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Polymer 53 (2012) 4128e4134

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Polymer

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Macroporous polymer monoliths with a well-defined three dimensional skeletalmorphology derived from a novel phase separator for HPLC

Ying Shen a,b, Li Qi a,*, Lanqun Mao a

aBeijing National Laboratory of Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences,No. 2 Zhongguancun Beiyijie, Beijing 100190, ChinabGraduate School, Chinese Academy of Sciences, 19A Yuquanlu, Beijing 100049, China

a r t i c l e i n f o

Article history:Received 6 April 2012Received in revised form3 July 2012Accepted 8 July 2012Available online 25 July 2012

Keywords:Poly (glycidyl methacrylate-co-ethylenedimethacrylate) monolithsWell-defined structureA novel phase separator

* Corresponding author. Tel.: þ86 10 82627290; faxE-mail address: qili@iccas.ac.cn (L. Qi).

0032-3861/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.polymer.2012.07.019

a b s t r a c t

Poly (glycidyl methacrylate) (PGMA) was successfully synthesized and proposed as a novel phaseseparator for fabrication of macroporous crosslinked poly (glycidyl methacrylate-co-ethylene dimetha-crylate) (poly (GMA-co-EDMA)) monoliths with well-defined three dimensional (3D) skeletal structure.The ratio of PGMA in porogenic system had significant influence on morphology development of themonolith. To gain insight into the effect of PGMA chain length on morphology formation, PGMAhomopolymers with different molecular weights were further synthesized for monolith preparation.Porous structure of the monoliths was characterized by scanning electron microscope, mercury intrusionporosimetry and gas absorption measurement. Additionally, the monoliths could maintain the well-defined 3D skeletal structure with a tunable amount of GMA monomer in polymerization system forimproved permeability and functionality while the content of other components is fixed. Meanwhile, thesuccessful application of the optimal monolith for separation of both small molecules and lagerbiomolecules was provided proof of its potential utilization in HPLC. Considering different requirements,the application of the novel well-defined 3D skeletal monolith could be easily widened via post-modification because of its possessing epoxy functionality. Hopefully, the newly synthesized PGMAwith different molecular weights could be developed as a novel candidate polymeric phase separator forfabricating various monoliths with well-defined structure, comparable to commercially available ones.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The development of macroporous monoliths with well-definedpores has been an ever-growing field over the last decade, moti-vated by their diverse applications, e.g., biological separation [1,2],catalysis [3,4] and adsorbents [5,6] due to their high permeability,fast mass transfer, high stability and ease of chemical modification[7]. Depending on the nature of materials, monoliths could bedivided into organic polymer monoliths and inorganic silicamonoliths. However, polymer based monoliths have importantadvantages over silica based monoliths in terms of pH stability,inertness of biomolecules and absence of deleterious effect fromsilanol [8], resulting in their particular appeal and preferableapplication. Generally, these polymer monoliths prepared by in situfree radical polymerization of monomer(s) with crosslinker inporogenic solvents [9,10] were featured with microglobulesaggregated structure, which inevitably contributed to their low

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permeability and limited molecular interaction sites [11]. Asa result, constructing novel polymer monoliths with well-definedthree-dimensional (3D) skeletal structure represents an intriguingavenue for exploring better total performance in their applications.

The last few years have witnessed increasing research efforts inthis avenue. Generally, the macroporous polymer monoliths couldbe obtained by polymerization induced phase separation (PIPS)[12,13], thermally induced phase separation (TIPS) [14] and non-solvent induced phase separation (NIPS). However, owing to exactdemands for monomers in the last two modes, PIPS has beenconsidered as the most excellent and promising candidate for thepreparation of polymer monolith up to now. As a result, themajority strategies for constructing monoliths with well-defined3D skeletal structure are based on this mode and could be grou-ped into two sorts. In the first approach, the alternation of poly-merization methods, such as polycondensation [15], high internalphase emulsion (HIPE) [16,17] and living radical polymerization(LRP) [18e22], has been proved to be the effective tool to achievethis goal. As an example, well-controlled 3D skeletal epoxy resin-based polymer monolith was prepared by using polycondensationmethod in Tsujioka’s study [15]. A novel glycidyl methacrylate-

Table 1Preparation and characterization of PGMA homopolymers.a

Type CuBr (mg) Bpy (mg) EBP (mL) DP PDI Averagemolecular weight

P1 54.0 176 48.9 80 1.3 1.1 � 104

P2 15.4 50.4 14.0 300 2.0 4.3 � 104

P3 3.86 12.6 3.49 1200 2.0 1.7 � 105

P4 0.772 2.52 0.699 5000 2.1 7.1 � 105

a The amounts of other compunds are fixed as follows: GMA 5.0 mL, EBP:-CuBr:bpy molar ratio is 1:1:3.

