Synthesis of Bimodal Porous Titania Beads and Their Potential in

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Synthesis of Bimodal Porous Titania Beads and Their Potential in Liquid Chromatography Kai-Feng Du,*,† Min Yan,‡ Hang Song,† and Yong-Kui Zhang† † ‡

Department of Pharmaceutical & Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu 610065, China Department of Computer, Sichuan TOP Vocational Institute of Information Technology, Chengdu 611743, China

bS Supporting Information ABSTRACT: Bimodal porous titania beads were synthesized from titania nanocrystals of about 34 nm by a method that combines particles agglomeration and sol
gel templating process. The structure properties of porous titania beads were investigated, and the results indicated that the porous titania beads in size of about 15 μm are pure anatase phase and featured by two types of pores with macropores centered at about 200 nm and mesopores smaller than 10 nm. The bimodal pore structure is attributed to hard aggregates of titania particles with different size. In particular, the larger titania nanocrystals agglomerate into macropores and the smaller ones from crystal growth of coated titania into mesopores. Finally, after cation modification, the bimodal porous adsorbents (BPA) were evaluated for investigating chromatographic performance and compared to microporous ones (MPA). It was observed that static and dynamic adsorption capacities of cytochrome C on BPA are both larger than that on MPA. Moreover, the BPA packed column shows better column efficiency than the MPA packed one in test flow rates. Therefore, we demonstrated that the BPA occupies more advantages over the other one and is expected for potential application in chromatography.

1. INTRODUCTION Chromatography serves as the most important technique in liquid separation due to its high selectivity, high efficiency, speed, and ruggedness.1 In view of this technique, the stationary phase is the kernel part that significantly determines the performance of the chromatography system.2 In the context, a variety of stationary phases with different chemical compositions including polysaccharides, organic polymer, and inorganic oxide (silica, titania, zirconia, and alumina) have been developed to satisfy the increased requirement for separation during the past decades.3
6 Among them, titania has excellent chemical/mechanical stability over silica and polymeric materials, easy modification with usual ligands, and unique retention behavior for different biomolecules. All of these properties make it ideal material as the stationary phase for chromatography.7
10 With the exception of chemical composition, control over the structure including outer shape, inner porosity, as well as surface area is the other important factor because it dictates the physical property of the stationary phase. From a viewpoint of chromatographic application, a spherical material with bimodal porosity is appreciated due to its superior adsorption property.11 Generally, the bimodal pore system containing both macropores and mesopores enhances the diffusion mass transport, and hence confers the improvement of adsorption property,12 while, despite of the popularity of titania-based stationary phase, the synthetic routes toward such bimodal porous beads are less common. The major obstacle is ascribed in part to the rapid sol
gel kinetic of titania precursor, which makes it difficult to control the structure development.13,14 Currently, relatively few techniques, such as polymerization-induced colloid aggregation (PICA) and spraydry method (SDM), have been used to fabricate the porous titania beads for chromatography.15,16 However, there exist still r 2011 American Chemical Society

more or less shortcomings associated with these techniques. For example, the PICA process often leads to the product with low repeatability and small pore size because the porous structure is seriously limited by preparation condition; the SDM technique always results in broad particle size distribution and high cost for operation. Thus, there is still a challenge to explore a novel method to produce the titania beads with desired bimodal porosity. A practical alternative route, named particles-agglomeration method, was provided to generate the porous beads.17,18 For this technique, the primary small particles aggregate to form larger ones, and hence construct the intraparticle pores. Because the inner pore size depends on the size of primary particles, it can be controlled by choice of the primary particles in suitable size. Additionally, we have used the customized agarose beads as shape templates to prepare the microporous titania beads by a sol
gel procedure.19 It is found that the sol
gel templating method occupies an obvious advantage in controlling the bead shape. Inspired by the existing studies above, a novel approach to fabricate bimodal porous titania beads is developed by combining particles-agglomeration and sol
gel templating method. In the synthesis, we utilized agarose and titania nanocrystals to manipulate the composite beads, followed by sol
gel process and calcination to form coated titania on nanocrystals. The fabrication process provides titania beads with both macropores for fast mass transfer and mesopores for high specific surface area. After synthesis and characterization, the bimodal porous titania were Received: September 27, 2010 Accepted: April 5, 2011 Revised: April 3, 2011 Published: April 05, 2011 6101

