Reactive & Functional Polymers 78 (2014) 32–37

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Reactive & Functional Polymers

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Glycidyl methacrylate and ethylhexyl acrylate based polyHIPEmonoliths: Morphological, mechanical and chromatographic properties

http://dx.doi.org/10.1016/j.reactfunctpolym.2014.02.0111381-5148/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: University of Maribor, Faculty of Chemistry andChemical Engineering, Smetanova 17, SI-2000 Maribor, Slovenia. Tel.: +386 2 2294422

E-mail address: peter.krajnc@um.si (P. Krajnc).

Simona Jerenec a, Mario Šimic b,a, Aleš Savnik b, Aleš Podgornik c,b, Mitja Kolar d,a, Marko Turnšek d,Peter Krajnc d,a,⇑a Centre of Excellence PoliMaT, Tehnološki Park 24, SI-1000 Ljubljana, Sloveniab BIA Separations d.o.o., Mirce 21, SI-5270 Ajdovšcina, Sloveniac Centre of Excellence COBIK, Velika pot 22, SI-5250 Solkan, Sloveniad University of Maribor, Faculty of Chemistry and Chemical Engineering, Smetanova 17, SI-2000 Maribor, Slovenia

a r t i c l e i n f o

Article history:Received 28 October 2013Received in revised form 5 February 2014Accepted 26 February 2014Available online 7 March 2014

Keywords:Glycidyl methacrylateEthylhexyl acrylatepolyHIPEMorphologyPorous polymersMonoliths

a b s t r a c t

Using water-in-oil emulsions with a high volume share of aqueous (droplet) phase as precursors (HighInternal Phase Emulsions; HIPEs), highly porous polymers (polyHIPEs) were prepared from glycidylmethacrylate (GMA) and ethylhexyl acrylate (EHA), their morphology investigated and mechanical andchromatographic characteristics evaluated. All polyHIPE monoliths had open cellular porous morphologywith primary pores (cavities) between 4.8 lm and 26.2 lm and secondary level of interconnecting pores.Introduction of EHA into the oil phase and consequently into the polymer matrix of polyHIPEs had a sig-nificant effect on the mechanical properties; both tensile strength and elasticity were increased. On theother hand, chromatographic properties, such as protein binding capacity and back pressure, did not dra-matically change.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Emulsions with a high volume fraction of internal phase areknown as HIPEs (High Internal Phase Emulsions) where the dropletphase occupies typically more than 74 vol.% [1,2]. By polymerisa-tion of a HIPE, with the continuous phase containing monomers,monoliths with a high level of porosity and open porous morphol-ogy are obtained (Scheme 1, Fig. 1). Such polymers, usually termedpolyHIPEs, can be prepared from either oil in water emulsions con-taining oil soluble monomers or from water in oil emulsions wherehydrophilic monomers are contained in the aqueous phase of theemulsion [3–5]. Among other applications of polyHIPEs, chroma-tography is a prospective field. Chromatographic monoliths are aparticular group of chromatographic stationary phases that consistof a single piece of highly porous material with interconnectedchannels which enable the flow of the mobile phase. Due to thisparticular structure several properties, such as flow unaffected res-olution and dynamic binding capacity, low pressure drop, and highdynamic binding capacity for very large molecules, are exhibited.

Polymer based monoliths are widely used for separation [6] andpurification of biologic macromolecules [7,8] due to their scalabil-ity [9,10] and chemical stability [11] required for sanitation. De-spite polymeric chromatographic monoliths which were preparedto exhibit various microstructures [12] there are very few reportsregarding polyHIPE materials applied for chromatography. Glyc-idyl methacrylate (GMA) is a reactive monomer, which is fre-quently used for the preparation of functional polymers. For thepreparation of monolithic polymeric chromatographic columns,especially, GMA has been extensively used [13]. Majority of porouscolumns, prepared from GMA, make use of the phase separationprocess with included porogenic solvents to induce the porousmorphology of polyGMA. On the other hand, preparation andapplications of GMA based polyHIPEs have also been demonstrated[4,14–19]. The possibility of preparation of monoliths with a highlevel of porosity (up to 90%) and pore size tuning make GMA basedpolyHIPEs good candidates for chromatographic applications. Theuse of GMA polyHIPEs for protein separation has already been re-ported, both in the form of discs [17] and membranes [18]. Espe-cially in the case of high levels of porosity, the brittleness of thematerial presents a drawback for chromatographic applications.We have therefore intended to influence the mechanical propertiesof GMA polyHIPEs by introduction of a co-monomer, namely

