Silica Microspheres with Fibrous Shells: Synthesis and Application in

Subscriber access provided by UNIV OF GEORGIA

Article

Silica Microspheres with Fibrous Shells: Synthesis and Application in HPLC Qishu Qu, Yi Min, Lihua Zhang, Qin Xu, and Yadong Yin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02511 • Publication Date (Web): 30 Aug 2015 Downloaded from http://pubs.acs.org on August 31, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

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

Analytical Chemistry

Silica Microspheres with Fibrous Shells: Synthesis and Application in HPLC Qishu Qu,1,2* Yi Min,3 Lihua Zhang,3 Qin Xu4 Yadong Yin,2* 1

Key Laboratory of Functional Molecule Design and Interface Process, School of

Materials and Chemical Engineering, Anhui Jianzhu University, Hefei 230601, China 2

Department of Chemistry, University of California, Riverside, CA 92521

3

National Chromatographic R. & A. Center, Key Laboratory of Separation Science for

Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China 4

College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou

225002, China Correspondence: Dr. Qishu Qu, Key Laboratory of Functional Molecule Design and Interface Process, School of Materials and Chemical Engineering, Anhui Jianzhu University, Hefei 230601, China. Prof. Yadong Yin, Department of Chemistry, University of California, Riverside, CA 92521 E-mail: Qishu Qu, [email protected] Yadong Yin, [email protected] Tel: +86-551-63828100. KEYWORDS: Fibrous shell; silica; synthesis; HPLC.

1

ACS Paragon Plus Environment


Table of Contents Graphic

0.08

0.06

mAU

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

Page 2 of 27

0.04

0.02

0.00 0

1

2

3

t (min)

2


4

5

Page 3 of 27

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


ABSTRACT Monodispersed silica spheres with solid core and fibrous shell were successfully synthesized using a biphase reaction. Both the thickness and pore size of the fibrous shell could be finely tuned by changing the stirring rate during synthesis. When stirring was adjusted from 0 to 800 rpm, the thickness of the shell could be tuned from 13 to 67 nm and pore size from 5 to 16 nm. By continuously adjusting the stirring rate, fibrous shells with hierarchical pore structure ranged from 10 to 28 nm and thickness up to 200 nm could be obtained in one pot. We demonstrate that fibrous shells with controllable thickness and pore size could be coated on silica cores with diameters from 0.5 to 3 µm while maintaining the monodispersity of the particles. As a result of the unique fibrous structure, the BET surface area could reach ~233 m2 g-1 even though the shell thickness was less than 150 nm. The core-shell particles were modified with C18, packed, and

then used in

high-performance liquid

chromatography (HPLC) separation, showing separation performance as high as 2.25×105 plates m-1 for naphthalene and backpressure as low as 5.8 MPa. These silica microspheres with fibrous shells are expected to have great potential for practical applications in HPLC.

3



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

Page 4 of 27

INTRODUCTION Core-shell spherical silica particles with solid cores and porous surfaces, initially developed by Horváth and Kirkland in late 1960s,1,2 have been increasingly used for high separation performance with fast flow rate and relatively low back pressure. Because the diffusion through the thin porous layer is faster than that of the whole particle, under favorable conditions there could be a reduction of the column height equivalent to a theoretical plate (HETP) in the column packed with core-shell particles.3

In 2006, a new generation of core-shell silica particles with 1.7-µm solid

silica cores and 0.5-µm-thick shells named HaloTM were developed by Kirkland.4 Compared with the core-shell particles developed 40 years ago, the size of modern core-shell particles is much smaller and their size distribution is much narrower (even narrower than any other conventional fully porous particles currently on the market). Therefore, the separation performance was greatly enhanced.

Compared with the

most popular fully porous particles, the core-shell particles have advantages including small C term due to the ease of external film mass transfer and small B term due to the solid core.5

Therefore, sub-3 µm core-shell particles achieved equal or even

higher chromatographic efficiency than sub-2 µm fully porous ones, but at significantly lower operating backpressures.5-9

As a result, the development of

core-shell particles has received considerable attention in recent years, and various kinds of core-shell particles with different particle sizes were commercialized by different companies including Kinetex from Phenomenex in 2009 and Poroshell 120 from Agilent in 2010.8,10,11 4


Page 5 of 27

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


Typically, these commercially available core-shell particles have sizes of 1.3 - 5 µm and shell thicknesses of 0.15-1.0 µm,12,13 and they were made of solid silica cores with a porous shell by a layer-by-layer growth process.

More than 50 steps were

needed for layer-by-layer coating and each step needs to be controlled very carefully to avoid the aggregation of the particles.14

Core-shell particles can also be prepared

by using the sol-gel method with CTAB as a soft template, however, with thickness of the shell typically less than 75 nm and the pore size less than 3 nm.15

Similar to the

layer-by-layer process, the sol-gel method cannot ensure high monodispersity of the particles.