Y. Shen et al. / Polymer 53 (2012) 4128e4134 4129

based monolith with sub-micron skeletons directed by the self-assembly of pluronic F127 has been proposed on the foundationof HIPE, as previously reported by us [17]. Nakanishi and coworkershave constructed several kinds of well-defined macroporousmonoliths by LRP in company with polymeric phase separator[18e21]. Also, our group has made great effort to fabricate poly(ethylene dimethacrylate) monoliths with 3D biocontinuous skel-eton by atom transfer radical polymerization (ATRP) method [22],which was conceived upon the principle that the nucleation andthe phase separation could be well controlled for forming uniformstructures in LRP method [23]. However, the adjustment of poly-merization methods is either restricted by monomer species oroperating condition, which would influence its applied range. Inthe second approach, employment of various phase separators hasemerged as a facile and general solution allowing for constructingdesirable 3D skeletal monoliths without any change in reactioncondition and chemical composition of the final polymer [23].However, conventional polymer monoliths were mostly preparedby using porogen consisting of small molecule solvents andinclined to shape monoliths with globular aggregated porousstructure. As a result, exploring new kinds of porogen has attractedconsiderable attention. Recently, the polymeric phase separatorappears to be a promising one [24e28]. Aoki and colleagues havereported the synthesis of glycerol dimethacrylate-based polymermonoliths with 3D continuous skeletal structure by using mono-disperse ultra high molecular weight poly (styrene) (PS) as a poro-gen on the basis of viscoelastic phase separation [24]. Tennikovaand co-workers have employed poly (dimethylsiloxane) (PDMS) asthe macromolecular porogen to construct macroporous poly-methacrylate monoliths for preparation of hydrophilic proteinmicroarrays [25,26]. Kubo and colleagues have prepared a novelhydrophilic monolithwith through-pores by adopting polyethyleneglycol (PEG) as a porogen [27]. Although all these polymeric phaseseparators including PS, PDMS and PEG could be obtained fromcommercial sources, most of themwere used to fabricate extremelyhydrophilic or hydrophobic polymer monolith with limitedmonomer category. More importantly, resulting from the lack offunctionalities, post-modification of these monoliths was difficult,leading to their finite applications. As a result, exploration of novelpolymeric phase separators is an important and arduous task todevelop monoliths with 3D skeletal structure for incorporatingmore polymerization system and widening their applications.

In this study, a novel polymeric phase separator, poly (glycidylmethacrylate) (PGMA), has been firstly used to construct well-defined 3D skeletal poly (glycidyl methacrylate-co-ethylenedimethacrylate) (poly (GMA-co-EDMA)) monoliths, which aregenerally regarded as one kind of the most extensively usedpolymer-based monolithic mediums due to its possession of reac-tive epoxy functionality for post-modification. Furthermore, theinfluence of PGMA molecular weight on morphology developmentof monoliths has been investigated. Meanwhile, to improve thepermeability and functionality for their preferable applications,monoliths prepared with different amounts of GMAmonomer havealso been studied. Finally, the optimal monolith with well-defined3D skeletal structure has been hydrolyzed and utilized to separatesmall molecules and lager biomolecules in high performance liquidchromatography (HPLC), which demonstrated its potential usage asthe new generation matrix in HPLC.

2. Experimental

2.1. Materials

Ethylene dimethacrylate (EDMA) and Glycidyl methacrylate(GMA) were purchased from Acros company (New Jersey, USA) and

purified before use. Azobisisobutyronitrile (AIBN) was produced byShanghai Chemical Plant (Shanghai, China) and refined by recrys-tallization from methanol before use. Cuprous bromide (CuBr)obtained from Beijing Chemical Plant (Beijing, China) was firstlywashed by acetic acid and subsequently washed by methanolbefore use. Ethyl-2-bromopropionate (EBP) was from Alfa Aesar(Lanes, England). 2,20-bipyridyl (bpy), tetrahydrofuran (THF),methanol, cyclohexanone, dimethylformamide (DMF) and benzenederivatives were all purchased from Beijing Chemical Plant (Beijing,China) and used as received. Medroxyprogesterone acetate andprednisone acetate were got ten from Zhejiang Xianju Pharma-ceutical Ltd. (Zhejiang, China). Hydrocortisone was gotten fromTianjing Jinyao amino acid Ltd. (Tianjing, China). Cytochrome C (CytC), lysozyme (Lys) and bovine serum albumin (BSA) were obtainedfrom Xinjingke Biotechnology Co., Ltd. (Beijing, China).

