinetic experiments, the initial concentrations used werethe same as those of pH tests. The samples were collected fromthe experimental tubes at pre-determined time intervals. For themeasurement of the adsorption isotherms, different initial concen-

V C C

286 S.M.L. dos Santos et al. /Microporous and Meq sol 0 eqmads

1

where Vsol represents the volume of biomolecule solution at equi-librium (typically 3 mL); C0 the initial concentration of the biomol-ecule (mg/mL); Ceq is the equilibrium concentration (mg/mL) andmads is the mass of adsorbent (typically 15 mg).

3. Results and discussion

3.1. Characterization of SBA-15 and SBA-16

The X-ray diffraction patterns were used to identify structuralordering of mesoporous materials under study. The results areshown in Fig. 2. In the case of SBA-15 SG, three main diffractionpeaks are present referring to the crystal planes corresponding toMiller indices (100), (110) and (200). These rst three peaks arecharacteristic of a two-dimensional hexagonal pore arrangement,commonly found in materials like SBA-15 [45,46], indicating a well

0 2 4 6 8 10

(a)

(b)Int

ensit

y

2 theta

(c)trations of BSA (1.015.0 mg/mL), LYS (1.015.0 mg/mL) and CEL(1.07.0 mg/mL) at xed pH and ionic strength were shaken for en-ough time to ensure equilibrium. The solid/liquid ratio used in thedetermination of adsorption isotherms was 5.0 mg/mL. In allexperiments, after a given time, the samples were collected andcentrifuged for 10 min at 10,000g (refrigerated microcentrifugeCientec CT-15000R, USA). The concentration in the supernatantwas analyzed with a UV spectrophotometer at 280 nm (UVVisspectrophotometer Biomate 3, Thermo Scientic, USA).

The adsorbed concentration of each biomolecule was calculatedusing a simple mass balance, according to Eq. (1):Fig. 2. X-ray diffraction patterns of SBA-15 SG (a), SBA-15 HD (b) and SBA-16 (c)samples.dened mesostructure. On the other hand SBA-15 HD is far less or-derly and practically shows only a well resolved peak at 2h 1.After 2h = 1 there are unresolved peaks which may indicate thatthere is some degree of mesoporous arrangement to be conrmedby TEM. SBA-16 also only shows one strong peak at low angle. Itsposition corresponds to the strong peak observed with polyhedralparticles and indexed by plane (110) [47]. Such arrangement ofmaterials may also be observed through TEM images illustratedin Fig. 3. The hexagonally arranged pore arrays of the pure-silicaSBA-15 samples can be clearly observed in both synthesis routes(Fig. 3A1, B1, A2 and B2). As expected, the TEM images of SBA-16do not show the hexagonal ordering as in SBA-15, but rather inter-connected cage-like pores.

Figs. 4 and 5 shows the N2 adsorption/desorption isotherm forSBA-15 and SBA-16 samples, which can be classied as type IVisotherms. This is characteristic ofmesoporousmaterialswith ahys-teresis 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. 4band 5b, the pore size distributions of the three samples reveal thatpore sizes are practically all beyond 2 nm (mesopores according toIUPAC classication). Considerably larger pores sizes were obtainedfor SBA-15 HD and this may be one of the reasons for the rather lessordered material. It is interesting to note that SBA-16 exhibits abimodal pore size distribution, which corresponds to the size ofthe cages (approximately 11 nm) and the pores interconnectingthem (approximately 3 nm).

The textural characteristics of the adsorbent were obtained fromN2 adsorption/desorption isotherm at 77 K and are summarized inTable 1. It is possible to observe that, despite having the lowest spe-cic surface area, SBA-15 HD shows the highest pore volume andpore diameter, interesting features for adsorption of biomolecules.

