Novel and facile synthesis of magnetic composites by a modified co-precipitation method

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Materials Chemistry and Physics 130 (2011) 624– 634

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Materials Chemistry and Physics

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Novel and facile synthesis of magnetic composites by a modified co-precipitationmethod

V.L. Lassallea,∗, R.D. Zyslerb, M.L. Ferreirac

a INQUISUR, Depto de Química, Avda Alem 1253, 8000, B. Blanca, Bs As, Argentinab Centro Atomico Bariloche – Instituto Balseiro, S. C. de Bariloche, RN, Argentinac PLAPIQUI-UNS-CONICET-Camino La Carrindanga Km 7, B. Blanca, Argentina

a r t i c l e i n f o

Article history:Received 8 March 2011Received in revised form 7 June 2011Accepted 10 July 2011

Keywords:ChitosanMagnetiteCelluloseBulkMagnetic composites

a b s t r a c t

A simple and effective method is proposed to synthesize chitosan/magnetite (CS/MAG) and cellu-lose/magnetite (CEL/MAG) composites. Biopolymers were employed in bulk during the co-precipitationof magnetite, avoiding the problems associated to the polymer’s solubility. FTIR and XRD analysis revealedthat the polymeric template was perfectly integrated to the crystalline structure of magnetite. Measure-ments of zeta (Z) potential indicated that new materials present different charged surfaces dependingon the biopolymer. SEM/TEM and dynamic light scattering (DLS) results demonstrated that particu-lated materials with average diameters lower than 1 �m were obtained. The magnetic properties of suchcomposites were qualitatively verified. The as-obtained hybrids composites were tested for proteinsadsorption using bovine serum albumin (BSA) and insulin (Ins) as models. It was found that CS/MAG wasa better protein adsorbent than CEL/MAG. One important drawback of CEL/MAG was the lack of stabilityunder the proteins adsorption conditions.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Investigation on novel materials composed of polymer andmagnetic particles are of great interest due to their potentialapplications in biomedical and biotechnological fields [1,2]. Ironoxide particles, such as magnetite (Fe3O4) and also maghemite(�-Fe2O3) structure, were incorporated in a wide variety of poly-meric networks, with the goal of to improve the biocompatibilityor bioactivity for specific applications, such as magnetic cell sep-aration, target drug delivery system and magnetic resonanceimaging of clinical diagnosis [3]. Encapsulation or dispersion ofmagnetic nano-particles into organic polymers to form magneticcomposites endows materials with some important propertiesthat bare, uncoated particles lack. Polymer coatings can furtherenhance compatibility with organic ingredients, reduce suscep-tibility to leaching and protect particle surfaces from oxidation.Consequently, encapsulation improves dispersion qualities, chem-ical stability and reduces toxicity [4].

Different approaches have been used to generate magnetic-polymeric materials such as cross-linking of polymer templates[5], emulsion-based methods [6,7] and co-precipitation methods[8]. Co-precipitation-based methods are the most typical ones. The

∗ Corresponding author.E-mail addresses: [email protected], [email protected]

(V.L. Lassalle).

procedure involves the use of stoichiometric ferrous and ferric ionsin the presence of polymeric templates (usually in solution), and theprecipitation of iron oxide with aqueous NaOH or NH4OH, addedquickly [9,10].

Within the enormous list of polymeric templates suitable to pre-pare composites with iron oxides, the biodegradable ones, such aschitosan (CS) and cellulose (CEL), have attracted many researchers’interest regarding to the final applications of the prepared materi-als.

Chitosan has excellent properties for the adsorption, principallydue to the presence of amino groups (–NH2), which can interactwith Fe ions in solution by ion-exchange and complexation reac-tions [11]. Therefore, the articles in the open literature regardingto the synthesis of magnetic CS composites using diverse meth-ods are abundant [12–14]. For instance, Wu et al. have preparedmagnetic Fe3O4–CS nanoparticles with different reagent additionorders in different temperature and concentration conditions bythe covalent binding of CS and sodium tripolyphosphate (TPP) onthe Fe3O4 nanoparticles by the one-step method. The as-preparedmaterials were used as support to prepare immobilized lipases[15]. Zhi et al. proposed a new method of in situ preparation ofmagnetic chitosan/Fe3O4 composite nanoparticles in microreactorsof tiny water pools of water-in-oil microemulsion. By addition ofthe solution of NaOH into the microemulsion containing solubi-lized chitosan and ferrous salt, the magnetic Fe3O4 and chitosannanoparticles were precipitated from the system [16]. This is avariant of the method here presented.

