Fabrication of Monodisperse Porous Silica Microspheres with a

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Fabrication of Monodisperse Porous Silica Microspheres with Tunable Particle Size and Pore Size for Protein Separation Jiwei Chen, Lili Zhu, Lianbing Ren, Chao Teng, Yong Wang, Biwang Jiang, and Jie He ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00088 • Publication Date (Web): 16 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 2018

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ACS Applied Bio Materials

Fabrication of Monodisperse Porous Silica Microspheres with Tunable Particle Size and Pore Size for Protein Separation §

§

Jiwei Chen†, , Lili Zhu⊹, , Lianbing Ren†, Chao Teng†, Yong Wang †, Biwang Jiang†, * , and Jie He †,‡,* †

Guangdong Key Laboratory of Nano-Micro Materials Research, Key Laboratory of Chemical Genomics, Shenzhen Graduate School of Peking University, Shenzhen, 518055, People’s Republic of China

‡

Shenzhen Weiguang Biological Products Co., Ltd., Shenzhen, 518107, People’s Republic of China

⊹

Key Laboratory for Heavy Metal Pollution Control and Reutilization, School of Environment and Energy, Peking University Shenzhen Graduate School, Shenzhen, 518055, People’s Republic of China

KEYWORDS: monodisperse, porous silica microspheres, polymer microspheres, hard template, functionalization, tunable, protein separation

ACS Paragon Plus Environment

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ABSTRACT: Monodisperse porous silica microspheres with tunable particle size and pore size were fabricated by utilizing porous polymer microspheres as a novel hard template during sol-gel process followed by calcination to remove the polymer. The particle size and pore size could be simply tuned by the feature of the polymer template and reaction conditions such as different functionalization of the parent polymer template, particle size of polymer template, amount of TEOS during sol-gel process. EDA (ethylenediamine), APTES (3-aminopropyl)triethoxysilane, and TMA (trimethylamine hydrochloride) functionalization of porous poly(GMA-co-EGDMA) microspheres were carried out to study their effect on the synthesized porous silica microspheres. The TMA functionalized polymer microspheres led to higher yield, smaller silica nanoparticles and no self-nucleation of TEOS due to their positive surface charge. Furthermore, no addition of NaOH during TMA functionalization and amount of TEOS during sol-gel process played key roles on determining the pore size and particle size of porous silica microspheres. Then, through poly(aspartic acid) coating of the APTES functionalized monodisprese porous silica microspheres, the modified monodisperse porous silica microspheres were explored as the stationary phase of HPLC for protein separation. The effects of particle size and pore size on chromatographic behavior were discussed. When the protein mixture composed of transferrin, hemoglobin, ribonuclease A, cytochrome C and lysozyme was used as the model analytes, the as-prepared silica microspheres exhibited excellent separation performance with high protein recovery and good reproducibility.

1. INTRODUCTION With rapid development of nanoscience and nanotechnology, porous materials have attracted widespread attention in applications of catalysis, drug delivery, adsorption, separation, and sensors, due to their high surface area, excellent chemical and physical properties, and


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controllable pore size and morphology.1-7 Especially, since Mobil company discovered M41S series (MCM-41, MCM-48 and MCM-50) of mesoporous silica in 1992, fabrication and applications of porous silica with different features have been widely explored.8,9 Due to high surface area, easy functionalization, unique biocompatibility and chemical stability, porous silica exhibited distinguished applications in various fields such as drug delivery, catalysis, and separation.10-12 As the structural characteristics of silica strongly determine its performance, controlling the morphology and pore structure would be of great significance. For example, for application in HPLC separation, porous silica with uniform spherical morphology and specific pore size is essential. As an ideal stationary phase of HPLC, porous materials should satisfy the following requirements: (1) variable phase ratio, i.e. sample retentivity and capacity; (2) long-term chemical stability; (3) high mechanical strength; (4) the particles should have high surface area and narrow size distribution; therefore, the morphology should be spherical, the pore must be open and suitable for the analytes to exhibit fast mass transfer; (5) the packing materials should be energetically homogeneous and easy to be chemically functionalized.13,14 According to the above requirements, silica is much more superior to other inorganic packing materials such as ZrO2, TiO2 and Al2O3.14 Furthermore, monodisperse porous silica microspheres with narrow pore size distribution are vital. For HPLC separation to achieve a perfect separation result, controlling particle size and pore size of porous silica microspheres is of great importance. Firstly, the particle size affects the column efficiency and backpressure. The smaller particle size leads to a higher column efficiency, but higher backpressure. Thus, the particle size from 3 µm to 10 µm would be suitable. Secondly, to decrease mass transfer resistance and ensure high surface area,


