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Journal of Chromatography A, 1342 (2014) 70–77

Contents lists available at ScienceDirect

Journal of Chromatography A

jo ur nal ho me pag e: www.elsev ier .com/ locate /chroma

reparation of polyhedral oligomeric silsesquioxane-based hybridonolith by ring-opening polymerization and post-functionalization

ia thiol-ene click reaction�

hongshan Liua,b, Junjie Oua,∗, Hui Lina,b, Hongwei Wanga, Jing Donga, Hanfa Zoua,∗

Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, ChinaGraduate School of Chinese Academy of Sciences, Beijing 100049, China

r t i c l e i n f o

rticle history:eceived 6 January 2014eceived in revised form 17 March 2014ccepted 20 March 2014vailable online 28 March 2014

eywords:ybrid monolithhiol-ene reactionisulfide bonding-opening polymerizationolyhedral oligomeric silsesquioxane

a b s t r a c t

A polyhedral oligomeric silsesquioxane (POSS) hybrid monolith was simply prepared by using octagly-cidyldimethylsilyl POSS (POSS-epoxy) and cystamine dihydrochloride as monomers via ring-openingpolymerization. The effects of composition of prepolymerization solution and polycondensation tem-perature on the morphology and permeability of monolithic column were investigated in detail. Theobtained POSS hybrid monolithic column showed 3D skeleton morphology and exhibited high columnefficiency of ∼71,000 plates per meter in reversed-phase mechanism. Owing to this POSS hybrid mono-lith essentially possessing a great number of disulfide bonds, the monolith surface would expose thiolgroups after reduction with dithiothreitol (DTT), which supplied active sites to functionalize with variousalkene monomers via thiol-ene click reaction. The results indicated that the reduction with DTT could notdestroy the 3D skeleton of hybrid monolith. Both stearyl methylacrylate (SMA) and benzyl methacrylate(BMA) were selected to functionalize the hybrid monolithic columns for reversed-phase liquid chro-matography (RPLC), while [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide(MSA) was used to modify the hybrid monolithic column in hydrophilic interaction chromatography(HILIC). These modified hybrid monolithic columns could be successfully applied for separation of smallmolecules with high efficiency. It is demonstrated that thiol-ene click reaction supplies a facile way to

introduce various functional groups to the hybrid monolith possessing thiol groups. Furthermore, due togood permeability of the resulting hybrid monoliths, we also prepared long hybrid monolithic columnsin narrow-bore capillaries. The highest column efficiency reached to ∼70,000 plates using a 1-m-longcolumn of 75 �m i.d. with a peak capacity of 147 for isocratic chromatography, indicating potentialapplication in separation and analysis of complex biosamples.

© 2014 Published by Elsevier B.V.

. Introduction

Up till now, monolithic column, as called continuous bed, haschieved much progress in preparation, characterization and appli-

ation in separation of small molecules and biomacromolecules1–14]. Generally, the monolithic columns are classified intorganic polymer-based [15–17], inorganic silica-based [18] and

� Presented at the 40th International Symposium on High Performance Liquidhase Separations and Related Techniques (HPLC 2013 Hobart), Hobart, Tasmania,ustralia, 18–21 November 2013.∗ Corresponding author at: Chinese Academy of Sciences, Dalian Institute of Chem-

cal Physics, Key Laboratory of Separation Science for Analytical Chemistry, Dalian16023, China. Tel.: +86 411 84379576/+86 411 84379610; fax: +86 411 84379620.

E-mail addresses: junjieou@dicp.ac.cn (J. Ou), hanfazou@dicp.ac.cn (H. Zou).

ttp://dx.doi.org/10.1016/j.chroma.2014.03.058021-9673/© 2014 Published by Elsevier B.V.

hybrid organic–inorganic [6] monolithic columns. Polymer-basedmonolithic columns are prepared by in situ polymerization usingorganic monomers and crosslinkers in the presence of porogenicsolvents to form organic polymers, such as polymethacrylates,polyacrylamides and polystyrenes [16]. Silica-based monolithiccolumns are commonly fabricated via sol–gel technique following achemical modification on surface of matrix with silylation reagents.