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2. EXPERIMENTAL SECTION 2.1. Chemicals. Agarose powder was obtained from Biowest (Shanghai, China). Polyoxyethylene sorbitanmonooleate (Tween 80) was purchased from Dengfeng Chemical Co. (Tianjin, China). Sorbitan trioleate (Span 85), ethylenediamine-N,N0 -tetramethyl phosphonic acid (EDTPA), pyridine, and potassium phosphate (PPS) were purchased from China National Pharmaceutical Industry Co. (Beijing, China). The metal oxide precursor, titanium(IV) tetraisopropoxide (TTIP, 99%), was purchased from Luchuan Chemical Co. (Shandong, China). Cytochrome C was obtained from Sigma (St. Louis, MO). Other reagents were all of analytical grade received from local sources. 2.2. Preparation of Titania Nanocrystals. The titania nanocrystals were prepared by sol
gel process of titanium isopopoxide (TTIP), and followed by hydrothermal treatment.20 During the procedure, 28.5 g of TTIP was dissolved in 300 mL of anhydrous ethanol and adjusted to pH = 3 by addition of hydrochloric acid. The obtained TTIP solution was then added dropwise to 200 mL of water/ethanol solution (50/50, v/v) to hydrolysis and precipitation at 20 °C with rigorous stirring. After being stirred for 2 h, the mixture containing hydrous oxide was transferred into a Teflon-lined autoclave and heated at 300 °C for 24 h. The resultant nanocrystals of titania were collected by centrifugation, washed with deionized water, and followed by drying in air at room temperature. 2.3. Preparation of Bimodal Porous Titania Beads. In a typical synthesis, 3 g of titania nanocrystals was well dispersed into 98 mL of aqueous solution by ultrasonic vibration. Next, 2 g of agarose powder was added into the suspension and heated to 95 °C in a water bath to completely dissolve agarose. Meanwhile, a 1000 mL three-neck flask was fed by a mixture of 200 mL of cyclohexane, 6 g of Span 85, and 2 g of Tween 80, and maintained at 60 °C in water bath. The well-mixed hot agarose/titania suspension was poured into the oil phase and stirred at 1000 rpm. After stirring for 30 min, the formed emulsification was rapidly cooled below 10 °C to solidify. The solidified ones were repeatedly washed by ethanol and distilled water to remove residual oil, and screened with 400 mesh and 500 mesh standard metal screens. The classified ones were dried and followed by soaking in 15 wt % TTIP 2-propanol solution in a closed vessel for 10 h at 20 °C. After the sol
gel process, the TTIP-filled composite beads were redispersed in 2-propanol to remove the unreacted TTIP precursors, and then dried in air at 50 °C. This step can contribute the chemical combination of the performed titania nanocrystals by the amorphous titania coating during the sol
gel process. Finally, the bimodal porous titania beads were obtained by calcination at 450 °C in air flow (50 mL min
1) by a tube muffler furnace. The temperature was raised at a rate of 2 °C min
1 to 450 °C and held for 2 h. 2.4. Surface Modification of Titania Beads with Ethylenediamine-N,N0 -tetramethyl Phosphonic Acid (EDTPA). The modification of titania beads is described as follows. First, 10 g of titania beads was dispersed into 100 mL of 0.1 mol L
1 EDTPA and followed by mixing by vacuum ultrasonic vibration. Second, the reaction suspension was placed into an oil bath at 100 °C for 6 h. Next, the reaction was terminated by decreasing temperature below 20 °C. Finally, the modified titania beads were obtained after the superfluous EDTPA was removed by

