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Introduction

Magnetic colloidal nano-particles are increasingly usedin bio-separation, in biosensor, and in diagnosis andtherapy in medicine including immunoassays, medicalimaging and targeted drug delivery [1 – 5]. Several kind of magnetic polymer nano-particles, which are usuallydispersed in a carrier liquid (usually water), thereforecalled also as ‘‘latex’’, with tailor-made propertiesdepending on the final application have been producedfrom both natural and synthetic polymers [2, 6 – 10].

Here, magnetic seed nano-particles (Fe3O4) are coatedwith a polymeric layer. Polymer coating prevents the

direct interaction of inorganic magnetic materials withthe biological system and therefore reduces toxicity andfurther increases biocompatibility of the nano-particles,and also create functional groups (coming from thepolymer chemical structure) on the surfaces of the nano-particles which would be available for modifications forspecific applications. In addition to their biocompati-bility, magnetic polymer nano-particles should fulfillsome criteria to fit further biomedical applications: no

F. SayarG. Gu ¨ venE. Pis ¸ kin

Magnetically loaded poly(methylmethacrylate-co -acrylic acid)nano-particles

Received: 8 April 2005Accepted: 7 July 2005Published online: 15 September 2005 Springer-Verlag 2005

Abstract Magnetically loaded poly-meric nano-particles carrying func-tional groups on their surface wereprepared by a two-stage process. Inthe first stage, super-paramagneticmagnetite (Fe3O4) nano-particleswere produced by a co-precipitationmethod from the aqueous solutionsof FeCl2Æ4H2O and FeCl3Æ6H2Ousing a NaOH solution. The smallestsize obtained was 40.9 nm with poly-dispersity index of 0.194 obtained byusing a Zeta Sizer. The effects of Fe2+/Fe3+ molar ratio, stirring rate,temperature, base concentration,and pH on the particle size/size dis-tribution and stability of the disper-sions were examined. Increasing therelative concentration of Fe2+ ionand decreasing the stirring rate andpH increased the particle size, whilethe concentration of NaOH andtemperature did not change theparticle size significantly. Polymercoating was achieved by emulsionpolymerization at high surfactant to

monomer ratio of methyl methacry-late (MMA) and acrylic acid whichwere used as comonomers (como-nomer ratio: 90/10 weight) with highsurfactant to monomer ratio. Thesurfactant and initiator were SDSand KPS, respectively. Nano-parti-cles in the range of 115 and 300 nmin diameter were produced depend-ing on recipe. Increasing the Fe3O4/monomer and surfactant/monomerratios, the KPS concentrationcaused a decrease in the averagediameter. Magnetic properties of thenano-particles were obtained byelectron spin resonance and vibrat-ing-sample magnetometer. Most of

the polymer-coated nano-particlesexhibited super paramagneticbehavior.

Keywords Magnetite nano-parti-cles Æ Poly(methyl methacrylate-co-acrylic acid) Æ Magnetically loadedpolymeric nano-particles Æ Emulsionpolymerization

Colloid Polym Sci (2006) 284: 965–978DOI 10.1007/s00396-005-1383-5 O R I G I N A L C O N T R I B U T I O N

F. Sayar Æ G. Gu ¨ ven Æ E. Piş kin (&)Department of Chemical Engineeringand TU ¨ B _ITAK-BiyomedtekCenter for BiomedicalTechnologies Beytepe,Hacettepe University,Ankara 06800, TurkeyE-mail: [email protected]


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sedimentation (stable latexes), uniform size and sizedistribution, high and uniform magnetic content, super-paramagnetic behavior, no toxicity, no metal-ions (e.g.,iron) leakage, etc.

Much attention has been paid to the preparation of different kinds of magnetic polymeric particles in thepast decades and various strategies have been performed

producing magnetic polymeric particles. Encapsulationof magnetic particles with preformed natural or syn-thetic polymers is the simple and classical method toprepare magnetic polymeric particles, but the particlesize and shape prepared by this method are random. Inorder to control the shape and structure of the magneticpolymeric particles, Ugelstad et al. [8] developed aneffective method through direct precipitation of iron saltinside the porous polystyrene particles. The developmentof emulsion polymerization, such as the conventionalmicro-emulsion polymerization, soap-free emulsionpolymerization, mini-emulsion polymerization and mi-cro-emulsion polymerization, has led to new synthesis

methods [2, 6, 7, 11, 12]. The typical method based onemulsion polymerization is to suspend magnetic parti-cles in the dispersed phase and then polymerize themonomer in the presence of the magnetic particles toform magnetic polymeric particles. Considering themechanism of different emulsion polymerizations, themini-emulsion polymerization is very suitable for mak-ing magnetic polymeric particles. In mini-emulsionpolymerization, the monomer droplets with magneticnano-particles act as ‘‘nano-reactors’’, the magneticpolymeric particles can be prepared in situ. Recently,Ramirez et al. [2] used a new three-step preparationroute including two mini-emulsion processes to encap-

sulate magnetic particles by polystyrene successfully,Wormuth [11] achieved 140–220 nm magnetic polymericparticles using monomers (2-hdroxyethylmethacrylateand methacrylic acid) and a diblock polymer emulsifier(poly(ethylene-co-butylene)-b-poly(ethylene oxide)), viainverse mini-emulsion polymerization. Polymethacry-late/poly(hydroxymethacrylate)-coated magnetite parti-cles could be also prepared by a single inverse micro-emulsion process, leading to particles with a narrow sizedistribution, but only with a magnetite content of 3.3 wt% [7]. Magnetite-containing nano-particles of 150–200 nm were also synthesized by seed precipitationpolymerization of methacrylic acid and hydroxyethylmethacrylate in the presence of magnetite particlescontaining tris(hydroxymethyl)aminomethane hydrox-ide in ethyl acetate medium [13].