Compared with the former two, the hybrid monolith maysomewhat combine the advantages of organic polymer-basedand silica-based monoliths, such as mechanical stability, lowshrinkage and controlling porous structure easily. Therefore, thehybrid monolith, mainly organic-silica monolith, has attracted

more and more attentions. Since Hayes and Malik [3] incorpo-rated organic functional moieties into inorganic silica monolithicmatrices via sol–gel chemistry, demonstrating good separationefficiency in capillary electrochromatography (CEC), various types

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Z. Liu et al. / J. Chroma

f organic-silica based monoliths have been reported by chang-ng the siloxanes with different organic moieties, such as allyl,ropyl, aminopropyl, vinyl, etc. [6,19–21]. However, the limitedypes of organic-trialkoxysilanes restricted the development ofybrid monolithic columns. To overcome this limitation, we haveeveloped the “one-pot” approach for incorporation of organicoieties into the silica monolithic matrix in our previous works

7,22]. To some extent, it turns out that the “one-pot” approach is aacile method to prepare various functionalized hybrid monolithicolumns.

In our recent works, we have introduced polyhedral oligomericilsesquioxanes (POSS) monomer, which can be regarded as themallest possible particles of silica with sizes of from 1 to 3 nmn diameter, into monolithic column matrix via free radical poly-

erization [10,23–25]. This method has some merits comparingith the sol–gel method, such as without hydrolysis, conden-

ation reactions of siloxane and good pH stability over a wideH range. Furthermore, different from free radical polymeriza-ion, we selected octaglycidyldimethylsilyl POSS (POSS-epoxy) andiamines as monomers to prepare a series of hybrid monolithsith well-controlled 3D skeletons via ring-opening polymeriza-

ion, gaining excellent chromatographic performance [11,26,27].Herein, we prepared hybrid monolithic column via ring-opening

olymerization using POSS-epoxy and cystamine dihydrochlorideCys·2HCl) as monomers. Due to the introduction of cystamine,he skeletal surface of monolithic column possesses a lot ofisulfide bonds. After simple reduction with dithiothreitol (DTT),hiol groups were generated as the active sites and subsequentlyeacted with methylacrylate monomers via thiol-ene click chem-stry, which is famous for its merits of high selectivity and highonversion under a variety of mild conditions [28–32]. The result-ng hybrid monoliths were successfully applied for capillary liquidhromatography (cLC).

. Materials and methods

.1. Chemicals and reagents

(3-Aminopropyl)triethoxysilane (APTES), POSS-epoxy,ys·2HCl, [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-mmonium hydroxide (MSA) and stearyl methylacrylate (SMA)ere purchased from Aldrich (Milwaukee, WI, USA). DTT,

etyltrimethyl ammonium bromide (CTAB), benzyl methacry-ate (BMA) and EPA610 were purchased from Sigma Chemicalo. (St Louis, Mo, USA). Dimethylphenylphosphine (DMPP) wasbtained from J&K Scientific Ltd. (Beijing, China). The fused-silicaapillaries with dimension of 50, 75 and 100 �m i.d. were obtainedrom the Refine Chromatography Ltd. (Yongnian, Hebei, China).enzene and butylbenzene were purchased from Beijing Chem-

cal Works (Beijing, China). Ammonium bicarbonate, sodiumydroxide (NaOH), ethylbenzene, dimethyl formamide (DMF),ydroquinone and other standard compounds were obtained

rom Tianjin Kemiou Chemical Reagent Co. Ltd. (Tianjin, China).PLC-grade acetonitrile (ACN) was used for mobile phase andbtained from Yuwang Group (Shandong, China). The water usedn all experiments was doubly distilled and purified by a Milli-Qystem (Millipore Inc., Milford, MA, USA). Other chemical reagentsere all of analytical grade.

.2. Preparation of POSS-based hybrid monoliths

Prior to use, the inner wall of fused-silica capillary was pre-reated and modified with a layer of amino groups for anchoring

onolith matrix according to the method described by Lin et al.11]. Briefly, the capillary was rinsed by 1.0 mol/L NaOH, water,

A 1342 (2014) 70–77 71

1.0 mol/L HCl and water, successively, which was later dried bynitrogen stream at room temperature. Then, the capillary was filledwith APTES solution in methanol (50%, v/v), sealed with rubbers atboth ends and submerged in water bath at 50 ◦C for 12 h. Finally,the capillary was rinsed with methanol to flush out the residualreagent and dried under nitrogen flow.

For preparation of POSS-based hybrid monolithic capillary col-umn, the prepolymerization mixture with different composition aslisted in Table 1 was introduced into the above-mentioned pre-treated capillaries with a syringe. After sealing both ends withrubbers, the capillary was immersed in a water bath at differenttemperature for 12 h. The obtained POSS-based hybrid mono-lith column was then flushed with methanol/H2O (60/40, v/v) toremove residuals.