Scheme 1. polyHIPE preparation.

S. Jerenec et al. / Reactive & Functional Polymers 78 (2014) 32–37 33

ethylhexyl acrylate which is known to influence the plastic behav-iour of polymers. Herein we report on the morphological, mechan-ical and chromatographic properties of GMA based polyHIPEmaterial, with included EHA.

2. Experimental section

2.1. Chemicals

Monomers glycidyl methacrylate (GMA, Sigma–Aldrich), ethyl-ene glycol dimethacrylate (EGDMA, Sigma–Aldrich), 2-ethylhexylacrylate (EHA, Sigma–Aldrich) were passed through a basicalumina column prior to use in order to remove the inhibitors.Potassium persulfate (PPS, Fluka), N,N,N0,N0-tetramethylethylenediamine (TEMED, Fluka), calcium chloride hexahydrate (Sigma–Al-drich), surfactant Synperonic PEL 121 (Sigma–Aldrich), toluene(Sigma–Aldrich) and diethyleneamine (DEA, Sigma–Aldrich) wereused as received. Proteins myoglobin, conalbumin from chickenegg white and soybean trypsin inhibitor (STI) were obtained fromSigma–Aldrich.

2.2. Preparation and functionalization of polyHIPE monoliths

Organic and aqueous phase were prepared separately. Organicphase consisted of monomers EGDMA, GMA and EHA, surfactantPEL 121 and toluene. Aqueous phase contained water, CaCl2 and

Fig. 1. SEM picture of polyHIPE material.

potassium persulphate. Aqueous phase was added dropwise tothe organic phase in a three necked flask within a half an hourperiod while stirred with an overhead stirrer at 250 rpm. Afterthe addition of aqueous phase stirring was continued for one hourcontinued by the addition of N,N,N0,N0-tetramethylethylenedi-amine (TEMED). The emulsion was transferred to a polypropylenecontainer and cured at 40 �C for 24 h. Monoliths were purified byextraction in Soxhlet apparatus with water and isopropanol for48 h. The amounts of components are presented in Table 1. Forchromatographic evaluation, polyHIPE monoliths were functional-ised with diethyleneamine introducing weak anion exchangediethylaminoethyl groups (DEAE) as described previously [17].

2.3. Structutral characterisation

SEM pictures were taken on a Quanta 200 3D (FEI Company;samples were gold sputtered (gold coating layer thickness under40 nm) and an acceleration voltage of 20 kV was used). Cavity sizedistribution was determined by SEM image analysis; measuringthe diameter of at least 100 cavities. Cavity sizes were adjustedfor random sectioning using the correction factor 2/

p3. Nitrogen

adsorption/desorption measurements were done on a Micromeri-tics TriStar II 3020 porosimeter using a BET model for surface areaevaluation. All samples were degassed and measured three times.

2.4. Determination of mechanical properties

Mechanical properties of monoliths were measured at a con-stant room temperature using Instron 3345 device (Norwood,USA). Cylindrical shaped monoliths had a diameter of 12 mm andheight of 12 mm for compression and approximately 100 mm fortensile experiments. To estimate monolith initial volume monolithdimensions were measured for compression test while for tensiletest monolith diameter and distance between two grips withinwhich monolith was fixed, were measured. Compression or pullingwas performed at a constant velocity of 1.0 mm/min till materialbreakage for determination of strain and stress at break. Fromthe linear part of stress–strain curve modulus was calculated.