There are several different synthetic methods to formmagnetite nano-particles. Synthetically, magnetite isformed by two fundamental processes: size reductionand aqueous precipitation. Ball mill grinding microme-ter-sized magnetite powder was one of the first methods,developed by Papell in the late 1960 s. Long grindingperiods of 500–1,000 h are required to form the nano-

particles, and therefore this method has been essentiallyreplaced by aqueous precipitation methods. Aqueousmethods for magnetite nano-particle formation include:oxidation of Fe2+; formation in water-in-oil micro-emulsion, vesicles, apoferritin; liposomes; and in thepresence of polymers. However, the most commonsynthetic route to magnetite is the co-precipitation of

hydrated divalent and trivalent iron salts in the presenceof a strong base. This process is more feasible to apply inthe production of more homogeneous magnetite parti-cles. This method results in the synthesis of a precipitate,which needs ulterior processing (centrifugation, washingand magnetic separation) before re-dispersion in waterand eventually an ulterior coating. Magnetite particlesmay also be synthesized at high temperature or byhydrolysis and oxidation of Fe(OH)2 suspensions atelevated temperatures. The majority of wet processesgive rise to products with a good crystallinity andmagnetization, but with bigger particles [14]. Recentlymagnetite nano-particles were synthesized by precipita-

tion with forced mixing in aqueous solution without anysurfactants. The method of the forced mixing has anadvantage of controlling a high product saturation de-gree and a constant pH value of the reaction systemduring the forced mixing process, which causes to formparticles with a small size and a narrow size distribution[15].

In this study, magnetically loaded polymeric nano-particles carrying functional group on their surface wereprepared by a two-stage process. First, magnetite(Fe3O4) nano-particles were produced by an aqueousphase co-precipitation method. The effect of Fe2+/Fe3+

molar ratio, stirring rate, temperature, pH, base con-

centration on the particle size, and size distribution of magnetite nano-particles were examined. Then thesenano-particles were coated with a polymeric layer bycopolymerization of methyl methacrylate (MMA) andacrylic acid in an emulsion at a high surfactant tomonomer ratio. The effect of the type of the initiator andsurfactant, the concentration of the initiator, the mag-netite/monomer ratio and surfactant/ monomer ratio onthe particle size and size distribution were examined.

Materials and methods

Materials

Ferric chloride hexahydrate (FeCl3Æ6H2O, 99%) andferrous chloride tetrahydrate (FeCl2Æ4H2O, 99%), te-tramethylammonium hydroxide (TMAOH) and sodiumhydroxide (NaOH) were purchased from Sigma (Tauf-kirchen, Germany) and used as received. Sodium dode-cyl sulfate (SDS), cetyltrimethylammonium bromide(CTAB), dodecyl-trimethylammonium bromide(DTAB), potassium persulfate (KPS), 2,2¢-az-

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obis(isobutyronitrile) (AIBN), 2,2¢-azobis(2-amidino-propane) dihydrochloride (V50) and sulfuric acid (98%)were obtained from Aldrich (Taufkirchen,Germany)and used as received. The main monomer MMA waspurchased from Fluka (Taufkirchen, Germany) and wastreated with an aqueous solution of NaOH (10%) toremove the inhibitor before use. Acrylic acid (AAc) was

also obtained from Fluka but used without any pre-treatment.

Synthesis of magnetite (Fe3O4) nano-particles

The magnetite nano-particles were produced by co-pre-cipitation of Fe2+ and Fe3+ from their aqueous solu-tions using NaOH as originally reported by Zhu et al.[15]. Our attempt was to optimize the reaction condi-tions to synthesize magnetite particles that are around50 nm or even smaller. In order to reach this task wehave conducted a series of preliminary experiments in

which we were able to decide the type of the parameterwhich would effective the size and stability of the mag-netite nano-particles, and their ranges to be changed.Then we designed the experiments given in Table 1 inwhich we changed Fe2+/Fe3+ mole ratio, stirring rate,temperature, precipitation agent (NaOH) concentration,and pH. Note that in order to increase the stability of the dispersions containing magnetite nano-particleswe introduced low polarizing positively charged ions,

i.e., N(CH3)þ4 ion of tetramethylammonium hydroxide

(TMAOH) as suggested by Massart [16].In a typical procedure, a 120 ml aqueous solution

containing Fe2+ and Fe3+ salts (total 1.25 M) and

120 ml of 5 M NaOH solution were added into the

reactor containing 160 ml distilled water at 80 C un-der N2 atmosphere, by vigorous agitation. The blackprecipitate was formed at the early phase, but themedium was continuously stirred for 2 h at a givenstirring rate and temperature followed by the slowaddition of 10 ml 25% (w/w) TMAOH to stabilize themagnetite particles.

Synthesis of magnetic polymeric nano-particles

The magnetite nano-particles produced with a recipe M1(see Table 1) with an average particle size of 40.9 nmwith a particle size distribution (as polydispersity index,PDI) of 0.194 were selected as the most suitable size forfurther studies, and used in the second step, in which theywere coated with acrylic polymers containing carboxylicacid functional groups (coming from the comonomeracrylic acid, AAc). Polymer coating was achieved byemulsion polymerization at a high surfactant to mono-

mer ratio conducted in an oil-in-water (o/w) system.Several parameters, including the magnetite nano-particles/monomer ratio, type of the surfactant (SDS,CTAB, DTAB), the type and the amount of the initiator(water-soluble KPS, oil-soluble AIBN, cationic V-50),monomer/surfactant weight ratio, were changed in dif-ferent ranges as given in the recipes in Table 2, and theparticle size and size distributions (PDI) were obtained.MMA and AAc were used as comonomers with a weightratio of 90/10. The surfactant and monomer concen-trations were 9.33 and 6.34% wt, respectively.