0254-0584/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.matchemphys.2011.07.035


V.L. Lassalle et al. / Materials Chemistry and Physics 130 (2011) 624– 634 625

In situ mineralization was another alternative route to achieveCS magnetic composites proposed in the open literature. It wassuggested that CS–Fe complex was used as a precursor for the min-eralization, and the chelation effect of CS–Fe complex may controlmagnetite mineralization. The mineralized magnetite nanoparti-cles were well characterized, and the mineralization mechanismwas discussed in the work of Wang et al. [17].

Cellulose-covered inorganic particles have emerged as a newkind of composite materials with suitable adsorbent properties, lowtoxicity and high biocompatibility [18]. Luo et al. prepared a sor-bent, in which the maghemite nanoparticles and activated carbonwere embedded in the cellulose matrix, to improve the adsorptioncapacity of organic dyes. Their structure, adsorption behaviours, aswell as desorption and regeneration properties were characterizedto evaluate the potential applications in the wastewater treatmentfield [19].

The preparation of cellulose-coated magnetite (CCM) nanopar-ticles, obtained by coagulation of an aqueous solution of cellulosecontaining magnetite nanoparticles, has been reported by Namb-deo et al. The as-obtained magnetic composites were efficientlyemployed as supports for the immobilization of amylase [20].

Although published articles regarding to the design of magneticcomposites-nano, micro and macroparticulated-using CS and CELare currently increasing, there are some drawbacks associated tothe polymers solubility, baffling its practical applications. It is well-known that chitosan is insoluble in most of conventional solventsand only dissolves below pH 5.5 [21]. On the other hand, cellu-lose is practically insoluble in common solvents due to its highlycrystalline structure and the presence of strong inter- and intra-molecular hydrogen-bonding interactions among hydroxyl groups[22].

The goal of this work is to synthesize magnetic composites,with sizes below 1 �m, by simple co-precipitation method, usingbiodegradable polymers (chitosan and cellulose) as supports ofmagnetite nanoparticles. The novelty of this proposal is that thepolymers are used in bulk, on the precipitation media of magnetite,with the aim to solve the drawbacks related to the polymers’s sol-ubility. To the best of author’s knowledge, the use of polymers insolid state to prepare nano, micro or even macromagnetic compos-ites has not been earlier reported in the open literature. Throughthis methodology, new hybrids materials are obtained, achievingthe biocompatibility and functionality of the polymeric templatesand the magnetic capability provided by the iron oxide. The appli-cation perspectives of materials with these characteristics are verypromissory. Here it was focused mainly in applications as supportsfor lipase immobilization in the design of biocatalysts, as biosen-sors or in the protein purification field. In view of this, the capabilityto adsorb model proteins, such as bovine serum albumin (BSA) andInsulin (Ins.), has been selected as an additional characterizationtool.

2. Materials and methods

Bi-distilled water was employed as protein solvents. Chitosan (CS), commer-cialized as Chitoclear, was provided by Primex (Iceland). Cellulose was �-cellulosefrom Sigma–Aldrich (Argentina). Analytical grade solvents provided by Dorwill(Argentina, SA) were used in all the described procedures.

Ins. was supplied by Betasint U-40 (Beta Laboratories, Argentina) as an aqueouscommercial solution of porcine neutral insulin. Bovine serum albumin (BSA) wassupplied by Laboratorios Wiener (Argentina).

2.1. Synthesis of magnetite

The co-precipitation method was used for the synthesis of magnetite [23].In brief, 3.254 g of FeCl3·6H2O (0.0121 moles of Fe3+) and 1.789 g of FeSO4

(6.46 × 10−3 moles Fe2+) were dissolved in 100 ml of distilled water. The solution wasstirred at 70 ◦C under atmosphere of N2 during 25 min. Then, 25 ml of NaOH 5 M wereadded to precipitate the oxide. The alkali addition was performed at controlled rate(approx. 1 ml min−1) to minimize the magnetite aggregation. The solution changed

from brown, at the initial addition stages, to black at the end of the NaOH addi-tion. The mixture was allowed to complete the magnetite formation during 30 min.The obtained dark solution was decanted and supernatant was extracted. Then,bidistilled water (approx. 80 ml) was added to remove the impurities by filtration.These washing cycles were repeated three times to ensure complete elimination ofthe ionic moieties. The solid was dried in oven at 45 ◦C overnight under vacuumrendering 1 g of magnetite.

2.2. Synthesis of the magnetic composites

The magnetite synthesis was adapted for the magnetic composites prepara-tion. The above described procedure was repeated using the same reagents ratios.The only difference was that the Fe2+/Fe3+ solution was in contact with 1 g of solidbiopolymer under stirring at temperature and N2 flow, during 25 min prior to thealkali aggregation.

Dark diluted slurry was formed immediately after NaOH was added. After purifi-cation process (idem than in the raw magnetite case) one uniform solid phase wasobserved.