Thus, developing a much simplified method for the synthesis of core-shell

particles with controllable shell thickness and pore size and high surface area is highly desirable.

In 2010, Polshettiwar et al. developed a method for the preparation of fibrous silica nanospheres (KCC-1) with high surface area, large pore size, and very good mechanical stability.16 Nanometer sized Fe3O4@SiO2 particles were coated with this type of fibrous shell by Yu et al. for removing dyes from wastewater.17

However, the

coating was achieved only on nanometer sized particles and the pore size was only 3.7 nm.

Very recently, Min et al. reported the preparation of Dandelion-like core-shell

silica microspheres with hierarchical pores.18 Although it has the potential to be used as stationary phase in HPLC separation, an etching process was involved to create the porosity in the shell which significantly reduces the mechanical stability of the structure. be tuned.

Furthermore, the pore sizes were still too small (< 7 nm) and could not

In this paper, we demonstrate the synthesis of novel core-shell silica 5



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

Page 6 of 27

microspheres with a fibrous morphology, tunable shell thickness and pore size, and high specific surface area using a convenient sol-gel process.

By simply controlling

the stirring rate, the thickness of the shell can be adjusted from 10 nm to 160 nm and the pore size can be tuned from 5 nm to 28 nm as desired. easy to control, highly reproducible, and easy to scale up.

The coating process is Thanks to the open pore

channels and high surface area which are essential features for mass transfer and separation, very high separation performance could be achieved by using this type of particles as the stationary phase in HPLC separation.

EXPERIMENTAL SECTION Materials.

Hexadecyltrimethylammonium bromide (CTAB, for molecular

biology, ≥ 99%) was purchased from Sigma-Aldrich.

n-Octadecyltrichlorosilane

(95%) was purchased from Alfa Aesar. Chlorotrimetylsiane and tetraethyl orthosilicate (TEOS) were purchased from Aladdin Chemistry Co., Ltd.

All other

reagents were purchased from Sinopharm Chemical Reagent Co., Ltd (analytical reagent grade).

All chemicals were used as received without purification.

Synthesis of core-shell silica particles. the procedure reported in literature.19,20

Silica cores were prepared according to Briefly, SiO2 spheres with an average

diameter of ~580 nm were prepared by mixing 3.4 mL of TEOS with 3.3 mL of NH3⋅H2O, 9.9 mL of H2O, and 33.4 mL of ethanol under vigorous stirring.

After 1-h

reaction, one eighth of the mixture was taken from the solution and used as seeds for the subsequent growth of 1.0-µm SiO2 spheres.

In turn, one fourth of the 1.0-µm

6


Page 7 of 27

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


SiO2 sphere sample was removed and used as seeds for the growth of 2.4-µm SiO2 spheres. The obtained nonporous silica particles were washed with water three times and dried at 60 oC for 3 h. In a typical process of forming a shell on silica core, 0.5 g dried silica particles, 0.5 g of CTAB, 0.3 g (5.0 mmol) of urea were dissolved in 15 mL of H2O.

Subsequently, 15 mL of cyclohexane, 0.46 mL (6.0 mmol) of

iso-propanol, and 0.5 mL of TEOS were added to the solution. magnetically stirred at a fixed rate (0, 150, or 800 rpm).

The mixture was

After stirring for 5 min at

room temperature, the reaction mixture was heated to 70 °C for 16 h.

For coating

the silica cores with continuously adjusting stirring rate, the reaction mixture was first heated at 70 oC for 14 h at stirring rate of 150 rpm.

Then 0.5 mL of TEOS was

added into the solution and the stirring rate was increased to a higher value (300, 500, or 800 rpm).

The reaction mixture was kept at 70 oC for another 16 h.

The

products were collected by centrifugation and washed three times with ethanol to remove the residual reactants, and subsequently dried at 60 oC for 3 h.

To be used as

the stationary phase, the surfactant was further removed by calcination at 550 oC for 2 o

C min-1, and then derivatized with

h with a initial heating rate of 1

octadecyltrichlorosilane (C18, Alfa Aesar) (see Supporting Information). Characterization.

The morphologies of the core-shell silica particles were

characterized by field emission scanning electron microscopy (FESEM, Hitachi S-4800 II, 15 kV) and Philips Tecnai 12 transmission electron microscope (TEM, 120 keV).

The surface area, pore volume and pore size of the particles were determined

by nitrogen adsorption-desorption measurements using a Micromeritics ASAP 2010

7



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

instrument.

Page 8 of 27

The Brunauer-Emmett-Teller (BET) surface area was obtained by

applying the BET equation to the adsorption data.

The pore size distribution was

calculated from the adsorption branch of the sorption isotherms using the Barrett-Joyner-Halenda (BJH) method. HPLC separation with UV was performed on a 1290 infinity UHPLC system (Agilent technology, Waldbroen, Germany) with 1 µL detection cell and 20 µL sample loop.