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washing repeatedly using deionized water. The resulting EDTPA-modified titania beads were dried at 50 °C in a clean vacuum oven overnight. 2.5. Characterization. Optical microscopy (OM) observations were conducted on a Nikon ECLIPSE E600 microscope. Scanning electron microscopy (SEM) was employed to observe the microscopic morphology of the samples on an XL 30 ESEM (Philips, Netherlands). The X-ray powder diffraction (XRD) patterns were collected on a Rigaku D/max 2500v/pc X-ray diffractometer (Rigaku, Japan) using Cu KR radiation (λ = 0.1542 nm). The pore size distribution was determined by mercury porosimeter (Quantanchrome Corp., U.S.). Specific surface area and mesopore volume were obtained from N2 adsorption measurement at
196 °C with a NOVA 2000 porosimeter (Quantachrome, U.S.). The thermogravimetric differential thermal analysis (TG-DTA) was performed using a Pyris Diamond TG-DTA (PerkinElmer Instruments, Boston, MA) at a heating rate of 10 °C min
1 and an air flow of 100 mL min
1, which can estimate the combustion of template in the agarose/ titania composite beads. Particle-size distribution was determined by a Mastersizer 2000 unit (Malvern Instruments, U.K.). The mass ratio of titania coating and titania nanocrystals (R) was calculated by mass balance, described briefly from the following equation: mi
m0 ð1Þ R ¼ mo where mi is the mass of bimodal titania beads calcined at 450 °C, and mo is the mass of titania nanocrystals that performed in the emulsion process. 2.6. Static Adsorption Equilibrium. The static adsorption property of EDTPA-modified titania adsorbents was determined by finite batch experiment.11 Briefly, as follows, the adsorbents were equilibrated with 20 mmol L
1 potassium phosphate (PPS) buffer (buffer A) with 4 mmol L
1 EDTPA (pH 5.5) and drained by a G3 sintered glass filter. Next, 50 mg of drained adsorbent was added into 15 mL polypropylene centrifuge tubes and mixed with 5 mL of cytochrome C standard solution of different concentrations (0
1.0 mg mL
1). The sealed tubes were gently shaken at 20 °C for 3 h on an incubator to achieve adsorption equilibrium. After the adsorption, they were centrifuged, and the absorbances of the supernatants at 280 nm were performed on Lamda 35 UV
vis spectrophotometer. On the basis of weight balance, the amounts of cytochrome C on absorbents were determined. The Langmuir isotherm was used to fit the equilibrium data by using the following equation: qm c ð2Þ q¼ Kd þ c where q is the adsorbed protein concentration, c is the free protein concentration in equilibrium with q, and qm and Kd denote adsorption capacity and dissociation constant, respectively. 2.7. Chromatography. The porous titania beads in size of about 15 μm were packed in a stainless steel column of 100 mm  4.6 mm i.d. by the stirred upward slurry method.21 The chromatographic experiments were performed with a Waters HPLC system (Waters, Milford, MA) companied with a model 600E multisolvent delivery system, a Rheodyne 7725i injector valve (Rheodyne, Cotati, CA), and a 2487 UV detector. The dynamic adsorption capacities of cytochrome C on packed columns were determined by breakthrough experiments. The column was first equilibrated with buffer A until a flat 6102

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Figure 2. XRD pattern of titania nanocrystals, along with the anatase reference (PDF card no. 21-1272).

Figure 1. TEM image of titania nanocrystals obtained by calcination at 300 °C.

baseline was achieved. Next, cytochrome C solution of 2 mg mL
1 was pumped into the column at flow rate of 1 mL min
1, and the outlet absorbance of the effluent at 280 nm was monitored. After adsorption, the packed column was washed with buffer A and followed by elution using buffer A with 1.0 mol L
1 NaCl (buffer B). The dynamic adsorption capacity was calculated from the breakthrough curve by using the following equation: q20% ¼

c0 uðt20%
t0 Þ m

ð3Þ

where q20% stands for the dynamic adsorption capacity of adsorbent at 20% breakthrough, c0 for the feed BSA concentration, u for the volumetric flow rate, t20% for the time at 20% breakthrough, t0 for the retention time under nonretained condition, and m for the mass of adsorbent. The column efficiency is expressed as the height of equivalent to a theoretical plate (HETP). The moment analysis was performed to determine the value of HETP by using the following equation: HETP ¼ L=5:54ðtR =tw0:5 Þ

