Formation of spherical iron(III) oxyhydroxide nanoparticles sterically stabilized by chitosan in aqueous solutions

Journal of Inorganic Biochemistry 95 (2003) 55–63 www.elsevier.com / locate / jinorgbio

Formation of spherical iron(III) oxyhydroxide nanoparticles sterically stabilized by chitosan in aqueous solutions ´ Sipos a,b , *, Otto´ Berkesi c , Etelka Tombacz ´ d , Tim G. St. Pierre e , John Webb b Pal a

Department of Inorganic and Analytical Chemistry, University of Szeged, P.O. Box 440, H-6701 Szeged, Hungary b Department of Chemistry, Murdoch University, Murdoch, WA 6150, Australia c Department of Physical Chemistry, University of Szeged, Szeged, Hungary d Department Colloidal Chemistry University of Szeged, Szeged, Hungary e Department of Physics, University of Western Australia, Nedlands, WA 6160, Australia Received 7 October 2002; received in revised form 3 February 2003; accepted 10 February 2003 ´ ´ Burger (1929–2000) The authors wish to dedicate this publication to the memory of Professor Kalman

Abstract The interactions between the cationic polymer chitosan (Chit) and iron(III) were investigated. The solution properties were studied by pH-metry, viscometry and dynamic light scattering. Solid state iron(III)–Chit samples were also prepared and characterized by IR spectroscopy and electron microscopy. In aqueous solutions, the precipitation pH of the iron(III) oxyhydroxide (FeOOH) is significantly shifted towards the higher pH values in the presence of Chit indicating that some interaction takes place between the iron(III) and the polymer. However, the additivity of the pH-metric titration curves, the lack of variation both in the viscometric and IR spectra of Chit in the presence and absence of iron(III), indicate the lack of direct complexation between the Chit and ferric ions. Isolated FeOOH nanospheres of 5–10 nm diameter were observed on the transmission electron microscopic pictures of samples obtained from solutions containing iron(III) and Chit, while from DLS measurements hydrodynamic units with a few hundred nm in diameter were identified. Our data support that Chit acts as steric stabilizer and inhibits the macroscopic aggregation of the subcolloidal FeOOH particles. Thus the iron(III)–Chit interactions offer a simple and economic way to fabricate nanometric size FeOOH spheres, morphologically similar to the core of iron(III)-storage protein, ferritin.  2003 Elsevier Science Inc. All rights reserved. Keywords: Iron(III); Chitosan; Ferritin; Polysaccharides; Nanostructure; pH-potentiometry; Viscometry; Dynamic light scattering; Electron microscopy; IR spectroscopy

1. Introduction Chitosan (Chit), one of the most abundant glycans, is a b-(1→4)-linked polymer consisting of D-glucosamine and N-acetyl-D-glucosamine monomeric units, and can be considered as mostly or completely deacetylated chitin [1,2]. While chitin is practically insoluble in water, Chit is soluble at pH#7, due to the protonation of its sugar-amino groups (the pKa of D-glucosamine is 7.52 [3]). Both mono- and polysaccharides are known to form stable complexes with transition metal ions (for the most *Corresponding author. Department of Inorganic and Analytical Chemistry, University of Szeged, P.O. Box 440, H-6701 Szeged, Hungary. Tel.: 136-62-544-054; fax: 136-62-420-505. E-mail address: [email protected] (P. Sipos).

recent review see Ref. [4]). Chit is able to ‘‘collect’’ transition metal ions selectively [2,5,6], and can be combined with metal ions through ion-exchange, sorption and chelation. Several examples can be found in the literature for each mode of interaction [1,2,5,6]. Recently the iron(III)–Chit interactions received considerable attention. The interaction of iron(III) with Chit is used for the treatment of iron overload [7] or for the removal of iron(III) (and other metal ions) from natural- or wastewaters [8–10]. Catalytic activity in various redox reactions of iron(III) immobilised to Chit [11,12] and a potential application of iron(III)–Chit as support for immobilized metal affinity chromatography [13] have also been reported. More recently an iron(III)–Chit complex was successfully utilized for the treatment of hyperphosphataemia [14–16].