2.5. Chromatographic experiments

Chromatographic experiments were performed on a gradientHPLC system consisting of two Pumps 64, an injection valve with20 ll sample loop, a variable wavelength monitor with a 10-mmoptical path set to 280 nm and HPLC hardware/software (dataacquisition and control station), all from Knauer (Berlin, Germany).

For separation of standard protein mixture and determinationof dynamic binding capacity loading buffer was 20 mM Tris–HCl,pH 7.4 and elution buffer 20 mM Tris–HCl + 1 M NaCl, pH 7.4. Flowrate was 4 ml/min.

Standard protein mixture consisted of myoglobin (c = 0.5 mg/ml), conalbumin (c = 1.5 mg/ml), soybean trypsin inhibitor(c = 2.5 mg/ml) dissolved in loading buffer. Linear gradient from0% to 100% elution buffer in 30 s was applied and retention timesof proteins were recorded. Dynamic binding capacity was mea-sured with bovine serum albumin (c = 1.0 mg/ml) dissolved inloading buffer. Capacity was determined at 50% of break throughcurve. All proteins were from Sigma–Aldrich (USA)

3. Results and discussion

3.1. Preparation of GMA/EGDMA/EHA monoliths

Preparation of polyHIPE monoliths with GMA as a functionalmonomer and crosslinked with ethyleneglycol dimethacrylate

Table 1Composition of polymer samples.

Samplea CaCl2 (g) PPS (g) H2O (mL) PEL 121 (g) EGDMA (mL) GMA (mL) EHA (mL) TEMED (lL)

A75 0.040 0.644 36 2.418 3.7 8.2 0 47.4A85 0.046 0.752 42 1.511 2.3 5.1 0 56.0A90 0.044 0.716 40 0.907 1.4 3.1 0 53.3B75 0.037 0.609 34 2.322 3.4 7.2 0.8 45.5B85 0.047 0.770 43 1.548 2.3 4.8 0.5 57.3B90 0.045 0.734 41 0.929 1.4 2.9 0.3 54.6C75 0.041 0.662 37 2.535 3.7 7.1 1.7 49.7C85 0.044 0.716 40 1.426 2.1 4.0 0.9 52.8C90 0.046 0.752 42 0.951 1.4 2.7 0.6 55.9D75 0.043 0.698 39 2.653 3.7 6.0 3.3 52.0D85 0.051 0.823 46 1.658 2.3 3.8 2.1 61.4D90 0.048 0.788 44 0.995 1.4 2.3 1.3 58.5

a Samples A are without EHA; samples B contain 5 mol% (with regards to monomers) EHA, samples C 10 mol.% and samples D 20 mol.% EHA. Numbers in sample ID standfor nominal porosity (vol.% of aqueous phase).

34 S. Jerenec et al. / Reactive & Functional Polymers 78 (2014) 32–37

(EGDMA) have been described previously [17]. For GMA/EGDMAmonoliths with high levels of porosity difficulties regarding chro-matographic applications in form of discs were reported due topoor mechanical properties. Addition of ethylhexyl acrylate(EHA) into the monomer mixture is known to increase the free vol-ume of resulting polymer therefore improving mechanical stabilityby increasing elasticity [20]. Firstly, the kinetical stability of HIPEscontaining EHA in addition to functional monomer and crosslinker(GMA and EGDMA) was investigated. Amount of EHA added tomonomer phase was varied, namely 5, 10 or 20 mol.% (with re-gards to monomer ratio). The mentioned amounts of EHA wereadded in the case of 75, 85 or 90 vol.% of aqueous phase (c.f. Tables1 and 2). The addition of EHA did not compromise emulsion stabil-ity in any of the experiments and monoliths could be obtainedfrom emulsions with aqueous phase ratio up to 90%. When com-paring SEM images of samples with added EHA to samples ob-tained previously without EHA, a structure with a narrowercavity size dispersion is observed. Furthermore, interconnectivityof cavities is also higher. Comparing morphology of samples inrelation to nominal porosity (volume share of aqueous phase inthe precursor emulsion), one can observe the increasing intercon-nectivity when porosity increases. This was expected as withincreasing share of droplet phase, the polymer film around thedroplets of internal phase is thinned and thus more interconnect-ing pores are formed. Mercury intrusion porosimetry results arein relatively good agreement with nominal porosity (volume shareof aqueous phase). For 75 vol.% of aqueous phase nominal porosityresults between 79 and 81 vol.% total porosity were obtained andfor 90% pore volume mercury intrusion resulted in 89% pore vol-ume. These measurements support the findings of experimentswhere we could not observe any significant shrinkage of the mono-liths after the polymerisation.