SBA-15 SG and SBA-15 HD show very distinct textural and struc-tural properties due to the different synthesis conditions, with re-gards to stirring time and temperature as well as the addition ofpore expanding agents. SBA-15HDwas synthesized hydrothermallywith the aid of octane as a pore expanding agent and ammoniumuoride as a solubility enhancer. According to Zhang et al. [50], theaddition of such reactants promotes the formation of organizedstructures at low temperatures, which is benecial, because PPO(propylene oxide) blocks tend to become more hydrophilic at lowtemperatures and to be easily hydrated, which impairs micelle for-mation and aggregation. In fact, even though hydrothermal synthe-sis usually leads quite ordered SBA-15withwell resolvedXRD (100)reection peaks, SBA-15 HD is fairly disordered, probably due to therelatively low temperature (298 K) in the condensation step. On theother hand, the presence of alkanes, such as octane, tends to sup-press the hydration of hydrophobic PPO, whereas the solubilizationof such alkane with the aid of ammonium uoride expands thehydrophobic nuclei of micelles [51]. The combination of all thesefactors (low condensation temperature and addition of poreexpanding agents) results in mesoporous silicas with larger porediameters (more than 10 nm); nevertheless, they tend to be moredisordered than those synthesized following a conventional proce-dure, such as SBA-15 SG. The latter was prepared without the addi-tion of pore expanders or solubility enhancers and the condensationsteps occurred at 323 K, which led to amore effectivemicelle aggre-gation and condensation and hence more ordered materials, butwith a narrower pore size.

3.2. Biomolecules adsorption

3.2.1. pH effect on the adsorption of BSA, LYS and CEL

soporous Materials 180 (2013) 284292Adsorption of BSA, LYS and CEL onto SBA-15 and SBA-16 sam-ples was investigated at different pH values and the results areshown in Fig. 6ac, respectively.

MeS.M.L. dos Santos et al. /Microporous andAccording to these results, the maximum adsorption uptake oc-curs at pH 4.8 for BSA, 10.6 for LYS and 4.0 for CEL. In the case ofproteins (BSA and LYS), these values are close to their isoeletricpoint (pIBSA = 4.8 and pILYS = 11.0), at which the net protein chargeis 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 minimalwhen pH equals pI. The charge difference can promote or not thebiomolecule interaction with the adsorbent and these interactionsmay be governed by hydrophobic forces, electrostatic interactionsor hydrogen bonding. In our case, the interactions between pro-tein/enzyme and SBA-15 are likely to be hydrophilic interaction,

Fig. 3. TEM images of samples SBA-15 SG (A1 and A2)soporous Materials 180 (2013) 284292 287due to the presence of hydroxyl groups on the surface of SBA-15and the functional groups of protein/enzyme [52].

Another explanation for favoring the adsorption at the isoelec-tric point is the change in chain conformation of the protein. In thecase of BSA and LYS, it is reported in the literature that these mol-ecules have the ability to fold or unfold their structure according tothe pH of the medium. BSA, for example, exists in a compact formbetween pH 4.3 and 10.5 [53,54] . The decrease of pH may lead tothe transition of BSA conformation from compact heart-shape (Nform) to unfolded cigar-shape (F form) at pH 4.5 and the conforma-tion changes are irreversible when pH < 4.0 [54,55], this transitionalways involves an expansion of the molecule. Gao et al. [54]

, SBA-15 HD (B1 and B2) and SBA-16 (C1 and C2).

0.01

0.02

0.03

dV/d

w(c

m3 /g

)

Me0.0 0.2 0.4 0.6 0.8 1.00

p/p0

2

4

6

8

10

12

dV/d

w(cm

3 /g)

(b)200

400

600

800

1000

1200

V ads (cm

3 /g)

(a)288 S.M.L. dos Santos et al. /Microporous andreported that when the pH value is decreased from 5.0 to 2.4, bothhydrodynamic diameters and the molecular volume of BSA in-crease signicantly. It implies that the observed decrease of theadsorption capacity with the decrease of pH could be consideredas a result of the unfolding and increase of the molecular volumeof BSA. Similar phenomenon should happens with the LYS, whichalso changes its structure with pH, according to the literature[56]. Literature records have not been found related to CEL withrespect to this matter, but we believe that similar changes in tridi-mensional conformation take place at pH lower them pI. It shouldbe taken into account that cellulase is a pool of three enzymes(cellobiohydrolase, endobeta-1,4-glucanase, and beta-glucosidaseor cellobiase), each one having its own pI [57]. Therefore, thismay explain the higher uptakes at pH 4 (Fig. 6c), a slightly lowerpH then reported pI of CEL.