The chromatographic column (100 mm × 2.1

mm) was packed with the home-made 2.7 µm C18-modified core-shell silica microspheres using an Alltech HPLC slurry packer.

The columns were packed using

10% (m/v) suspensions of the C18-modified core-shell silica particles in a mixture of toluene and acetone (2/3, v/v) at 40 MPa using methanol as propulsion solvent. After packing, the column was connected to a high pressure pump (Peeke Scientific SSI UHP Pump, Scientific Systems Inc.) and flushed with mobile phase by applying a pressure of ~124 MPa for 12 h. Separation of peptides and proteins. The mixture of 5 peptides, composed of R-V-Y-H-P-I (883.1 Da), R-V-Y-V-H-P-F (917.1 Da), A-P-G-D-R-I-Y-V-H-P-F (1271.4 Da), D-R-V-Y-V-H-P-F-H-L (1282.5 Da), D-R-V-Y-I-H-P-F-H-L (1296.5 Da), each with concentration of 1 mg mL-1, was separated using the following conditions: (1) H2O with 0.1% trifluoroacetic acid (TFA) as mobile phase A and acetonitrile (ACN) with 0.1% TFA as mobile phase B; (2) a linear mobile phase gradient (18-30% B) applied at 5 min at 50 oC with a flow rate of 0.3 mL min-1; and (3) an injection volume of 1 µL.

A mixture of 5 intact proteins, composed of ribonuclease A (1 mg

mL-1, RNase A, 14 kDa), cytochrome c (1.2 mg mL-1, Cyt c, 12.4 kDa), lysozyme (0.5

8


Page 9 of 27

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


mg mL-1, Lys, 14 kDa), brovine serum albumin (2 mg mL-1, BSA, 66 kDa), and ovalbumin (7.7 mg mL-1, OVA, 45 kDa), was prepared by taking 10 µL of each sample except 20 µL of Lys and 7 µL of BSA. The proteins were separated using a linear mobile phase gradient (25-65%, B) applied at 3 min with a flow rate of 0.6 mL min-1. The injection volume was 0.5 µL. to a reported protocol.21

The BSA digests were prepared according

The separation was carried out with linear gradients from 5

to 33 % B in 10 min, and a step to 80% B in 2 min. and the injection amount was 5 µL.

The flow rate was 0.6 mL min-1,

The separation temperature was 50 oC.

Column Evaluation. The overall extra-column volume of this UHPLC system, related to the injection system, connecting tube, and detection cell was measured by replacing the column with a zero dead volume connector.

The extra-column peak

dispersion ( σ ν2,ext ) was determined in µL2 as follows:

σ ν2, ext = Fν2

( t1r/ 2 − t1f/ 2 ) 2 5.545

where Fv was the flow rate (µL min-1), and ( t1r/ 2 − t1f/ 2 ) was the measured peak width at half height without the column, respectively. increased from 0.1 to 0.8 mL min-1.

The flow rate of mobile phase was

Naphthalene was used as the test analyte.

The separation performance was investigated by constructing its Knox plots and by estimating their reduced plate height minimum values (hmin). The following equations were used for the calculations: 1

h = Aν 3 +

h=

H dp

B

ν

+ Cν

(1)

(2)

9



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

ν=

ud p DM

Page 10 of 27

(3)

where H is the theoretical plate height, dp the particle size of the column packing material, u the chromatographic linear velocity of the mobile phase, DM the analyte diffusion coefficient (1.4 × 10−5 cm2/s)22 and A–C are constants accounting for band broadening. The linear velocity was experimentally determined (u =L/t0 where L is the column length and t0 is the column dead time) and column dead time was measured with uracil.

RESULTS AND DISCUSSION In this work, it was found that both the thickness and pore size of the fibrous shell were strongly affected by the stirring rate.

Figure 1 shows the TEM images of

core-shell silica particles prepared at different stirring rate.

The clearly core-shell

structure demonstrated that spherical silica cores were uniformly coated with a well-defined fibrous silica shell in all directions. the curved silica surface.

All the fibers run perpendicular to

Furthermore, the TEM images show well separated

monodispersed spherical core-shell particles which are very useful for silica-based catalysts, stationary phases, and adsorbents.

Fibrous shell could form at any stirring

rate, however, the exact shell thickness and pore size varied with the stirring rate. Figure 2 shows the typical nitrogen adsorption/desorption isotherms and pore size distribution curves at 77 K for the core-shell silica particles synthesized using different stirring rates.

As summarized in Table 1, the pore size and thickness of the

shell synthesized without stirring were the smallest.

The pore size was only 5 nm

10


Page 11 of 27

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


when the solution was kept in static, while it increased to ~10 nm at stirring rate of 150 rpm and further to ~16 nm at 800 rpm. The shell thickness obtained under stirring was overall larger than that without stirring.