2.2. Synthesis of PGMA

A typical procedure to prepare the PGMA by ATRP method isdescribed as follows: CuBr and bpy were added into the pre-diredflask, which was then sealed under vacuum after evacuated andback-filled with nitrogen thrice. A mixture of GMA, cyclohexanoneand EBP was added into the flask via syringe after being purgedwith nitrogen for 15 min. The polymerization was carried out ina pre-heated oil bath at 55 �C for 24 h under the protection ofnitrogen. The resulted crude product was purified by precipitatinginto a large amount of methanol and water mixture for three times.The concrete reactant quantity for preparation of PGMA withdifferent molecular weights was listed in Table 1. The molar massevaluated by monomer conversion and the polydispersity index(PDI) determined by size exclusion chromatography (SEC) withstandard PS calibration were also showed in Table 1.

2.3. Preparation of macroporous polymer monoliths

The polymer monolith was prepared by a facile moldingprotocol using PGMA as a phase separator. For fabricating mono-liths with well-controlled 3D skeletal structure, given amounts ofAIBN, PGMA, EDMA, GMA and DMF in Table 2 were added intoa glass vial in the listed order and blended under ultrasonic. Thenthe polymerization mixture was thermally initiated by AIBN at60 �C for 24 h. In order to remove the polymer template and theunreacted monomers, the resultant bulk samples were placed inthe Soxhlet extracter by using THF for 48 h, followed by dryingunder vacuum at 50 �C overnight. As a control experiment toconfirm the effect of PGMA on phase separation, PGMA homopol-ymers with different molecular weights were used as phase sepa-rators for the preparation of polymer monoliths.

For extending the application, monoliths fabricated withincreased amount of GMA monomer from 0.5 mL to 0.8 mL wereinvestigated for improving their permeability and degree of func-tionality, while the content of other components in the

Table 2Starting compositions and sample notations.a

Monolithb GMA (mL) DMF (mL) P2 (g)

G0.5eD2.0eP20.10 0.5 2.0 0.10G0.5eD2.0eP20.15 0.5 2.0 0.15G0.5eD2.0eP20.20 0.5 2.0 0.20G0.5eD2.5eP20 0.5 2.5 0G0.5eD2.5eP20.10 0.5 2.5 0.10G0.5eD2.5eP20.15 0.5 2.5 0.15G0.5eD2.5eP20.20 0.5 2.5 0.20G0.5eD3.0eP20.10 0.5 3.0 0.10G0.5eD3.0eP20.15 0.5 3.0 0.15G0.5eD3.0eP20.20 0.5 3.0 0.20G0.6eD3.0eP20.15 0.6 3.0 0.15G0.7eD3.0eP20.15 0.7 3.0 0.15G0.8eD3.0eP20.15 0.8 3.0 0.15

a The amounts of other compunds are fixed as follows: EDMA 0.5 mL, AIBN30.0 mg.

b For directviewing, monoliths are called according to the name and the amountof major component in polymerization system. Namely, “G”, “D” and “P2” representGMA, DMF and the kind of PGMA homopolymers respectively.

Y. Shen et al. / Polymer 53 (2012) 4128e41344130

polymerization system was fixed as shown in Table 2. The poly-merization and post-treatment process was the same as describedabove.

For chromatography evaluation, the monolith was prepared ina stainless steel column (50 mm � 4.6 mm I.D.). After polymeri-zation at 60 �C for 24 h, the column was connected with an HPLCpump to completely remove the polymer template and the othersoluble compounds by THF. Then, the GMA epoxide moiety on thesurface of the monolithic column was hydrolyzed to hydroxylgroups for 8 h at 60 �C by using 0.25 M sulfuric acid at a flow rate of0.1 mL/min. After completion of the reaction, the resultanthydroxylatedmonolithic columnwas washedwith deionizedwaterto neutral pH, followed by methanol before chromatographic use.