0162-0134 / 03 / $ – see front matter  2003 Elsevier Science Inc. All rights reserved. doi:10.1016 / S0162-0134(03)00068-0

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In our previous publications [17–19] we have attempted to describe the solution (and some aspects of the solid) structure of the complex compounds formed between iron(III) and various anionic polysaccharides such as hyaluronate, heparin, dextran-sulfate and chondroitin sulfates A and C. Formation of water soluble compounds containing unexpectedly high numbers of iron(III) per repeating polymeric units have been observed [17] and it has been concluded that the polysaccharide backbone unfolds as iron(III) is coordinated to them and nanometric size (subcolloidal) particles with rod-like morphology are formed [17]. Based on these results, we concluded that coordination chemical interactions govern the nanometric level assembly of the FeOOH particles and the morphology of the composite compounds (both in solution [17,19] and in the solid state [18,19]). Other iron(III)–polysaccharide complexes showing unexpectedly high stability towards hydrolytic decomposition have also been observed for other macromolecular ligands such as k-carrageenan [20–22], alginic acid [23,24], Chit [25–28] and a range of other neutral or anionic polysaccharides (see Ref. [17] and the references therein). To describe the structure of the composite materials formed in these solutions, there are two main approaches in the literature. The first assumes that iron(III) is bound through the binding sites of the saccharide moieties (e.g., carboxylate, sulfonate, alcoholic hydroxylate or amine) and forms spatially separated iron(III) centers along the polymeric backbone (site binding model) [11,12,25–27]. The second proposes that iron(III) forms FeOOH precipitate, which is covered by the polysaccharide and inhibits their aggregation of the subcolloidal FeOOH particles [18,20– 24]. In the latter model only non-specific interactions are believed to be responsible for the high solubility of the metal hydroxides (colloidal model). In the combination of these two [18,19] the donor groups of the polysaccharide act as nucleation sites for the metal ions, which then bind further metal ions through the formation of hydroxide bridges and a variety of ‘‘nanostructures’’ are formed in situ, with shape and size, that depends on the type of the polysaccharide and the properties of the solution (pH, temperature, etc.). The way iron(III) interacts with Chit is not yet well understood. Ershov [25] suggests that Chit is bound to the isolated iron(III) atoms through the hydroxyl and nitrogen containing sites in the iron(III)–Chit complex. From ¨ Mossbauer and IR spectroscopic measurements, Nieto et al. [26] concluded that ferric ion is coordinated with two chitosan residues, and four coordination sites are occupied by water and chloride ions. Bathia and Ravi [27] came to very similar conclusions, and inferred a direct coordination of nitrogen to iron from IR measurements. On the contrary, Gamblin et al. [28] established a diminished importance of nitrogen containing groups in the bonding of iron and suggested the formation of iron(III) clusters in these compounds.

The general aim of the present paper is the description of the interactions between iron(III) and Chit in dilute aqueous solutions and the characterization of the compounds isolated from these systems, using a range of experimental techniques, such as pH-potentiometry, viscometry, dynamic light scattering, Fourier transform (FT) IR spectroscopies and electron microscopy. Our specific goal was to establish whether the amino nitrogens are directly involved in the iron(III)–Chit interactions. We have attempted to decide if the site binding or the colloidal model, or the combination of the two gives the best description for the complexes of iron(III)–Chit. Based on these investigations, some comparison of anionic and cationic polysaccharides’ behavior towards iron(III) also become possible.