2.3. Stability and adsorption assays

Stability of magnetic composites in the media of protein adsorption has beenevaluated prior to the contact with BSA and Ins. 20 mg of magnetic supports or puremagnetite were dispersed in 5 ml of bidistilled water under magnetic stirring at 37 ◦Cduring 180 min. Aliquots of 200 �l were withdrawn at different intervals of time anddiluted to 3 ml with bi-distilled water. The dilutions were measured by UV/vis spec-trometry to evaluate the presence of decomposition moieties or Fe2+/Fe3+ species insolution interfering in the proteins UV quantification.

BSA and Ins. adsorption assays were conducted following the above describedprotocol. About 6 or 12 mg of proteins (Ins. and BSA, respectively) were added to20 mg of the supports dispersed in 5 ml of bi-distilled water at 37 ◦C under vigor-ous magnetic stirring during 180 min. The decrease of the protein amount in thesolution is a measure of the protein adsorbed on the solid supports. The absorbancevalue recorded at the end of the incubation period (180 min) was considered tocalculate the loading efficiency using calibration curves and a protocol previouslyimplemented for protein quantification [24]. In brief, the response to UV/vis of dif-ferent proteins was evaluated focusing on two main points: (i) the possibility ofthe proteins aggregation in solution, considering that the aggregations levels arestrongly dependent on protein structure and composition. The study was done tryingto reproduce the experimental conditions fixed during protein adsorptions, speciallythe stirring; (ii) the use of the same protein as standard in the place of other suchas BSA (that is the most common protein used as standard). Within this work wehave obtained a simple protocol to quantify proteins and enzymes avoiding mistakesderived from the above mentioned and more experimental facts (such as ionic force,between others) [24].

The loading efficiency was calculated from Eq. (1), as the percentage of proteinadsorbed relative to the initial amount of protein added to the adsorption media:

Loading efficiency (%) = mg initial protein − mg final protein in solutionmg initial protein

× 100

(1)

Mg initial protein refers to the amount of BSA or Ins. initially added. Mg finalprotein in solution is calculated by UV/vis applied to the supernatant of adsorption.Similarly, adsorption of the same proteins on bare magnetite and biopolymers wasperformed for comparison.

3. Characterization techniques

3.1. FTIR spectroscopy

FTIR spectroscopy was employed to corroborate the presenceof polymeric moieties on the magnetite as well as to confirmthe adsorption of proteins on magnetic supports. A FTIR (DRIFTS)Thermo Scientific Nicolet 6700 spectrometer was used for record-ing spectra in the range 4000–400 cm−1. A few milligrams of thesamples (roughly 10–20 mg) were mixed in a mortar manually withnear 50 mg of KBr powder. The mixture was placed in the sampleunit of FTIR-DRIFTS.

3.2. Electron microscopy

Scanning electronic microscopy (SEM, JEOL 35 CF 1983, TokioJapan) from CRIBABB (Bahía Blanca, Argentina) and transmission


626 V.L. Lassalle et al. / Materials Chemistry and Physics 130 (2011) 624– 634

electronic microscopy (TEM, JEOL 100 CX II, JEOL, TOKIO, Japón, 1983)were used to examine the morphology of the carriers.

3.3. X-ray diffraction (XRD)

XRD analysis was performed to confirm the crystalline pat-terns of magnetite. The assays were recorded by a PHILIPS PW1710diffraction spectrometer with anode of Cu and a curve monochro-mator of graphite from CRIBABB–CCT (Bahía Blanca, Argentina).

3.4. UV/vis spectrophotometry

A UV/visible spectrophotometer Shimadzu 160 Japan, equippedwith a computer system for data acquisition, was used to evaluatethe stability of the composites and to quantify the adsorbed protein.

3.5. Z potential and particle size measurements

A Malvern Zetasizer was employed to measure the Z poten-tial and the average particle diameter. Dispersions of the magneticcomposites, magnetite and pure polymers were prepared in mix-ture ethanol/bi-distilled water 50/50 (pH approx. 7.6–8) to measurethe Z potential. The same dispersions were ultrasonicated during60 min to measure the average particle sizes (nm).

3.6. Area BET and pore size

The area BET, the pore size and volume were measured withArea BET Nova 1200e equipment (PLAPIQUI, Argentina).

3.7. Magnetic measurements

Magnetic properties were measured with a commercial vibrat-ing sample magnetometer (VSM) at room temperature with amagnetic field in the −10 kOe to 10 kOe range.