2.4. Characterization

For SEM observation of the microstructures, the monoliths werepre-dried at 50 �C under vacuum overnight and snapped apart intopieces, and then placed onto sticky copper foil, whichwas adhered toa standard aluminum specimen tub. Microscopic analysis was per-formedon aHitachi (HitachiHigh Technologies, Tokyo, Japan) S-4300scanning electron microscopy (SEM) instrument after the sampleswere sputter coated with gold nanoparticles by Eiko IB-3 sputtercoating (Eiko, Tokyo, Japan). The macropores of polymer monolithwere characterized by mercury porosimetry (AUTOPORE IV 9510,Micromeritics, USA), while the meso- and micropores were charac-terized by nitrogen absorptionedesorption (ASAP-2020, Micro-meritics, USA). For mercury porosimetry, the pore size wascharacterized using the Wasburn equation assuming a cylindricalshape of the pores. For nitrogen adsorption, the adsorption anddesorption isothermwas calculated by theBrunauereEmmetteTeller(BET)method and the pore size distributionwas calculated by theBJHmethod using the adsorption branch of each isotherm. Chromato-graphic experiments were performed on a Shimadzu LC-20A HPLCsystem (Shimadzu, Japan) consisting of a binary LC-20AT HPLC pumpand an SPD-20A-vis detector. Data processing was carried out witha HW-2000 chromatography workstation (Nanjing Qianpu Software,Nanjing, China).

3. Results and discussion

3.1. Synthesis and characterization of PGMA homopolymers

To investigate the influence of PGMA chain length on porousmorphology of the polymer monolith, four PGMA homopolymers

with different molecular weights were synthesized by ATRPmethod, in which EBP and CuBr/bpy were used as the initiator andthe catalyst respectively. The average degree of polymerizationðDPÞ was evaluated by monomer conversion which was confirmedby 1HNMR. All characteristics of the polymers are listed in Table 1.These results demonstrated that PGMA with various molecularweights was obtained, providing a solid foundation for thefollowing preparation of well-defined 3D skeletal poly (glycidylmethacrylate-co-ethylene dimethacrylate) monoliths.

3.2. Preparation of the 3D skeletal monolith

Macroporous poly (GMA-co-EDMA)monoliths were prepared insitu by using PGMA as a new phase separator. It has been reportedthat the well-defined biocontinuous poly (1,3-glycerol dimetha-crylate) monoliths could be prepared by using PEO with middlemolecular weight as a phase separator [20], thus we supposed thatthe PGMA with moderate molecular weight might contribute toappropriate phase separation tendency in consideration of wellcontrol of pore properties. As a result, in this study, we investigatedthe phase separation behavior of the polymerization system, inwhich P2 (Mn ¼ 4.3 � 104) was employed as a phase separator forcomprehending its influence on the morphology of polymermonoliths. Monoliths prepared without PGMA (G0.5eD2.5eP20,Table 2) did not show any porosity after soxhlet extraction andevaporative drying. However, by incorporating P2, monoliths withporous structure were obtained and could be varied with thealteration of starting composition as displayed in Table 2. It could befound that from left to right in each row in Fig. 1, pore size becamelager with increasing amount of P2 when GMA/DMF was fixed.Whereas, by comparing G0.5eD2.0eP20.15, G0.5eD2.5eP20.15 andG0.5eD3.0eP20.15 (from upper middle to lower middle in Fig. 1),increasing the amount of solvent, DMF, gave a smaller macroporesize. As a result, it could be deduced that the original single phasesolution should be separated into poly (GMA-co-EDMA)-rich phaseand PGMA-rich one in the polymerization process rather than poly(GMA-co-EDMA)-PGMA complex and DMF in consideration of theaffinity interaction between PGMA and poly (GMA-co-EDMA),because the latter one was only special to a strong hydrogen-bonding system of polymer blends and of inorganic system [20].Therefore, in phase separation, with the increased concentration ofP2, more PGMA should distribute into the PGMA-rich phase to fillthe larger pores [18], while DMF should act as a cosolvent andretard the onset of phase separation resulting in smaller domainswith its increasing amount. Additionally, except for pore sizechange, the morphology of the monolith also varied with theaddition of P2. From each column in Fig. 1, the macroporousstructure became finer with increasing amount of DMF while theGMA/P2 ratio was fixed, and those microglobules tended to trans-form into a well-defined 3D skeletal structure. However, for row 3in Fig. 1, from G0.5eD3.0eP20.10 to G0.5eD3.0eP20.20, this 3Dskeletal structure broke up and transformed into particle aggre-gates to decrease the interfacial energy. These results demonstratedthat the ratio of PGMA/DMF had significant influence on theformation of well-defined 3D skeletal monolith.