For preparing of bulk hybrid monoliths, to a centrifuge tubethe prepolymerization mixture was added and reacted at 50 ◦C for12 h. Then the bulk hybrid monolith was cut into smaller piecesand extracted with methanol/H2O (60/40, v/v) in a Soxhlet appara-tus and dried in a vacuum. For the following reduction of disulfidebonds and modification, the monolith was immersed in 0.2 mol/LDTT in 0.1 mol/L aqueous ammonium bicarbonate for 2 h, and thenrinsed with methanol/H2O (60/40, v/v). The monomer solutions(SMA, BMA or MSA, the precise proportion as seen below) wereadded to the above monolith for modification. The bulk hybridmonoliths were rinsed with methanol and dried for measuring inFT-IR.

2.3. Modification of hybrid columns via reduction of disulfidebonds and thiol-ene click reaction (Fig. 1)

A 0.2 mol/L DTT in 0.1 mol/L aqueous ammonium bicarbon-ate was flushed through POSS-based hybrid monolith columnby nitrogen pressure with 4 MPa for 2 h, and then rinsedwith methanol/H2O (60/40, v/v). Then the monomer solution ofSMA/DMPP/ethanol (10/1/100, v/v/v) was flushed through hybridmonolith under nitrogen pressure for 2 h, and rinsed with methanolfor chromatographic experiments.

Similarly, the hybrid columns were also functionalizedwith BMA and MSA according to aforementioned proce-dures. The precise proportions of monomer solution wereBMA/DMPP/ethanol = 10/1/100 (v/v/v) for BMA modification andMSA/DMPP/ethanol/H2O = 20/2/400/100 (w/v/v/v) for MSA modi-fication, respectively.

2.4. Instruments and methods

The microscopic morphology of hybrid monoliths was obtainedby scanning electron microscopy (SEM) (JEOL JSM-5600, Tokyo,Japan). Fourier-transformed infrared spectroscopy (FT-IR) charac-terization was carried out on Thermo Nicolet 380 spectrometerusing KBr pellets (Nicolet, Wisconsin, USA).

The cLC experiments were performed on LC system equippedwith an Agilent 1100 micropump, a K-2501 UV detector (Knauer,Berlin, Germany) and a 7725i injector with a 20 �L sample loop. AT-union connector was used as a splitter, with one end connectedto a blank capillary (200 cm × 50 �m i.d.) and the other connectedto the monolithic column. The detection window was made byremoving the polyimide coating of fused-silica capillary tubing. Allchromatographic data were collected and evaluated using the soft-ware program HW-2000 from Qianpu Software (Shanghai, China).

For illustrating the effects of all parameters more intuitively,permeability was calculated according to Darcy’s law [33] by the

following, B0 = F�L/(�r2�P), where F (m3/s) is the flow rate ofmobile phase, � is the viscosity of water (1.0 × 10−3 Pa s), L and r(m) are effective length and inner radius of the column, �P (Pa) isthe pressure drop of column. The data of �P and F was measured

72 Z. Liu et al. / J. Chromatogr. A 1342 (2014) 70–77

Table 1Detail composition of prepolymerization mixture for preparing POSS-based hybrid monolithic columns.

Column POSS-epoxy (mg) CTAB (mg) Cys·2HCl/NaOH/H2O (�L)# Ethanol (�L) Permeability/10−14 m2

45 ◦C 50 ◦C 55 ◦C

A 50 14 130 125 13.7 3.1 2.4B 50 16 130 125 12.2 3.0 2.0C 50 18 130 125 0.001 – –D 50 16 125 125 0.15 0.07 –

2%, w

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E 50 16 135

# Cys·2HCl/NaOH/H2O: 1080/383/7200, mg/mg/�L. (Cys·2HCl, 12.47%; NaOH, 4.4

n an Eksigent one dimensional Plus Nano-HPLC system (Eksigent,ublin). The mobile phase was water, and flow rate was set at00–1500 nL/min.

. Results and discussion

.1. Preparation of POSS-based hybrid monolithic column

The preparation of POSS hybrid monolith is outlined in Fig. 1. Asxpected, the morphology and permeability of hybrid monolithsere significantly affected by composition of prepolymerization

olution and polycondensation temperature, which were inves-igated in detail. The mixture of propanol/1,4-butanediol andEG10,000 was initially selected as porogenic solvents followinghe approach in our previous works [11,26,27]. However, it wasound that the commercial reagent of Cys·2HCl could not reactith POSS-epoxy because of low reaction activity and insolubil-

ty of Cys·2HCl in this porogenic system. We have attempted usingnhydrous triethylamine and sodium hydroxide to generate freeystamine, and finally chose sodium hydroxide solution to neutral-ze hydrogen chloride in Cys·2HCl and form Cys·2HCl/NaOH/H2O

olution (Cys·2HCl/NaOH/H2O, 1080/383/7200, mg/mg/�L), andhe ring-opening polymerization between the free cystamineCys) and POSS-epoxy could be carried out in the ethanol-TAB-Cys·2HCl/NaOH/H2O solution. However, the content of