4. Results and discussion

4.1. Characterization of magnetic composites

4.1.1. FTIR analysisThe binding of Fe3O4 (MAG) to biopolymers was confirmed by

FT-IR analysis. FT-IR spectrum of iron oxide exhibited strong bandsin the low-frequency region (1000–500 cm−1) due to the iron oxideskeleton. This pattern is consistent with the magnetite (MAG) spec-trum since the band located at 570 cm−1 is typical of MAG and couldbe useful to distinguish it between other Fe oxides [25]. In Fig. 1a thespectra of raw CS is compared with the corresponding to CS/MAGcomposite. The bands assigned to the polymer functional groups,i.e. at 3400(�NH and OH), 1640(� N–H) and near 1100 cm−1 (�COC)are observed in the spectra of the magnetic composite. The char-acteristic band of Fe–O at 573 cm−1 also appears in the spectrumof CS/MAG in Fig. 1a. The spectrum of CEL is compared with thecorresponding to CEL/MAG composite in Fig. 1b. Typical CEL. bandsappear at 3500 (�OH), 1620(�COH) and near 1100 cm−1 (�COC).The spectrum of the composite presents those bands although aslight shift of the C–OH signal is appreciated and may be attributedto interactions with iron oxides. The band corresponding to Fe–Ois also evident in this spectrum.

4.1.2. XRDX-ray diffraction was used to identify the crystal structure of

the iron oxide dispersed in biopolymeric matrix. It is worth notingthat XRD pattern of MAG is the same than maghemite (�-Fe2O3),therefore it is not possible to discern between the crystalline struc-tures of both oxides using XRD. However it is possible to identify

Fig. 1. (a) DRIFTS spectra of CS and CS/MAG and (b) DRIFTS spectra of CEL andCEL/MAG.

between them by simple visual inspection of the sample imme-diately after precipitation. In the case of ferric oxide obtainedwithin this work the colour was black (characteristic of MAG)while in the maghemite case a brownish solid should be obtained.Therefore it is possible to assume that MAG was predominantlyobtained.

XRD patterns of formed composites (a) with CS and (b) with CELare shown in Fig. 2. The X-ray diffraction patterns of raw Fe3O4 werecomparable to standard Fe3O4 included in Table 1. The characteris-tic peak of crystalline chitosan should appear at 2� of approximate20.01◦ but is not observed in the diffractogram corresponding toCS/MAG shown in Fig. 2, suggesting that only amorphous CS ispresent. As a difference the peak associated to crystalline CEL isobserved at almost 2� = 22.5◦ in the diffractogram correspondingto CEL/MAG in Fig. 2 [26]. Sharp diffraction peaks appearing at2� = 30.31, 35.71, 43.31, 57.61 and 62.81◦ in both composites cor-respond to the scattering from (2 2 0), (3 1 1), (4 0 0), (5 1 1) and(4 4 0) of magnetite, respectively. The fact that XRD patterns forCS/MAG, CEL/MAG and raw magnetite were mostly similar, meansthat hybrids materials are formed and that the crystal structure ofFe3O4 has not been modified. Then, the iron oxide existing in theformed composite is exactly magnetite.

4.1.3. Morphology of magnetic compositesVisual inspection of magnetic composites reveals that com-

pletely new materials with aspect greatly different to the polymeric



Fig. 2. XRD of magnetic composites.

precursors have been obtained through this procedure. To illus-trate, images of raw CS and CEL are compared with the resultingmagnetic composites in Fig. 3.

Electronic microscopy was used to analyze the compositesmorphology. TEM micrographies comparing raw biopolymers,magnetite and magnetic composites are included in Fig. 4. The rawmagnetite synthesized by co-precipitation presents highly aggre-gated nanoparticles whose individual sizes are lower than 20 nm(see Fig. 4a). As a difference the biopolymer’s morphology appearsto be less uniform as it is expected from typical amorphous biopoly-mers (see Fig. 4b and c). The particle shape of CEL/MAG compositesis quite comparable to the magnetite one. A homogeneous struc-ture is observed where the CEL domains are not distinguishedfrom the iron oxide ones (see Fig. 4d). The morphology of theCS-based magnetic composite is more heterogeneous. A bipha-

Table 1Crystallographic pattern of magnetite.

Name: magnetite, synFormula: FeFe2O4

d value Angle Rel. lnt.

4.8520 18.270 82.9670 30.095 302.5320 35.423 1002.4243 37.053 82.0993 43.053 201.7146 53.392 101.6158 56.944 301.4845 62.516 401.4192 65.745 21.3277 70.926 41.2807 73.950 101.2659 74.962 41.2119 78.931 21.1221 86.704 41.0930 89.620 121.0496 94.428 60.9896 102.228 20.9695 105.222 60.9632 106.209 40.9388 110.273 40.8952 118.741 20.8802 122.124 60.8569 128.038 80.8233 138.659 40.8117 143.244 60.8080 144.857 4

JCPDS (Joint Committee on Powder Diffraction Standards) card number for mag-netite: 19-629.

Fig. 3. (a) Photographs of raw CS and MAG/CS and (b) photographs of raw CEL andMAG/CEL.

sic structure composed of magnetic and polymeric amorphousdomains is clearly distinguished (see Fig. 4e).