However, the largest thickness

of the shell was obtained by using moderate stirring rate (150 rpm).

In the static

reaction system, the hydrolysis rate of the precursors and the diffusion rates of resultant silicates from oil phase to aqueous phase were relatively lower, resulting in a thinner shell with smaller pore size.

When stirring rate increased to 150 rpm, oil and

water phases remained well separated while the diffusion and hydrolysis rates of silicates increased significantly, promoting the growth of the fibrous mesostructure into a thicker shell with larger pore size.

When stirring rate was increased to 800

rpm, the mixture changed from a well separated biphase to a microemulsion phase, in which the diffusion and hydrolysis rates of silicates would be the highest. Interestingly, the thickness of the shell decreased almost linearly with increasing stirring rate from 150 to 800 rpm (Figure S1).

It indicates that both too fast and too

slow mass transfer are not good for the growth of the fibrous structure.

11



Figure 1. TEM images of core-shell silica particles.

The stirring rate was set at (a,b)

0, (c,d) 150 rpm, and (e,f) 800 rpm.

a

120

0 150 rpm 800 rpm

90

b

0.5

0 150 rpm 800 rpm

0.4

dV/dD (cm3 g-1 nm-1)

3

Adsorbed Pore Volume (cm g)

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

Page 12 of 27

60

30

0.3

0.2

0.1

0.0

0 0.0

0.2

0.4

0.6

0.8

0

1.0

20

40

60

80

100

120

140

Pore Diameter (nm)

Relative Pressure (p/p0)

Figure 2. Nitrogen adsorption-desorption isotherms and pore size distribution of 12


Page 13 of 27

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


core-shell particles obtained at different stirring rates.

Table 1. Structure Properties of the Calcined Core-shell Silica Particles Obtained at Different Stirring Rates shell thickness

mean size of

BET surface area

volume of

(nm)

pores (nm)

(m2 g-1)

pores (cm3 g-1)

0

13

5.0

43

0.066

150

67

10

102

0.215

300

55

11

98

0.198

500

42

14

86

0.173

800

30

16

76

0.147

stirring rate (rpm)

Shen et al. previously reported that the formation of fibrous mesoporous silica particles in a biphase reaction system took place at the interface of aqueous and oil phase.23

However, the success of forming a fibrous shell onto the surface of silica

particles of micrometer size in a static solution demonstrates that the coating reaction might take place in the aqueous phase as in this case the silica cores were settled at the bottom of the flask during reaction.

If the reaction only takes place at the

water/oil interface, deposition of fibrous shell onto the surface of silica cores is unlikely to occur.

Considering that shell structures can form in solvents with or

without stirring, the existence of two phases in solution might play the key role in the formation of shell structure.

In this case, the function of the oil phase is to control

13



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

Page 14 of 27

the release rate of the TEOS to the aqueous phase, consequently to control the assembly and growth of the silica mesostructure.

The change in thickness and pore

size with the stirring rate could be attributed to the changed released rate of the TEOS to the aqueous phase with different stirring rates. mechanism.

Here we propose a possible

Without stirring or at low stirring rates, the oil/water interface is small

so that the majority of CTAB is dissolved in water to help transport the micelles containing TEOS and its derivatives to the core surface.

In the static condition,

however, the growth is limited due to the low diffusion rate of the micelles.

Stirring

promotes the diffusion of micelles and subsequently the growth of the fibrous shells. However, a too high stirring rate results in significantly increased oil/water interfacial area which consumes more surfactant CTAB, leading to a decreased concentration of micelles that contains TEOS and its derivatives and subsequently, a relatively slower growth of fibrous shells.

When less CTAB and silicate species are available during

growth, they pack on the silica surface in a relatively lower density, leading to increased pore sizes. Since both the thickness and pore size of the shell were varied with different stirring rates, we further demonstrate the formation of hierarchically fibrous shell structures by varying the stirring rate in a still one-pot synthesis.

As shown in Figure

S2(a-c) where the core-shell structures were prepared by changing the stirring rate to 800 rpm after 14 h of initial reaction at 150 rpm, in the presence of additional precursor, the fibrous shell structure was kept while the shell thickness increased from ~67 nm to ~145 nm.

The pore size shows the bimodal distribution with two major

14


Page 15 of 27

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


peaks at ~10 and ~21 nm, suggesting the close connectivity and continuous pore channels between the first layer and the second layer of the shell. By using this method, three-dimensional fibrous core-shell particles with finely tunable shell thickness and pore size ranged from 10 to 28 nm could be achieved simply by continuously adjusting the stirring rate (Figure S2).

Since the stationary was

designed for separating both small and large molecules, the unique hierarchical pore structure of our core-shell particles would be of great value for separating complex samples with different molecular sizes.

It should be noted again that the pore size of

other commercially available core-shell particles is not easy to be tuned. Normally, a post treatment is needed to enlarge the pore size.