In a typical polymerization procedure, surfactant wasadded to the magnetite suspension previously synthe-

sized (2% wt) in a 100 ml pyrex reactor. Then, theTable 1 Co-precipitation recipe for production of magnetite nano-particlesa

Experiment no. Fe2+/Fe3+ ratio (mol/mol) Stirring rate (rpm) Temperature (C) NaOH conc. (M) pH

M1 0.5 3,000 80 5 12M2 1.0 3,000 80 5 12M3 1.5 3,000 80 5 12M4 2.0 3,000 80 5 12M5 0.5 1,000 80 5 12M6 0.5 1,500 80 5 12M7 0.5 2,000 80 5 12M8 0.5 3,000 80 5 12M9 0.5 2,000 20 5 12M10 0.5 2,000 40 5 12

M11 0.5 2,000 60 5 12M12 0.5 2,000 80 5 12M13 0.5 2,000 80 2.5 12M14 0.5 2,000 80 5.0 12M15 0.5 2,000 80 7.5 12M16 0.5 2,000 80 10.0 12M17 0.5 2,000 80 5 3M18 0.5 2,000 80 5 6M19 0.5 2,000 80 5 9M20 0.5 2,000 80 5 12

aThe reaction (stirring) time was 2 h in all these experiments.

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comonomer mixture was added to this dispersion whichwas mixing in an ultrasonic bath for about half an hourin order to ensure dispersion of the magnetite nano-particles and the monomers. Prior to polymerization,initiator was added to the solution and nitrogen gasblown through the medium for about 1–2 min to removedissolved oxygen. Polymerizations were carried out in a

constant temperature shaking bath at 65 C, undernitrogen atmosphere for 24 h. After the polymerization,the magnetic polymeric nano-particles were cleaned bywashing with methanol and water several times to re-move the surfactant and the unreacted monomers. Themagnetic nano-particles were collected with the help of amagnet and washed with distilled water several times.Then these particles were immersed in 0.1 M of H2SO4solution for 48 h to separate the magnetite nano-parti-cles, which were not coated (naked) with a polymerlayer. After that polymer-coated particles were furtherwashed with deionized water.

Characterization

The average particle size, size distributions, and surfacecharges were determined by Zeta Sizer (MalvernInstruments, Model 3000 HSA, France).

The crystalline structure and particle sizes wereinvestigated with powder X-ray diffractometer (XRD,Rigaku RU-200, Japan). The XRD patterns were takenfrom 10 to 80 (2h value) using Cu Ka radiation at roomtemperature. The XRD graphics were compared to theASTM XRD graphics in order to deduce the crystalstructure of the product.

FTIR spectra of the polymer-coated nano-particleswere obtained by using an FTIR spectrophotometer

(Shimadzu, Model: FTIR-8000 series) with KBr (IRgrade)–nano-particle mixture in the powder form.

Thermal transitions were obtained by using a differ-ential scanning calorimeter (DSC) (Shimadzu, ModelDSC-50, Japan). Nitrogen was used as the sweeping gas.The samples (5–10 mg) were heated at a scan rate of 10 C min)1 from 25 C to 500 C followed by rapid

cooling.TGA thermograms were also obtained by thermo-

gravimetric analysis (TGA) (Setaram, Labsys, France).About 10 mg sample was placed into aluminia cruciblewith increasing temperature (10 C min)1) in a con-trolled, flowing air atmosphere, and heated from 25 Cto 550 C.

Electron spin resonance (ESR) spectra of themagnetite nano-particles and their polymer-coatedforms transferred into sealed tubes of 4 mm internaldiameter were recorded using a Varian E-line 9 spec-trometer (Germany) with TE102 type double cavityresonator. ESR spectra of the samples were displayed

in the form of the first derivative of the absorptionpeak plotted against magnetic field. All measurementswere made at room temperature. Spectrometer oper-ating conditions are 9.3 GHz, 3,300 field set, 100 Hzfield modulation, 1 G peak-to-peak modulationamplitude and 1 mW microwave power to determinesignal intensities, line widths, and g-values. All ESRsignal intensities were performed using a DPPH stan-dard sample.

Magnetic properties of magnetite nanoparticles andtheir polymer-coated forms were determined using PAR-150A parallel field vibrating sample magnetometer(USA) in conjunction with a Fieldial Mark II Varian

(Germany) electromagnet assembly that can provide amaximum magnetic field of 2.0 T. The sample was

Table 2 Emulsion polymerization recipea

Experiment no. Surfactant type Initiator type Conc. (mM) Magnetite/monomerratio (wt%)

Surfactant/monomerratio (wt/wt)

P1 SDS KPS 1.5 25 1.47P2 CTAB KPS 1.5 25 1.47P3 DTAB KPS 1.5 25 1.47P4 SDS KPS 1.5 25 1.47P5 SDS AIBN 1.5 25 1.47P6 SDS V-50 1.5 25 1.47

P7 SDS KPS 1.5 25 1.47P8 SDS KPS 2.0 25 1.47P9 SDS KPS 2.5 25 1.47P10 SDS KPS 1.5 5 1.47P11 SDS KPS 1.5 25 1.47P12 SDS KPS 1.5 50 1.47P13 SDS KPS 1.5 25 1.11P14 SDS KPS 1.5 25 1.47P15 SDS KPS 1.5 25 2.94

aThe monomer concentration, polymerization temperature, and polymerization time were 6.34% wt, 65C, and 24 h, respectively, in allthese experiments.

