Synthesis of Nanospheres-on-Microsphere Silica with Tunable ShellMorphology and Mesoporosity for Improved HPLCAdham Ahmed, Peter Myers, and Haifei Zhang*

Department of Chemistry, University of Liverpool, Oxford Street, Liverpool L69 7ZD, United Kingdom

*S Supporting Information

ABSTRACT: Core−shell particles have a wide range of applications.Most of the core−shell particles are prepared in two or multiple steps.Core−shell silica microspheres, with solid core and porous shell, have beenused as novel packing materials in recent years for highly efficient liquidchromatography separation with relatively low back-pressure. These core−shell silica microspheres are usually prepared by the time-consuming layer-by-layer technique. Built on our previous report of one-pot synthesis ofcore−shell nanospheres-on-microspheres (termed as SOS particles for“spheres-on-spheres”), we describe here a two-stage synthesis for theintroduction of shell mesoporosity into SOS particles with tunable shellmorphology by co-condensation of tetraethyl orthosilicate (TEOS) with 3-mercaptopropyltrimethoxysilane (MPTMS) in the presence of surfactantin the second stage. With MPTMS as the primary precursor at the firststage, some other silica precursors (apart from TEOS) are also employedat the second stage. Expansion of the surfactant-templated mesopores with swelling agents during the reaction and byhydrothermal postsynthesis treatment is then performed to allow the pore sizes (> 6 nm) suitable for separation of smallmolecules in liquid chromatography. Compared to the standard SOS silica (both the nanospheres and microspheres containnearly no mesopores), the introduction of mesoporosity into the nanosphere shell increases the separation efficiency of smallmolecule mixtures by 4 times as judged by the height equivalent plate number, while the separation of protein mixtures is notnegatively affected.

1. INTRODUCTION

Core−shell particles, either nanoparticles or microparticles,have been extensively investigated and have wide applicationsin catalysis,1 battery,2 drug delivery,3 biomedical area,4 andchromatography.5 Core−shell particles are usually synthesizedby a two-step or multiple-step process. The core particles aresynthesized first, and the shells are then formed on the coreparticles via different methods, depending on the type of coreand shell materials and their morphologies.6 While core−shellnanoparticles are mostly investigated,1−4 core−shell micro-spheres have found unique applications as novel packingmaterials for chromatography.5 In liquid chromatography orhigh performance liquid chromatography (HPLC), silicamicrospheres are the mostly used packing materials. TheHPLC performance can be normally improved via the use ofsmall and monodispersed silica microspheres. Currently, sub-2μm silica microspheres are the state-of-the-art for HPLC.Nanospheres are rarely used for HPLC. It is not that onecannot prepare smaller microspheres or nanospheres but thatpacking the smaller spheres into a column will considerablyincrease the column back-pressure which is a huge burden forchromatography instrumentation. Halving the particle size maydouble the separation performance (in terms of theoreticalplate numbers) but can also quadruple the back-pressure (ΔP∝ 1/d2), which is highly unfavorable.7

A type of porous shell silica particles, with solid core andporous shell, has been employed as an alternative to theconventional porous silica microspheres.5 In the late 1960s,several core−shell pellicular sorbent particles were commer-cialized. But these particles had low surface areas in the range of5−15 m2 g−1, which resulted in very low loading capacities andpoor analyte retention.8,9 The new generation of core−shellparticles prepared by layer-by-layer (LBL) techniques couldoffer improved performance due to a high surface area ofaround 150 m2 g−1 and narrow particle size distribution.10,11 InHPLC, these core−shell particles are also known as fused-core,porous shell, or superficially porous particles. The widely used2.7 μm core−shell particle has a 1.7 μm core and 0.5 μm shellwith 9 nm mesopores in the shell. These particles yieldefficiency closer to that of sub-2 μm particles but with a columnpressure closer to that of 3 μm particles.12 The advantage withthe core−shell particles as packing materials is that the smallersuperficial pore volume reduces the volume present for peakbroadening from longitudinal diffusion (B term in the vanDeemter equation). The short diffusion path length can reducethe contribution of the C term due to the fast mass transfer.13