3.2.2. Kinetic and adsorption isotherm of BSA, LYS and CEL onto SBA-15 and SBA-16

In Fig. 7, the uptake curves as a function of time are shown forBSA (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 thebiomolecules concentration in liquid phase. The equilibrium wasreached in about 240 min (4 h) for BSA and LYS in both SBA-15samples and 120 min (2 h) in SBA-16. In the case of CEL, the equi-librium was reached in 240 min for all adsorbents. This informa-tion is useful for the knowledge of the contact time required foradsorption equilibrium to be established.

Figs. 810 show the adsorption isotherms of BSA, LYS and CEL,respectively, obtained in stirred tanks with SBA-15 SG, SBA-15HD and SBA-16. The curves shown in all gures represent the

0 5 10 15 20 25 30 35 400

Pore size (nm)Fig. 4. N2 adsorption isotherm (a) and NLDFT pore size distribution (b) for SBA-15SG (j) and SBA-15 HD (s).0.0 0.2 0.4 0.6 0.8 1.00

80

160

240

320

400

480

560

640

Va

ds( cm

3 /g)

p/p0

(a)

0.04

0.05(b)

soporous Materials 180 (2013) 284292regression of experimental data, according to the Langmuir (L),Henry (H) and LangmuirFreundlich (LF) models, as expressed be-low (Eqs. (2)(4)). The estimated parameters of these isothermmodels are shown in Table 24:

q qmaxkLCeq1 kLCeq 2

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

q kH Ceq 3where kH is the constant of Henrys law (mL/g) and Ceq is the con-centration of the uid phase (mg/mL).

q qmaxkLFCeqb

1 kLFCeqb4

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

0 5 10 15 20 25 30 350.00

Pore size (nm)Fig. 5. N2 adsorption isotherm (a) and NLDFT pore size distribution (b) for SBA-16.

Table 1Textural 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 609 16.7 1.65SBA-15 SG 777 9.1 0.92SBA-16 755 11.7 0.91

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

Me40

80

120

160

200(a)

q (m

g/g)

S.M.L. dos Santos et al. /Microporous andequilibrium (mg/mL), kLF (mL/mg)1/b is the LangmuirFreundlichconstant associated with the energy of adsorption, b is the Lang-muirFreundlich heterogeneity constant. LF trends to the Langmuirisotherm when the heterogeneity constant b is close to unity.

The adsorption isotherms of BSA in SBA-15 SG and HD and LYSin all adsorbents show a considerable initial increase, suggesting ahigh afnity between these biomolecules and the surface of theadsorbent until nally the isotherm reaches a plateau (type Lang-muir isotherm) [58]. It is observed that the value of qmax for bothBSA and LYS is relatively high as compared to data reported inthe literature [9,5860], particularly for SBA-15 HD. This materialshows the largest pore diameter and pore volume and will there-fore accommodate more biomolecules per unit mass. The adsorp-tion capacity of proteins/enzymes on the mesoporous molecularsieves is known to be strongly inuenced by the specic area and

3.2 3.6 4.0 4.4 4.8 5.2 5.6 6.00

pH

6 7 8 9 10 11 12 130

40

80

120

160

200(b)

q (m

g/g)

pH

3.2 3.6 4.0 4.4 4.8 5.2 5.60

40

80

120

160

200

q (m

g/g)

pH

(c)

Fig. 6. Inuence of pH on adsorption of BSA (a), LYS (b) and CEL (c) in SBA-15obtained by solgel (j), hydrothermal (s) and SBA-16 (N).0.2

0.4

0.6

0.8

1.0

1.2(a)

C/C

0

soporous Materials 180 (2013) 284292 289volume of pores [61]. Since both BSA and LYS have been adsorbedat their isoelectric point, it is likely that adsorption uptake is drivenmajorly by size exclusion effects.

