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Atrazine removal by covalent bonding to piperazinefunctionalized PolyHIPEs

Irena Pulko, Mitja Kolar, Peter Krajnc ⁎

Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova 17, 2000 Maribor, Slovenia

Received 13 November 2006; received in revised form 12 June 2007; accepted 19 June 2007Available online 27 July 2007

Abstract

The removal of atrazine from water by a solid phase extraction technique using insoluble polymers is described. Porouscrosslinked polymers bearing piperazine moieties were prepared in a one step reaction from the precursor 4-nitrophenylacrylateincorporating polymers (PolyHIPE type prepared by the polymerization of the continuous phase of a high internal phase emulsionand polymer beads prepared by suspension polymerization). Polymers were applied to sequester atrazine from aqueous solutionswith a concentration of 33 ppb and irreversible covalent bonding to the polymers was achieved. GC/MS/MS was used to monitorthe dynamics of atrazine uptake and it was found that almost complete removal of atrazine was acomplished with an excess ofpolymer after 48 hours at room temperature. For comparison, polymer beads of identical chemistry but lower porosity were alsoused and showed significantly slower action (near complete removal after 72 hours).© 2007 Elsevier B.V. All rights reserved.

Keywords: Atrazine; Water purification; Polymer scavengers; PolyHIPE; Piperazine; Solid phase extraction

1. Introduction

The presence of atrazine in ground, surface anddrinking water presents a serious problem regardingwater supplies especially in regions with intenseagricultural activities. Despite convincing evidence oftoxicity towards animals (Alvarez and Fuiman, 2005;Frampton et al., 2006; Pelletier et al., 2006) and humans(Barlow, 2005; Rodriguez et al., 2005; Zeljezic et al.,2006) this chlorotriazine herbicide remains in use in theUnited States while its use is banned in European Unioncountries since 2003 (Sass and Colangelo, 2006). Thereare efforts of complete removal of atrazine and othertriazines from drinking water. The European Union

legislation allows only a very low amount of atrazine(0.1 ppb) present in the drinking water, while in theUnited States this limit is set to 3 ppb (Sass andColangelo, 2006). However, in some cases much higheramounts of atrazine found in streams and ground water,as high as 200 ppb, are reported (U.S. GeologicalSurvey, 2003).

Most of the methods for the removal of atrazine fromwater so far are using different forms of sorbents, whichrely on the adsorption–desorption equilibrium. Amongthe sorbents, carbon based materials are most commonlyused (Li et al., 2003). There are reports of carbon fibers(Martín-Gullón and Font, 2001) being used for atrazineremoval and the use of electrochemical regenerations ofcarbon based sorbents (Brown et al., 2004). Apart fromcarbon, polymers have also been used for the adsorptionof atrazine from water. Carboxy benzoyl derived

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⁎ Corresponding author. Tel.: +386 222 944 22; fax: +386 225 277 74.E-mail address: peter.krajnc@uni-mb.si (P. Krajnc).

0048-9697/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.scitotenv.2007.06.032

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crosslinked polymers, starting from commerciallyavailable polystyrene beads have been prepared,however only 11% of the sites were functionalized(Masqué et al., 1998). Another method frequentlystudied is the use of nanofiltration with the appropriateporous membranes (Zhang et al., 2004). The reports ofusing polymers for the removal of atrazine by covalentbonding to the polymer matrix are scarce. Whilesorbents can be reused after desorption of the molecule,polymers which bind the molecule covalently, need achemical transformation in order to be reused. However,desorption cannot occur during the removal processeven if the conditions change since the molecule iscovalently attached to the supporting polymer. Hollinket al. (2005) have used polystyrene crosslinked withdivinylbenzene to prepare piperidine functionalizedpolymer beads for the covalent attachment. Thepreparation of piperidine functionalized resins startingfrom modified polystyrene using either a one step or atwo step protocol is reported.