Therefore, the pore size of most

commercially available core-shell particles is about 10 nm which is not optimal for the separation especially for the separation of biomacromolecules.24 As the presence of two phases is the key factor to obtain fibrous morphology, we further studied the effect of cosolvents and the catalyst (in this case, urea) on the silica morphology.

As mentioned before, the role of oil phase was to control the release

rate of TEOS to the aqueous phase.

In this work, we used alkyl alcohols as the

cosolvents to control the release rate of the TEOS precursor to water.

According to

the “like dissolves like” principle, TEOS tends to dissolve in nonpolar solvents, such as cyclohexane which is used as oil phase in this work.

The addition of alkyl alcohol

is expected to change the solubility of TEOS in the oil phase.

Since alkyl alcohols

also has considerably high solubility in water than the original oil phase, the hydrolysis rate of TEOS in water would be affected and consequently change the

15



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

growth of the fibrous morphology.

More importantly, the incorporation of alcohol

can swell the surfactant aggregates and cause larger pores in the deposited silica shells.25

These predictions have been confirmed experimentally, as shown in Figure

3 which compares the SEM and TEM images of core-shell silica particles prepared with and without alkyl alcohol cosolvents. properties of these particles.

Table 2 summarizes the structure

As expected, very loose shells with significantly larger

pores were formed when n-butanol and n-pentanol were added into the reaction.

Figure 3. SEM (a-c) and TEM (d-f) images of the core-shell particles without cosolvent (a,d) or with identical molar amounts of cosolvents n-butanol (b, e) and n-pentanol (c, f).

Table 2. The Effect of Cosolvents to the Structural Characteristics of the Calcined Core-shell Silica Particles.

16


Page 16 of 27

Page 17 of 27

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


type of the

amount

shell thickness

BET surface

mean size of

volume of

cosolvent

(mmol)

(nm)

area (m2 g-1)

pores (nm)

pores (cm3 g-1)

-

0

63

83

8.6

0.171

n-butanol

6.0 (0.55 mL)

44

92

13

0.273

n-pentanol

6.0 (0.65 mL)

40

33

15

0.145

The effect of the catalyst on the growth of fibrous morphology was shown in Figure S3.

When no urea was used, a thin fibrous shell was obtained.

The BET surface

area and mean pore size were measured to be ~43 m2 g-1 and 9 nm, respectively. reason for that could be attributed to the slow hydrolysis of TEOS.

The

When the

amount of urea was increased to 30 mmol, compared to the particles synthesized under standard condition with only 5 mmol of urea, no significant change in the fibrous morphology and thickness of the shell was observed.

In this case, the BET

surface area and mean pore size were measured to be ~103 m2 g-1 and 10 nm, respectively which were nearly the same as those obtained by using 5 mmol of urea. All these results demonstrate that the key to the formation of fibrous shell structure is the two phases reaction system.

However, the thickness and pore size of the shell

could be affected by changing the amount/type of the cosolvents and catalysts as well as the stirring rate. To further study the mechanism of shell formation, core-shell silica particles were prepared by using different reaction time.

Same as that reported before,26 the

formation of the fibrous shell structure does not follow a linear growth scheme with reaction time.

From the TEM images shown in Figure S4, it can be found that shell

17



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

Page 18 of 27

structure was not formed after 10 h reaction. The BET surface area of ~4.3 m2 g-1 which can be regarded as nonporous particles confirmed that not even a very thin shell was formed on the surface of the silica core.

A thin layer of shell structure

(~43 nm) was obtained after 12 h reaction and the growth of the shell completed after 14 h reaction (~71 nm).

The BET surface areas were measured to be ~65 m2 g-1 and

~101 m2 g-1 for the reaction time of 12 and 14 h, respectively.

The results of these

experiments indicate that the formation of a fibrous shell structure might include two steps: formation of mesophase and its assembly onto silica core.

The formation of

mesophase is an important step which involves a long reaction time.

If the

mesophase forms too fast, very tight mesoporous spheres instead of fibrous mesoporous spheres would be obtained. Moon et al. also found that the fibrous structure gradually disappeared with decreasing amount of oil in the fixed amount of water,26 confirming our understanding of the critical importance of the two phases as the oil/water ratio and the interface area could determine the release rate of TEOS to the aqueous phase. As a stationary phase for chromatography separation, the silica particles should have high enough surface area and sufficiently large pores.

We synthesized

core-shell particles by starting with dense silica cores and then coated with porous silica layer under continuously adjusted stirring (200 to 600 rpm). the TEM images of thus obtained core-shell silica spheres. considerably thick shell was formed by using this method.

Figure 4 shows

It can be seen that a The shell thickness and

BET surface area were measured to be 160 nm and 137 m2 g-1, respectively.

18


The

Page 19 of 27

pore size distribution shows typical bimodal mesopores at the mean values of ~8.4 and 14 nm.