The solid and dash lines in Fig. 8 represent the ts of L and LFmodels for BSA adsorption on SBA-15 HD and SBA-15 SG. Accord-ing to results presented in Table 2, BSA adsorptions in both SBAswere well tted by LF. The maximum amount of BSA adsorbed inSBA-15 HD was 317.38 mg/g and 74.69 mg/g in SBA-15 SG. BSAis a large ellipsoid-shaped protein with molecular mass of 69 kDaand molecular size of 4 4 14 nm [61]. Its cross section is closerto the size of the average pore diameter of SBA-15 SG (9 nm) thanto the pore diameter of SBA-15 HD (17 nm). Additionally, the porevolume 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 for-mer adsorbent is much lower than in the latter. Based on the same

0 1 2 3 4 5 6 7 8 90.0

Time (h)

0 1 2 3 4 5 6 7 80.0

0.2

0.4

0.6

0.8

1.0

1.2(b)

C/C

0

Time (h)

0 1 2 3 4 5 60.0

0.2

0.4

0.6

0.8

1.0

1.2(c)

C/C

0

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

Me200

250

300

350

g/g)

(a)

290 S.M.L. dos Santos et al. /Microporous andreasoning, BSA is hardly adsorbed in SBA-16 (Fig. 8b), which has aclearly bimodal pore size distribution (see Fig. 4b). Even though thecages of such adsorbent are spacious enough to accommodate BSA(between 10 and 13 nm), the size of the interconnecting pores issmaller than 4 nm, which interposes serious steric hindrances.These data are in agreement as reported by Diao et al. [61], whoinvestigated the effect of the pore size of mesoporous SBA-15 onadsorption of BSA and LYS. It was also observed that the equilib-rium adsorption capacity of BSA on SBA-15 increased with increas-ing pore size.

0 2 4 6 8 1 0 1 2 1 4 1 60

50

100

150

q (m

Ceq (mg/mL)

0 2 4 6 8 10 12 14 160

20

40

60

80

100

q (m

g/g)

Ceq (mg/mL)

(b)

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 LangmuirFreundlich model and dotted lines (---) represents Henrymodel.

0 1 2 3 4 5 6 7 80

100

200

300

400

500

600

700

800

q (m

g/g)

Ceq (mg/mL)Fig. 9. LYS adsorption isotherms at 295 K in SBA-15 SG (j), SBA-15 HD (s) andSBA-16 (N). Solid lines (__) represents Langmuir model and dash lines (. . .)represents LangmuirFreundlich model.50

100

150

200

250

300

350

q (m

g/g)

soporous Materials 180 (2013) 284292LYS is a quite smaller biomolecule than BSA, with dimensions3 3 4.5 nm [9]. It is adsorbed in all three silicas with highermaximum uptakes than those measured for BSA. In SBA-16, LYSis the only biomolecule to show sharp isotherms of Langmuirbehavior, although the uptake is the lowest as compared to theother silicas. This may be explained again by the small size of theaccess pores to the cubic cages. As seen in Fig. 4b, the average sizeof such pores is around 3 nm, which may also limit the adsorptionof LYS, despite the relatively large cubic cage. For the case of LYSadsorption on SBA-15 HD, SBA-15 SG and SBA-16 (Fig. 9), the solidlines and dashed lines represent the ts of L and LF models. Asshown in Table 3, unlike BSA adsorption, L model was more suit-able to represent the experimental data in all adsorbents. Althoughvisually showed a better t, LF model presented large standarddeviations for the estimated parameters.

0 1 2 3 4 5 6 70

Ceq (mg/mL)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 LangmuirFreundlich model.

Table 2Parameters of the Langmuir, Henry and LangmuirFreundlich equation for adsorptionof BSA in mesoporous silica.

Sample Model Parameters R2

qmax (mg/g) kL (mL/mg),kLF (mL/mg)1/b

b

SBA-15 SG Langmuir 65.02 1.35 70.52 17.62 0.9682LFa 74.69 2.22 70.42 12.05 0.39 0.05 0.9973

SBA-15 HD Langmuir 328.74 14.86 23.10 4.98 0.9195LFa 317.38 10.54 25.17 2.19 2.21 0.47 0.9501

SBA-16 Langmuir Henry 6.20 0.10 0.9951

a LangmuirFreundlich.