Fig. 1. Preparation of POSS-based hybrid monolith via ring-opening polyme

125 142 55 12

t%)

Cys·2HCl/NaOH/H2O solution has a great influence on the mor-phology and permeability. A little change of Cys·2HCl/NaOH/H2Ocontent could result in a transformation of monoliths from denseto loose (Fig. 2d, b, e), which was also confirmed by column perme-ability changing from 1.5 × 10−15 to 1.42 × 10−12 m2 with increasecontent of Cys·2HCl/NaOH/H2O solution at 45 ◦C. Therefore, theCys·2HCl/NaOH/H2O solution was selected at 130 �L for the fol-lowing experiments.

Owing to the hydrophobic property of POSS-epoxy, CTAB wasneeded to form a homogenous mixture. As discussed above, waterwas a poor solvent that led to earlier phase separation, super-vening with large pores in monolithic material (Fig. 2e). CTABmay form micelles and enhance the uniformity and stability ofprepolymerization mixture due to the interaction between watermolecules and hydrophilic heads of CTAB. As a result, the phaseseparation may be postponed, and the average pore diameterof monolith is decreased with an increase of CTAB content. Asshown in Table 1 (Columns A–C), increasing CTAB content from14 to 18 mg, the permeability of monolithic columns was remark-ably decreased from 1.37 × 10−13 to 1.0 × 10−17 m2, which hardlyallowed the mobile phase to pass through (Table 1, Column C).The SEM micrographs also verified that the pore size became

smaller, especially when CTAB content was up to 18 mg in Fig. 2c.What’s worse, high CTAB content would lead to monolith matrixdetaching from the inner wall of fused-silica capillary. This phe-nomenon was also observed as raising the content of ethanol in

rization and post-functionalization based on thiol-ene click reaction.

Z. Liu et al. / J. Chromatogr. A 1342 (2014) 70–77 73

F n A,

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toluene as model analyte (thiourea as the void time marker). The

ig. 2. SEM micrographs of POSS-based hybrid monolithic columns with (a) Columolumn E prepared at 50 ◦C, (g) Column E prepared at 55 ◦C. The composition of pre

repolymerization mixture (Fig. S1). Therefore, the CTAB contentf 16 mg was controlled.

Another important effect on formation of monolithic columnas polycondensation temperature. In fact, prepolymerizationixture would transform to opaque monolithic materials in 2 h

t temperatures above 45 ◦C, which implied ring-opening reac-ion basically finished. As indicated in Table 1, the permeabilityas decreased with an increase of polycondensation tempera-

ure for all prepolymerization mixtures with different ratios. Forxample, as for column A, the permeability was decreased from.37 × 10−13 to 2.4 × 10−14 m2 as polycondensation temperature

ncreasing from 45 to 50 ◦C. Additionally, they all exhibited more

niform and smaller pore size at high temperature (Fig. 2e–g).his trend may result from high temperature accelerating the ring-pening polymerization, which lead to little diffusion of monomers

ig. 3. The retention factor (k) of toluene on unmodified and SMA-modified hybridonolithic columns with different DTT reduction time.

(b) Column B, (c) Column C, (d) Column D and (e) Column E prepared at 45 ◦C, (f)erization mixture as shown in Table 1.

and phase separation in situ rapidly. Concretely, low polycon-densation temperature made the column good permeability butlow column efficiency, while too high temperature resulted in themonolith hard to flush through and lowered the reproducibility.As for Column B (Table 1) being prepared at 55 ◦C, the val-ues of reproducibility were 9.4% (column-to-column) and 12.1%(batch-to-batch), respectively. So we finally controlled the polycon-densation temperature at 50 ◦C, which also significantly improvedcolumn reproducibility.

The reproducibility of hybrid monolith was evaluated throughthe relative standard deviation (RSD) for the retention factor (k) of

run-to-run (n = 4), column-to-column (n = 4) and batch-to-batch(n = 4) were 0.17%, 0.33% and 0.50%, respectively. All results aboveindicated the good reproducibility of POSS-based hybrid monolith.

Fig. 4. FT-IR spectra of (a) unmodified monolith, (b) SMA-modified monolith and(c) MSA-modified monolith.