Received: July 29, 2014Revised: September 22, 2014Published: September 24, 2014

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These particles are commonly prepared by the time-consumingLBL technique due to the repeating deposition and rinsingsteps. A fast preparation method would be highly beneficial. Arecent study by Dong et al. showed the possibility of improvingthe traditional LBL assembly into a multilayer-by-multilayer(MLBML) approach, which showed a more efficient depositionof up to 6−7 layers of silica nanoparticles in each coating.14 Theresulting porous shell indicated the presence of around 9 nmpores with up to 255 m2 g−1 surface area. Even though theMLBML procedure is more efficient than the current LBLmethods, it still requires up to five coatings to achieve thedesired morphology to be comparable to current superficiallyporous particles.The sol−gel synthesis approach has also been employed to

produce core−shell silica particles. In an early report, sequentialaddition of tetraethyl orthosilicate (TEOS) and TEOS/octadecyltrimethoxysilane (OTMS) in one-pot synthesis wasemployed to produce solid-core mesoporous shell particles.The condensation of TEOS in the presence of OTMS resultedin the formation of mesoporous silica shell with an average porediameter of 3.8 nm after removal of the porogen bycalcination.15 The two-step synthesis has been more widelyused to produce such type of core−shell particles. Mono-dispersed nonporous silica spheres are commonly prepared firstby a Stober method, which are then used as the core particlessuspended in a mixture also containing Si precursors andstructure directing agents for the sol−gel synthesis of the shell.The removal of the directing agent from the shell (e.g., bywashing or calcination) produces mesoporosity.16−20 Thesetwo-step procedures may offer better control on shell thicknessand mesopore morphology in the shell. However, thesesurfactant-templated mesopore are normally in the range of<5 nm. Mesopores of such sizes are usually too small for liquidchromatography separation where minimal pore sizes around 6nm are required for low molecular weight analytes.7

Recently, a unique type of core−shell particle, nanospheres-on-microsphere or known as spheres-on-sphere (SOS) silica,has been prepared in one-pot synthesis from a single precursor3-mercaptopropyltrimethoxysilane (MPTMS).21 MPTMS wascritical for the SOS morphology because the use of other silicaprecursors in the one-pot synthesis did not produce SOSparticles. The presence of hydrophilic polymer poly(vinylalcohol) (PVA) and surfactant cetyltrimethylammonium bro-mide (CTAB) was also important for the SOS morphology.However, both PVA and CTAB did not participate in MPTMScondensation to form surfactant-templated pores. This is nothighly surprising. Although MPTMS has been used for thepreparation of organosilica microspheres and nanospheres forheavy metal removal or further oxidized to produce acidsites,22−27 MPTMS was required to co-condense with TEOS toform templated mesopores or covalently attached to preformedmesoporous silica.26−30 For the SOS particles, both thenanospheres and microspheres are nearly mesopores free.The interstitial porosity generated from the packing ofnanospheres provides surface porosity, which is the platformfor liquid phase separation. Indeed, the SOS particles-packedcolumn showed fast and efficient separation of small moleculesand large protein mixtures.21,31 Furthermore, controlled growthof metal-organic framework nanocrystals (providing intrinsiccrystalline microporosity) on SOS silica particles wasperformed. The resulting composite particles were demon-strated as a stationary phase for fast separation of xyleneisomer.32 The presence of mesopores in silica spheres is

beneficial for many applications1−5 and particularly for theseparation of small molecules in HPLC.7 Therefore, introduc-tion of mesopores into SOS particles is investigated whilemaintaining the superficial macroporosity. Because surfactant-templated silica can be formed by co-condensation of MPTMSwith TEOS,22−26 we have decided to adopt a two-stagesynthesis: (1) in the first stage, MPTMS is used to maintain theSOS morphology, and (2) in the second stage, TEOS (or someother precursors) is added so that surfactant templates can beincorporated to introduce the mesopores into the materials.CTAB present in the reactions is responsible for the surfactant-templated mesopores (2.1 nm). In order to be suitable forHPLC separation,7 the CTAB-templated mesopores are furtherexpanded with swelling agents during the reaction or viahydrothermal post-treatment. The SOS particles with shellmesoporosity show significantly improved separation of smallmolecules by HPLC while maintaining the protein separationefficiency.

2. EXPERIMENTAL SECTION2.1. Materials. 3-Mercaptopropyltrimethoxysilane (MPTMS,

95%), tetraethyl orthosilicate (TEOS, 97%), tetramethyl orthosilicate(TMOS), (3-glycidyloxypropyl)trimethoxysilane (GPTMS), 3-amino-propyltriethoxysilane (APTES), cetyltrimethylammonium bromide(CTAB, ≥98%), octadecyltrimethoxysilane (OTMS), ammoniumhydroxide solution (reagent grade, 28−30% NH3 basis), poly(vinylalcohol) (PVA, Mw 10K), N,N-dimethyldecylamine (DMDA, 98%),1,3,5-trimethylbenzene (TMB), trifluoroacetic acid (TFA), acetonitrile(ACN), and chloro(dimethyl)octylsilane (C8) were purchased fromSigma-Aldrich and used as received. All the solvents were analytical orHPLC grade.