2

ð4Þ

where L is the length of the column, tR is the retention time of pyridine, and tw0.5 is the peak width at half-height for pyridine by elution with buffer A.

3. RESULTS AND DISCUSSION 3.1. Titania Nanocrystals. The titania nanocrystals were prepared via sol
gel processing of titanium isopropoxide and followed by calcination, as described in section 2.2. To elucidate the crystal structure, these titania particles were studied by TEM and X-ray diffraction (XRD), respectively. The external morphology of titania particles is illustrated by TEM (Figure 1). It is observed that the sample obtained is composed of nanocrystals with a relatively uniform distribution in size of about 30 nm, and their lattice fringes from TEM image display an anatase phase (inset in Figure 1). The XRD spectrum also shows that the nanocrystals are considered as monophasic crystalline anatase according to PDF index lines (card no. 21-1272) (Figure 2). By Scherrer’s equation on anatase (101) diffraction, the crystalline sizes of titania particles are determined to be about 34 nm, which

Figure 3. OM images of agarose beads (a,b) and agarose/titania composite beads (c,d) after (a,c) and before (b,d) swelling in water.

match well with the TEM observation. On the basis of these results, it can be stated that the obtained titania nanocrystallines are eligible for the fabrication of porous titania beads. 3.2. Agarose/Titania Composite Beads. The agarose gel studied for templating strategy is made up of a loosely interwoven network with spherical shape and serves as both microcontainers for encapsulation of titania nanocrystals and spherical control. For better understanding of the role of agarose gel as template, the comparison of agarose beads and agarose/titania composite ones was carried out, as shown in Figure 3. It is found that two types of beads in the wet state all exhibit perfectly uniform spherical shape with an average diameter of about 30 μm (Figure 3a,c). Differently, because the solid particles are encapsulated in the gel network, the composite beads are rather darker and harder as compared to the wet pure agarose beads. Meanwhile, the encapsulation of solid particles also leads to less contraction in volume after drying. For example, the composite beads reduce only to about 18 μm, apparently larger than the dried agarose ones (10 μm) (Figure 3b,d). Although it is difficult to see the inner portion of the composite beads, from the results we can deduce that the agarose fibers led to the aggregation of a large amount of titania nanocrystals, and then formed the agarose/titania composite beads. The structure characteristic 6103

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Figure 4. Thermogravimetric differential analysis of titania/agarose composite beads.

endows its potential application for the fabrication of porous titania beads. 3.3. Porous Titania Beads and Structure Characterization. The porous titania beads were manufactured by crystallization and removal of template by calcination in air, as described in section 2. It is worth noting that the titania skeleton comes from two resources. One is originated from the performed titania nanocrystals in the agarose solution; the other is from the titania coating on the original composite beads during the sol
gel process. Before calcination, thermogravimetric differential thermal analysis (TG/DTA) was first carried out to give both the temperature at which the agarose template was removed and the content of titania component in composite beads. Herein, the typical TG/DTA curves are shown in Figure 4. From the TGA curve on composite beads, there are four zones of weight loss from room temperature to 450 °C, with a total weight loss of about 33% in mass. As seen in Figure 4, first two slight weight drops were determined to be about 3.5% and 3% in mass, which were attributed to releasing residual solvents (water and ethanol) below 105 °C and the further condensation of titania oligomers and unreacted titania precursors between 105 and 230 °C, respectively. With increasing temperature up to 450 °C, the further distinct weight drops of 14% and 12% in mass were observed and companied by two obvious exothermic peaks (DTA curve in Figure 4). It is estimated by that the larger peak at 320 °C arises from the imperfect combustion of organic groups and the smaller one at 380 °C from the complete combustion of the organic residues. The result is in accordance with our previous report.19 Apparently, the residual inorganic fraction is very high and consistent with the entrapment and coating of titania component during the fabrication process. On the basis of the TG/DTA result, the composite beads were calcined to obtain the porous titania beads, and their size is further decreased to be about 15 μm with a geometric standard deviation of around 1.1, determined by particle size measurement. The shrinkage can be regarded as a result of further aggregation of nanocrystals and agarose burn off together during the calcination process. By using eq 1, the mass ratio of coated titania and preformed titania nanocrystals was determined to be about 1.2. The high content for coated titania would afford both large specific surface area and mesopore volume for these bimodal porous titania beads. That is significant for their application in