By incorporating PGMA as a polymeric phase separator, poly(GMA-co-EDMA) monoliths with well-defined 3D skeletal struc-ture could be constructed, which was different from these mono-liths prepared by using conventional porogen consisting of smallmolecule solvents and featuredwith aggregated particles structure.This difference could be ascribed to the distinct phase separationprocess as depicted in Scheme 1. In the process of monolith prep-aration with conventional porogen, the gellike nuclei are formed inearly stage of polymerization and gradually swollen with themonomers due to the higher local monomer concentration than

Fig. 1. SEM imaging of the obtained monoliths (the starting compositions and notations were shown in Table 2).

Y. Shen et al. / Polymer 53 (2012) 4128e4134 4131

that in the surrounding solution. With the proceeding of poly-merization, the nuclei would associate into microgels by polymerchains that cross-link the neighboring nuclei, and then the largerenough microgels would further contact with some of theirneighbors. Finally, the earlier phase separation contributed to themacroporous morphology consisting of aggregated particles aftersolvent drying as shown in Scheme 1(a) [29]. However, with theincorporation of PGMA as a phase separator, the monolith withwell-defined 3D skeletal structure was obtained, which could beexplained by mean-field theory [30]. The occurrence of phaseseparation in polymer solution should depend on its free energychange of mixing, which can be defined as follows:

Scheme 1. The comparative mechanism illustrations of (a) monolith prepared by using porocontaining a polymeric agent.

DGmixfRT�f1P1

lnf1 þf1p2

lnf2 þ c12f1f2

�

where fi and Pi represent volume fraction and polymerizationdegree of component i (i ¼ 1 or 2), c12 is the interaction parameterof the two component, which is defined as negative or positivedepending upon whether the interaction between component 1and 2 is attractive or repulsive respectively, and proportional to thedifference between the solubility parameters of this two compo-nents.We assumed that the EDMA-co-GMAderived species and theporogenic system corresponded to component 1 and 2 respectively.In the equation, the first and the second term relate to the entropy,

gen composed of small molecule solvents and (b) monolith prepared by using porogen

Fig. 2. SEM imaging of the obtained monoliths prepared by using PGMA with different molecular weights as the polymeric phase separator (the notations indicate their statingcompositions as shown in Table 2).

Y. Shen et al. / Polymer 53 (2012) 4128e41344132

both of which have negative values because 0 < fi < 1. Withincreasing P1 of GMA-co-EDMA, the first entropy term on the rightside of the equation should become less negative while P2 and f2kept constant in the process of polymerization. Whereas, theincorporation of PGMA as a polymeric phase separator, which wasconstituted of the monomer GMA in the polymerization system,would incline to strengthen the interaction between the growingpolymer chains and the phase separator compared with smallmolecule solvents due to its preferable affinity with GMA-co-EDMAderived polymer, resulting in more negative c12, and the lastenthalpy term c12 f1 f2 lowered. As a result, DGmix decreased andthe phase separation was delayed, which was favorable for a well-defined 3D skeletal structure as displayed in Scheme 1(b). All theseresults demonstrated that incorporation of polymer as a phaseseparator could open a new way to construct 3D skeletal p (GMA-co-EDMA) monolith.

3.3. The effect of PGMA homopolymers with different molecularweights on morphology development of the monolith

To gain further insight into the influence of PGMA on the porousproperty of the monolith, the PGMA homopolymers with differentmolecular weights (P1, P3, P4 in Table 1) were used as phase sepa-rators for monolith preparation, in which the starting compositionwas the same as that in G0.5eD3.0eP20.15 preparation, and theobtained monoliths were named as G0.5eD3.0eP10.15,G0.5eD3.0eP30.15 and G0.5eD3.0eP40.15 respectively. Bycomparing these four monoliths (G0.5eD3.0eP20.15 in Fig. 1 andthe other three in Fig. 2), the pore size was largened with theincrease of PGMA molecular weight, which was further confirmedby mercury porosimetry as shown in Fig. 3. The results demon-strated that the average pore diameter of these polymer monoliths