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the pore size should be in the range of 6 nm to 50 nm. Therefore, it is essential to fabricate monodisperse porous silica microspheres (MPSM) with tunable particle size and pore size. Since micrometer-sized monodisperse nonporous silica microspheres were fabricated using TROS/H2O/NH3·H2O/ethanol system through sol-gel process by Stöber in 1968, the door of micrometer-sized silica microspheres have been opened up.15 Through adding the soft template during Stöber’s sol-gel process, porous silica microspheres were obtained by the template removal.16-18 Extensive efforts have been devoted to fabricate porous silica microspheres with different pore size and particle size. Initially, traditional surfactants were used to fabricate porous silica microspheres with pore size less than 4 nm.19-26 By using alkyltrimethyl ammonium chloride as the soft template, Yano and Fukushima have prepared monodisperse porous silica microspheres (MPSM) with pore size of about 2 nm and particle size of hundreds of nanometers.18 Chen used Gemini surfactants to fabricate MPSM with pore size from 2.2 to 3.4 nm and particle size in the range from 70 to 460 nm.27 To make the pore size more than 4 nm, pore-expanding agents were added during the synthetic process, such as long-chain amines and aromatic compounds.28-30 However, porous silica microspheres prepared by this method would exhibit poor stability and the pore size distribution became wider. Another method to expand the pore size was using the block copolymer surfactants with molecular weight more than 2000 such as P123 and F127.31 Through using block copolymers, Mesa, Katiyar and Khanrahan all obtained porous silica microspheres with pore size of about 10 nm. However, they showed wide pore size and particle size distribution.32-34 Li’s group developed new cationic block copolymers (d-D400 and q-D400) as the soft template to fabricate MPSM with pore size of 20 nm. And the obtained MPSM could be used for HPLC separation of nucleotides. However, the particle size of MPSM was only 1.7 µm, which resulted in a high operating backpressure.35 Besides adding the soft


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template during the sol-gel process, the hard-templating method and polymerization-induced colloid aggregation were also used to prepare porous silica microspheres.36,37 Despite their remarkable work on porous silica microspheres, it is still challenging to fabricate monodisperse micrometer-sized porous silica microspheres with tunable particle size and pore size. In our previous reports, monodisperse porous silica, magnetic silica, carbon, zirconia and nickel microspheres were fabricated through utilizing uniform porous polymer microspheres as the hard template.38-43 Previously, monodisperse porous silica microspheres were fabricated by using EDA-functionalized porous poly(GMA-co-EGDMA) microspheres to proceed sol-gel process with TEOS in H2O/isopropanol system. The EDA-functionalized polymer microspheres tend to form aggregates during sol-gel process and tetra-n-butylammonium bromide (TBAB) was added to avoid aggregation.39 However, when the concentration of the template and TEOS was increased, the aggregation still existed. Bai’s group adopted tetraethylenepentamine (TEPA) functionalized polymer microspheres to enlarge the production scale and the obtained porous silica microspheres were then modified with octadecyltrichlorosilane to execute protein separation as the reverse phase chromatographic materials.44,45 Herein, we report another simple and efficient strategy to accomplish controllable fabrication of monodisperse porous silica microspheres through TMA functionalization of the polymer template microspheres. The particle size, pore size, surface area and pore volume could be tuned by the porous polymer hard template and the reaction conditions. The as-prepared monodisperse porous silica microspheres were explored as the weak cationic-exchange stationary phase of HPLC and exhibited excellent performance for protein separation.

2. EXPERIMENTAL SECTION 2.1 Chemicals and Materials.


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The silica precursor tetraethylorthosilane (TEOS) was purchased from Alfa Aesar. Trimethylamine

hydrochloride

(TMA·HCl),

Ethylenediamine

(EDA),

(3-

aminopropyl)triethoxysilane (APTES), sodium hydroxide, ammonium hydroxide (28% NH3·H2O), D, L-aspartic acid, NEt3, β-alanine, human transferrin,

human hemoglobin,

ribonuclease A (from bovine pancreas), cytochrome C and lysozyme were purchased from Sigma-Aldrich. The hard template porous polymer microsphere named poly(GMA-co-EGDMA) is a polymer of glycidyl methacrylate (GMA) cross-linked with ethylene glycol dimethacrylate (EGDMA) supplied by Nano-Micro Technology Company, China. Other chemicals were all purchased from Sigma-Aldrich and anhydrous solvents were distilled by standard procedure. Water was purified by distillation followed by deionization using ion exchange resins. All chemicals were analytical grade and used without further purification. 2.2 Preparation of Monodisperse Porous Silica Microspheres. Monodisperse porous silica microspheres (MPSM) were fabricated by utilizing the functionalized porous poly(GMA-co-EGDMA) microspheres to proceed sol-gel process with TEOS followed by calcination to remove the template. EDA functionalization of porous poly(GMA-co-EGDMA) polymer microspheres: Poly(GMA-co-EGDMA) microspheres of 3 g were dispersed in 200 ml of water and sonicated for 0.5 h before 6 g of EDA was added and the mixture was mechanically stirred at 80 °C for 24 h. The resulting EDA functionalized poly(GMA-co-EGDMA) microspheres were then washed repeatedly with distilled water till the filtrate was neutral and dried at 50 °C. The EDA functionalized porous poly(GMA-co-EGDMA) polymer microspheres of 3.24 µm were named “F. T. 1”.