Figure 5. SEM images of the agarose/nanocrystal composite bead (a), the bimodal porous titania bead (b), and the corresponding broken debris (c).

chromatography. To clearly elucidate the bead formation, the nanocrystal/agarose composite beads and bimodal porous titania beads were evaluated by SEM (Figure 5). Obviously, the former is characterized by loose aggregate of nanocrystals entangled with agarose fiber (Figure 5a). Also, the latter contains a wellconsolidated structure of solid particles tightly attached to each other, and the transecting macropores are clearly visible in the surface and interior of the single bead (Figure 5b,c). Additionally, the particles seem to be much larger than the performed titania nanocrystals from the SEM image. The increased particle size might be due to the titania coating on the nanocrystals. Generally, the titania coating can improve the mechanical strength and provide a large specific surface area of the final titania beads after the sintering process (Table 1). 6104

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Table 1. Physical Properties of Bimodal Porous Titania Beads Calcined at Different Temperatures samples a

crystalline size (nm) specific surface areab (m2 g
1) mesopore volumec (mL g
1) macroporosityd (%)

titania beads

titania beads

(450 °C)

(600 °C)

42 ( 2 73.5

51 ( 3 57.4

0.3721 29

0.2140 31

mesopore sizee (nm)

6(1

10 ( 1

macropore sizef (nm)

200 ( 6

200 ( 7

a

Crystallite size calculated from XRD patterns based on the Scherrer formula. b Specific surface area taken from the linear part of the BET plot (P/P0 = 0.05
0.225). c Mesopore volume estimated from the volume of N2 adsorbed at P/P0 = 0.986. d Macroporosity is the volume fraction of the macropores determined by mercury porosimeter with respect to the bead volume. e Mesopore size obtained from the desorption branch of the isotherm using Barrett
Joyner
Halenda (BJH) method. f Macropore size determined by mercury porosimeter.

Figure 7. XRD patterns of bimodal porous titania beads obtained by calcination at different temperatures: 450 °C (a), 600 °C (b).

Figure 8. Adsorption isotherm of cytochrome C to the cation adsorbents: (4) BPA, (0) MPA. The solid lines are calculated from eq 2. Figure 6. Pore size distribution of bimodal porous titania beads obtained by calcination at different temperatures: 450 °C (O), 600 °C (4).

To more accurately elucidate the pore structure, the titania beads were further characterized by mercury intrusion measurement and N2 adsorption, and the results are listed in Figure 6 and Table 1. From mercury intrusion measurement, the titania beads exhibit typical bimodal pore distribution, with mesopores smaller than 10 nm and macropores centered at about 200 nm. Generally, the hard aggregate of particles serves as the pore formation, and the particle size is related to the pore size and pore volume.22 Thus, it is easy to understand that the mesopores and macropores are ascribed to crystal growth of coated amorphous titania and the further accumulation of preformed larger titania nanocrystals, respectively. Different from the unchanged macropores, there is an obvious increase from 6 to 10 nm in mesopore size when increasing temperature from 450 to 600 °C. Correspondingly, the BET surface area and pore volume of titania beads monotonically decreased from 73.5 to 57.4 m2 g
1 and from 0.3721 to 0.2140 mL g
1, respectively. This is caused by the crystal growth of titania coating at high temperature. In addition, the titania beads have larger surface area over the titania nanocrystals. This might be ascribed to the macroporous channels inside titania beads, which contribute high permeation for