Fig. 3. Pore size distributions of the monoliths prepared by using PGMAwith differentmolecular weights as the polymeric phase separator, measured by mercury intrusionporosimetry.

increased from 15.4 nm to 137 nm and the total surface areadecreased from 150 m2/g to 45 m2/g with the increase of PGMAlength, which implied that PGMA with higher molecular weightwas favorable for larger pore size but led to smaller surface area.This is in accord with the inverse relationship between porediameter and surface area. However, we found that the molecularweight of PGMA didn’t only influence the pore size distribution ofthe resulted polymer monolith, but also played a significant role inmorphology development, whichmight be influenced by the phaseseparation behavior relying on the DGmix in mean-field theory asdescribed above. However, the first two entropy term remainedconstant (note that P1, P2, f1 and f2 is constant owing to the sameamount of the starting compositions) while only the third enthalpyterm might change due to the different c12 values which furtherinduced to the distinct DGmix values. Thus, we assumed thatcompared with P2, PGMA with too lower and higher molecularweight (P1, P3, P4) might denote to inappropriately matched solu-bility between the porogenic system and EDMA-co-GMA derivedpolymer (more or less negative c12 values), which resulted in tooslow or too fast phase separation and then the aggregated globularmonolith structure.

In addition, for further exploring the porous property of theobtained monolith with well-defined 3D skeletal structure, thenitrogen adsorptionedesorption was performed. The resultantadsorptionedesorption isotherm (Fig. 4) exhibited adsorptionhysteresis, indicative of the presence of mesopores, which wasconsidered as a significant advantage of monolithic medium formany applications.

3.4. Application of the monolith to HPLC

The poly (GMA-co-EDMA) monolith with well-defined 3Dskeletal structure exhibits interesting features which make itsuperior to the conventional monolith consisting of aggregated

Fig. 4. Nitrogen adsorptionedesorption isotherm of the monolith (G0.5eD3.0eP20.15)with 3D skeletal structure.

Fig. 5. SEM imaging of the obtained monoliths consisting of GMA with different amounts (the starting compositions and notations were shown in Table 2).

Fig. 6. Mercury intrusion porosimetry (a) and nitrogen adsorption-desorption isotherm (b) of the monolith (G0.8eD3.0eP20.15) with 3D skeletal structure.

Y. Shen et al. / Polymer 53 (2012) 4128e4134 4133

particles in many applications. However, to improve the perme-ability and functionality of this monolith for its preferable appli-cation, the polymerization system with increasing content of GMAmonomer from 0.5 mL to 0.8 mL was applied to construct themonoliths (Table 2). In Fig. 5, it could be observed that with theincreased amount of GMA monomer in the polymerization system,the pore size enlarged in order for better permeability [31,32]. Thisphenomenon could be explained by the fact that with the increaseof GMA monomer, the relative ratio of the cross-linker decreased,which would lead to increased pore size [23,33]. In addition, fromG0.5-D3.0-P20.15 to G0.8-D3.0-P20.15, although these monolithshad different morphology, they could still maintain the well-defined 3D skeleton, and the increased amount of GMA monomercould provide more interaction sites resulting from epoxy groups.All these results indicated that the monolith prepared by using P2as a polymeric phase separator could not only get the well-defined3D skeletal structure, but also maintain this structure in a tunablerange of GMA monomer with the fixed amount of cross-linker and

Fig. 7. Separation of small molecules including (a) steroids and (b) benzene derivatives on aat 254 nm; (a) mobile phase: water; sample: hydrocortisone (peak 1), prednisone acetate (pwater; mobile phase B, methanol; step gradient: 0e3.0 min, 100% A; 3.0e18.0 min, 10% B; 1

porogen, which could potentially widen its applied range accordingto different requirement in various degrees. Finally, in consider-ation of good permeability and high functionalities,G0.8eD3.0eP20.15 was selected as the separation medium forHPLC. Its pore size distribution was characterized by the mercuryporosimetry and nitrogen adsorptionedesorption isotherm asdepicted in Fig. 6. The mercury porosimetry demonstrated thepresence of macropores (>50 nm) and the nitrogenadsorptionedesorption isotherm exhibited hysteresis loopbetween adsorption and desorption branches which suggested theexistence of mesopores in G0.8eD3.0eP20.15. These resultsensured its lager pores for convection and mesopores for highcapacity, which further inspired its great promise for usage in HPLC.