Recently, another type of insoluble polymers, with amuch higher porosity, has been used for the sequestrationprocedures and proved to be very suitable (Krajnc et al.,2002). Highly porous polymers prepared from concen-trated emulsions are termed PolyHIPEs and have alreadybeen prepared with various chemistries (Cameron, 2005).Since the introduction of PolyHIPE materials by Unileverresearchers (Barby and Haq, 1985), these monolithicporous polymers have been used for various applications,such as the supports for organic synthesis (Brown et al.,2005), separation (Krajnc et al., 2005a), for theimmobilization of enzymes (Pierre et al., 2006), for theremoval of arsenic from water (Katsoyiannis and

Zouboulis, 2002) and as tissue engineering matrices(Busby et al., 2002; Akay et al., 2004; Hayman et al.,2005). High fractions of the discontinuous internal phaseyield the material with a very high pore volume (typicallybetween 75% and 90%). Good permeability, the result ofan open cellular architecture (see Fig. 1), is advantageousespecially in flow through applications (Mercier et al.,2001; Hodge, 2003). The pattern of larger pores (voids),smaller pores (windows) that connect the larger ones andthe porosity of the continuous phase make this kind ofmaterial more versatile than usual macroreticular resins interms of solvent compatibility.

In this report, a simple one step procedure tofunctionalize 4-nitrophenylacrylate based crosslinkedporous polymers (PolyHIPEs and beads) and their usefor the removal of atrazine from aqueous solutions withlow concentrations is described.

2. Materials and methods

2.1. Materials

4-nitrophenol (Aldrich), acryloyl chloride (Merck),triethylamine (Fluka), dichloromethane (Merck), sodi-um hydrogen carbonate (Kemika), magnesium sulphate(Kemika), chlorobenzene (Aldrich), methanol (Fluka),N,N-dimethylformamide (DMF, Carlo Erba Reagenti),methanol (Fluka), piperazine (Aldrich), atrazine(Merck), ethyl acetate (Aldrich), potassium peroxodi-sulfate (Fluka), α,α′-azoisobutyronitrile (AIBN, Fluka),calcium chloride hexahydrate (Merck), tetrahydrofurane(THF, Merck), hexane (Merck), sorbitan trioleate (Span85, Merck), sorbitan monooleate (Span 80, Merck) andpoly(vinylpyrrolidone) (M=40,000, Aldrich) were usedas received. Divinylbenzene (DVB, Aldrich, a mixtureof 20% ethylstyrene and 80% of isomeric divinylben-zene) and styrene (Aldrich) were filtered through Al2O3

to remove the inhibitors. 4-nitrophenylacrylate wasprepared by literature methods (Narasimhaswamy et al.,1993). FTIR spectra were taken on a Perkin-Elmer FT-IR 1720X spectrometer (KBr disks). Combustionelemental analyses were done on a Perkin-Elmer 2400CHN analyzer. Scanning electron micrographs weretaken on Quanta 200 3D (ESEM mode).

2.2. Polymer supports

PolyHIPE material incorporating 4-nitrophenylacry-late (PHP) was prepared by polymerizing the continuousphase of a high internal phase emulsion containingstyrene (0.78 g, 7.5 mmol), 4-nitrophenylacrylate(1.45 g, 7.5 mmol), DVB (0.65 g, 5 mmol), Span 80Fig. 1. Scanning electron micrograph of PHP.

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(0.36 g), Span 85 (0.22 g) and chlorobenzene (1.27 g) asthe organic (continuous) phase and potassium perox-odisulfate (0.05 g, 0.18 mmol) dissolved in 0.5%aqueous solution of CaCl2 (25.6 mL) as the water(discontinuous) phase. The aqueous phase was addeddrop wise to the organic phase while stirring with anoverhead stirrer at 350 rpm, for 1.5 h. The emulsion wastransferred to the mold and cured at 70 °C for 48 h. Theresulting monolithic polymer was purified via Soxhletextraction with water (24 h) and methanol (24 h).

Poly (styrene-co-4-nitrophenylacrylate) beads (BDS;diameter between 25 and 100 μm) were prepared by asuspension polymerization procedure already described(Pulko and Krajnc, 2005).