74 Z. Liu et al. / J. Chromatogr. A 1342 (2014) 70–77

Fig. 5. (a) and (b): separation of alkylbenzenes on the hybrid monolith by cLC. Analytes: (1) thiourea, (2) benzene, (3) toluene, (4) ethylbenzene, (5) propylbenzene and (6)butylbenzene. (c) and (d): Dependence of the plate height of analytes on the linear velocity of mobile phase by the hybrid monolith capillary column. (e) and (f): The effectof ACN content in mobile phase on retention factor of alkylbenzenes. Experimental conditions: effective length of 40.7 cm × 100 �m i.d.; off column detection, 6 cm × 50 �mi split,f

3

utbw

.d.; mobile phase, ACN/H2O (55/45, v/v, for a, b, c, d); flow rate, 160 �L/min (beforeor SMA-modified monolithic columns, respectively.)

.2. Modification and characterization of hybrid monoliths

In recent reports, Svec et al. has prepared polymeric monolith

sing glycidyl methacrylate and ethylene dimethacrylate, to whichhiol groups were introduced via post-modification and disulfideond reduction of cystamine dihydrochloride [30,34]. In presentork, POSS-based hybrid monolithic column, which intrinsically

for a, b, e, f); detection wavelength, 214 nm. (Note: a, c, e for unmodified and b, d, f

contained disulfide bonds, was facilely prepared by “one-step”method. Thiol groups were produced on surface of hybrid mono-lith after reduction with DTT [35]. Considering that the amount of

thiol groups seriously affected the following functionalization, wehave investigated the reduction time with DTT using the retentionfactor (k) of toluene as standard. It is suggested from Fig. 3 that twohours of treatment would meet the following experiments because

Z. Liu et al. / J. Chromatogr. A 1342 (2014) 70–77 75

Fig. 6. Separation in HILIC mode on the MSA-modified hybrid monolith. Analytes:(1) dimethyl formamide, (2) benzene and (3) thiourea. Experimental conditions:emw

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Fig. 8. Separation of phenols on SMA-modified monolithic column by cLC. Ana-lytes: (1) hydroquinone, (2) resorcinol, (3) pyrocatechol, (4) phenol and (5) 4-cresol.Experimental conditions: effective length of 30 cm × 100 �m i.d.; off column detec-

Fou

ffective length of 40 cm × 100 �m i.d.; off column detection, 6 cm × 50 �m i.d.;obile phase as noted in Fig. 4a; flow rate, 150 �L/min (before split); detectionavelength, 214 nm.

he retention factor of toluene did not further increase after 2 heduction with DTT.

It should be noted here that thiol-ene click reaction is gener-lly conducted under radical conditions or nucleophilic catalysis.ccording to the reported method [36], the methacrylate is loweactive towards hydrothiolation under radical-mediated due tots electron-deficient feature, but such thiol-ene click reaction cane mediated under nucleophilic catalysis using primary/secondarymines or phosphines. What’s more, catalysis-mediated thiol-ne click reaction would be accomplished faster even at roomemperature with insensitive to oxygen. So based on this point,MPP was selected to catalyze thiol-ene click reaction betweenethacrylate and thiol group [37]. The grafting reaction was

erified by FT-IR spectroscopic analysis (Fig. 4), which clearlyhowed the presence of an intense absorption band at 1734 cm−1

C O), enhanced absorption at 2870 and 2950 cm−1 ( CH2 , CH3)Fig. 4b), 1377 cm−1 (C N) (Fig. 4c) originating from the introduc-ion of SMA or MSA.

Due to the hydrophobicity of POSS-epoxy, the separation abilityf POSS hybrid monolithic column was investigated in reversed-hase mode using alkylbenzenes as probes. As shown in Fig. 5a,

ig. 7. The chromatograms of 1-m-long columns with (a) 50 and (b) 75 �m i.d. using alkylbff column detection, 6 cm × 50 �m i.d.; mobile phase, ACN/H2O (50/50, v/v); flow rate, (nmodified and (b) SMA-modified monolithic columns, respectively.

tion, 6 cm × 50 �m i.d.; mobile phase, ACN/H2O (55/45, v/v); flow rate, 150 �L/min(before split); detection wavelength, 214 nm.

five alkylbenzenes were baseline-separated with good peak shapesunder the mobile phase of ACN/H2O (55/45, v/v). To evalu-ate the column efficiency of POSS monolithic column, the plateheight–linear velocity curves were depicted in Fig. 5c. The low-est plate height 14 �m was obtained, corresponding to ∼71,000plates per meter. The influence of ACN content on the retentionfactors of alkylbenzenes is shown in Fig. 5e. The retention factorsof alkylbenzenes decreased with an increase of ACN content from45 to 75%, which suggested that the separation of these soluteson POSS hybrid monolithic column was based on typical reversed-phase mechanism. However, when the ACN content was higherthan 90%, the elution order of thiourea and benzene was reversed(Fig. S2). This result demonstrated a typical hydrophilic interactionretention mechanism, which may be attributed to the hydroxyl andamino groups on hybrid monolith surface.