Range of cavities sizes (primary pores) in the polyHIPE monolithsize is from 4.8 to 26.2 lm. As seen from the Table 2, the largestcavities were obtained by the presence of 5 mol.%EHA (with re-gards to total monomer composition). In this case average cavitiesdiameter increases from 15.4 lm to 26.2 lm by the increasing ofaqueous phase from 75 vol % to 90 vol.%. Also in the presence of90 vol.% of aqueous phase monoliths with more open cellularstructure were prepared (Fig. 2: Samples C90, D90). By usingEHA (10 and 20 mol.%) and 90 vol.% of aqueous phase samples witha more pronounced polyHIPE morphology were obtained (SEMimages of samples C90 and D90).

Nitrogen adsorption/desorption experiments were also per-formed for polyHIPE monoliths in order to determine the possibleformation of smaller pores, in the meso and micro range (nanopores). Results of surface area using BET model indicated that nosignificant amount of nano pores are present in the material

(results were between 2 and 10 m2/g) suggesting that gel typepolymer film was formed in the continuous phase of the HIPemulsion.

Since mechanical properties of monoliths produced from ahighly porous polymeric material can be of critical importancefor chromatographic applications, monoliths with 75% and 90%nominal porosity were subjected to mechanical testing, namelytension and compression experiments. A series of samples with25% of EGDMA and 0%, 5% and 10% of EHA in the monomer mix-ture, were prepared and tested. Results are collected in the Table 3.From stress/strain behaviour for both compression and tensile test-ing it is evident that the introduction of 5% of EHA into the mono-mer mixture significantly increases the elasticity of the monoliths.In the case of both 75% and 90% nominal porosity the modulus (cal-culated from initial, linear part of stress–strain curve) decreaseswith the addition of EHA (from 34 MPa to 23 MPa in the case of75% porosity and from 13.5 MPa to 4.6 MPa in the case of 90%porosity for tensile testing; from 10.4 MPa to 6.0 MPa in the caseof 75% porosity and from 6.1 MPa to 2.4 MPa in the case of 90%porosity for compression testing). The changes in monomer com-position are similarly evident through sample compression orelongation; introduction of 5% of EHA into monomer mixtureresulted in increased compression or elongation before break;the increase is between 16% and 100%, being more evident attensile experiments (however the specimen length changes aresignificantly smaller at tensile testing therefore the accuracy ofmeasurement is lower). In most cases, addition of 10% EHA didnot increase elasticity further; the samples exhibited similar oreven less elastic behaviour than samples with 5% EHA in the mono-mer mixture. Bulkier side chains of ethylhexyl acrylate increase thefree volume of the polymer and furthermore, a larger number ofrotatable bonds have a similar effect. Strain and stress at breakare significantly lower in the case of tensile testing; this wasexpected as the morphology of the material is cellular and in thecase of contraction open space accommodates the geometricaldistortion before the break (c.f. Table 3).