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APTES functionalization of porous poly(GMA-co-EGDMA) polymer microspheres: Poly(GMA-co-EGDMA) microspheres of 7.5 g were dispersed in 200 ml of anhydrous ethanol and sonicated for 0.5 h. Afterwards, APTES of 10 ml was added and the mixture was mechanically stirred at 60 °C for 18 h. The obtained APTES functionalized poly(GMA-coEGDMA) microspheres were washed repeatedly with distilled water till the filtrate was neutral and dried at 50 °C. The APTES functionalized porous poly(GMA-co-EGDMA) polymer microspheres of 3.24 µm were named “F. T. 2”. TMA functionalization of porous poly(GMA-co-EGDMA) polymer microspheres: Poly(GMA-co-EGDMA) microspheres of 4 g were dispersed in 50 ml of methanol and 100 ml of water through sonication for 0.5 h. Then TMA·HCl of 8 g and 3.34 g of NaOH were added. Afterwards, the mixture was mechanically stirred at 50 °C for 24 h. The obtained TMA functionalized poly(GMA-co-EGDMA) microspheres were washed repeatedly with distilled water till the filtrate was neutral and dried at 50 °C. The TMA functionalized porous poly(GMAco-EGDMA) polymer microspheres of 3.24 µm were named “F. T. 3”. Preparation of monodisperse porous silica microspheres: In a typical synthesis, the functionalized poly(GMA-co-EGDMA) microspheres of 0.5 g were dispersed in 20 ml of water and 80 ml of ethanol through sonication for 0.5 h. 28% NH3·H2O of 2 ml was added and then 1.5 g of TEOS dissolved in 20 ml of ethanol was added dropwise by peristaltic pump. The reaction was carried out at room temperature for 24 h. Afterwards, the silica/polymer composite microspheres were washed repeatedly with distilled water till the filtrate was neutral and dried at 50 °C. The dried microspheres were then calcined at 600 °C for 6 h to form monodisperse porous silica microspheres. 2.3 Preparation of the Modified MPSM Stationary Phase.


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To explore the performance of MPSM in HPLC for protein separation, MPSM-a (7.25 µm, 30 nm), MPSM-b (7.78 µm, 14.4 nm) and MPSM-c (3.24 µm, 30 nm) were chosen and their synthesis processes are shown in Experimental Section, Supporting Information. For protein separation in HPLC, silica microspheres could interact with protein by nonspecific irreversible adsorption due to the silanol group, which leads to some problems such as low recovery and protein denaturation. Therefore, porous silica microspheres should be coated by hydrophilic polymer to cover the residual silanol. Herein, the hydrophilic poly(aspartic acid) coating of MPSM was carried out according to Alpert’s work [46]. Synthesis of Poly(succinimide): D, L-aspartic acid of 20 g was polymerized in oven at 190 °C for 48 h. The obtained pale brown power was heated to be dissolved in 80 ml DMF. Then the mixture was centrifuged to remove the white precipitate. The supernate was recrystallized by diethyl ether. Through continuous stirring, the brown precipitate was formed. The precipitate was then washed by diethyl ether repeatedly to exchange DMF. All of the obtained poly(succinimide) precipitate was dried in oven at 50 °C. APTES Functionalization of MPSM: Before APTES functionalization, MPSM was acidized by 0.1 M HCl. After filtration, the acidized MPSM was washed repeatedly by H2O and ethanol. Afterwards, in a 250-ml three-necked rounded-bottomed flask, the dried acidized MPSM of 15 g was dispersed in 120 ml of anhydrous toluene and then 6 g of APTES was added. The mixture was refluxed for 24 h. After the reaction, the mixture was filtrated and the microspheres were washed by toluene and ethanol. The obtained APTES functionalized MPSM was then dried in oven at 50 °C. Poly(aspartic acid) coating of APTES-MPSM: The APTES functionalized MPSM of 2.5 g was dispersed in 30 ml of DMF and poly(succinimide) of 1.875 g dissolved in 20 ml of DMF