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placed into magnetic field vibrating with a constantfrequency. Magnetic fields of 0–2.0 T were applied to thesamples for the determination of magnetization. Themagnetic properties of the particles were expressed inelectron mass units (emu).

Results and discussion

In this study, we attempted to prepare ‘‘magnetic poly-meric nano-particles’’, or in other terms ‘‘polymer-coated magnetite nano-particles’’ which are carryingfunctional group on their surfaces for further treatmentsand therefore related applications. A two-step processwas applied in which; first, Fe3O4 (‘‘magnetite’’) superparamagnetic particles were prepared by an aqueousphase co-precipitation, and then these nano-particleswere coated with a polymer layer containing functionalgroups by emulsion polymerization at a high surfactantto monomer ratio. In both the steps, our main aim wasto find out the effects of several parameters on particlesize and size distribution, and of course to reach long-term stable latexes. The results of these two groups(steps) of studies are given and discussed below sepa-rately.

Magnetite nano-particles

Particle size and size distribution of magnetitenano-particles

The average particle size and size distribution of themagnetite nano-particles prepared in the first step weremeasured by Zeta Sizer. Table 3 gives these values ob-

tained at different conditions. Note that in each grouponly one parameter was changed (given in bold in Ta-ble 3) the others were the same (see Table 1 for recipe).The average particle size was an average of minimum 30measurements, and the size distributions (or the so-called PDI) were recorded automatically by the softwareof these repeated measurements.

Effects of Fe2+/Fe3+ mole ratio

In this study, Fe2+/Fe3+ ratio in the initial mixturewas selected as one of the parameters, which is effec-

tive on the particle size of magnetite nano-particles tobe produced by co-precipitation following the state-ments given in the related literature [15 – 17]. Thechemical reaction of magnetite precipitation is ex-pected as follows:

Table 3 Effects of severalparameters on particle size andsize distribution of magnetitenano-particlea

aThe reaction conditions ineach experimental group aregiven in Table 1.

Experiment no. Average particlesize (nm)

Particle sizedistribution (PDI)

Fe2+/Fe3+ ratio (mol/mol)

M1 0.5 40.9 0.194M2 1.0 91.6 0.326M3 1.5 112.3 0.308M4 2.0 130.0 0.577

Stirring rate (rpm)M5 1,000 325.6 0.637M6 1,500 224.9 0.414M7 2,000 130.7 0.517M8 3,000 61.9 0.256

Temperature (C)M9 20 130 0.234M10 40 136.7 0.294M11 60 145 0.326M12 80 150.2 0.341

NaOH Conc. (M)M13 2.5 148.5 0.520

M14 5.0 143 0.324M15 7.5 138.2 0.317M16 10.0 134.9 0.414

pH M17 3 391 0.167M18 6 274.7 0.153M19 9 217.4 0.362M20 12 134 0.335

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Fe2þ þ 2Fe3þ þ 8OH ! Fe3O4 þ 4H2O ð1Þ

It means that the co-precipitation reaction requires1 mol of Fe2+ ions and 2 mol of Fe3+ ions (or anFe2+/Fe3+ ratio of 0.5) for stoichiometric conversion.However, the Fe2+ salts are oxidatively unstable, andare oxidized to akaganite forms (FeOOH), as indicated

by color change in the medium. Storing the reagentsunder nitrogen and careful deoxygenation of all solu-tions prior to use are pre-requirements to increase thestability and therefore to prevent these undesirablechanges. It is also recommended that the mixtureshould be prepared freshly under nitrogen atmosphere

just before the co-precipitation procedure and usedimmediately to prevent oxidation or other side reac-tions. Note that addition of base for co-precipitationshould be also rapid (1–2 s) with intense agitation. Aslow base addition not only causes formation of theoxidation but also may create homogeneous regions of the hydrated ion species, in which leads to nonmagnetic

iron compounds.Therefore, oxidative instability of the Fe2+ ion has

led some researchers to the use of molar ratio of Fe2+/Fe3+ larger than 0.5 [17, 18]. However, as also stated inthe related literature, the following reactions may occurwhen there is excess of Fe2+ ions in the medium byaddition of base for precipitation. The Fe(OH)2 formedis converted to ferric oxide, which then slowly producesferric (Fe3+) ions that interact with ferrous compoundsto yield magnetite usually with larger size [15].

Fe2þ þ NaOH ! FeðOHÞ2 ð2Þ

FeðOHÞ2

! ‘‘green rust’’ ! Fe3O4 ð3Þ

Following these statements given in the literature,we also change the Fe2+/Fe3+ ratio between 0.5 and 2.Note that it was not possible to produce magnetitelatexes stable enough if the ratio was lower than 0.5.The smallest size obtained was 40.9 nm (the sampleM1) with PDI of 0.194 in which the Fe2+/Fe3+ ratiowas 0.5. It should carefully be noted that co-precipi-tation results in magnetite nano-particles mostlyaround 8–12 nm. Therefore, most likely the nano-par-ticles (even those smallest ones) contain a number of magnetite primary particles, in other words there areaggregates.

When we increased the relative concentration of Fe2+ions in the solution, the particle size also increased, mostprobably due to side reactions mentioned above. Notethat in the preliminary studies even at these conditionsthe particle sizes were quite large and size distributionwas broad. In some recipe the nano-particles werenonmagnetic, and latexes were not stable. We were ableto prevent these undesirable events only with properlydeoxygenated solutions, working under nitrogen atmo-

sphere, and applying very rapid base addition withextensive agitation.