The SEM images of raw polymers and the corresponding to themagnetic composites are presented in Fig. 5. Comparing the imagesof CS and CS/MAG including in Fig. 5a and b, respectively, the mor-phology of the composites does not seem different enough from theoriginal polymeric one. In the case of the CEL, the fibrous charac-teristic morphology of the original polysaccharide is fully missingin the magnetic composite structure (see Fig. 5c and d). This find-ing suggests that CEL undergoes fragmentation and de-aggregationin contact with Fe2+/Fe3+ solutions. After alkalinization and co-precipitation of magnetite, the structure observed in the figure isobtained. According to the final morphology and size of MAG/CELobserved in Fig. 5, the mechanism of CEL/MAG formation could bedescribed in two steps: (i) the first: where CEL is in contact withferric/ferrous solutions and undergoes a great change in its typi-cal morphology due to a process of mechanical nature and (ii) thealkali addition where the coprecipitation properly takes place. Inthis last, interactions between iron oxide nucleolus (in formation)and surface functional groups of CEL are of physical nature.

4.1.4. Area BET and pore sizeMeasurements of BET area pore size and pore volume, were

performed on the raw materials (oxide and polymers) and on themagnetic composites. The obtained data, as well as the estimatedMAG content in the composites, are included in Table 2. Huge dif-ferences are appreciated in the evaluated parameters comparingthe raw polymers and the magnetite with the magnetic compos-ites. Typical low BET area of raw CS is modified by the compositeformation. The BET area of CS/MAG is about 23 times higher thanthe corresponding to the original biopolymer.

In the case of CEL the increment is greater since the BET area ofCEL/MAG is almost 32 times higher than the pure polysaccharide.



Fig. 4. (a) TEM micrography of MAG (scale 1 cm = 220 nm); (b) TEM micrography of pure CEL (scale 1 cm = 220 nm); (c) TEM micrography of pure CS (scale 1 cm = 220 nm);(d) TEM micrography of MAG/CEL (scale 1 cm = 110 nm); (e) TEM micrography of MAG/CS (scale 1 cm = 220 nm).

Table 2Area BET, size pore distribution and pore volume of magnetic composites, magnetite and biopolymers.

BET (m2 g−1) Average pore size from PSDa (A) Pore volume (cm3 g−1) %MAG content b

CS 0.50 900 6.51 × 10−4 –CEL 1.40 573 2.73 × 10−3 –CS/MAG 12.0 13 1.82 × 10−2 55CEL/MAG 46.2 44 0.12 60MAG. 80.0 – – 100

a Pore size distribution.b Theoretical content based on the yield of magnetite reaction expressed as %MAG (w/w).



Fig. 5. (a) SEM micrography of pure CS; (b) SEM micrography of CS/MAG; (c) SEM micrography of pure CEL; (d) SEM micrography of CEL/MAG.

However, the BET area of the magnetic composites appears to belower than the corresponding to raw magnetite (80 m2 g−1).

An important reduction of the pore size is evidenced in themagnetic composites comparing with the data from the rawbiopolymers, following the BJH method. However, it is probablethat these reported pore sizes are effectively inter-particle’s spaces,looking at the very low BET area obtained for CS and CEL. In the caseof the MAG/CEL and MAG/CS, the BET area obtained is related tothe supported magnetite, whereas the pore volume may be corre-lated with the small particle size of the supported magnetite. MAGresults are very difficult to analyze (and not reported) because of theproblem of particle aggregation and lack of access to its micropores.

The results arising from the BET area analysis demonstrated thatthe polymeric template is highly affected by the precipitation ofthe magnetic moieties. Hybrid materials with improved adsorptionproperties are obtained within this route.

The MAG content in the composites was estimated consideringthe magnetite reaction yield [23]; expressed as %Fe3O4 (w/w). Thedata suggest that magnetic and polymeric components are almostin the same proportion. The identity of the polymeric template hasnot major influence on the amount of MAG incorporated since thedifferences are of less importance when using CEL or CS as poly-meric template.

4.1.5. Z potential and particle sizesTable 3 shows the values of Z potential for the magnetic com-

posites, magnetite and biopolymers. The Z potential of magnetitedispersed in mixture bidistilled water/ethanol (1:1) is negative.The pH of the dispersion prepared to measure the Z potential of

raw magnetite was about 7.6–8. It has been reported that in acidicpH, the dominating surface species in magnetite is tentatively Fe(II,III)OH2+, implying positive zeta potentials. With increasing pH, thezeta potential decreases and Fe (II, IIIOH) becomes the dominatingspecies around pH pzc. At alkaline pH, the surface species Fe (II, III)O− is mainly responsible for the negative zeta potential [27–29].