Table 3Parameters of the Langmuir and LangmuirFreundlich equation for adsorption of LYSin mesoporous silica.



b



SBA-16 Langmuir 76.07 0.58 9.35 0.79 0.9994LFa 75.03 1.60 0.08 0.04 1.20 0.38 0.9994


MeTable 4Parameters of the Langmuir and LangmuirFreundlich equation for adsorption of CELin mesoporous silica.



b



SBA-16 Langmuir 473.38 122.64 0.06 0.02 0.9922LFa 412.81 6.62 0.07 0.00 1.00 0.00 0.9930


0 2 4 6 8 10 12 14 160

20

40

60

80

100

120

140

q (m

g/g)

S.M.L. dos Santos et al. /Microporous andIn the case of CEL, all adsorbents showed appreciable adsorp-tion capacity at 295 K and only slightly non-linear behavior. Thetting parameters of the L and LF equations are summarized in Ta-ble 4. According to results showed in Table 4, L model was alsomore suitable to represent the experimental adsorption of CEL inall SBAs. It is the biomolecule best adsorbed by SBA-16, possiblydue to the different conformations and sizes of the individual en-zymes that compose the cellulase pool, which leads to a less pro-nounced exclusion effect than that observed for BSA and LYS.This may be best appreciated in Fig. 11, in which the isothermsof the three studied biomolecules are shown for SBA-16. In fact,it is reported in Hartono et al. [57] that cellulase may assumeeither spherical (2.47.4 nm diameter) and ellipsoidal(1.3 7.9 nm to 4.2 25.2 nm). Taking into account that the poremouth diameter of the synthesized SBA-16 is around 3 nm (seeFig. 5b), smaller enzymes or thin ellipsoidal ones will have free ac-cess to the cubic cages.

4. Conclusions

SBA-15 and SBA-16 were successfully synthesized by solgeland hydrothermal routes. All three synthesized mesoporous silicaswere capable of adsorbing the studied biomolecules (BSA, LYS andCEL) at pHs close to their pIs with relatively high capacities in mostcases. Among the silicas with parallel cylindrical pores (SBA-15),the material synthesized hydrothermally had superior texturalproperties (pore size and specic pore volume) and showed thehighest 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 solgel route showeda larger adsorption capacity for cellulase (957 mg/g) as comparedto the other materials. Not only it is the material with better mes-

Ceq (mg/mL)Fig. 11. Adsorption isotherms at 295 K in SBA-16: BSA (N), LYS (}) and CEL ().oscopic pore arrangement (Fig. 2), but it also exhibits a narrowerpore size distribution (Fig. 4b) as compared to the other adsor-bents, which suggests that cellulase is likely to be more denselypacked in SBA-15(SG) than in SBA-15(HD). Regarding SBA-16, low-er adsorption capacities were expected for all studied biomoleculesdue to the nature of his own structure, since it presents a face-cen-tered cubic structure connected by narrow pores. Nevertheless, theuptake of cellulase by this material (473 mg/g), reported for therst time in this paper, far exceeds the uptakes of other biomole-cules, and this may be an interesting feature to be exploited inthe design of chromatographic separation processes of thisenzyme.

Acknowledgments

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

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292 S.M.L. dos Santos et al. /Microporous and Mesoporous Materials 180 (2013) 284292

Synthesis and characterization of ordered mesoporous silica (SBA-15 and SBA-16) for adsorption of biomolecules1 Introduction2 Experimental part2.1 Reactants2.2 Synthesis2.3 Characterization2.4 Protein adsorption experiments

3 Results and discussion3.1 Characterization of SBA-15 and SBA-163.2 Biomolecules adsorption3.2.1 pH effect on the adsorption of BSA, LYS and CEL3.2.2 Kinetic and adsorption isotherm of BSA, LYS and CEL onto SBA-15 and SBA-16

4 ConclusionsAcknowledgmentsReferences

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