The same experiments were also performed on SMA-modifiedhybrid monolithic column (Fig. 5b,d,f). It was clear that the reten-tion factors of alkylbenzenes were all increased significantly,

confirming that SMA was successfully modified. The columnefficiency was also enhanced after functionalization with SMA,especially in high linear velocity zone according to Fig. 5c and d.

enzenes as probes. The elution order is the same to Fig. 2a. Experimental conditions:a) 120 �L/min and (b) 200 �L/min before split; detection wavelength, 214 nm; (a)

76 Z. Liu et al. / J. Chromatogr. A 1342 (2014) 70–77

Fig. 9. Separations of EPA 610 on the (a) SMA- and (b) BMA-modified monolithic column by cLC with the same gradient elution. Solutes: (1) naphthalene, (2) acenaphthylene,( e, (8)b nzo(g,l e, mor

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3) fluorene, (4) acenaphthene, (5) phenanthrene, (6) anthracene, (7) fluoranthenenzo(k)fluoranthene, (13) benzo(a)pyrene, (14) dibenzo(a,h)anthracene, (15) be

ength of 33 cm × 100 �m i.d.; off column detection, 6 cm × 50 �m i.d.; mobile phasate, 150 �L/min (before split); detection wavelength, 254 nm.

or instance, when the linear velocity was at 1.0 mm/s, the columnfficiencies were increased by 58% (benzene), 73% (toluene), 86%ethylbenzene), 102% (propylbenzene) and 96% (butylbenzene),espectively. Additionally, the permeability decreased 1.6% afterMA modification. These results indicated the above modificationould not destroy the 3D skeleton of POSS-based hybrid monolith.

MSA as a zwitterionic monomer has been used for preparationr post-modification of hybrid monolithic column and success-ully utilized in hydrophilic interaction liquid chromatographyHILIC). So we also attempted to modify MSA on surface of POSS-ased hybrid monolithic column for HILIC separation. As shown

n Fig. 6, the elution order of thiourea and benzene was reversedn MSA-modified monolithic column with mobile phase of 70%CN, though thiourea was firstly eluted on unmodified POSS-basedonolithic column with ACN content from 45% to 75% (as shown

n Fig. 5e). According to peaks under mobile phase of 80% ACN inig. 6, the column efficiencies were calculated about 58,200 (DMF),7,200 (benzene) and 76,700 (thiourea). However, the retentionime of thiourea was obviously deferred with increasing ACNontent. It’s worth noting that the analyte of DMF was alwaysrstly eluted before benzene even ACN content higher than 90%.his phenomenon may result from the intrinsic hydrophobicity ofOSS-based hybrid monolith, and that the hydrophilic interactionas still weak even though the POSS-based hybrid monolith wasodified with MSA.In the LC separation and analysis of complex mixtures, there

re often two different ways to improve the resolution power ofolumns by achieving extremely high column efficiency and usingradient elution instead of isocratic conditions. However, the gen-ral practice for the former method is to increase column lengthnd/or decrease the particle sizes, which supervenes with highnlet pressure and long dead time [38–40]. Hybrid monolithic col-mn would possess the potential for high efficiency separationsy increasing its length, because hybrid monolithic column withhrough pores makes mobile phase at reasonable pressure drop9]. Besides, using smaller i.d. columns could improve sensitivity inC–MS analysis of enzymatic digests [41]. So achieving a long lengthnd narrow-bore hybrid monolithic column would meet the sep-ration of complex components to some extent. In this work, we

emonstrated a 1-m-long column of 50 �m i.d. with the highest col-mn efficiency of ∼85,000 plates and a dead time of about 32 mint 24 MPa (Fig. 7a). By adjusting the proportion of prepolymeriza-ion mixture to improve the permeability, a 1-m-long column of

pyrene, (9) benzo(a)anthracene, (10) chrysene, (11) benzo(b)fluoranthene, (12)h,i)perylene and (16) indeno(1,2,3-cd)pyrene. Experimental conditions: effectivebile phase A, water; mobile phase B, ACN; gradient, 60% B to 85% B in 25 min; flow

75 �m i.d. was also prepared, which exhibited a dead time of 9 minat 24 MPa (Fig. 7b).