The surface charge of CS dispersion in the same media suggeststhat the amine groups are protonated leading to a positive valueof Z potential. The surface charge of chitosan magnetic compos-ite greatly differs from the magnetite. Although data in the tableindicate that the composite exhibits cationic surface charge, themagnitude of the Z potential is considerable lower than the corre-sponding to pure CS.

In the case of cellulose dispersion, the negative Z potentialreveals that the surface charge is negative under the assay con-ditions, as the surface of CEL/MAG.

Table 3Z potential, average particle diameter and polydispersion index of magnetic com-posites, magnetite and biopolymers.

Composite Z-potential Average particle diameter (nm) PDIa

MAG −7.88 417 0.252CS/MAG 3.77 610 0.456CEL/MAG −9.67 498 0.337CS 11.9 >5 �m –CEL −19.9 1725 1.00b

a Polydispersity index.b The quality of the assay was not suitable to validate the results due to the

presence of large particulate aggregates.



The data of Z potential is easy to interpret, considering that NH2groups are present in chitosan, whereas only OH groups are presentin cellulose. At the precipitation pH, NH2 groups are protonated asNH3

+; in CS/MAG. Changes in the Z potential arise from the par-tial neutralization of the positive charges of ammonium with thenegatively charged magnetite structure. The magnitude of the zetapotential, which should correspond to the charge density, expect-edly conforms to the extent of surface modification by interactionwith magnetite surface [30].

In CEL/MAG, OH groups are not available for deprotonation dueto the interaction with MAG and the surface charge in the compositeshows the impact of the magnetite [20].

The average particle diameter (nm) and the polydispersityindex (PDI) of the prepared composites, biopolymers and MAG areincluded in Table 3. The measurements were performed using dis-persions prepared in a mixture bi-distilled water/ethanol (1:1) atpH approx. 7.6–8. Comparing the values in Table 3 it is clear that thegreatest particle size is found using CS as polymeric template. Thehigh PDI reveal that the particle size distribution is quite heteroge-neous, in agreement with the images derived from TEM analysis.The particle size distribution of CEL/MAG is more homogeneous,with lower PDI values. This implies a rather narrow particle sizedistribution is reached, which is also in agreement with the TEMcharacterization.

The diameter of the magnetic composites particles is on thesame order than pure magnetite. This could be attributed to thestrong aggregation of the magnetite particles under the disper-sion conditions even when ultrasonic and vortex treatments wereimplemented. According to TEM results the average diameter of theiron oxide particles was almost 20 nm. On the other hand, huge dif-ferences are observed between the particle’s size of composites andraw polymers. Biopolymer’s dispersions render large aggregateswhen testing with DLS. Their high particle size and polydispersityare not suitable for DLS assays leading to unreliable data due toerrors related to the quality of the measurement.

It is hypothesized that a partial dissolution of lower molecu-lar weight CS fractions occurs during the composite formation;probably induced by the ionic force of Fe2+/Fe3+ solution and the co-precipitation conditions, contributing to the difference in particlesize of CS and CS/MAG [31]. Besides this, a segregation or disaggre-gation of CS flakes is also possible and may promote the final shapeand size of the CS/MAG [32].

In the CEL case the differences observed in the sizes as well asin the morphology of the particles could be attributed to the frag-mentation or decomposition of the CEL fibbers induced by metalion–polysaccharide interactions [33].

It was found that cellulose acts as an adsorbent for metal saltsin the cellulose–metal salt system. According to the available lit-erature, the interaction of cellulose with metal salts takes placethrough weak chemical adsorption, which keeps the metal ions inthe homogeneous state during the transformation [34]. The com-posite’s sizes obtained within this work are comparable with thoseprepared with more sophisticated methods, such as emulsion basedones [5,35,36].

4.2. Possible mechanism for the composite’s formation

Different hypothesis have been reported in the open literatureregarding to the nature of biopolymers–iron oxide interactions toexplain the formation of magnetic composites [17,37]. According tothe synthetic procedure the Fe2+/Fe3+ aqueous solution is in contactwith bulk biopolymers at pH ∼6, under N2 atmosphere with mag-netic stirring at 70 ◦C. At these conditions, the available functionalgroups of CS and CEL were present as NH3

+ and OH, respectively. CSor CEL are as solids. Even when the solubility tests shown that onlyat lower acidic pHs the pure CS dissolves, a partial dissolution of

fractions of low molecular weight cannot be discarded, especiallyin the presence of Fe2+/Fe3+. With the addition of the alkali, the pHincreases to near 11, the precipitation of magnetite takes place (andprobably also of the fraction of dissolved CS). Therefore, the hypoth-esis proposed, in agreement with published articles in the openliterature, is that – at least for the major insoluble fraction – thestructure of the biopolymers acts as a reaction vessel. In the case ofthe CS it is thought that NH2 groups may form chelates with the Fe2+

and Fe3+ aqueous solutions under N2 atmosphere, controlling thediffusion of the iron ions [38]. The formation of complex Fe–CS havebeen previously reported in the open literature [10]. Therefore inthe presence of OH-moieties the ferric and ferrous ions chelated bythe amino groups [(chitosan-NH2)2–Fe2+, (chitosan-NH2)2–Fe3+]provided nucleation sites for magnetite crystals growth [17]. Thepostulated mechanism could justify the results from FTIR, XRD andSEM/TEM. The shifts observed in the FTIR spectrum of MAG/CSwith respect to raw CS may be ascribed to the mentioned Fe–CSinteractions.