Effects of stirring rate

In this part of the study, effect of stirring rate was

examined on the basis of related literature [19]. Moraiset al. [19] reported the effect of stirring rate on the size of cobalt ferrite nano-particles. They synthesized cobaltferrite nano-particles by using different stirring range inthe range 2,700–8,100 rpm by co-precipitation method.It was found that increasing the stirring speed from2,700 to 8,100 rpm reduces the average particle diameterfrom 15.1 to 11.4 nm. Following this information, in ourstudies we changed the stirring rate in the range of 1,000–3,000 rpm. Smaller particles were produced athigher stirring rates as expected. The average particlesize reduced very significantly, from 325.6 to 61.9 nm byincreasing the stirring rate from 1,000 to 3,000 rpm. The

narrowest particle size distribution was observed withthe smallest particles which synthesized with the higheststirring rate (Table 3).

The decrease of the particle size as the stirring rateincreases may indicate that the nano-particle growthprocess is mainly dominated by the nano-particle diffu-sion throughout the medium instead of the diffusion of the aqueous cations into the particle surface. As thestirring rate increases, the time the nano-particle speedduring a sequence of short flies also increases, allowingthe onset of a higher density of nucleation sites, withconsequent reduction of the particle size [19].

Effects of temperature

In the related literature it was pointed out that thepreparation temperature has an important effect on theproperties of the Fe3O4 particles [20, 21]. They reportedthat it is necessary to work at high temperatures to reachoptimal crystal growth and particle formation. Thepreparation temperature, not only alters the particle sizebut also the morphology of Fe3O4 from spherical tocubic. With increasing preparation temperature thegrains grow rapidly, with different growth rates for thedifferent crystal planes that result in the changes in theshape of the products. They suggested also that the

temperature of formation should not exceed 353 K,where the quality of the particle is affected negatively.

Considering previous reports in the related literature,in this study magnetite nano-particles were synthesizedat four different temperatures, 20 C, 40 C, 60 C, and80 C. Table 3 shows the particle size and size distri-bution for the Fe3O4 nano-particles, which was preparedat observable (but not very significant) particle size in-creases were noted when the reaction temperature was

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increased. The maximum particle size (150.2 nm) wasachieved at 80 C, while the particle size distribution wasbroader (0.341) than those observed at lower tempera-tures (the sample M12 in Table 3).

Effects of NaOH concentration

Several bases including KOH, NaOH, LiOH, andNH4OH have been used in the production of magnetitenano-particles by co-precipitation method, and sug-gested that the type and concentration of the base affectsboth the size and magnetic properties of the magnetiteparticles [20, 22 – 24]. Kim and coworkers synthesized themagnetite nano-particles under different conditions byco-precipitation, and reported that when the concen-tration of precipitating NaOH solution was increasedfrom 0.9 to 1.5 M at pH 14, the particle size was in-creased from 13 to 30 A ˚ [22]. Gribanov et al. showedthat the saturation magnetization increased in order of

the following base strengths: KOH>NaOH>LiOH>NH4OH in the co-precipitation of magnetitenano-particles [20]. Babes et al. formed magnetite nano-particles using NH4OH with different concentrationsfrom 0.25 to 2.0 M and suggested that there were nosignificant effects on the magnetic properties of theparticles [23].

After preliminary studies, as suggested by Jiang et al.[24], we decided to use NaOH as the co-precipitationagent. As seen in Table 3, with the change in the NaOHconcentration in a wide range (from 2.5 to 10 M), we didnot observe a decrease in the size but not very signifi-cantly, which was around 135–140 nm. The PDI value

of magnetite nano-particle obtained by co-precipitationusing NaOH with different concentrations were in therange of 0.324–0.520.

Effects of pH

The pH value of the solution for precipitation plays animportant role in controlling the particle size and mag-netic properties [25, 26]. According to Eq. 1 given above,a complete precipitation of Fe3O4 should be expectedbetween pH 7–14 while maintaining a molar ratio of

Fe2+

/Fe3+

=0.5 under a nonoxidizing environment.Nonmagnetic iron oxyhydroxides (e.g., Goethite, Lepi-docrocite) were synthesized at very high or low pHs [25].It was proposed that pH values should be higher than 7to produce magnetite nano-particles. It was also re-ported that stability of ferro-fluid depends on pH. BelowpH 7 ferro-fluids become unstable, and resulting infaster sedimentation as the pH is decreased.

In order to observe the effects of pH on co-precipi-tation and product stability, we also studied both below(3–6) and above (9–12) neutral pH. As seen in Table 3(and also Fig. 1), increase in the pH from 3 to 12 hasresulted in a significant decrease in particle size from 391

(M17) to 134 nm (M20), and a significant increase inPDI from 0.167 to 0.362.In this group of experiments we measured also zeta

potentials of the magnetite nano-particles produced. Thezeta potentials dropped significantly when the pH wasincreased, in parallel to the decrease in particle size.Positive values were observed in acidic region, however,zeta potentials were negative above pH 7, as also re-ported by Cornell and Schertmann [26]. Note also thatnano-particles produced below pH 7 were not stable andexhibit magnetic properties.

Properties of magnetite nano-particles

FTIR spectra of magnetite (Fe3O4) nano-particle wereobtained. Two typical bands at 678.2 and 408 cm)1 were

Fig. 1 Effects of pH on particlesize and size distribution andzeta potentials of nano-particlesproduced

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observable, which correspond to the stretching vibrationof Fe–O group of magnetite nano-particles observed asalso stated in the related literature [1, 27, 28].

Crystallographic structure of the magnetite particlewas analyzed from XRD patterns [28]. Experimental d spacings obtained from these patterns are given in Ta-ble 4. Note that the XRD graph of the magnetite nano-

particles produced in this study were very similar to theASTM XRD graphics of Fe3O4, which exhibit the sameinverse spinal structure. XRD results exhibited that theiron oxide particles are composed of magnetite (Fe3O4).