2.2. Preparation of SOS Silica. PVA (0.25 g, 5 wt %) and CTAB(0.1 g, 2 wt %) were dissolved in distilled water (5 g). (Theconcentrations were based on the amount of water.) To this solution,methanol (8 cm3) was added while stirring. The purchased ammoniasolution (28%) was diluted with water to the concentration of 5.6 wt%. The ammonia solution (2 cm3) was added into the reactionmixture. After stirring for 15 min, MPTMS (0.5 cm3) was addeddropwise over a period of 30 s. The reaction was stirred at roomtemperature until the solution became cloudy. Then 0.5 cm3 Siprecursor (MPTMS or TEOS) was added after different reaction time(15 min, 30 min, 60 min, and 24 h, but typically 30 or 60 min, andexpressed as t30 min or t60 min) to increase MPTMS concentration orallow co-condensation of MPTMS and TEOS. The solution wasallowed to react for further 24 h. The resulting silica microsphereswere collected by centrifuging the suspensions at 3000 rpm for 2 min.This would allow the separation of microspheres from unattachednanospheres. The procedure was repeated three times to purify thesilica microspheres. These silica microspheres were calcined in afurnace with this procedure: heated at 1 °C min−1 in air to 600 °C,held for 300 min, and then cooled down to room temperature at 5 °Cmin−1.

2.3. Mesopore Expansion. The expansion of mesopores wasperformed with two swelling agents TMB and DMDA during the one-pot synthesis (method 1) or by hydrothermal post-treatment of theSOS particles still containing CTAB templates (method 2).

Method 1. During the reaction as described in section 2.2, at 60min after adding MPTMS, 0.5 cm3 TEOS was added into the reactionand was homogenized for 15 min. 0.1 cm3 DMDA or TMB wassubsequently added and stirred for further 15 min. The resultingsolution was placed in autoclave and heated at 120 °C for 24 h. Theparticles were then washed and collected by centrifugation.

Method 2. (a) DMDA as a swelling agent: dry TEOS-modified SOSparticles (0.4 g) were added in a mixture of 15 cm3 water and 0.1 cm3

DMDA. The mixture was stirred for 30 min and then heated inautoclave at 120 °C for 3 days. (b) TMB as a swelling agent: dryTEOS-modified SOS particles (0.25 g) were treated with 15 cm3 of

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water/ethanol (50/50, v/v) solution and 0.1 cm3 TMB. The mixturewas stirred for 30 min and then heated in autoclave at 120 °C for 7days.2.4. Characterization. The morphologies were observed by a

Hitachi S-4800 SEM equipped with an EDX detector. The sampleswere coated with gold using a sputter-coater (EMITECH K550X) for2 min at 25 mA before imaging. The Brunauer−Emmett−Teller(BET) surface area and pore size by N2 sorption at 77 K weremeasured using a Micromeritics ASAP 2420 adsorption analyzer. Thesamples were degassed for 10 h at 120 °C before N2 sorption analysis.The micropore size distributions were calculated by the nonlocaldensity functional theory (NLDFT) method which is typically used formicropores. Mesopore size distributions were commonly calculated bythe Barrett−Joyner−Halenda (BJH) method from the desorption data.Both methods were used to calculate the pore sizes. Thermal stabilityof the materials was investigated by thermal gravimetric analysis (TGA,Model Q5000IR TGA, TA Instruments). The sizes of the silicamicrospheres were measured using a Malvern (Malvern, Worcester-shire, UK) Mastersizer 2000 equipped with a dispersion unit. The d10/d90 data were obtained from the Mastersizer data. Zeta potentialmeasurements were performed on a Zetasizer Nanoseries (Nano-SZ)(Malvern instruments). UV−vis analysis was conducted with a UVplate reader (μQuant, Bio-Tek instruments Inc.).2.5. HPLC Test. The calcined SOS particles were used for the

normal phase HPLC test. For the reverse phase HPLC test, thecalcined SOS particles (5 g) were modified with C8 ligand(chlorodimethyloctysilane, 2.50 g) and imidazole (1.65 g) in toluene(50 cm3) at 100 °C for 24 h. The particles were then thoroughlywashed with toluene, methanol, methanol/water (1:1), methanol, anddiethyl ether in turn and dried.To pack the column, the SOS particles (0.3 g) were suspended in

15 cm3 methanol by ultrasonication for 30 min (sonication bath,Fisherbrand FB11021). The slurry was poured into a 15 cm3 reservoirand packed into a 2.1 mm (i.d.) × 50 mm (L) stainless steel columnusing the in-house packing method at 350 bar with Knauer K-1900.The column was flushed with heptane before testing.For the HPLC tests, the column was fitted into an Agilent 1200

series HPLC, comprising a vacuum degasser, quaternary pump, ALSautosampler, heated column compartment, and UV−vis detector. Allthe tests were carried out at 20 °C unless otherwise stated. Dataanalysis was performed using Agilent Chemstation software, versionB.02.01 (Agilent Technologies). The plate numbers were calculated bythe equation N = 5.54(tR/W1/2)

2, where tR is the retention time andW1/2 is the half-height peak width of the peak concerned. Theretention factor k was calculated by the equation k = (tr − t0)/t0, wheretr is the retention time of the compound in concern and t0 is theretention time of the solvent peak.