2.3. Functionalizations

For solid phase extraction experiments, both Poly-HIPE support (PHP) and beads (BDS) were furtherfunctionalized with piperazine:

1.0 g of PHP (powdered, 2.8 mmol of ester groups)or 1.0 g of BDS (2.8 mmol of ester groups) weresuspended in 50 mL of N,N-dimethylformamide and723 mg of piperazine was added (8.4 mmol; three times

excess in relation to ester groups). The reaction mixturewas stirred for 8 hours at 50 °C and another 16 hours atroom temperature. The polymer was filtered, washedwith DMF (3×10 mL), a mixture of DMF and NEt3(1:1; 3×10 mL), a mixture of DMF and MeOH (1:1;3×10 mL), MeOH (5×5 mL), THF (5×5 mL) and airdried. 0.773 g of piperazine derived PolyHIPE (PHP-PZ) and 0.934 g of piperazine derived polymer beads(BDS-PZ) were obtained. For elemental analysis datasee Table 1.

Table 1Elemental analysis data

Polymer Calculated Found mmol of reactive groups/g a

C H N C H N

BDS

74.0 5.7 3.7 73.1 5.8 3.9 2.8

PHP

74.0 5.7 3.7 71.7 5.7 3.9 2.8

BDS-PZ b

78.6 8.1 8.5 73.6 8.2 7.8 2.2

PHP-PZ b

78.6 8.1 8.5 73.3 8.0 7.4 1.9

a Calculated from elemental analysis data.b Calculated data expecting one molecule of piperazine replacing one molecule of nitrophenol.

Table 2Atrazine uptake dynamics

t (h) % atrazine sequestered

PHP-PZ a BDS-PZ a PHP-PZb

6 68 15 3112 89 23 7818 95 48 8224 96 62 8848 98 84 9372 99 97 95144 99 98 98a Atrazine concentration: 33 ppb.b Atrazine concentration: 330 ppb.

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2.4. Solid phase extraction experiments

26 mg of ground PolyHIPE piperazine derivative(PHP-PZ) or micro beads piperazine derivative (BDS-

PZ) were placed in a 200 ml flask and 100 ml of atrazinesolution (33 ppb or 330 ppb) added. Samples of solutionwere taken according to the timescale (see Table 2) andanalyzed using the procedure described below. Polymer

Fig. 2. FTIR spectra of functional PHP supports.

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samples before the sequestration of atrazine andafterwards were subjected to FTIR spectroscopy andCHN elemental analysis (Table 1).

2.5. Atrazine concentration determination

Stock solution of atrazine was prepared by weighing3.30 mg of atrazine into a 50 mL volumetric flask anddissolved in methanol. It was stored at 4 °C. Standardand working solutions of atrazine were prepared dailyfrom stock solution by diluting 2000 times with twicedistilled water obtaining a 33 ppb aqueous solution ofatrazine.

For GC/MS analysis water samples spiked withatrazine were prepared by solid phase extraction usingBond Elut LRC C18 (500 mg) Varian cartridges. Thecartridges were activated with 2 mL of methanol and5 mL of distilled water at a flow of 1 mL/min. Then125 mL of sample was passed through at a flow of10 mL/min. After drying under vacuum for 15 min theatrazine was eluted with 5 mL of ethyl acetate, theextract concentrated under vacuum for 30 min andadjusted to the final volume of 1 mL with hexane.

The atrazine concentrations were determinated byGC/MS (GC 3900, MS Saturn 2100 T— ion trap, 8410Auto sampler; Varian). A modified GC/MS/MS methodreported for the determination of different triazines(Sandra et al., 1995) was used for the quantification ofatrazine. Helium as the carrier gas at the flow rate of1 mL/min and a low bleed capillary column VF 5 ms(30 m×0.25 mm, df=0.25 μm; Varian) were used. Thetemperature of the injector was 250 °C and splitlessinjection of sample (1 μL) was performed. The GC ovenprogram started at 50 °C for 1 min, then the rate 10 °C/min was applied up to 220 °C and the final temperaturewas set for 1 min. The transfer line temperature was280 °C and ion trap temperature was 250 °C. Mass rangewas scanned from 80–250 m/z at a scan time 1 s/scan(background mass 50 m/z). The multiplier delay was setto 5 min and a multiplier offset of ±200 V was used.Molecular peak for atrazine was recorded at 215 m/z.Using MS/MS mode atrazine fragments at 173 m/z and200 m/z were used for quantification using an isolationwindow of 3 m/z.