For the calculation of particle size distribution, 500 individual particles

were surveyed (Figure S5a). S5b).

The distribution is presented in histogram (Figure

The mean diameter of core-shell particles was determined as 2.70 µm

(RSD=6.63%).

Thus prepared particles were bonded with C18 and packed into

stainless steel column for the chromatographic evaluation.

The particles were

pressure-tested using a high pressure pump with a continuous pressure of 124 MPa for 12 h.

For comparison, the highest pressure can be used for other commercially

available core-shell particles is 100 MPa.24

SEM imaging of the pressure-tested

particles revealed their excellent mechanical stability and therefore appropriateness for high-pressure applications.

c

0.6

dV/dD (cm3 g-1 nm-1)

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


8.4 nm

0.4

14 nm 0.2

0.0 0

20

40

60

80

100

Pore Diameter (nm)

Figure 4. TEM images of core-shell particles with ~2.4 µm silica core and 160 nm fibrous silica shell and corresponding pore size distribution.

For separation using narrow bore column, the extra-column volume affects the column efficiency significantly.

The overall extra-column volume was 17.8 µL

measured by injecting naphthalene with a zero-dead-volume connector instead of the column at the flow rate of 0.1 mL min-1.

This volume represents about 7% of the

19



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

Page 20 of 27

holdup volume of the compared 10 cm × 2.1 mm columns.

The extra-column

variance was found to be mainly between 8 and 9 µL2 (Figure 5), indicating that the dispersion of the system was very low.27

Plot of the reduced HETP of naphthalene

versus the reduced interstitial velocity from the column packed with ~2.7 µm core-shell particles was displayed in Figure 6 (actual data points are represented by markers).

The results were fit to the Eq. 1 using a nonlinear regression analysis and

displayed as curves through the data points.

The optimal reduced plate height for

naphthalene from the fitted Knox data was 1.64.

This value is smaller than the data

obtained from the columns of 2.7 µm Halo-C18 (hmin=1.8) and Poroshell 120-C18 (hmin=2.5) core-shell particles and is only slightly larger than that of the column of 2.6 µm Kinetex core-shell particles (hmin=1.5).7

However, the small C term suggested

that the rate of mass transfer of this stationary phase was very high compared to other core-shell particles (~0.1 for 2.7 µm Halo core-shell particles) indicating that this sort of stationary phase is more suitable for the fast separation.11

The small C term could

be attributed to the pore channels perpendicular to the particle surface. shows the chromatogram of separation of test mixtures.

Figure 7

Due to the unique shell

structure, the column demonstrated good separation efficiency with a plate number of 2.25×105 m-1 (calculated from the sample of naphthalene) with a back pressure of only 5.8 MPa at a flow rate of 0.1 mL min-1.

Compared to the commercially

available columns packed with sub-2 µm fully porous silica particles (Waters Acquity BEH C18, 2.1×100 mm, 1.7 µm; Agilent Zorbax Extended-C18, 2.1×100 mm, 1.8 µm; Thermo Hypersil Gold, 2.1×100 mm, 1.9 µm) and core-shell silica particles (Halo

20


Page 21 of 27

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


C18, 2.1×100 mm, 2.7 µm; Supelco Ascentis Express C18, 2.1×100 mm, 2.7 µm), higher separation performance was obtained.28 Moreover, the back-pressure of our columns was much lower than the columns packed with sub-2 µm fully porous silica particles and other commercially available 2.7 µm core-shell particles, although it was still much larger than the monolithic columns.29

Figure S6 shows that the

back-pressure of the column was as low as 35 MPa at the flow rate of 1.0 mL min-1. For comparison, the back-pressures of the columns packed with Accucore 2.6 µm and Halo 2.7 µm core-shell particles were about 50 MPa29 and 55 MPa30, respectively, when the same separation conditions were used.

The total porosity of the column

packed with Halo 2.7 µm core-shell particles was 0.521,31 while it was measured to be 0.480 for our core-shell column.

The relative low total porosity indicates that the

low backpressure of our column could be attributed mainly to the unique fibrous structure of the shell.

The low back-pressure is beneficial for enhancing the column

performance by using long column. The column with core-shell particles was further tested for the separation of biomolecules. Figure 8 shows that the full separation of 5 peptides was observed within 4 min with a maximum back-pressure of 21.7 MPa.

The peaks were sharp

and symmetrical because of the large pore sizes of the particles.32

A selection of

proteins with sizes ranging from 12.4 to 66 kDa were also analyzed (Figure 9).

All

analytes were well separated in 7 min with a maximum back-pressure of 27.6 MPa. Sharp and symmetric peaks were obtained for most of the peaks.

Peak tailing was

only observed in the case of OVA, indicating that it was held quite strongly by the

21



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

stationary phase.

Page 22 of 27

A complex sample of a tryptic digest originating from bovine

serum albumin was also separated.