Peak capacity (Pc) is the maximum number of componentsresolvable under given condition as defined by the followingequation [42]: Pc = 1 + √

N/4 ln tn/t0 for isocratic chromatography,where N is the average number of theoretical plates of all analytes,tn is the retention time of the nth component, and t0 is the reten-tion time of void time marker. As noted above, the same separationconditions (column length, mobile phase and flow rate, etc.) werecontrolled for unmodified and SMA-modified monolithic columnsin Fig. 5a and b. The peak capacities were calculated about 26 forunmodified and 73 for SMA-modified hybrid monolithic columns,respectively. The simultaneous increases of N and tn/t0 after SMAfunctionalization could account for an increase of peak capacity.Additionally, the ratio tn/t0 keeps a constant at a particular mobilephase ratio for SMA-modified monolithic column. So the change inPc is due solely to the change in N, which could be regarded as lengthdependent. A 1-m-long SMA-modified POSS-based hybrid mono-lithic column of 75 �m i.d. was evaluated using alkylbenzenes asprobes under mobile phase of ACN/H2O (50/50, v/v) (Fig. 7b). Thepeak capacity was calculated about 147.

3.3. Application of hybrid monolithic columns

For further separation in reversed phase, the SMA-modifiedmonolithic column was used to separate the mixture of phenols,exhibiting a column efficiency of 64,000–97,000 plates/m (Fig. 8).EPA 610, which consists of 16 priority pollutant PAHs, presentspotential health hazards because of their toxic mutagenic and car-cinogenic properties. Fig. 9a shows that the separation of EPA 610on SMA-modified monolithic column by cLC with gradient elution.Except that benzo(a)anthracene (analyte 9) and chrysene (analyte10) could not be separated, the others were well separated. In addi-tion, the POSS-based hybrid monolith column was also modifiedwith BMA to separate EPA 610 under the same gradient elution. Asindicated in Fig. 9b, the result was similar to that on SMA-modifiedmonolithic column.

4. Conclusions

A facile approach to prepare a POSS-based hybrid monolithwas successfully developed using POSS-epoxy and Cys·2HCl viaring-opening polymerization, in which the disulfide bonds were

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imply and directly introduced. The thiol groups were producedn the porous surface of hybrid monolith after reduction of disul-de bonds, and subsequently reacted with various methacrylateonomers via thiol-ene click chemistry. The results demonstrated

hat the thiol-ene reaction was a facile and effective approach tontroduce various methacrylate monomers to POSS-based hybrid

onolith with thiol groups. In fact, thiol group can also react withlkene, epoxy, alkynyl, isocyanate etc. via click chemistry, whichroadens the scope of modification of thiol-containing hybridacroporous materials. Furthermore, the obtained hybrid mono-

ithic column has advantages in separation of complex biosamplesue to its good permeability and high peak capacity. So we would

ike to focus attention on application of longer hybrid column innalysis of complex biosamples in our future works.

cknowledgments

Financial support is gratefully acknowledged from the Chinatate Key Basic Research Program Grant (2013CB-911203,012CB910601), the National Natural Sciences Foundation ofhina (21235006), the Creative Research Group Project of NSFC21321064), and the Knowledge Innovation program of DICP to H.ou as well as the National Natural Sciences Foundation of ChinaNo. 21175133) and the Hundred Talents Program of the Daliannstitute of Chemical Physics of Chinese Academy of Sciences to J.u.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.chroma.014.03.058.

eferences

[1] F. Svec, J.M. Frechet, Anal. Chem. 64 (1992) 820.[2] H. Minakuchi, K. Nakanishi, N. Soga, N. Ishizuka, N. Tanaka, Anal. Chem. 68

(1996) 3498.[3] J.D. Hayes, A. Malik, Anal. Chem. 72 (2000) 4090.

[[[

[

A 1342 (2014) 70–77 77

[4] F. Svec, E.C. Peters, D. Sykora, J.M.J. Fréchet, J. Chromatogr. A 887 (2000) 3.[5] F. Svec, J. Sep. Sci. 27 (2004) 1419.[6] H. Colón, X. Zhang, J.K. Murphy, J.G. Rivera, L.A. Colón, Chem. Commun. (2005)

2826.[7] M. Wu, R. Wu, F. Wang, L. Ren, J. Dong, Z. Liu, H. Zou, Anal. Chem. 81 (2009)

3529.[8] O. Núnez, K. Nakanishi, N. Tanaka, J. Chromatogr. A 1191 (2008) 231.[9] K. Miyamoto, T. Hara, H. Kobayashi, H. Morisaka, D. Tokuda, K. Horie, K. Koduki,

S. Makino, O. Nuın˜ez, C. Yang, T. Kawabe, T. Ikegami, H. Takubo, Y. Ishihama,N. Tanaka, Anal. Chem. 80 (2008) 8741.

10] M. Wu, R.a. Wu, R. Li, H. Qin, J. Dong, Z. Zhang, H. Zou, Anal. Chem. 82 (2010)5447.