3. RESULTS AND DISCUSSION

3.1. Shell Morphology. In spite of the one-pot synthesis,the SOS particles were generated in a two-stage nucleationprocess.21 The first stage was the formation of the microspheresand lasted for a period of about 30 min. In the second stage,nanosphere nucleation occurred on the surface of themicrospheres. Controlling the number or the density ofnanospheres on the surface is important because this can varythe surface porosity and influence the subsequent applications.As observed in the previous study, pH of the reaction solutionshad a big impact on the number of nanospheres on the surface.The lower the pH (but still under the basic condition), thehigher density of nanospheres formed on the surface.21 There isanother convenient way to tune the shell morphology and thenumber of the nanospheres by simply adding more MPTMS atthe later stage of the reaction. After initial addition of MPTMSat the start of the reaction, 0.1, 0.3, or 0.5 cm3 of MPTMS wasfurther added at the reaction time of 30 min. This resulted infurther growth in the shell nanosphere coverage (Figure S1). Asmore MPTMS was added, double shell morphology started toform, which is clearly seen by SEM imaging (Figure 1A).However, the stability of the second shell is rather weak andmay detach off the surface. The double-shell morphology maybe maintained/improved by careful handling and highertemperature sintering (e.g., at 1000 °C).21

The concentration of MPTMS in solution could bemonitored by UV−vis spectroscopy during the reaction (Figure1B). MPTMS showed a UV absorbance peak around 218 nm inthe reaction solution (Figure S2). The growth was clearlyobserved by a two-step drop in MPTMS concentration,reflecting the two stages of nucleation (Figure 1B). In thesecond stage when more MPTMS added, there is an initialincrease of UV absorbance. This is because the completehydrolysis/dissolution of MPTMS in the reaction solution tooksome time. Further addition of MPTMS in solution suppliedthe reaction with more monomers to induce denser shellgrowth. The surface charges for the SOS particles remainedclose to neutral and constant throughout the reaction at around+0.05 mV (Figure 1C). This was due to the presence of CTABduring the reaction. In the absence of CTAB the particlesurface charge became negative (−35 mV), and smoothsurfaced particles were formed.

3.2. SOS Particles with Mesoporous Shell. BothMPTMS and surfactant CTAB were involved in the synthesisof SOS particles. It is known that mesoporous silica with high

Figure 1. (A) Silica microspheres with double shell morphology synthesized with further 0.5 cm3 MPTMS addition in the reaction at t30 min. (B)UV−vis monitoring of unreacted MPTMS in the reaction. (C) Zeta potential measurement of the resulting SOS particle suspensions after thereaction.

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surface areas has not been formed by the sol−gel process ofMPTMS only, while co-condensation of MTPMS with TEOS(or other alkane silanes) and modification of mesoporous silicawith MPTMS have been frequently reported.22−30 For ourcurrent method, although it was essential to include CTAB inthe reaction (no CTAB, no SOS morphology), CTAB and/orPVA were not encapsulated within the SOS as evidenced byFTIR measurement. Very low levels of mesopores weredetected by N2 sorption in the calcined SOS particles.21 In aprevious study, the mixture of MPTMS and TEOS at differentvolume ratio was employed to replace MPTMS. This mixturehad a significant effect on the SOS morphology. WhenTEOS:MPTMS was 4:1 v/v, the SOS morphology was lost.31

However, it is worth to investigate how the surface areas havechanged with the TEOS:MPTMS mixture. It was found thatthe surface areas of calcined SOS particles increased linearlywith the increased ratio of TEOS:MPTMS. At the ratio ofTEOS:MPTMS 4:1, the specific BET surface was measured tobe around 760 m2 g−1 (Figure S3). Despite this impressivesurface area, the SOS morphology was not retained.A two-stage reaction was thus proposed where MPTMS was

used at the start of the reaction to maintain the SOSmorphology, and TEOS was added at the second stage tohelp encapsulate the CTAB templates. Addition of TEOS (0.5cm3) at different reaction times was performed. The count ofreaction time started when MPTMS was initially added, and thesolution became cloudy. This was done in order to co-condenseTEOS with MPTMS. 0.5 cm3 TEOS was added at differenttimes during the reaction t0, t15 min, t30 min, t60 min, and t24 h.When TEOS was added at 0 min, aggregated and some smoothmicrospheres were formed, and SOS morphology was onlyobserved occasionally (Figure S4A). When TEOS was addedduring the reaction times of 15, 30, and 60 min, SOS particleswere formed with the surface nanospheres becoming denserwith later addition of TEOS (Figure S4B and Figure 2). Withthe addition of TEOS at 24 h, the SOS particles became moreaggregated/fused together (Figure S4C). This shows it ispossible to maintain the SOS morphology with the co-condensation of TEOS (added at 30 or 60 min) at the secondstage. The size of the TEOS-modified SOS particles increasedslightly when TEOS was added after longer reaction time; themean particle size changed from 5.4 to 6.3 μm (Figure 3).However, the size distributions were similar when TEOS wasadded after 10 min, with a polydispersity d90%/d10% of 3.9(Figure 3).The effect of adding TEOS at different reaction times on