The separation performance of the column under

gradient conditions can be assessed by conditional peak capacity, which is defined as: nc =

tR , n − t R ,1 W

(4)

where tR,n is the retention time of the last eluted chromatographic band, while tR,1 is the first eluted one and W is the average 4σ peak width.

It can be seen from the

Figure 10 that most peptides were well separated with the average 4σ peak width as 0.056 min (nc=159).

As a reference, previously published data on trypsin digested

BSA peptide maps showed that the nc was only 58 when a column packed with 2.6 µm Kinetex C18 core-shell particles was used for separation.33

The high peak

capacity could be attributed to the low C term value as well as the wider pore size of our core-shell particles with unique fibrous structure.

It should be noted that the

back-pressure was only 28 MPa indicating that higher resolution separation of any other real-life samples could be achieved by using longer columns.34 These experimental results demonstrate the great potential of the fibrous core-shell particles in HPLC applications.

22


Page 23 of 27

12

2

Extra-column variance (µL )

10 8 6 4 2 0 0.0

0.2

0.4

0.6

0.8

1.0

-1

Flow rate (mL min )

Figure 5. Plot of extra-column variance versus mobile phase flow rate. Instrument: 1290 infinity HPLC system (Agilent), mobile phase: acetonitrile-water (80/20, v/v), temperature, 22 oC, injection: 0.2 µL, detection: 254 nm. 2.5 Equation

Reduced plate height, h

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


H=A*x^(1/3)+B/x+C*x

Adj. R-Square

0.9866

H

A B C

Value 1.51 0.12 0.031

Standard Erro 0.02 0.02 1E-3

2.0

1.5 0

3

6

9

12

15

18

Reduced linear velocity, ν

Figure 6. Knox plot for the column packed with 2.7 µm core-shell particles. The reduced plate height is plotted for naphthalene.

Mobile phase: acetonitrile-water

(50 : 50, v/v).

23



3

160

2

4

120

mAU

1 80

40

0 0

3

6

9

12

t (min)

Figure 7. Chromatogram of separation of mixtures.

Back pressure: 5.8 MPa.

Column: 100 × 2.1 mm I. D. packed with 2.7 µm core-shell C18 particles; column temperature: 25 oC; UV detection: λ = 254 nm; mobile phase: acetonitrile-water (50/50, v/v) at flow rate 0.1 mL min-1. Peaks: 1, uracil (6.02×104/m); 2, benzene (1.53 ×105/m); 3, toluene (1.96×105/m); 4, naphthalene (2.25×105/m). 200

0

150

mAU

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

Page 24 of 27

100

1

2

3

4

50

5

0 0

1

2

3

4

5

t (min)

Figure 8. Separation of a mixture of five peptides. Back-pressure, 21.7 MPa.

Peaks:

0, solvent; 1, R-V-Y-H-P-I (883.1 Da); 2. R-V-Y-V-H-P-F (917.1 Da); 3.

24


Page 25 of 27

A-P-G-D-R-I-Y-V-H-P-F (1271.4 Da); 4. D-R-V-Y-V-H-P-F-H-L (1282.5 Da); 5.D-R-V-Y-I-H-P-F-H-L (1296.5 Da).

100

1 2 3

80

4

mAU

60

40

0

20

5

0 0

1

2

3

4

t (min)

Figure 9. Separation of intact proteins. Back-pressure: 27.6 MPa.

Peaks: 0, solvent;

1. RNase A; 2, Cyt c; 3. Lys; 4, BSA; 5, OVA. 100

80

60

mAU

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


40

20

0 0

2

4

6

t (m in) Figure 10. Separation of BSA digests. Back-pressure: 28 MPa.

25


8

10


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

In summary, we have developed an effective biphase method for the synthesis of monodispersed fibrous core-shell silica spheres, with convenient control over the thickness and pore size of the shells, and significantly simplified process compared to that of commercial products.

Even with a thinner shell than the commercial Halo

particles which were made by layer-by-layer coating method (145 vs. 500 nm), the BET surface area of our particles was still larger than the latter one (137 vs 125 m2 g-1).10 With their unique fibrous structure, large surface area and pore size, and high uniformity in particle size, the current core-shell particles showed high performance in chromatography separation.

We also expect the fibrous core-shell mesoporous

silica particles to have potential applications in other areas such as catalysis and adsorption.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (NSFC, No. 20975090, 21275125).

Yin also thanks a gift from Agilent Technologies, Inc.

for partially supporting this research through its University Relations Program.