Thermal behavior of the magnetite was examined byDSC. From the DSC thermograms we observed that thephysically adsorbed water leaves the structure at71.5 C. The broad exothermic transition at 193.87 Cwas attributed to the magnetite (Fe3O4) to maghemite(c-Fe2O3) transition. It is reported that temperaturesgreater than 300 C magnetite (Fe3O4) is oxidized tohematite (a-Fe2O3) [29]. Therefore, a sharp exothermicpeak was observed at 308.7 C which shows the ma-

ghemite to hematite transition.

Magnetic polymeric nano-particles

Particle size and size distribution

In this second step, for production of magnetic poly-meric nano-particles, the magnetite (Fe3O4) nano-parti-cles with an average size of 40.9 nm synthesized in theprevious step (see Table 3) were used as seed particles. Aseries of magnetic polymeric particles (called here as‘‘poly(MMA/AAc-90/10)’’copolymers) were preparedby emulsion polymerization of comonomers at a high

surfactant to monomer ratio, i.e., MMA and AAc atdifferent experimental conditions. The comonomer ra-tio, MMA/AAc was 90/10 in all cases. Note that it wasnot possible to produce emulsions when the AAc initialcontent was more than 10%, also according to ourpervious expertise [30]. In order to ensure the polymercoating these magnetic polymeric nano-particles weretitrated with 0.05 N NaOH to determine the COOHgroups on the particle surface. Calculated number of moles of COOH groups is found as 1.2 mmol g)1

polymer (for the particles in P4, see Table 2). We

changed the following parameters and investigated theireffects on mainly polymeric particle size and its distri-bution: surfactant and initiator type, magnetite (Fe3O4)/monomer ratio, surfactant (SDS)/monomer ratio, andinitiator (KPS) concentration. The findings and discus-sion are given below in the separate subsections.

Effects of surfactant and initiator type

In this section magnetic poly(MMA/AAc-90/10)copolymeric nanoparticles were produced by using dif-ferent type of surfactants (i.e., SDS, CTAB, and DTAB)and/or initiators (i.e., KPS, AIBN, and V-50) withconstant initial concentrations of 9.3 wt% and 1.5 mM,respectively. Note that we used KPS when we changedthe type of surfactant, while SDS was used in theexperiments in which the initiator type was changed.Other polymerization conditions were the same in allthis group of experiments (see Table 2 for experimental

design).Note that we were not successful to produce nano-particles/stable latexes neither with CTAB nor DTAB;instead we observed precipitations in most of the cases.This negative effect was most probably due to electro-static interactions between the surfactant and initiatormolecules. Note that CTAB and DTAB are positivelycharged due to ammonium, which attracted electro-statically the free negative ions of KPS and formed alayer on the surfaces of the micelles which reduced theeffectiveness of the initiator and neutralized surfacecharges of the forming micelles/particles that causedstability lost and therefore resulted. Similar electrostatic

attractions and their negative effects were also reportedin earlier studies in which DTAB and AOT (sodiumbis(2-ethylexylsulfosuccinate)) were used for the emul-sion polymerization of MMA [31].

In contrast, we were able to produce magnetic poly-meric nano-particles and their stable latexes when weused a negatively charged surfactant, i.e., SDS. Mostprobably, there was no adsorption of the initiator mol-ecules on the micelles; KPS radicals stayed in aqueousphase and successfully initiated emulsion polymeriza-tions at high surfactant to monomer ratio. Consideringthese results, we decided to use SDS as surfactant in therest of the experiments discussed below.

Three different initiators, two water-soluble hydro-philic (i.e., KPS and V-50) and organic phase-solublemore hydrophobic (i.e., AIBN) were used as initiator asin the emulsion polymerization at high surfactant tomonomer ratio. Note that SDS was the surfactant in allof these experiments. Table 5 gives the nano-particle sizeand its distribution obtained using different types of initiators. The smallest size and the narrowest size dis-tribution, and also more stable latexes were obtainedwith KPS, as also mentioned in similar experiments [2].

Table 4 Summary data of experimental d (exp) (A ˚ ) spacing ob-tained from the X-ray patterns and from ASTM data cards [8] foriron oxide (d (Fe3O4))

2h (exp) d (exp) d (Fe3O4)

17.460 5.07 4.85230.200 2.96 2.09956.720 1.62 1.61562.500 1.48 1.484

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For this reason, in this study KPS is selected as theinitiator and used in the rest of the polymerization rec-ipe.

Effects of initiator (KPS) concentration

We performed a series of emulsion polymerization athigh surfactant to monomer ratio with the selected ini-tiator (i.e., KPS) by changing its concentration. In thisgroup of experiments, we used KPS with three differentconcentrations (1.5, 2.0, and 2.5 mM) in the feed andproduced magnetic poly(MMA/AAc-90/10) copoly-meric particles were prepared to investigate the effect of KPS concentration on the particle size and size distri-bution. Table 5 indicates that the higher initiator con-centration results in a decrease in the particle size. Notethat KPS is a water-soluble initiator. When its concen-tration is high the initiator (KPS) molecules diffusewithin the dispersion phase (i.e., water) faster and find

the micelles swollen with monomer (typical in micro-emulsions) and or magnetite particles carrying monomeron their surfaces (most probably), and start polymeri-zation in more points which leads smaller particles, asalso reported by Macias et al. [31]. Xie and coworkershave also prepared magnetic poly(St-BA-MMA) nano-particles with different concentrations of KPS [12]. Theyreported that an increase in the KPS content may lead toa higher number of primary particles, and thereforesmaller particle size are achieved, however, the increasein the electrolyte concentration in the polymerizationsystem may depress the electrical double layer of theprimary particle surface which may cause an increase in

the particle size. When one of these two factors waspredominant over the other one, the particles were big-ger or smaller. Increasing the ionic strength (due to in-crease in the KPS concentration) may cause a decreasein the Debye length which might cause coagulation orcoalescence process as the particles can approach close.In our case, since we have not seen any coagulation(neither microscopically nor by Zeta Sizer), most prob-ably increasing KPS concentration leads to the forma-

tion of higher number of primary particles and also thenumber of polymeric particles containing no magnetiteparticles, as a result, smaller particle sizes were observed.