However the data from Z potential reveal that the compositeexhibit a positive charged surface. Therefore electrostatic inter-actions between CS and magnetite are not discarded and wouldcontribute to the iron oxide linkage on polymeric networks.

In the CEL case, the iron oxide remained closely linked to the cel-lulosic chemical structure though active OH groups forming intra-and intermolecular H-bridges [34,39]. A similar kind of coordina-tion was reported in polystyrene latex and magnetite by means ofcarboxylic groups [40].

A general representation of pathways involved in compositesformation is illustrated in Scheme 1. The whole composite could beschematized as a particulated structure whose sizes are lower than1 �m.

4.2.1. Dispersion properties and magnetic behaviour of magneticcomposites

To evaluate hydrophilic/hydrophobic character, dispersions ofthe magnetic composites and magnetite were prepared using dif-ferent solvents and solvents systems such as bi-distilled water,acetone/water and ethanol/water (EtOH/H2O; 1:1). CEL/MAGformed stable dispersion in acetone/water (1:1) whereas pure mag-netite, CEL, CS and CS/MAG were not well dispersed in such amedia. As a difference, CS/MAG composite is slightly dispersedin water as the raw magnetite while the CEL/MAG is not. Finallyboth composites may form stable dispersions in EtOH/H2O (1:1)mixture. Fig. 6a shows dispersions of CS/MAG and CEL/MAG inan acetone/water (1:1), where it is seen the different hydropho-bic/hydrophilic behaviour of each composite.

These experiences revealed that CEL-based materials presentmore hydrophobic character than the CS based one. Hence theselection of the polymeric template to prepare magnetic com-posites will depend on the desired properties regarding to theapplications media of these materials.

The magnetic behaviour of CS and CEL composites has beenqualitatively evaluated in terms of their response to a magneticfield. The image in Fig. 6b shows a dispersion of CEL/MAG in ace-tone/water mixture exposed to a weak magnetic field created bya laboratory stirrer. The accumulation of the materials particlesaround the stirrer is a clear proof that the materials herein preparedare magnetically active. Similar responses were achieved using theCS based magnetic composites.

Quantitative measurements of magnetism were obtained fromvibrating sample magnetometer (VSM). From this assay the magne-tization (M) of each composite as a function of the applied magneticfield (H) at room temperature has been obtained. The magnetiza-tion curves reveal that the nanoparticles have superparamagneticbehaviour at this temperature, with reversible curves followinga Langevin like function. MAG/QUIT and MAG/CEL magnetization



Scheme 1. Steps involved in composites formation.

curves are compared with the corresponding to pure magnetite inFig. 7. The data in the figure suggest that both composites presentsuitable magnetic properties for the desired applications. CEL/MAGexhibits a reduction of M with respect to MAG and QUIT based

Fig. 6. (a) Photographs of dispersions of CS/MAG and CEL/MAG in acetone/watermixture (1:1) and (b) photograph of dispersion of CEL/MAG in acetone/waterexposed to a magnetic field.

composites. Such difference could be attributed to the differentmechanism of formation.

4.3. Stability of composites and protein adsorption assays

UV/vis spectra of CS/MAG, CEL/MAG and bare magnetiterecorded during its incubation in similar conditions than theadsorption assays are included in Fig. 8a, b and c, respectively. Eachspectrum in the figure corresponds to an interval of time along theincubation period. In Fig. 8a a flat plot is observed at any selectedassay time; meaning that the CS/MAG composite remain stableunder explored conditions.

In Fig. 8b, bands appearing at 240 nm suggest that partial disso-lution/decomposition of the CEL/MAG or CEL/MAG derivates occurduring the period that the adsorption takes place. The cause forthe lack of stability is the different mechanism of composite for-mation between CS and CEL-based magnetic composites. Intra andintermolecular H-bridges formed between CEL and magnetite seem

Fig. 7. Magnetization curves at room temperature of MAG, MAG/QUIT andMAG/CEL.



Fig. 8. UV/vis spectra of composites, recorded at different intervals of time during incubation under the adsorption conditions. (a) CS/MAG; (b) CEL/MAG; (c) MAG.

not to be enough strong to ensure the stability of the compositesduring in vitro incubation. In Fig. 8c it is seen that magnetite ispartially dissolved under the assay conditions leading to Fe moi-eties in solution that absorb in the region of UV/vis. According tothe existent literature, magnetite may undergo surface metal ionhydrolysis at pH > pzc (pH = 6), leading to different species in solu-tion such as those are detected by UV/vis and shown in Fig. 8c[29].