3. Results and discussion

3.1. Preparation of 4-nitrophenylacrylate PolyHIPEsand beads

As PolyHIPE polymer supports have already provedtheir versatility in the usage as scavengers in batch and

flow through applications and acrylate based reactivePolyHIPEs have already been successfully prepared, the4-nitrophenylacrylate groups in the polymer matrix waschosen for the one step functionalization with piperazinein order to obtain insoluble porous polymers withpiperazine moieties. It has been found previously thatreactions of 4-nitrophenylacrylate based PolyHIPEs inaqueous medium required no cosolvent while in the caseof less porous beads of similar chemistry a cosolventwas needed. Furthermore, the kinetics of hydrolysis wasdependent on the degree of porosity (pore volume of thepolymer) (Krajnc et al., 2005b). Therefore, as the basisfor functionalizations and sequestration experiments, ahigh internal phase emulsion including 4-nitrophenyla-crylate and divinylbenzene (a crosslinking agent) in theorganic phase has been prepared. The emulsion wassuccessfully stabilized by a mixture of sorbitan mono-oleate and sorbitan trioleate to allow curing by thermalradical initiation using potassium peroxodisulphate.Enough of reactive acrylate was incorporated into theemulsion to obtain the loading of ester groups of2.8 mmol per gram of polymer. An appropriate amountof divinylbenzene was added to yield the degree ofcrosslinking approx. 20% (by mole). A typical Poly-HIPE structure, which is the open cellular architectureconsisting of voids (these are in place due to the dropletsof the internal phase of the emulsion) and connectingpores (windows) through the walls of the polymerizedcontinuous phase of the emulsion, can be seen on theSEM image of the produced material (PHP, Fig. 1). Theaverage diameter of the voids is approximately 32 μmand the average diameter of the windows approximately1.5 μm. As batch type experiments were used for furtherfunctionalization and the removal of atrazine fromwater, the monolithic polymers were powdered bygrinding the cylinders obtained after the polymerization

Fig. 3. Optical micrograph of BDS.

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Fig. 4. FTIR spectra of functionalized BDS supports.

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and purification. The chemistry of obtained polymerswas confirmed by FTIR spectroscopy (Fig. 2) andcombustion elemental analysis from which (from thepercentage of nitrogen) the actual loading of estergroups was determined (2.8 mmol of ester groups pergram; Table 1).

In order to compare the novel type of support with amore standard type of polymer support normally usedfor the purpose of scavenging solutions for reagents orproducts, polymer beads of the same reactive chemistryand similar degree of crosslinking were prepared by astandard suspension polymerization technique. Using anoverhead stirrer set at 750 rpm 4-nitrophenylacrylatebased beads of diameter between 25 μm and 100 μmwere obtained (see Fig. 3). It had been previously foundthat the type and the degree of the crosslinker and thestirrer speed have a strong effect on the bead sizedistribution (Pulko and Krajnc, 2005) and used onlybeads prepared via one conditions for the purpose of thisresearch (750 rpm, 20% of DVB). As with thePolyHIPE support, the chemistry of the beads wasconfirmed by FTIR (Fig. 4) and combustion elementalanalysis (Table 1). From the nitrogen percentage theloading of ester groups was calculated to be 2.8 mmolper gram of polymer beads.