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was added. Then the mixture was mechanically stirred at room temperature for 24 h. The modified microspheres were washed repeatedly with DMF and ethanol before being dried at 50 °C. Afterwards, the poly(succinimide)-modified MPSM of 2.6 g was dispersed in 26 ml of DMF. 6.5 ml of H2O, 0.406 ml of NEt3 and 0.536 g of β-alanine were added. After being stirred mechanically for 24 h, the mixture was filtrated and the microspheres were washed by DMF and 0.1 M HCl. Then they are washed repeatedly by water and ethanol. The obtained poly(aspartic acid)-coated MPSM was dried in oven at 50 °C. The poly(aspartic acid)-coated MPSM-a, MPSM-b and MPSM-c are named the stationary phase SP-1, SP-2 and SP-3 respectively. 2.4 The Modified MPSM Stationary Phase for Protein Separation. Chromatographic Procedure HPLC was performed on an Äkta purifier 10 system manufactured by GE company with Monitor UV-900 and pH/C-900 as the detectors. The modified MPSM stationary phases were slurry-packed into 50 mm × 4.6 mm I. D. stainless steel columns. Samples were applied with the injection valve equipped with a 25 µL-loop. The protein mixture consisted of transferrin (4 mg/ml), hemoglobin (5 mg/ml), ribonuclease A (10 mg/ml), cytochrome C (4 mg/ml) and lysozyme (2.5 mg/ml) in 20 mM PBS (pH 6.8). Typically, 20 µL of the protein mixture were injected. The starting buffer A was 20 mM PBS (pH 6.8) and the B buffer was 20 mM PBS + 0.5 M NaCl (pH 6.8). The proteins were eluted by a linear gradient from 100% A to 100% B with 25 CV. The flow rate was 0.5 ml/min. And the proteins were monitored at 280 nm. 2.5 Characterization Techniques. A field emission scanning electron microscope (SEM) Hitach S4800 was applied to determine the morphology and structure of the microspheres. The particle hydrodynamic size was measured through using a Beckman Coulter Counter laser size analyzer (Multisizer 3). Measurement of N2 adsorption/desorption isotherms were carried out at


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77 K on a Micromeritics Tristar 3020. The zeta potential of the samples was measured on Zetasizer Nano Series (Nano-ZS90). Fourier transform infrared (FT-IR) spectra of the microspheres were obtained on a Shimadzu IR Prestige-21 with resolution of 4 cm-1. Thermogravimetric analysis (TGA) of the polymer microspheres and polymer/silica composite microspheres was performed on a Du Pont TGA 2050, with a terminal temperature of 600 °C and a heating rate of 10 °C·min−1.

3. RESULTS AND DISCUSSION Scheme 1. Fabrication of Monodisperse Porous Silica Microspheres

3.1 Preparation and Characterization of MPSM As shown in Scheme 1, monodisperse porous silica microspheres were fabricated by utilizing porous polymer microspheres as the novel hard template to proceed sol-gel reaction with TEOS followed by calcination to remove the template. Herein, porous poly(GMA-co-EGDMA) microspheres were chosen as the template because of their easy functionalization due to the existence of epoxy groups. Based on the previous work on applying porous poly(GMA-coEGDMA) as the hard template to fabricate porous silica microspheres39,44,45, the polymer properties was of vital importance. Different functionalization and different particle size of the polymer microspheres led to different features of MPSM. Furthermore, during sol-gel process, the synthesis conditions played a key role on determining the properties of MPSM. Firstly, EDA, APTES, and TMA functionalization of the parent template were carried out to obtain F. T. 1, 2,


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and 3 (Figure 1). The reaction conditions and the particle size of the as-prepared MPSM are shown in Table 1. The pore size, BET surface area and pore volume of MPSM 4-12 are exhibited in Table S1, Supporting Information. In our article published in 2012, EDA functionalization of the polymer microspheres was carried out, the solvents used during sol-gel process were water and isopropanol and tetra-n-butylammonium bromide (TBAB) was added as a dispersant to avoid aggregation39. In this maunuscript, we found that the EDA functionalized microspheres

Figure 1. EDA, APTES, and TMA functionalization of porous poly(GMA-co-EGDMA) microspheres. during sol-gel process were much more stable in water/ethanol system with no need of adding TBAB. As seen from Table 1, when the mass ratio of F. T. : TEOS increased, the particle size increased. Compared with the F. T. 1 and F. T. 2, the F. T. 3 led to the larger MPSM, indicating that the TMA functionalized polymer microspheres could interact with much more TEOS and the yield was higher. Another phenomenon should be pointed out that silica nanoparticles were easily formed due to self-nucleation of TEOS and the obtained composite microspheres were