The proteins loading efficiency for each composite, biopolymersand bare magnetite are presented in Table 4 and were calculatedaccording to Eq. (1). From the data in Table 4, both proteins may sat-isfactory adsorb in magnetic substrates through a relatively simpleroute leading to suitable loading efficiencies.

It is important to highlight that these experiences were devel-oped as preliminary assays to evaluate the applicability of theprepared materials. Therefore the assays were performed in a

Table 4Loading efficiency of model proteins on magnetic and biopolymeric supports, Gexpressed as percentage of adsorbed proteins related to the initially added amountand calculated according to Eq. (1).

BSAa Insulinb

CS 48 70CS/MAG 50 75CEL. 80 76CEL/MAG 27c 55c

MAG 72d 71d

a Initial amount of BSA 12 mg.b Initial amount of Ins. 6 mg.c Value obtained after correction of the spectra affected by the decomposition

products from CEL/MAG.d Value obtained after correction of the spectra affected by the decomposition

products from MAG.

unique condition in terms of the BSA concentration (BSA/compositeratio), pH, incubation media and temperature. More complete stud-ies regarding to the adsorption process are currently in progressand will be object of a next work focused exclusively on proteinadsorption.

Comparing the values obtained with raw polymers and mag-netic composites it is clear that both CS and CS/MAG lead to similarlevels of adsorbed proteins. On the other hand, very different load-ing efficiencies were recorded from CEL and MAG/CEL. The datarecorded using MAG/CEL and MAG as supports indicate that bothproteins bind satisfactorily to them. However the lack of stabilityof MAG/CEL and MAG under the adsorption conditions seriouslyaffects the quality of the results. Therefore correction steps had tobe implemented on the UV/vis spectra tending to eliminate theinterference of Fe2+/Fe3+ ions in solution from MAG segregatedfrom CEL/MAG; and the presence of colloidal particles of magnetitethat broad the bands of the spectra and affects the baseline. Fromthe profile of the CS, CEL and CS/MAG these problems are by far lessimportant than in the case of CEL/MAG.

The trend found in the BSA/Ins adsorption could be explained by:(i) the lack of stability of the magnetic composite in MAG/CEL as itwas earlier demonstrated and (ii) the mechanism of protein adsorp-tion and/or interaction with supports surfaces. Through measuringthe zeta potential of BSA solution and the supports, it was foundthat under the adsorption conditions the surface of BSA took thenegative charge [41], but both CS and CS/MAG supports took thepositive charge (see Table 3). A similar situation occurred in thecase of Ins. whose pzc is about 5.30. Therefore electrostatic inter-actions are expected to govern the BSA and Ins. adsorption on CSbased supports [30,42].

As a difference, the raw CEL as well as the CEL/MAG compos-ite, in bi-distilled water renders negatively charged surface, hence



Scheme 2. Possible pathways for protein adsorption on magnetic composites [42].

not electrostatic but mainly physical and hydrophobic interac-tions (plus other Van der Waals forces such as H-bonding) areexpected to maintain the proteins linked to those supports [41].Also in this sense, the interaction CEL/MAG is not expected to beas strong as in the case of CS/MAG. To illustrate Scheme 2 showsthe possible pathways for protein adsorption on magnetic compos-ites.

5. Concluding remarks

CS/MAG and CEL/MAG composites have been prepared though anovel route involving the co-precipitation of the iron oxide on bulkpolymeric templates.

The as-obtained materials were adequately characterized bytechniques like FTIR (DRIFTS), XRD, BET area, SEM/TEM, potentialZ and DLS measurements.

The magnetic properties have been verified as well as thehydrophobic/hydrophilic behaviour. It was determined that possi-ble route for the magnetic/polymeric composites formation involvethe disaggregation, segregation and partial dissolution (in thecase of low molecular weight CS) of polymeric templates. Asa result, composites with sizes lower than a 1 �m could beobtained.

It was verified that CS/MAG could be satisfactorily used asprotein adsorbent while the CEL/MAG was not stable under theadsorption assay’s conditions.

The hybrids materials prepared within this work could beutilized in several biotechnological applications under properexperimental conditions. This involves bioseparation or purifica-tion of proteins and cells as well as adsorbents of contaminants inremediation of environmental problems and immobilization sup-ports for enzymes.

Acknowledgements

The authors acknowledge the financial support of CONICET,the Secyt-ANPCyT (PICT Redes 2006-729), and the UNS (PGI24/MQ022).

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Novel and facile synthesis of magnetic composites by a modified co-precipitation method

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