mesoporosity was investigated by N2 sorption measurements,after calcining these particles. As shown in Figure 4A, the BETsurface area increases from 200 to about 550 m2 g−1 whenTEOS was added at the later stage. With time, more MPTMS

was consumed; more TEOS could then condense into the shellstructure with higher mesoporosity and surface area. Themesoporosity was attributed to the CTAB templating withTEOS co-condensation process. At addition time of 24 h,although the highest surface area was attained, some level ofSOS aggregation was observed (Figure S4C). A step increasewas also observed in N2 uptake at lower pressures which is afeature for CTAB-templated pores close to the boundary ofmesopores and micropores. This became more defined with theaddition of TEOS at 30 min and later (Figure S5), which wasreflected by an increase in mesopore volume (Figure 4A).The pore size distributions generated from BJH calculation

show all the pores are below 3 nm and peaked at around 2.1nm (Figure 4B). After TEOS was added at 30 min, the surfaceareas increase substantially but the micropore volumes decreaseconsiderably. This is believed to be caused by blockage of themicropores as the mesoporous shell is further formed on SOSsurface (Figure 4A). To demonstrate the SOS particles withmesoporous shell were produced (other than the whole SOSparticles were mesoporous), a control experiment was carriedout where the calcined t60 min SOS particles were sonicated inwater at two stages for a total of 16 h. The large microsphereswere precipitated from the suspension by gravimetricsedimentation on the bench for 24 h and then collected. TheSEM imaging showed the microspheres with smooth surface;i.e., the nanospheres were removed from the microspheresduring sonication (Figure S6). The nitrogen sorption analysison these collected microspheres showed a surface area of 238m2 g−1, a micropore volume 0.083 cm3 g−1, and a mesoporevolume 0.014 cm3 g−1. Compared to the sample beforesonication (Figure 4A), this represents a significant loss ofmesoporosity from 0.18 cm3 g−1 while the increased micro-porosity may be attributed to the loss of blockage due to theloss of the mesoporous shell. This shows that after TEOS co-

Figure 2. SOS particles are formed with the co-condensation of TEOS, added at the reaction time of (A) 30 min and (B) 60 min.

Figure 3. Average particles size and particle size distribution of theparticles formed when TEOS was added at different reaction times.

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condensation the mesoporosity is formed in the shellnanospheres of the SOS particles. The calcined t30 min SOSparticles were measured by Hg intrusion porosimetry. Amacropore distribution around 0.81 μm was characterized withan intrusion pore volume of 0.875 cm3 g−1 (Figure S7). Themacropores included interstitial spaces from the assembly ofsurface nanospheres and between the SOS particles. Thissuggested that macroporosity of the modified particles was alsomaintained.3.3. Effect of Templates on SOS Morphology and

Mesoporosity. Co-condensation of TEOS at different timeshad clear effect on shell morphology and porosity. At thereaction time of 30 min (t30 min), both micropores in the coremicrosphere and the increased mesoporosity in the surfacenanospheres were observed (Figure 4A). This mesoporosityresulted from the CTAB templating during TEOS co-

condensation. There was no additional CTAB added. TheCTAB was from the initially added at the start of the reaction.Further addition of CTAB was investigated to see whether

this would increase the mesoporosity. 0.1 g of CTAB was addedinto the reaction with 0.5 cm3 TEOS at t30 min. The SOSstructure was observed although the nanospheres were slightlyfused together (Figure 5A). The measured surface area reached600 m2 g−1 with 0.265 cm3 g−1 pore volume, and the pore sizepeaked at around 2.1 nm (Figure 5B). This showed that themesopores were mainly contributed from CTAB templateswithin the reaction mixture. The TGA analysis showed a 15%increase in organic content, suggesting the entrapment of moreCTAB within the structure (Figure S8).OTMS is an organosilica precursor with a long carbon chain

and was used before to synthesize silica spheres withmesoporous shell.15 In order to see the effect of OTMS astemplate, the synthesis was performed with addition of 0.1 g of

Figure 4. Porosity of the calcined SOS particles by co-condensation of TEOS and MPTMS at different TEOS addition times. (A) BET surface areaand micropore/mesopore volumes vary with the TEOS addition time. (B) Pore size distributions of the samples prepared by adding TEOS atdifferent reaction times, calculated by the BJH method from the desorption data.