References (1) Kirkland, J. J. Anal. Chem. 1969, 41, 218-220. (2) Horvath, C. G.; Preiss, B. A.; Lipsky, S. R. Anal. Chem. 1967, 39, 1422-1428. (3) Knox, J. H.; Vasvari, G. J. Chromatogr. 1973, 83, 181-194. (4) DeStefano, J. J.; Langlois, T. J.; Kirkland, J. J. J. Chromatogr. Sci. 2008, 46, 254-260. (5) Guiochon, G.; Gritti, F. J. Chromatogr. A 2011, 1218, 1915-1938. (6) Carr, P. W.; Stoll, D. R.; Wang, X. Anal. Chem. 2011, 83, 1890-1900. (7) Fekete, S.; Olah, E.; Fekete, J. J. Chromatogr. A 2012, 1228, 57-71. (8) Vanderheyden, Y.; Cabooter, D.; Desmet, G.; Broeckhoven, K. J. Chromatogr. A 2013, 1312, 80-86. (9) Chester, T. L. Anal. Chem. 2013, 85, 579-589. (10) Omamogho, J. O.; Hanrahan, J. P.; Tobin, J.; Glennon, J. D. J. Chromatogr. A 2011, 1218,

26


Page 26 of 27

Page 27 of 27

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


1942-1953. (11) Olah, E.; Fekete, S.; Fekete, J.; Ganzler, K. J. Chromatogr. A 2010, 1217, 3642-3653. (12) Kirkland, J. J. Anal. Chem. 1992, 64, 1239-1245. (13) Sanchez, A. C.; Friedlander, G.; Fekete, S.; Anspach, J.; Guillarme, D.; Chitty, M.; Farkas, T. J. Chromatogr. A 2013, 1311, 90-97. (14) Kirkland, J. J.; Langlois, T. J. US. Pat. 0189944 A1 2007. (15) Yoon, S. B.; Kim, J.-Y.; Kim, J. H.; Park, Y. J.; Yoon, K. R.; Park, S.-K.; Yu, J.-S. J. Mater. Chem. 2007, 17, 1758. (16) Polshettiwar, V.; Cha, D.; Zhang, X.; Basset, J. M. Angew. Chem. Int. Ed. 2010, 49, 9652-9656. (17) Yu, K. J.; Zhang, X. B.; Tong, H. W.; Yan, X. Y.; Liu, S. M. Mater. Lett. 2013, 106, 151-154. (18) Min, Y.; Yang, K. G.; Liang, Z.; Zhang, L. H.; Zhang, Y. K. RSC Adv. 2015, 5, 26269-26272. (19) Chen, S.; Dong, P.; Yang, G.; Yang, J. J. Colloid Interface Sci. 1996, 180, 237-241. (20) Zhang, T.; Zhang, Q.; Ge, J.; Goebel, J.; Sun, M.; Yan, Y.; Liu, Y.; Chang, C.; Guo, J.; Yin, Y. J. Phys. Chem. C 2009, 113, 3168-3175. (21) Qu, Y. Y.; Xia, S. M.; Yuan, H. M.; Wu, Q.; Li, M.; Zou, L. J.; Zhang, L. H.; Liang, Z.; Zhang, Y. K. Anal. Chem. 2011, 83, 7457-7463. (22) Gritti, F.; Omamogho, J.; Guiochon, G. J. Chromatogr. A 2011, 1218, 7078-7093. (23) Shen, D. K.; Yang, J. P.; Li, X. M.; Zhou, L.; Zhang, R. Y.; Li, W.; Chen, L.; Wang, R.; Zhang, F.; Zhao, D. Y. Nano Lett. 2014, 14, 923-932. (24) González-Ruiz, V.; Olives, A. I.; Martín, M. A. TrAC-Trend. Anal. Chem. 2015, 64, 17-28. (25) Muñoz, M. a.; del Mar Graciani, M. a.; Rodrı́guez, A.; Moyá, M. a. L. J. Colloid Interface Sci. 2003, 266, 208-214. (26) Moon, D. S.; Lee, J. K. Langmuir 2012, 28, 12341-12347. (27) Fekete, S.; Fekete, J. J. Chromatogr. A 2011, 1218, 5286-5291. (28) Cunliffe, J. M.; Maloney, T. D. J. Sep. Sci. 2007, 30, 3104-3109. (29) Hayes, R.; Ahmed, A.; Edge, T.; Zhang, H. J. Chromatogr. A 2014, 1357, 36-52. (30) Ali, I.; Gaitonde, V. D.; Grahn, A. J. Chromatogr. Sci. 2010, 48, 386-394. (31) Gritti, F.; Tanaka, N.; Guiochon, G. J. Chromatogr. A 2012, 1236, 28-41. (32) Schuster, S. A.; Wagner, B. M.; Boyes, B. E.; Kirkland, J. J. J. Chromatogr. Sci. 2010, 48, 566-571. (33) Fairchild, J. N.; Walworth, M. J.; Horváth, K.; Guiochon, G. J. Chromatogr. A 2010, 1217, 4779-4783. (34) De Vos, J.; Stassen, C.; Vaast, A.; Desmet, G.; Eeltink, S. J. Chromatogr. A 2012, 1264, 57-62.

27


Silica Microspheres with Fibrous Shells: Synthesis and Application in

Recommend Documents