N2 and then lead to more effective surface area accessible for N2 adsorption. Figure 7 shows the XRD patterns of the porous titania beads by calcination at 450 and 600 °C, respectively. From the crystalline peaks, both titania beads display the crystal phase of anatase, and the intensity of the crystal phase is amplified slightly when increasing temperature from 450 to 600 °C. It indicates that the porous titania beads had barely undergone a phase transformation during calcination below 600 °C with the exception of crystal growth of the titania nanocrystals (Table 1). Needless to say, it would be favorable to have a pure anatase crystal phase that is associated with a high surface homogeneity. 3.4. Static Protein Adsorption. The static adsorption isotherms of cytochrome C on the cation absorbents (denoted bimodal porous adsorbent as BPA and microporous one as MPA) are presented in Figure 8. Langmuir model fitting the isotherm data to eq 2 gave the static protein adsorption capacity (qm) and the dissociation constant (Kd) (Table 2). The results from Table 2 show that two types of adsorbents have similar dissociation constants. It might be deduced the fact that there are similar ligand densities available for cytochrome C adsorption on these absorbents. Moreover, the protein adsorption capacity based on BPA reaches about 53.1 mg g
1, 55.8% higher than that of the MPA (34.3 mg g
1). The increased adsorption capacity on the 6105

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Table 2. Adsorption Properties of the Cation Adsorbents for Cytochrome C cation adsorbents

qm (mg g
1)

Kd (mg mL
1)

q20% (mg/g)

MPA

34.3 ( 1.7

0.015 ( 0.003

16.0 ( 1.1

BPA

53.1 ( 2.0

0.017 ( 0.002

32.2 ( 1.3

Figure 10. Column efficiency detected with pyridine as a function of flow rate: (0) BPA, (O) MPA. Conditions: column 100 mm  4.6 mm i.d.; mobile phase, 20 mmol L
1 potassium phosphate, pH 5.5.

Figure 9. Breakthrough curves of cytochrome C on columns packed with the cation adsorbents: (- - -) BPA, (
) MPA.

former lies in both large macroporosity and wide pores through the BPA. As expected, the macropore structure provides more transport channels and more adsorption sites accessible for protein approach, thus leading to high static adsorption capacity. 3.5. Breakthrough Behavior and Dynamic Adsorption Capacity. It is more practical for an adsorbent to present rather high dynamic adsorption capacity than high static adsorption capacity in the chromatography process. Frontal analysis can provide the information about dynamic adsorption capacity and mass transfer behavior, and thus can account for the benefit from the bimodal porous structure of adsorbents. Figure 9 shows the breakthrough curves of BPA and MPA at 360 cm h
1, respectively. The dynamic adsorption capacities at 20% breakthrough were calculated by using eq 3. It is found that the column packed with BPA reaches about 32.2 mg g
1 determined by weight balance, 2 times higher than that (16.0 mg g
1) of MPA, whereas the slope of breakthrough curve from BPA was less steep than other ones. These results suggest that more adsorption sites are available for absorbing protein in less time by introducing macropores in adsorbents, and thus lead to an increased dynamic adsorption capacity. Apparently, the bimodal porous titania beads are more suitable for chromatography. 3.6. Column Efficiency. The structure feature of EDTPAmodified adsorbent can be illustrated by the Van Deemter model, which describes the variation of column efficiency (HETP) with flow rate. In this way, the relationships of HETP versus flow rate for columns packed with different adsorbents were measured using pyridine as tracer (Figure 10). As seen in Figure 10, best column efficiencies were reached to be 0.141 mm (plate number 7092 m
1) and 0.120 mm (plate number 8333 m
1) for MPA and BPA, respectively, at flow rate of 180 cm h
1. Moreover, as compared to MPA, the column efficiency of column packed with BPA is higher and somewhat less dependent on flow rate in a wide flow rate range from 180 to 1080 cm h
1. According to the Van Deemter model, it is considered by that macropores in BPA