Figure 5. Effect of organic templates on surface morphology and pore size distribution: (A, B) 0.1 g of CTAB + 0.5 cm3 TEOS; (C, D) 0.1 g ofOTMS + 0.5 cm3 TEOS, both added at t30 min.

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OTMS with TEOS instead of CTAB at t30 min. The reactionresulted in SOS particles with a denser nanosphere shell andsome degree of aggregation (Figure 5C). The BET surface areafor the calcined particles was 390 m2 g−1, and the pore sizedistribution peaked at 1.5 and 2.1 nm, calculated using NLDFTand BJH methods, respectively (Figure 5D). The surface areasand mesoporosity were not as high as those of the samplesprepared with CTAB. Although OTMS produced SOS particleswith mesoporous shells, a broad particle size distribution wasobserved. CTAB was thus the preferred template for thesynthesis of SOS particles with mesoporous shells.3.4. Effect of Other Silica Precursors. MPTMS was

always the precursor that was added at the start of the reactionin order to maintain the SOS morphology. However, differentsilica precursors could be used at the second stage of thereaction in order to provide addition functionality into the SOSparticles. In addition to the TEOS described above, theprecursors including TMOS, APTES (introducing −NH2group), and GPTMS (presence of epoxy group for furthereasy functionalization) were also investigated. 0.5 cm3 ofdifferent precursors was added at t30 min under the standardconditions. SOS particles with fewer nanospheres wereproduced when APTES and GPTMS were added (Figure S9)while the use of TMOS produced SOS particles with roughnanosphere shell morphology (Figure 6A). For the particlesprepared in the presence of APTES and GPTMS, infrared (IR)and microanalysis data did not show any distinctive differencebetween the original SOS particles and the co-condensed SOSparticles. This suggested APTES and GPTMS were not reactedwith MPTMS to form the SOS particles.TMOS modification showed a similar behavior as compared

to TEOS in terms of porosity but produced nanosphere shellwith rough surface morphology. The shell morphology changedwith the addition of 0.1 g of CTAB + 0.5 cm3 TMOS, which

resulted in the formation of two different sized nanospheres(about 50 and 200 nm in size) on the surface (Figure 6B). Thesmaller nanospheres were mainly on the surface of themicrospheres, and the larger nanospheres formed anotherlayer above. This was accompanied by an increase in surfacearea from 230 to 680 m2 g−1 and mesopore volume from 0.02to 0.14 cm3 g−1, while the micropore volume staying at thesame level of 0.10 cm3 g−1. This is attributed to the increasedCTAB templates, like the case in TEOS (Figure 5B). The poresize distributions showed the population of mesopores at 0.9and 2.1 nm in diameter, calculated using the NLDFT and BJHmethods, again consistent with the SOS particles prepared fromTEOS (Figure 5C,D). In both cases, broad particle sizedistributions were observed as well. It can therefore besummarized that the choice of silica precursor is critical forthe condensation reaction with MPTMS and the formation ofmesoporous shell SOS particles. TEOS and TMOS are theprecursors that can produce SOS silica particles withmesoporous shells.

3.5. Expansion of CTAB-Templated Mesopores. TheSOS particles were recently employed for highly efficientseparation of proteins. The separation was attributed to theSOS morphology and the superficial shell porosity generatedfrom the interstitial space between the nanospheres.21 Thedistinct feature of the newly prepared SOS particles withmesoporous shells in this study is that both macroporosity andmesoporosity are present within the particles. However, theCTAB-templated mesopores are around 2.1 nm in diameter.This is very small in terms of chromatographic separation, andlarger mesopores (>6 nm) are required for separation of evensmall molecules.7

Surfactant-templated mesopores can be expanded byintroducing a swelling agent into the structure-directingtemplate, either in the sol−gel preparation step or by the

Figure 6. SOS particles prepared by (A) replacing TEOS with 0.5 cm3 TMOS and (B) 0.1 g of CTAB + 0.5 cm3 TMOS. Pore size distributions by(C) BJH and (D) NLDFT calculations.