As the separation media in HPLC, the poly (GMA-co-EDMA)monolith could serve its purpose by tailoring surface chemistryrequired for desired applications. A GMA epoxide moiety on themonolith could be converted into diol groups [34] for performancein a hydroxylated chromatographic mode. The mechanical stability

monolithic column. Conditions: column 50 mm � 4.6 mm I.D.; flow rate, 1 mL/min; UVeak 2), medroxyprogesterone acetate (peak 3). (b) mobile phase: mobile phase A, pure8.0 min, 60% B; sample: benzoic acid (peak 1), toluene (peak 2), naphthalene (peak 3).

Fig. 8. Separation of a protein mixture on a monolithic column. Conditions: column50 mm � 4.6 mm I.D.; flow rate, 1 mL/min; UV at 280 nm; mobile phase: mobile phaseA, 10.0 mM phosphate buffer, pH 7.0; mobile phase B, 2.0 M (NH4)2SO4 in 10.0 mMphosphate buffer, pH 7.0; step gradient: 0e4.0 min, 100% B; 4.0 min, 50% B; sample, CytC (peak 1); Lys (peak 2); BSA (peak 3).

Y. Shen et al. / Polymer 53 (2012) 4128e41344134

of this monolith was evaluated by the linear relationship betweenpressure drop and flow velocity as displayed in Fig. S1. The resultdemonstrated that thematerial was rigid and resistant to swell. Thechromatographic permeability (K) of the monolith, which indicatedits flow-through property, was calculated as from Darcy’s law [35]:

K ¼ uLh=DP

where u stands for superficial velocity (m/s), L for column length(m), h for mobile-phase viscosity (Pa s) and DP for back pressure(Pa). The K value of the monolith was calculated to be5.2 � 10�14 m2, which was better than that of particle-packedcolumn (particle size �5 mm) [36].

To evaluate the separation performance of the monolith afterhydrolysis in HPLC, its application for the separation of both smallmolecules and large biomolecules has been presented. As displayedin Fig. 7, a mixture of three steroids as well as a standard benzenederivatives mixture, using as small molecular probes, was effec-tively separated according to their hydrophobicity. Also, goodseparation of proteins containing cytochrome (Cyt C), lysozyme(Lys) and bovine serum albumin (BSA), using as macromolecularprobes, was achieved under mild condition (Fig. 8). All these resultsdemonstrated the improved performance of the monolith featuredwith well-defined 3D skeletal structure in comparison with themicroglobules-aggregated monolith prepared using conventionalporogen (Figs. S2 and 3), and indicated its latent capacity in theseparation of both small molecules andmacromolecules which wasone of the bottlenecks in monolith for HPLC aplication. Moreover,the monolith surface could be further tailored for improving itsperformance and widening its application range via post-modification in future [37].

4. Conclusions

This work has indicated that PGMA could be employed asa novel phase separator to construct poly (GMA-co-EDMA) mono-lith with well-defined 3D skeletal structure.With the adjustment ofPGMA/DMF ratio and PGMAmolecular weight, the porous structurecould be varied from aggregated microglobular structure to well-defined 3D skeletal structure. Moreover, the monolith couldpreserve this 3D skeletal structure in an adjustable range of GMAmonomer in polymerization system for improved permeability andfunctionality. Meanwhile, this hierarchically porous structure couldafford the monolith with good mass transfer and large absorbentcapacity, which was confirmed by its application in HPLC for the

separation of both small molecules and large molecules. Notably,the applied range of the well-defined 3D skeletal monolith, pos-sessing reactive epoxy functionality, could be easily widened bypost-modification in future. More importantly, the synthesizedPGMA with different molecular weights could be potentiallydeveloped as a promising alternative or supplement to thesecommercially available counterparts for fabrication of monolithswith well-defined structure over a wide range.

Acknowledgments

We gratefully thank the financial support from NSFC (No.21175138 and No. 20935005), Ministry of Science and Technologyof China (No. 2007CB714504), and Chinese Academy of Sciences.We appreciate Dr. Peiyong Xin for his kind help.

Appendix A. Supplementary material

Supplementary material associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.polymer.2012.07.019.

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