3.2. Functionalizations with piperazine

Piperidine and other nucleophiles have been shown tocovalently bond atrazine and therefore remove it from the

solutions (Acosta et al., 2004). When such a reagent isimmobilized to an insoluble polymer, the material iscapable of removal of atrazine from the solution by eithera simple “tea bag” principle or by pumping the solutioncontaining atrazine through a column containing thepermeable polymer. It is known that the morphology ofthe supporting polymer can have a major influence on thekinetics of the process, especially when diffusion is themajor process of mass transfer (Hodge, 1997). With lessporous material, swelling in the reaction medium isessential for the process to run. That is one of the reasonsfor applying the PolyHIPE type of support for ourinvestigation. The reason for choosing 4-nitrophenyla-crylate as the reactive monomer is its already provencapability for functionalization with nucleophiles. Thepossibility of functionalization of 4-nitrophenylacylategroups in polymer beads (gel type, 2 or 4% crosslinkedwith DVB) with piperazine has already been demonstrat-ed (Zupan et al., 1996). It was shown that the reactionproceeded well in DMF however some immobilizedamine groups were not available due to additional cross-linking that occurred when both groups of piperazinereacted with ester groups. In this and another study by ourgroup it was demonstrated that the temperature, theconcentration of the amine, and the degree of crosslinkingare the factors affecting the type of the product (Scheme 1)(Zupan et al., 1998; Pulko and Krajnc, 2005). An excessof piperazine (approx. 3 times) and lower temperature(50 °C) was therefore used in order to achieve thefunctionalization with as low additional crosslinking as

Scheme 2. Sequestration of atrazine by functionalized polymers.

Scheme 1. Functionalization with piperazine.

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possible. For the same reason, PolyHIPE (PHP) and beads(BDS) with a high level of initial crosslinking (20%) wasused.

Simanek et al. used polystyrene based beads for theimmobilization of piperidine groups (Hollink et al.,2005). Two protocols for functionalizing the polymersyielding supported piperidine groups were described. Ourmethod is an alternative, using a more polar and a muchmore porous support and immobilizing the amine directlywith a nucleophilic substitution. PHP support is also verysuitable for the preparation of monolithic version of thereagent, appropriate for placement in a column for flowthrough applications. A direct nucleophilic displacementprocedure was used for the reaction of both types ofpolymers (PHP and BDS) with piperazine. After thesuspension of beads or powdered PHP in solvent (DMF)and the addition of an excess of piperazine (3 times)reaction was allowed to proceed at 50 °C for at most24 hours (checking every 4 hours by FTIR until thedisappearance of acrylate carbonyl and nitro groupsignals). FTIR spectroscopy for both BDS-PZ (pipera-zine immobilized on beads) and PHP-PZ (piperazineimmobilized on PolyHIPE) confirmed the change inchemical structure. Signals at 1760 cm−1 and at1345 cm−1, corresponding to acrylate carbonyl andnitro group, respectively, disappeared and amide carbon-yl signal at 1640 cm−1 can be seen (Figs. 2 and 4). Thecombustion elemental analysis data for piperazinederived beads (BDS-PZ) suggested that some additionalcrosslinking (calculated approx. 22%) occurred, since theactual nitrogen amount is lower than calculated for aproduct with one molecule of piperazine replacing onenitrophenol molecule (calculated 8.5% of nitrogen, found7.8% of nitrogen, Table 1). Similarly, 33% of additionalcrosslinking was calculated for PHP-BZ (calculated 8.5%of nitrogen, found 7.4% of nitrogen, Table 1).

Reactive polymer supports prepared in such amanner were subsequently used for the scavenging ofatrazine from aqueous solutions.

3.3. Solid phase extractions of atrazine

To mimic the actual polluted water, a low concen-tration solution of atrazine in water, namely 33 ppb, wasprepared. However, prior to scavenging experiments, wewished to check if the atrazine in water will covalentlybond to our polymers. We therefore firstly set upexperiments, suspending PHP-PZ or BDS-PZ in theaqueous solution of atrazine (Scheme 2), this time usinga much higher concentration of atrazine (13 mmol/L).FTIR spectroscopy of the polymers after 24 hoursuspension in the solution of atrazine revealed thesignal at 1550 cm−1 proving the presence of atrazineskeleton in the polymer matrix (C_N bond in theatrazine skeleton). The combustion elemental analysisgave results of 9.9 % of nitrogen for beads and 8.8% ofnitrogen for the PolyHIPE support confirming anincrease of nitrogen comparing to starting BDS-PZand PHP-PZ (7.8% and 7.4% respectively).