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hydrothermal post-treatment. Both approaches have beenadopted in this study. For the incorporation during thesynthesis, the swelling agent could get into the surfactantmicelles and enlarge the size of the micelles.33 For thehydrothermal post-treatment, the silica/surfactant compositespheres were treated with the swelling agents in water or inwater/ethanol at elevated temperatures.34,35 The flexible silicastructure could allow the incorporation of the swelling agentinto the surfactant templates. Generally, hydrophobic agentscan be used as the swelling agents to expand the pores. Thesecan include aromatic hydrocarbons, long-chain alkanes, alkyl-amines, and auxiliary alkyl surfactants.33 Both TMB34 andDMDA35 have been widely used. Particularly, the use of TMBcould significantly enhance the surfactant-templated meso-pores.36,37 TMB and DMDA were therefore chosen as theswelling agents in this study.For the use of swelling agent during the reaction, TEOS and

one swelling agent were added in sequential at the reactiontime of 60 min. The whole reaction was then subjected tohydrothermal treatment. TEOS only was added as well with theresulting particles as a control sample. SOS particles wereproduced under all the conditions with the samples of TEOSand TEOS + TMB showing similar SOS morphologies (denselycoated nanospheres around 200 nm, Figure S10A−C). Thenanosphere growth was strongly affected when DMDA wasutilizedlarger nanospheres and some smooth microsphereswere formed (Figure S10B). After calcination at 600 °C, the N2isotherm showed the formation of small hysteresis loop for thesample with TEOS only added, which is reflected by themesopores around 2.8 nm with BET surface area 370 m2 g−1

and a total pore volume of 0.12 cm3 g−1 (micropore volume0.061 cm3 g−1 and mesopore volume 0.062 cm3 g−1) (Figure7A,B). For the samples produced by the addition of DMDA or

TMB with TEOS, increased hysteresis loops are observed,indicating the formation of larger mesopore (Figure 7A).Indeed, the peak pore sizes are enlarged from 2.8 to 3.9 and 4.2nm by using DMDA and TMB as swelling agents, respectively(Figure 7B). With the expansion of the pores, both the surfaceareas and the pore volumes are increased (540 m2 g−1 and 0.53cm3 g−1 for DMDA; 417 m2 g−1 and 0.64 cm3 g−1 for TMB)compared to the TEOS-only sample.For the post-treatment approach, the as-prepared CTAB-

templated t60 min SOS particles were treated with DMDA orTMB under the hydrothermal conditions. After treatment, theSOS morphology remained intact and the nanospheres werestill attached to the microsphere surface (Figure S10D). Figure7C shows the N2 sorption isothermal profiles for the treatedsamples after calcination. An increasing size of hysteresis loopcan be seen for SOS particles post-treated with DMDA andTMB. The pore size distributions in Figure 7D show that thepores are expanded from 2.1 to 4.3 and 6.9 nm by using DMDAand TMB as swelling agents, respectively. With the expansionof the pores, the pore volumes were increased accordingly (0.21cm3 g−1 of which 0.03 cm3 g−1 for micropore volume for theuntreated sample, and the pore volumes of 0.37 cm3 g−1 forDMDA and 0.44 cm3 g−1 for TMB). The BET surface areaswere decreased from 476 to 385 m2 g−1 for DMDA and to 337m2 g−1 for TMB; a similar trend was observed before.37 Themicropore volumes were kept at the same level for the treatedsamples.

3.6. HPLC Evaluation of SOS Particles with Meso-porous Shells. After calcination at 600 °C, the standard SOSparticles gave a surface area of around 245 m2 g−1, which wasresulted from the micropores (about 1.4 nm) formed due tothe loss of organic component during calcination. Thesuperficial macroporosity from the interstitial space of the

Figure 7. N2 sorption results for the calcined pore-expanded CTAB-templated SOS particles by (A, B) one-pot modification with swelling agentsTMB and DMDA and (C, D) two-step post-treatment both under hydrothermal conditions with TMB and DMDA. (A, C) Sorption isothermprofiles and (B, D) BJH pore size distributions.

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surface nanospheres made this material efficient for largemolecule protein separation. The lack of mesoporosity couldlimit separation efficiency for small molecules.21 Under thenormal phase conditions, the optimal column efficiencyachieved was 16 270 plates/m based on p-nitroaniline.21 Forthe SOS particle with expanded mesopores, it is expected thatthe column efficiency for separation of small molecules wouldbe improved. Here, the calcined TMB post-treated SOS particlewith 6.9 nm mesopores were assessed in a normal phase HPLCfor separation of toluene, nitrobenzene, and nitroanilineisomers. Figure 8A shows five well-resolved peaks within 6min, with a back-pressure of 39 bar. The column efficiency wascalculated to be 68 000 plates/m based on p-nitroaniline,calculated by retention time and half peak width. Thisrepresents an increased efficiency of about 4 times. The resultsshowed that this modified SOS column (retention factor k =1.56 for the first two peaks) had a higher retention time thanthe SOS column (k = 0.50) due to the increase in surface area.Peak broadening is expected for totally porous microsphereswith 6.9 nm mesopores. However, for the SOS particles withmesoporous shells, the eluents only moved through a shorterdistance of the thickness of the shell (the size of nanospheresfor the SOS particles, ca. 200 nm), which minimizes bandbroadening effect. A van Deemter plot was obtained for thenitroaniline isomers (Figure S11). The plate height of the p-nitroaniline was only modestly increased with increasing linearvelocity. This suggests that the separation may be made fasterby increasing flow rates without significant loss of columnefficiency. Another normal phase test mixture containingtoluene, 2,4-di-tert-butylphenol, o-nitroaniline, and cinnamylalcohol (TM1 test mixture, from Thermal Fisher Scientific) wasalso evaluated. Baseline separation with symmetrical peakshapes was achieved using a mobile phase of isopropanol:hep-tane (85:15 v/v) within 5 min (Figure S12). The theoreticalplate number based on cinnamyl alcohol was calculated to be59 000 plates/m.With the unique core−shell property and the superficial