Figure 11. Effect of flow rate on back pressure of the column packed with (0) MPA and (O) BPA. Conditions: column 100 mm  4.6 mm i.d.; mobile phase, distilled water.

decrease the mass transfer resistance and cause faster diffusion within the pool of the stagnant mobile phase located in the pores than do the MPA. 3.7. Physical and Chemical Stability. The titania supported adsorbent occupies the advantage of physical and chemical stability. To confirm the stability, flow hydrodynamics was examined in terms of the relationship between back pressure and flow rate (Figure 11). As seen here, the reasonably linear relationship was observed for flow rate up to around 1080 cm h
1. It proves that the BPA in column did not crumble even at high back pressure, indicating their excellent physical stability. Regardless of this, in case of BPA, the column shows a lower back pressure than does the column packed with MPA in the tested flow rate. Because two types of beads have similar particle size distribution, the lower back pressure can be explained by the combination of their surface roughness and larger macroporosity, which leads to a more open and more permeable column. The chemical stability and the reproducibility are studied as follows. The column packed with BPA was subject to continuous flushing of 1.0 mol L
1 NaOH, and the integrity of stationary phase was tested through the retention of cytochrome C after 6106

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’ AUTHOR INFORMATION Corresponding Author

*Tel.: þ86-28-85405221. Fax: þ86-28-85405221. E-mail: kfdu@ scu.edu.cn.

’ ACKNOWLEDGMENT This work was supported by the Foundation of Sichuan University for Young Teachers and the Doctoral Fund of the Ministry of Education of China (No. 20090181110043). ’ REFERENCES Figure 12. Effect of column volume on cytochrome C retention on column packed with BPA. Conditions: column 100 mm  4.6 mm i.d.; mobile phase, 20 mmol L
1 potassium phosphate with 0.2 mol L
1 KCl, pH 5.5; flow rate, 1 mL min
1.

equilibrium with 20 mmol L
1 potassium phosphate with 0.2 mol L
1 KCl (Figure 12). As seen in Figure 12, the retention time for cytochrome C remains unchanged even after flushing 5000 column volumes (CVs) of 1 mol L
1 NaOH solution at 1 mL min
1. Moreover, there was no significant difference in dynamic binding capacity for protein in the course of 6 months. Therefore, the titania supported adsorbent is chemically stable at such a corrosive condition.

4. CONCLUSIONS In summary, we have demonstrated a facile method that combines particles agglomeration and sol
gel templating process to prepare the bimodal porous titania beads. For bead formation, the agarose gel serves as shape templates to determine the spherical morphology and the nanocrystals in different sizes aggregate into the bimodal pore system. The bimodal pore structure and corresponding properties were characterized in terms of external morphology, crystal phase, pore size distribution, specific surface area, flow hydrodynamics, static/dynamic adsorption capacity, and column efficiency. It is found that, after calcination at 450 °C for 2 h, the fabricated beads are pure anatase phase and occupy both macropores and mesopores. After cation modification, the adsorbent of bimodal porous beads possesses more advantages, like higher static and dynamic adsorption capacity, as well as better column efficiency, as compared to the microporous ones. All of the properties demonstrate that the bimodal porous titania beads can act as a novel stationary phase for use in chromatography. In addition, the method developed here is conveniently extendable to fabricate the other porous metal oxide beads, which would provide more extensive applications from chromatography to photocatalysis. ’ ASSOCIATED CONTENT

bS

Supporting Information. Details of the nitrogen adsorption desorption isotherm of bimodal titania beads calcined at 450 °C and separation of proteins on the columns packed with EDTPA-modified beads (BPA and MPA). This material is available free of charge via the Internet at http://pubs.acs.org.

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