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severely aggregated when the EDA and APTES functionalized microspheres were applied as the template. Figure 2, 3 and 4 show SEM images of porous poly(GMA-co-EGDMA) microspheres and MPSM 4-12. According to SEM images of MPSM 4-12, MPSM consisted of many silica nanoparticles and the size of nanoparticles became larger with the increasing amount of TEOS. The silica nanoparticle size distribution diagrams of MPSM 4-12 were obtained by calculating the nanoparticle size from SEM images using the size marking function of SEM (Figure S1, Supporting Information). As seen from Figure S1, whatever kind of functionalization was adopted, with increase amount of TEOS, the silica nanoparticles became larger. Compared to F. T. 1 and F. T. 2 as the template, F. T. 3 resulted in much smaller silica nanoparticles. During solgel process of MPSM 6, 8 and 9, the filtrate of the reaction mixture was a little muddy, indicating that self-nucleation of TEOS occurred. Monodisperse porous silica microspheres fabricated by Bai’s group through using TEPA functionalization polymer microspheres consisted of silica nanoparticles from 50 nm to 130 nm when the mass ratio of TEPA functionalized polymer microspheres : TEOS was 1 : 2.33, which is much larger than that of using TMA functionalized polymer microspheres.44,45 Therefore, the TMA functionalized polymer microspheres led to higher yield, smaller silica nanoparticles and no self-nucleation of TEOS. Aditya reported that different electrostatic interaction due to different surface charge played a key role in nucleation.47 Therefore, it’s perhaps because the polymer microspheres can interact much better with the negative silica network during sol-gel process due to the surface positive charge of F. T. 3. The zeta potential of each template and TEOS in the reaction system was measured to support this argument (Table S2). The zeta potential of F. T. 1, F. T. 2 and F. T. 3 was -0.016 mV, -0.348 mV and 0.321 mV, respectively. And the zeta potential of hydrolysis and condensation silica network of TEOS in the reaction system was negative (-3.35 mV). Therefore,


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due to the electrostatic attraction interaction, the TMA functionalized template interacted much better with TEOS and the silica nanoparticle size was much smaller. Table 1. Fabrication of Monodisperse Porous Silica Microspheres with Different Functionalized Template

Entry

a

a

TEOS/ F.T.

Particle Size

(g/g)

(mean ± SD) (µm)

F.T.

MPSM

1

1

3

3.47 ± 0.124

4

2

1

6

3.93 ± 0.117

5

1

10

4.01 ± 0.114

6

2

3

3.62 ± 0.136

7

2

6

4.12 ± 0.126

8

2

10

4.13 ± 0.098

9

7

3

3

3.85 ± 0.112

10

8

3

6

4.28 ± 0.118

11

9

3

10

4.38 ± 0.129

12

3

b

4 5 6

b

b

a

Reaction conditions: F.T. (0.5 g), NH3·H2O (2 ml), H2O/C2H5OH: 20 ml/100 ml, 24 h, r.t., 200 rpm. b During sol-gel process, silica nanoparticles were formed due to self-nucleation of TEOS. The composite microspheres were severely aggregated.


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Figure 2. SEM images of the parent template and MPSM 4-6.

Figure 3. SEM images of the parent template and MPSM 7-9.


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Figure 4. SEM images of the parent template and MPSM 10-12. The TMA functionalized polymer microspheres (F. T. 3) were used to explore further research due to their excellent performance. The effect of the amount of TEOS on the properties of MPSM has been further studied. The reaction conditions and the properties of the as-prepared MPSM 10-18 were shown in Table 2. The particle size of MPSM ranges from 2.33 µm to 4.49 µm with 3.24 µm of the polymer microspheres as the template. Figure 5 exhibits their pore size distribution. As shown in Figure 5, with increase of the amount of TEOS, the main pore size of MPSM firstly increased from 3.55 nm to 8.79 nm. Secondly, the main pore size transferred from 8.79 nm to 4.10 nm. When the amount of TEOS was small, the composite microspheres collapsed severely during calcination at 600 °C and the pores of 3.55 nm could be ascribed to the gap between silica nanoparticles. With increase of the amount of TEOS, the composite


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microspheres could gradually endure calcination and thus the pores became larger. However, when the amount of TEOS was sufficient, the polymer channel was mainly filled with silica nanoparticles and due to the high density of silica, the composite microspheres collapsed severely once again. Thus, the pore size was changed to 4.10 nm, which belonged to the gap between silica nanoparticles. As mentioned above, the pore size, BET surface area and pore volume of MPSM could be tuned by the amount of TEOS. Furthermore, it’s worthwhile pointing out that the pore size of porous poly(GMA-co-EGDMA) template microspheres of 3.24 µm range from 20 nm to 100 nm and the pore distribution is broad. The main pore size is about 5060 nm. MPSM also possesses this kind of pores, indicating the macropores of MPSM are ascribed to the polymer microsphere pores. With increase amount of TEOS, during the sol-gel process, the pores of porous polymer template were gradually filled with silica nanoparticles. Therefore, the macropores of the obtained MPSM gradually disappeared. When porous poly(GMA-co-EGDMA) microspheres of 7.46 µm were used as the template, the result was similar (please see the following result and discussion on Page 18). This phenomenon makes it possible that porous silica microspheres with hierarchical pores (mesopores and macropores) could be fabricated by applying different pore size of porous polymer microspheres, which shows fascinating prospect in various applications. Table 2. Effect of the Amount of TEOS on the Properties of MPSM TEOS/F.T.