Effects of magnetite (Fe3O4)/monomer ratio

As mentioned in the related literature, the ratio of magnetite (the seed particles) to the monomer used inthe feed is one of the most important parameters whichare effective not only on the size and distribution butalso on the quality (sphericity and mono dispersity) of the latex particles synthesized and the product (latex)stability [8, 9, 25, 32]. For instance, Kondo and Fukudaprepared magnetic poly(St/NIPAM/MAA) latex parti-cles using two-step emulsifier-free emulsion polymeri-zation and reported that the hydrodynamic diameter of the magnetic latex particles decreased with increasingweight ratio of magnetite to monomers [32].

In this group of studies, we copolymerized MMA and

AAc in the media containing different amounts of magnetite and comonomers. The weight percentage of magnetite (considering only amounts of magnetite andtotal monomers), which is the so-called ‘‘magnetite/monomer ratio’’, was 5%, 25%, and 50%. As seen here,the average particle size and size distribution decreasedfrom 136.5 nm to 120.9 nm and from 0.3 to 0.183,respectively, when we increased the weight ratio of magnetite/monomer from 5 to 50 (Table 6), which wereconsiderable changes. This is expected, because when themagnetite content is increased there will be more seedparticles in the reaction medium, if we assumed thatpolymerization will occur homogeneously (evenly)

throughout the reaction vessel, each particles will get lessamount of polymer (means thinner polymer coating),and therefore particles with smaller sizes will be formed.Note that we did attempt to use less magnetite (less than5%) or more (more than 50%) in the preliminaryexperiments. However, in the first case the copolymericparticles were not magnetic (not observable magnetism),and in the later case aggregation and incomplete poly-mer coatings were observed.

Table 5 Effects of initiatortype on the particle size and sizedistribution of magneticpoly(MMA/AAc-90/10)copolymeric particles



Initiator typeP4 KPS 125.2 0.193P5 AIBN 150.0 0.218P6 V-50 148.8 0.273KPS concentration (mM)

P7 1.5 151.9 0.217P8 2.0 130.1 0.195P9 2.5 125.2 0.183

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Table 6 Effects of severalparameters on the particle sizeand size distribution of magnetic poly(MMA/AAc-90/10) copolymeric particles



Magnetite (Fe3O4) /monomer ratio (wt%)P10 5 136.5 0.300P11 25 125.2 0.183P12 50 120.9 0.158KPS concentration (mM)

P13 1.11 159.7 0.251P14 1.47 125.2 0.183P15 2.94 117.5 0.206

Fig. 2 Representative DSCthermograms: (a) plain nano-particles and (b) magnetic nano-particles (containing 25%Fe3O4)

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Effects of surfactant (SDS)/monomer ratio

Note that the surfactant (SDS) concentration, especiallythe ratio of SDS/monomer is a very important param-eter in general in emulsion polymerization. It was re-ported that if the SDS/monomer mass ratio is lowerthan about 1.0, the polymerization shifts from micro-

emulsion to mini-emulsion or even to classical emulsionlimits (at lower values). It is also recommended that inorder to obtain stable latexes the SDS/monomer ratio inmicro-emulsion formulation should be around 1.0–3.0[33 – 35]. Note that this is still quite high. High concen-trations of SDS mean pollution of latexes which maycause problems in further applications. Since we haveseed magnetite nano-particles in the feed, we decided to

use lesser amounts of SDS (in the range of 1.1–2.94 wt/wt).

The effects of surfactant (SDS)/monomer mass ratioon the particle size and the PDI of the nano-particles aregiven in Table 6. Particle diameters and the PDI of thesepolymer latexes were in the range of 117.5–159.7 nm(P13, P14, and P15) and 0.183–0.251, respectively. As

expected that the average size of the nano-particles de-creased with increasing SDS concentration, which issimilar to those reported by Xie for the magnetic poly(St/BA/MAA) nano-particles [12]. More SDS means moremicelles/particle are supported by the surfactant mole-cules which leads smaller particles and also more stablelatexes due to surface-adsorbed SDS molecules thatcause electrostatic repulsion between the latex particles.

Fig. 3 Representative TGA

thermograms: (a) plain nano-particles and (b) magnetic nano-particles (containing 25%Fe3O4)

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Properties of magnetic polymeric nano-particles

FTIR spectra of poly(MMA/AAc-90/10) copolymericnano-particles produced both without using (‘‘plain’’ or‘‘nonmagnetic polymeric nano-particles’’) and withusing magnetite seed particles (‘‘magnetic polymericnano-particles’’) were taken. Both spectra were very

similar and the following peaks were identified: at3,550 cm)1 OH stretching in carboxylic acid groups, at2,853–2,922 cm)1 CH stretching in CH2 and CH3groups, at 1,736 cm)1 C=O stretching in carbonylgroups, at 1,466 cm)1 CH2 bending vibration, at1,392 cm)1 CH3 bending vibration, at 1,223–1,080 cm

)1

C–O–C vibration in ester groups, at 991 cm)1 C–Cstretching of main chain, at 845 cm)1 CH out of plaindeformation, at 756 cm)1 CH2 rocking. There was onlyone new signal at 671 cm)1 , specific for the stretchingvibration of Fe–O groups from the magnetite nano-particles, which confirmed the coating on the magnetiteseed particles with copolymer (note that we have sepa-rated nonmagnetic and magnetic polymeric particleseffectively), as also reported in the related literature[1, 27] .