macroporosity, the packed mesoporous SOS particles can alsobe good stationary phase for separation of large proteins. Themesoporosity within the shell structure would provide verylimiting access to proteins; hence, the separation should mainlyoccur in the shell interstitial space. The silica was modified withC8 ligands and assessed under the reversed phase mode forseparation of a mixture of proteins including ribonuclease A,

cytochrome c, lysozyme, trypsin, and bovine serum albumin(BSA). The mobile phase was a mixture of 0.1% v/v TFA inacetonitrile (ACN) and water. A three-step gradient profile wasused in order to achieve the separation of these proteins in justover 2.5 min (Figure S13)a similar performance as thenonmesoporous SOS particles.21 However, the presence ofmesopores did increase the back-pressure (recorded as 251 barat the end of the gradient). The column efficiency wascomparable to the standard SOS particles as the peak capacitywas calculated to be 17.4 (SOS peak capacity = 19.1).21 A morecomplex mixture containing seven proteins including ribonu-clease A, insulin, cytochrome c, lysozyme, myoglobin, BSA, andcarbonic anhydrase was further examined. The separation of theproteins was performed within 3 min with a back-pressure of355 bar (Figure 8B), which is the average pressure for astandard narrow bore silica column. Thus, the HPLC testresults demonstrate the capability of the SOS particles withshell mesoporosity for improved separation of small moleculesand large molecules such as proteins with no loss inperformance.

4. CONCLUSIONOne-pot two-stage synthesis has been developed to prepareSOS silica particles with tunable surface morphology andimportantly shell mesoporosity. MPTMS was the precursoradded at the start of the reaction to generate the SOSmorphology. Among the precursors added in the second stage,TEOS and TMOS could produce the desired SOS particles.Amount of precursors, addition time, and concentration ofsurfactant CTAB in the reactions can be varied to tune surfacemorphology and produce SOS particles with high surface areaand large mesoporosity in the nanosphere shells. Themesopores resulted from CTAB templates via the co-condensation reactions of MPTMS and TEOS/TMOS in thesecond stage. The CTAB-templated mesopores could beenlarged by swelling agents DMDA and TMB via one-potmodification process during the synthesis or hydrothermalpost-treatment of the SOS particles containing CTABtemplates. The mesopores could be expanded from 2.1 up to6.9 nm with TMB post-treatment, the pore sizes suitable forliquid chromatographic separation of small molecules. Thecalcined SOS particles with shell mesopores of 6.9 nm wereevaluated for HPLC separation of small molecules undernormal phase, which showed a higher efficiency up to 68 000

Figure 8. Chromatograms obtained from the calcined mesoporous SOS column (2.1 mm i.d. × 50 mm L) with 5 μL injection and UV detection at254 nm at 25 °C. (A) Analysis of the test mixture under the normal phase condition. Mobile phase: heptane:isopropanol (95:5 v/v). Flow rate: 0.5cm3 min−1. (B) Separation of seven proteins with C8-bonded SOS particles under the reversed phase condition. Mobile phase: the mixture of ACNsolution (containing 0.1% TFA) in water (also containing 0.1% TFA) with a gradient change: 0 min: 1% v/v ACN, 0.5 min: 20% v/v ACN, 4 min:70% v/v ACN. Flow rate: 3 cm3 min−1.

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plates/m (∼4 times higher) based on p-nitroaniline comparedto the standard nonmesoporous SOS particles. After bondingwith C8 ligands, the particles were further assessed for proteinseparation without any loss in resolution and peak capacity.This study demonstrated that SOS particles with mesoporousshell could be used for highly efficient HPLC separation formixtures of both small molecules and biomacromolecules.

■ ASSOCIATED CONTENT

*S Supporting InformationMore SEM images, N2 sorption data, UV−vis data, TGA data,and HPLC graphs. This material is available free of charge viathe Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Tel +44 151 7943545; Fax +44 151 7943588; e-mailzhanghf@liv.ac.uk (H.Z.).

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The authors are grateful for the access to the facilities in theCentre for Materials Discovery at the University of Liverpool.

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