Pore Volume (cm3/g)

Particle Size (mean ± SD) (µm)

MPSM

(g/g)

BET Surface Area (m2/g)

1

1.25

453

0.75

2.33 ± 0.09

13

2

2

609

0.93

2.60 ± 0.09

14

3

3

531

0.85

3.11 ± 0.09

10

Entry

a


16

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4

4

402

0.81

3.55 ± 0.10

15

5

6

283

0.56

4.03 ± 0.10

11

6

7

219

0.43

4.18 ± 0.10

16

7

8

199

0.34

4.30 ± 0.11

17

8

9

169

0.29

4.34 ± 0.11

12

9

10

167

0.28

4.49 ± 0.12

18

a

Reaction conditions: F.T. 3 (0.5g), NH3·H2O (2 ml), H2O/C2H5OH: 20 ml/100 ml, r.t., 24 h, 200 rpm. The composite microspheres were calcined at 600 °C.

Figure 5. Pore size distribution of MPSM 10-18 and 3.24 µm porous polymer template.. According to the above results, the polymer functionalization is one of the most important factors to determine the feature of MPSM. We found that without addition of NaOH during the TMA functionalization, the obtained MPSM 19 exhibited different properties. The preparation of


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MPSM 19 was shown in Experimental Section, Supporting Information. Figure 6 exhibits pore size distribution, BET surface area and pore volume of MPSM 19. The pore size of MPSM 19 at 20.51 nm is much larger than that of MPSM 10 at 5.37 nm. It’s perhaps because the strong basicity due to adding NaOH leads to the breakage of the ester groups of the polymer skeleton in some way. The influence of different particle size of the polymer microspheres on MPSM has also been studied. Monodisperse porous poly(GMA-co-EGDMA) microspheres of 7.46 µm were used as the parent template. Through the TMA functionalization, F. T. 4 was formed (Experimental Section, Supporting Information). After sol-gel reaction and calcination, MPSM with different properties was obtained (Table 3). As seen from Table 3, with increase of the amount of TEOS, the particle size of MPSM increased from 5.56 µm to 9.01 µm. And BET surface area and pore volume of MPSM mainly decreased. The pore size distribution of MPSM 20-26 has similar regularity with that of MPSM 10-18 (Figure S2, Supporting Information). The pore size could be tuned from 4 nm to 12.7 nm. The macropores derived from the polymer microspheres gradually decreased with increase amount of TEOS. Figure 7 exhibits SEM images of F. T. 4 and MPSM 20-26. All of these microspheres show well-defined spherical morphology and excellent monodispersity. The commercial porous silica microspheres of 10 µm with pore size of about 10 nm have been purchased from well-known companies such as Daisogel, Fuji and Kromasil. They exhibit much poorer monodispersity than our prepared silica microspheres (Figure S3, Supporting Information) and are very expensive. Herein, it’s important to note that the higher concentration of the polymer microspheres and TEOS will also lead to higher pore size of MPSM.


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Figure 6. Pore size distribution, BET surface area and pore volume of MPSM 19. FT-IR spectra of the microspheres are shown in Figure S4, Supporting Information. After EDA, APTES, and TMA functionalization of poly(GMA-co-EGDMA) microspheres, the band at 909 cm-1 of epoxide stretching vibration disappears and the bands at 1160 cm-1 and 1264 cm-1 are assigned to the vibration of C-N bond.38 The new band at 1480 cm-1 of the TMA functionalization polymer microspheres is ascribed to the methyl group of ammonium. As seen from the spectrum of porous silica microspheres, the bands of the polymer all disappeared indicating the removal of the polymer template through calcination. The bands at 418 cm-1, 806 cm-1 and 1065 cm-1 correspond to the bending vibration of the Si-O bond, the symmetric and asymmetric stretching vibration of the Si-O-Si bond, respectively.


19


Page 20 of 36

Table 3. Effect of the Particle Size of the Polymer Template. TEOS/F.T.

Pore Volume (cm3/g)

Particle Size (mean ± SD) (µm)

MPSM

(g/g)

BET Surface Area (m2/g)

1

2

560

0.88

5.56 ± 0.18

20

2

3

419

0.94

6.56 ± 0.19

21

3

4

360

0.84

7.20 ± 0.18

22

4

5

248

0.54

7.75 ± 0.17

23

5

6

212

0.42

7.92 ± 0.17

24

6

7

188

0.38

8.13 ± 0.19

25

7

8

183

0.39

9.01 ± 0.20

26

8

-

56

0.41

7.46 ± 0.15

Polymer Template

Entry

a

a

Reaction conditions: F. T. 4 (0.5g), NH3·H2O (2 ml), H2O/C2H5OH: 20 ml/100 ml, r.t., 24 h, 200 rpm. The composite microspheres were calcined at 600 °C.