3.2. Chromatographic evaluation

To evaluate chromatographic performance of prepared mono-liths, especially the effect of EHA addition, polyHIPE monolithswere tested for their separation efficiency and binding capacityof proteins. Since for the isolation of biological molecules ion-exchange groups (IEX) are normally implemented, epoxy groups,present on the monolith after polymerisation, were convertedusing diethylenediamine into primary and secondary aminegroups exhibiting anion exchange character therefore beingcharged positively around neutral pH value. This chemical

Fig. 2. SEM pictures of samples A75 (upper left, bar is 20 lm), A90 (upper right, bar is 10 lm), B75 (middle row left, bar is 20 lm) B90 (middle row right, bar is 20 lm), C75(lower left, bar is 20 lm) and C90 (lower right, bar is 10 lm).

S. Jerenec et al. / Reactive & Functional Polymers 78 (2014) 32–37 35

modification enabled the use of standard set of proteins normallyused for evaluation of anion-exchangers – mioglobin, conalbuminand trypsin inhibitor to test separation efficiency and bovine ser-um albumin to determine the dynamic binding capacity.

While methacrylate polyHIPE monoliths were already evalu-ated for chromatography there is no information regarding the ef-fect of EHA addition. In particular, there are two parameters whichmight influence chromatographic performance when EHA isadded: substitution of GMA with EHA results in a decrease ofepoxy groups density thus affecting also the amount of formedIEX groups. Because of that protein retention and capacity mightbe affected if their content is too low [21]. However, since the bind-ing capacity depends also on the available surface which mightalso be affected by EHA addition area, it is therefore not so repre-sentative for this evaluation. Second effect of EHA addition mightbe due to the fact that EHA affects monolith mechanical propertiesby increasing its elasticity what increases monolith compressibilityand consequently accessibility of macromolecules to the bindingsites. As a result, chromatographic performance might change,

Table 2Morphological properties of polyHIPE monoliths.

EGDMA (mol.%) GMA (mol.%) EHA (mol.%) Aque

A75 25 75 0 75A85 25 75 0 85A90 25 75 0 90B75 25 70 5 75B85 25 70 5 85B90 25 70 5 90C75 25 65 10 75C85 25 65 10 85C90 25 65 10 90D75 25 55 20 75D85 25 55 20 85D90 25 55 20 90

again by changes in the retention times of retained proteins. Toverify if the addition of EHA has an impact, experiments with thesame protein sample and mobile phase were performed on variouspolyHIPE EHA monoliths. Results are summarized in Tables 2 and4, while the representative chromatogram is shown in Fig. 3.

From Fig. 3 it can be concluded that there is a good separation oftest proteins. Under applied conditions, mioglobin is non-retained,weakly retained is conalbumin while trypsin inhibitor is the moststrongly retained. Efficient separation demonstrates the potentialof prepared polyHIPE EHA monoliths for chromatography and alsoenables to accurately determine the retention time for particulartest protein as shown in Table 4. As it can be seen, most of theretention times are comparable. Retention time of mioglobin,which is non-retained, indicates changes of system dead- volumeand might be slightly influenced by the changes in monolith poros-ity. However, since the retention time is a consequence of themonolith porosity but also of the dead volume of the housing inwhich monolith is packed as well as of system tubing, this changeseems to be too small in comparison to the total dead volume. On

ous phase (vol.%) Cavities, d (lm) Interconnecting pores, d (lm)

14.2 0.815.0 2.415.9 1.415.4 2.219.8 1.926.2 2.224.2 2.320.6 2.914.4 1.121.4 0.717.9 1.3

4.8 1.3

Table 3Mechanical properties of GMA based polyHIPEs.

Samplea Compression Tensile testing

Modulus (MPa) Stress at break (kPa) Compression at break (%) Modulus (MPa) Stress at break (kPa) Elongation at break (%)

A75 10.4 ± 0.3 630 ± 17 7.5 34 ± 3 35 ± 6 0.3B75 6.0 ± 0.3 560 ± 10 11.1 23 ± 2 60 ± 7 0.5C75 9.5 ± 0.6 593 ± 12 8.5 34 ± 5 87 ± 6 0.4A90 6.1 ± 0.9 330 ± 20 15.5 13.5 ± 2 30 ± 2 0.5B90 2.4 ± 0.7 140 ± 20 18.0 4.6 ± 0.2 30 ± 1 1.0C90 1.2 ± 0.2 110 ± 10 13.0 1.3 ± 0.06 10 ± 0.5 0.9

a Samples A contain no EHA, samples B contain 5 mol% EHA, samples C contain 10 mol% EHA (to monomers). 75 or 90 refers to nominal porosity (vol% of aqueous phase).