11] H. Lin, J. Ou, Z. Zhang, J. Dong, H. Zou, Chem. Commun. 49 (2013) 231.12] Y. Liang, L. Zhang, Y. Zhang, Anal. Bioanal. Chem. 405 (2013) 2095.13] L. Xu, H.K. Lee, J. Chromatogr. A 1195 (2008) 78.14] I. Nischang, J. Chromatogr. A 1287 (2013) 39.15] E.C. Peters, M. Petro, F. Svec, J.M.J. Fréchet, Anal. Chem. 70 (1998) 2288.16] H. Zou, X. Huang, M. Ye, Q. Luo, J. Chromatogr. A 954 (2002) 5.17] D. Hoegger, R. Freitag, Electrophoresis 24 (2003) 2958.18] O. Núnez, T. Ikegami, W. Kajiwara, K. Miyamoto, K. Horie, N. Tanaka, J. Chro-

matogr. A 1156 (2007) 35.19] R. Roux, G. Puy, C. Demesmay, J.-L. Rocca, J. Sep. Sci. 30 (2007) 3035.20] L. Yan, Q. Zhang, J. Zhang, L. Zhang, T. Li, Y. Feng, L. Zhang, W. Zhang, Y. Zhang,

J. Chromatogr. A 1046 (2004) 255.21] Y. Tian, L. Zhang, Z. Zeng, H. Li, Electrophoresis 29 (2008) 960.22] H. Lin, J. Ou, Z. Zhang, J. Dong, M. Wu, H. Zou, Anal. Chem. 84 (2012) 2721.23] J. Ou, Z. Zhang, H. Lin, J. Dong, M. Wu, Electrophoresis 33 (2012) 1660.24] J. Ou, Z. Zhang, H. Lin, J. Dong, H. Zou, Anal. Chim. Acta 761 (2013) 209.25] Z. Liu, J. Ou, Z. Liu, J. Liu, H. Lin, F. Wang, H. Zou, J. Chromatogr. A 1317 (2013)

138.26] H. Lin, J. Ou, S. Tang, Z. Zhang, J. Dong, Z. Liu, H. Zou, J. Chromatogr. A 1301

(2013) 131.27] H. Lin, Z. Zhang, J. Dong, Z. Liu, J. Ou, H. Zou, J. Sep. Sci. 36 (2013) 2819.28] H.C. Kolb, M.G. Finn, K.B. Sharpless, Angew. Chem. Int. Ed. 113 (2001) 2056.29] A. Shen, Z. Guo, L. Yu, L. Cao, X. Liang, Chem. Commun. 47 (2011) 4550.30] Y. Lv, Z. Lin, F. Svec, Analyst 137 (2012) 4114.31] K. Wang, Y. Chen, H. Yang, Y. Li, L. Nie, S. Yao, Talanta 91 (2012) 52.32] M.-L. Chen, J. Zhang, Z. Zhang, B.-F. Yuan, Q.-W. Yu, Y.-Q. Feng, J. Chromatogr.

A 1284 (2013) 118.33] R.D. Stanelle, L.C. Sander, R.K. Marcus, J. Chromatogr. A 1100 (2005) 68.34] Y. Lv, F.M. Alejandro, J.M.J. Fréchet, F. Svec, J. Chromatogr. A 1261 (2012) 121.35] A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Anal. Chem. 68 (1996) 850.36] A.B. Lowe, Polym. Chem. 1 (2010) 17.37] J.W. Chan, B. Yu, C.E. Hoyle, A.B. Lowe, Chem. Commun. (2008) 4959.38] R.P.W. Scott, P. Kucera, J. Chromatogr. A 169 (1979) 51.

39] H.G. Menet, P.C. Gareil, R.H. Rosset, Anal. Chem. 56 (1984) 1770.40] K.E. Karlsson, M. Novotny, Anal. Chem. 60 (1988) 1662.41] Q. Luo, Y. Shen, K.K. Hixson, R. Zhao, F. Yang, R.J. Moore, H.M. Mottaz, R.D. Smith,

Anal. Chem. 77 (2005) 5028.42] E. Grushka, Anal. Chem. 42 (1970) 1142.