20

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Figure 7. SEM images of MPSM 20-26 and F. T. 4. Thermogravimetric analysis (TGA) of the microspheres is exhibited in Figure S5, Supporting Information. The polymer microspheres are burned out completely at 480 °C. For the polymer/silica composite microspheres, there are three stages of weight loss, 25-200, 200-400, and 400-600 °C. The weight loss below 200 °C is assigned to the gasification of small molecules.

The

weight

loss

between

200

and

400

°C

are

attributed

to

the

decomposition/dehydration of polymer chain and further condensation of silica in the composite microspheres. When the temperature is higher than 400 °C, the gradual weight loss is ascribed to further condensation of silica. Overall, through applying the hard-templating method, monodisperse porous micrometer-sized silica microspheres with well-defined spherical morphology were fabricated and the particle size


21


Page 22 of 36

and pore size could be simply controlled by the feature of polymer template, different functionalization of the template, the amount of TEOS and other reaction conditions. It should be pointed out that during sol-gel process, the reaction temperature, type of base, the ratio of H2O/C2H5OH and concentration of the polymer template could also affect the formation of the composite microspheres and the properties of MPSM. These conditions shown in this article have already been screened and proved to be relatively optimal. The details were not given here. 3.2 Application of MPSM in HPLC for Protein Separation The hydrophilic poly(aspartic acid) coating of MPSM was carried out according to Alpert’s work[46]. As shown in Scheme 2, the acidized MPSM was firstly functionalized by APTES to possess the amine group. Secondly, APTES-modified MPSM reacted with poly(succinimide) through the amino group. Subsequent treatment with base catalyzed the hydrolysis of unreacted succinimide rings to produce the poly(aspartic acid)-coated MPSM. Scheme 2. Fabrication of the Poly(aspartic-acid)-coated MPSM.


22

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MPSM-a (7.25 µm, 30 nm), MPSM-b (7.78 µm, 14.4 nm) and MPSM-c (3.24 µm, 30 nm) were chosen to explore their behavior in HPLC for protein separation due to the difference of their particle size and pore size. For application in protein preparative separation, modification of porous silica microspheres is of vital importance to keep protein activity during chromatographic separation. As is known to all, silica could interact with protein by nonspecific irreversible adsorption due to the silanol group, which leads to some problems such as low recovery and protein denaturation. Therefore, porous silica microspheres should be coated by hydrophilic polymer to cover the residual silanol and improve their biocompatibility. Therefore, poly(aspartic acid) coating was chosen because poly(asparatic acid) was produced from D, Laspartic acid (one kind of amino acid), which is highly biocompatible. Through poly(aspartic acid) coating, the weak cationic-exchange stationary phases SP-1, SP-2 and SP-3 were obtained, respectively. After poly(aspartic acid ) coating, the pore size, BET surface area and pore volume


23


Page 24 of 36

of porous silica microspheres decreased slightly (Table S3 and Figure S6, Supporting Information). Ribonuclease A, cytochrome C, lysozyme, transferrin and hemoglobin were employed as the model analytes to evaluate the separation performance. The molecular weight, diameters of gyration and pI values of the model proteins are given in Table 4. The separation results using the as-prepared stationary phases SP-1, SP-2 and SP-3 as the weak cationic-exchange chromatographic packing materials are shown in Table 5 and Figure 8. The protein mixture was well resolved on our poly(aspartic acid)-silica columns using linear gradient similar to those employed with CM-type materials. Under our chromatographic condition, human transferrin with pI value of 5.9 didn’t combine with the poly(aspartic acid) functionalized silica spheres and flowed through directly. The retention times of the proteins were lysozyme > cytochrome C > ribonuclease A > hemoglobin > transferrin. The effect of pore size of the packing materials was illustrated by comparing the separation data of SP-1 with that of SP-2. The pore size should be large enough for the proteins to enter the inner pores and keep well-defined mass transfer. Otherwise, the restricted diffusion would lead to broader peaks and reduced column performance.48 SP-1 with pore size of 26.7 nm exhibited sharper peaks (smaller peak width at half height) and better column performance (larger separation coefficient Rs) than SP-2 with pore size of 12.7 nm. The pore size of 26.7 nm was large enough for proteins with diameter of about 3 nm such as ribonuclease A and cytochrome C to access pores without restricted diffusion. The broad peak of lysozyme was probably due to its high pI value, which resulted in strong combination with the solid phase. The broad peaks of hemoglobin could be ascribed to restricted diffusion due to its large size. The effect of particle size was demonstrated by comparing the chromatographic results of SP-1 and SP-3. The peak width at half height of SP-3 with particle