Thermal properties and behavior of the plain andmagnetic copolymeric nano-particles were examined byDSC and TGA. Representative DSC and TGA ther-mograms are given in Figs. 2 and 3, respectively. Theglass transition (T g), melting temperature (T m), anddecomposition temperatures (T decom) for both nano-particles are given in Table 7. As seen here, there arevery significant increases in the T g, T m, and T decom val-ues when magnetite particles incorporated within thecopolymeric nano-particles as also reported by Denget al. [10]. The decomposition temperatures obtainedwith TGA were both higher and the temperature rangeswere wider comparing those obtained with DSC. How-ever, in both cases, thermal stability increases were veryprofound. Most probably, polymerization on magnetiteaggregates and it did cause formation of differentstructures at molecular level (different entanglement,crystallinity, and even may be orientation), which inturn, resulted those changes. In general, sense may beimportant in the application of these nano-particles at

Table 7 Thermal properties of plain and magnetic poly(MMA/AAc-90/10) copolymeric nano-particles

T g (C) T m (C) T decom (C)a

Plain poly(MMA/AAc-90/10) copolymeric nano-particles94.1 117.7 195–210 (225–395)Magnetic poly(MMA/AAc-90/10) copolymeric nano-particles126.9 208.7 300–375 (235–410)b (260–425)c

aThe values in the parentheses were found from TGA thermograms.bThe values for the nano-particles containing 15% magnetite.cThe values for the nano-particles containing 25% magnetite.

Fig. 4 Representative ESR spectra: (a) magnetite nano-particles(Fe3O4); (b) magnetic polymeric nano-particles (containing 25%Fe3O4); and (c) plain polymeric nano-particles

Fig. 5 Representative magnetization curves: (a) magnetite nano-particles (Fe3O4); (b) magnetic nano-particles (containing 25%Fe3O4)

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elevated temperatures. The TGA results also show thatthe increase in the magnetite content further improvesthe thermal stability of the nanoparticles. The magneticcopolymers containing 15 and 25% Fe3O4 particles weredecomposed at 235–410 and 260–425 C, respectively.As seen in the TGA thermographs (Fig. 3), decompo-sition occurs in two steps. Up to 600 C, the mass loss of

plain copolymeric nano-particles is 75%, while it is 72%and 66% for magnetic nano-particles containing 15%and 25% Fe3O4, respectively.

The presence of magnetite particles in the copoly-meric nano-particles was confirmed by ESR. Represen-tative ESR spectra of the plain copolymeric nano-particles, magnetic copolymeric nano-particles, andmagnetite (Fe3O4) particles are given in Fig. 4. As seenhere, magnetite particles alone typically show intensityof 100 around 3,300 G applied magnetic field, whilerelative intensity of the magnetic copolymeric nano-particles decreases to about 45 at the maximum whichshifts to around 3,800 G due to the effects of the poly-

meric coating on the magnetite seed particles. Note alsothat the plain poly(MMA/AAc) copolymer nano-parti-cles do not exhibit any magnetic property, as expected.

The magnetic properties of magnetic polymeric par-ticles were also studied by using a vibrating-samplemagnetometer. Here, applied magnetic field (tesla) waschanged and magnetization (M , emu g)1) was measured.A representative magnetization curves for magnetitenano-particles (Fe3O4) and magnetic copolymeric nano-particles are shown in Fig. 5. Note that both the mag-netite and the magnetic copolymeric nano-particles weresuper-paramagnetic and no remanence was observedwhen the magnetic field is removed. The saturation

magnetizations obtained for magnetite and magneticcopolymeric nano-particles are 6.601 and 3.190 emu g)1,respectively.

Conclusions

Super paramagnetic poly(MMA/AAc-90/10) nano-par-ticles were synthesized in situ via two steps: (1) prepa-ration of magnetite (Fe3O4) nano-particles by co-precipitation method and (2) emulsion polymerization athigh surfactant/monomer ratio of MMA and AAc

monomers in the presence Fe3O4 nano-particles. Studieson particle size and size distribution of the magnetitenano-particles revealed that the change in Fe2+/Fe3+

molar ratio, stirring rate, and pH plays an importantrole in controlling the particle size and magnetic prop-erty. On the other hand, the concentration of NaOH andtemperature does not affect the size significantly. Thesmallest size obtained was 40.9 nm. Conventionalemulsion polymerization at high surfactant/monomerratio technique was used for the coating of the magnetitenano-particles with this size. The results showed thatSDS and KPS are the most convenient surface activeagent and initiator for this system. Depending on the

reaction parameters the size range obtained was between115 and 300 nm for the polymer-coated nano-particles.Increasing magnetite, surfactant content, and KPSconcentration decreased the particle size. Zeta potentialof the smallest particle was )20.1 mV, which indicatesthe carboxylic acid groups on the particle surfaces.These magnetically loaded polymeric nano-particles didnot loose their magnetic property after the coating. Theystill showed super-paramagnetic property. Applicationsof these novel magnetically loaded polymeric nano-particles for diverse biomedical applications includingelectrochemical-based DNA sensors and separation/purification of stem cells are under investigation.

Acknowledgement Prof. Erhan Piskin was supported by TurkishAcademy of Sciences as a full member.

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