Table 4Chromatographic evaluation data.

Aqueous phase (vol.%) Protein binding capacity (mg/mL) Retention time (min) Back pressure (bar)

Mioglobin Conalbumin Trypsin

A75 75 8.4 0.10 0.34 0.55 0.77A85 85 7.1 0.14 0.30 0.47 0.37B75 75 10.6 0.13 0.28 0.54 0.29B85 85 6.1 0.14 0.28 0.47 0.28B90 90 6.9 0.15 0.29 0.47 0.39C75 75 6.5 0.13 0.28 0.52 0.37C90 90 10.6 0.14 0.27 0.51 0.16D75 75 8.1 0.13 0.28 0.48 0.83

36 S. Jerenec et al. / Reactive & Functional Polymers 78 (2014) 32–37

the other hand, retention time of conalbumin and trypsin inhibitordepends on the strength of interaction. Also in this case however,there is no pronounced effect. There might be slight increase intrypsin inhibitor retention time for samples B75, C75 and D75.They have same content of aqueous phase and EDMA but decreas-ing amount of EHA (20%, 10% and 5% respectively). This trend canbe explained by an increase of the amount of active groups, whichis not observable. However, for conalbumin indication the interac-tion has to be very strong for any difference to occur. Based on thisdata it can be concluded that the addition of EHA has no significanteffect on the chromatographic performance in terms of interaction.However, as seen from Fig. 2, and Table 4 it has a substantial im-pact on the monolith structure. Structural differences can also benoticed from the pressure drop data, which differ among samplesup to five-fold.

Due to a complex structure of polyHIPE EHA monolith it wasdemonstrated that the pressure drop cannot be described by Koze-ny–Carman relation but rather pressure drop model for catalyticfoams has to be used [22]. Therefore it seems to be difficult to pre-dict the chromatographic behaviour from this information. This isfurther confirmed since there is no correlation between dynamicbinding capacity (DBC) and pressure drop. For conventional meth-acrylate monoliths it was recently shown, that there is a linear

Fig. 3. Chromatogram for separation of standard protein mixture with sample D75.

correlation between DBC and square root of the pressure drop onthe monolith [23]. In the case of EHA polyHIPE monoliths however,there is no such trend, since for example samples B75 and B85 exhi-bit the highest and the lowest DBC having practically identical pres-sure drop. This can be explained by a more complex structureshown in Fig. 1, where besides interconnecting pores there are alsocavities contributing substantially to overall porosity and conse-quently the utilization of volume for protein binding. It can there-fore be concluded that there are more degrees of freedom duringthe optimization of polyHIPE methacrylate monoliths and ratherhigh binding capacity can be achieved at low pressure drops.Nevertheless, DBC of polyHIPE monoliths is still twice lower in com-parison to conventional methacrylate monoliths (www.biasepara-tions.com) therefore further optimization is required.

4. Conclusions

We have shown that the copolymers of glycidyl methacrylateand ethylhexyl acrylate in a form of highly porous polyHIPE mono-liths can be prepared with the use of an emulsion templatingapproach. Application of EHA as a comonomer improves themechanical stability of polyHIPEs in terms of both tensile strengthand elasticity (deformation at break); monoliths have thus reducedbrittleness, which is a welcome feature especially for chromato-graphic applications. At the same time the introduction of EHAdid not significantly affect the chromatographic properties suchas back pressure and protein binding capacity.

Acknowledgements

The financial support of the Ministry of Science, Higher Educa-tion and Technology of the Republic of Slovenia (Centre of Excel-lence PoliMaT) through the contract No. 3211-10-000057 isgratefully acknowledged.

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