For the actual sequestration of atrazine from a 33 ppbconcentration, a large excess of polymer immobilizedpiperazine groups (26 mg of polymer for 100 mL ofsolution) was used. The kinetics of the polymer actionwas tested by taking the samples every 6 hours until wecould no longer detect the atrazine by the GC/MS/MStechnique. The morphology of the polymer supportproved of vital importance for the dynamics of atrazineuptake. PolyHIPE based atrazine scavenger performedmuch faster when compared to chemically analogouspolymer beads. After 6 hours, PHP-PZ removed 68% ofatrazine from the solution, while BDS-PZ removed 15%(Table 2). Near complete removal (more than 98%) wasachieved after 48 hours with the PolyHIPE support (PHP-PZ) and after 144 hours with polymer beads (BDS-PZ).We attributed this effect to a much higher porosity of thepolyHIPE (PHP) support and subsequent higher acces-sibility of reactive sites. Better compatibility of PHParylacrylate supports with aqueous media in comparisonFig. 5. Dynamics of atrazine uptake by PHP-PZ and BDS-PZ.

Fig. 6. Dynamics of atrazine uptake by PHP-PZ (atrazine conc. 33 ppbor 330 ppb).

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with less porous supports has also been observed in thecase of hydrolysis of ester groups (Krajnc et al., 2005b).The uptake dynamics are presented in Fig. 5.

As the PolyHIPE type of supported piperazine per-formed significantly better than beads prepared by sus-pension polymerization,we used only PolyHIPE (PHP-PZ)for further experiments regarding the influence of theexcess of polymer supported piperazine groups on thedynamics of uptake. Simanek et al. used a very high excessof polymer supported reactive groups (20,000 times) whenremoving atrazine from water (Hollink et al., 2005). Wealso used a high excess, namely approx. 3200 times, for thefirst set of experiments. The excesswas then lowered to 320times (using the same amount of polymer but a higherconcentration of atrazine, 330 ppb) and the dynamics ofatrazine removal measured. The results show a slightlyslower uptake, as after 12 hours 78% of atrazine from thesolution had been removed and near complete removalafter 72 hours, compared to 48 hours in the case of a largerexcess. These results are presented in Fig. 6. In the case ofthe method scale up it would be interesting to test evenlower excesses of polymer. Such experiments are currentlyunder investigation.

3.4. Control experiments

In order to establish whether any of the removedatrazine could be attributed to adsorption rather thancovalent bonding the experiments were repeated withPHP and BDS prior to the functionalization withpiperazine. Similarly large excesses of polymers wereused. Using the same detection method, namely GC/MS/MS, no reduction in atrazine concentration wasfound neither for PHP nor for BDS. We thereforeconcluded that all sequestered atrazine was due to thecovalent binding to the polymers PHP-PZ or BDS-PZ.

4. Conclusions

Piperazine derivatized PolyHIPE proved to be veryeffective for the removal of atrazine from aqueoussolutions with very low concentrations, less than 40 ppb,and performed better than polymer beads. It has beenshown that 4-nitrophenylacrylate based crosslinkedpolymers, especially the PolyHIPE version, can be anappropriate precursor for the one-step preparation ofimmobilized piperazine groups. PolyHIPE supportedpiperazine is effective also in the case of lower excess ofreactive groups in relation to atrazine which suggestsgood accessibility of reactive sites.

We therefore believe that the reactive ester approach andPolyHIPE architecture are very suitable for the preparation

of functional polymers for atrazine removal. Furthermore,the nature of the reaction between the supported piperazineand the pesticide molecule makes this approach attractivealso for the removal of other triazine type pesticides.

Currently, investigations for the preparation ofpiperazine derived columns with the monolithic versionof PolyHIPEs and applications for the removal of othertriazines by means of a flow through setup, are ongoingin our laboratories.

Acknowledgment

This work was supported by the Slovenian Ministryof Science, Technology and Higher Education. We aregrateful to Prof. B. Stanovnik from Ljubljana Universityfor the combustion elemental analyses.

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