24

Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


size of 3.26 µm was a little smaller than that of SP-1 with particle size of 7.26 µm, indicating better column efficiency (smaller peak width at high height). This result was in consistent with the traditional theory that smaller particle size would lead to higher column efficiency.49 However, smaller particle size caused higher operating pressure. The operating pressure of SP-3 (3.9 MPa) was much higher than that of SP-1 (0.94 MPa) and SP-2 (0.83 MPa). Based on consideration of practical application, SP-1 would be much more suitable for preparation separation of proteins. Furthermore, the as-prepared stationary phases exhibit excellent protein recovery and reproducibility (Table 5 and Figure S7, Supporting Information). Overall, for protein separation, controlling pore size and particle size of the stationary phase is essential to ensure excellent separation efficiency and low operating pressure. Table 4. The Properties of the Model Proteins a Proteins

M. W. (kDa)

pI value

Diameter of Gyration (nm)

Ribonuclease A

13.8

7.8

2.86

Cytochrome C

14.3

10.1

3.06

Lysozyme

18.7

11.2

3.31

Transferrin

76

5.9

5.65

Hemoglobin

64.5

7.07

4.69

a

the molecular weight and diameter of gyration was obtained from PDB database and website: https://www.iitm.ac.in/bioinfo/pdbparam/computenew.html,respectively.


25


Page 26 of 36

Table 5. The Separation Parameters and Protein Recovery of the Model proteins using SP-1, 2 and 3 as Packing Materials. Packing Materials

SP-1

SP-2

SP-3

Proteins

Rs

Peak width at half height (ml)

Protein Recovery

Transferrin

-

0.25

98%

Hemoglobin

Ra-b=5.56

0.75

97%

Ribonuclease A

Rb-c=3.08

0.45

96%

Cytochrome C

Rc-d=2.70

0.425

94%

Lysozyme

Rd-e=4.27

0.625

98%

Transferrin

-

0.235

97%

Hemoglobin

Ra-b=3.2

1.5

92%

Ribonuclease A

Rb-c=1.75

0.7

96%

Cytochrome C

Rc-d=1.45

0.5

95%

Lysozyme

Rd-e=2.55

0.875

97%

Transferrin

-

0.5

98%

Hemoglobin

Ra-b=5.41

0.5

98%

Ribonuclease A

Rb-c=4.71

0.25

96%

Cytochrome C

Rc-d=2.71

0.225

95%

Lysozyme

Rd-e=5.14

0.3

98%


26

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Figure 8. HPLC separation of protein mixture using SP-1, SP-2 and SP-3 as the stationary phases. The protein mixture consists of (a)-transferrin, (b)-hemoglobin, (c)-ribonuclease A, (d)cytochrome C and (e)-lysozyme.

4. CONCLUSION In this manuscript, the hard-templating method was demonstrated to be an efficient and facile approach to fabricate monodisperse porous micrometer-sized silica microspheres with tunable particle size and pore size. Monodisperse porous polymer microspheres were employed as the novel template. The particle size and monodispersity of MPSM could be mainly tuned by the polymer size and the amount of TEOS during sol-gel reaction. And the pore size could be tuned by different functionalization of the polymer template and the reaction conditions of sol-gel process. Compared with EDA and APTES functionalization, TMA functionalized polymer microspheres led to higher yield, smaller silica nanoparticles and no self-nucleation of TEOS due


27


Page 28 of 36

to their positive surface charge. Through poly(aspartic acid) coating, the obtained poly(aspartic acid)-MPSM was employed as the weak cationic-exchange stationary phase of HPLC for protein chromatography. Different particle size and pore size of silica microspheres have been explored to study the effects of particle size and pore size. SP-1 with 7.25 µm porous silica microspheres of pore size at 30 nm coated by poly(aspartic acid) displayed superior separation performance and low operating pressure indicating that the particle size and pore size are vital to ensure favorable separation efficiency and low operating pressure. Furthermore, high protein recovery and excellent reproducility could be gained. This manuscript may provide more opportunities to investigate wider applications of porous silica microspheres and a facile strategy to fabricate more porous inorganic materials with different features.

ASSOCIATED CONTENT Supporting Information. FT-IR curves, TGA curves, experimental section, pore size, BET surface area and pore volume of the samples have been given. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] * E-mail: [email protected] Author Contributions §

Equal contributors

Jiwei, Chen and Lili, Zhu are the co-first authors. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


28

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Notes The authors declare no competing financial interest. Funding Sources Jie, He received funding from The Shenzhen Science and Technology Innovation Committee programs for Tackling Technique Key Problems (JSGG20160331173111649) and Personal Innovation (GRCK2017042414315012). Lili, Zhu received funding from The Shenzhen Science and

Technology

Innovation

Committee

program

for

Fundamental

Research

(JCYJ

20170818091708114).

ACKNOWLEDGMENT This work is financially supported by grants of the Shenzhen Science and Technology Innovation Committee research program JSGG20160331173111649, GRCK2017042414315012 and JCYJ 20170818091708114. The authors appreciate the financial support of Shenzhen Government

(JSGG20160331173111649,

GRCK2017042414315012,

and

JCYJ

20170818091708114).

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Fabrication of Monodisperse Porous Silica Microspheres with a

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