2. Experimental

2.1. Materials Technical-grade Chit (Sigma–Aldrich, USA) was purified via dissolution in HCl and precipitation with NaOH, which was performed three times. The purified solid Chit was washed with distilled water and was cooled to 270 8C prior to lyophilization. The obtained solid powder was stored in vacuo over P2 O 5 . The degree of deacetylation of the Chit from pH-metric titrations [1] was found to be 78%, which is very close to the result obtained by Neugebauer for the same product from the same supplier [29]. The molar mass of the Chit from the limiting viscosity of 570 g ml 21 (determined at 25 8C, 0.15 M NaCl, pH 4.6), using the data of Berth et al. [30] was estimated to be (661)310 4 Da. FeCl 3 reagent solutions ([Fe(III)] T |0.25 M, [HCl] T |1 M) were prepared by diluting a concentrated stock solution (Ajax UNIVAR, Australia, 60%, w / v, analytical grade) and were standardized by EDTA titration using Variamin Blue as indicator. For the solution preparation, Millipore MilliQ water was used. All other chemicals used were of analytical grade.

2.2. Methods pH-metric titrations were carried out in the pH range of 2–11, in N 2 at 25.060.1 8C and at constant ionic strength (0.15 M NaCl). A Radiometer M64 type pH-meter equipped with a Radiometer AX-1 D64 calibrated combination glass electrode was used for the measurements. The titrant solutions ([NaOH] T 50.050 M and [HCl] T 50.025 M) were prepared from DBH standard ampoules and their concentrations were checked and measured by the Gran method [31]. During the pH-metric titrations a total volume of 25–40 ml of initially acidic solution was titrated with NaOH from a Metrohm 665 Model Dosimat unit. Potential readings were recorded when the displayed pH

P. Sipos et al. / Journal of Inorganic Biochemistry 95 (2003) 55–63

value was stable for several minutes in the third decimal place (i.e., within 60.1 mV). Viscosity measurements were performed using both rotational and capillary viscometers with solutions containing #1 g l 21 (or 5310 23 M in monomeric units) of Chit. Ferric concentrations were 0#[Fe(III)] T / [Chit] T #2. The pH of the solutions was kept at 4.6 and the ionic strength was constant (0.15 M NaCl). Sample solutions were prepared from mixing of a Chit stock solution (2 g l 21 , containing [HCl] T 50.01 M) and iron(III) (0.25 M, containing [HCl] T 51 M) and solid NaCl. The pH of the solution after thorough mixing of the constituents and 10 min ultrasonication, was brought up to 4.6 with NaOH and then quantitatively transferred to a volumetric flask. The solutions were passed through a folder filter to remove dust particles. For the low shear rotational viscometric measurements, a Haake Rotovisco RV-20, CV-100 instrument (ME-30 type head, PG-242 programmable unit) was used. The linearity of all the shear stress vs. shear rate curves of Chit containing solutions and the lack of any shear hysteresis both in presence and in absence of iron(III) over the above concentration ranges indicated that these systems are Newtonian. The exact viscosities of the solutions were determined with an Ostwald type capillary viscometer (Poulten, Seife and Lee Ltd., basic efflux time 111.660.1 s for a 0.15 M NaCl solution) in a circulated water bath (25.060.05 8C). The efflux times were measured manually performing at least five measurements for each sample and were accepted only when they were reproducible to within 60.10 s. For the Chit containing samples, only the data in the efflux time range of t.120 s were used for the calculations. Thus the maximum uncertainty of the specific viscosity values is expected to be always #2%. Since low polyelectrolyte concentrations were used (as recommended in Ref. [32]) density corrections were unnecessary. Dynamic light scattering (DLS) measurements were performed at 25.060.1 8C using a ZetaSizer 4 (Malvern, UK) apparatus operating at 633 nm. Clear solutions containing 2 and 4 mM Fe(III) and 4 mM Chit (I50.15 M NaCl, pH 4.6) were tested in parallel with Chit solution without iron(III). Since the scattered light intensity of solutions especially that of pure Chit was very low, long data collection time was chosen in both automatic and manual operation mode at different scattering angles from 60 to 1208 to get satisfactory correlation functions.

2.3. Preparation and characterization of the solid samples For the preparation of the solid iron(III)–Chit samples, a Chit solution of 5.43 g l 21 concentration (with [HCl] T 5 0.025 M) was mixed with the necessary amount of a ferric solution to set up the iron(III):monomeric Chit unit ratios of 0, 1, 2 or 3. The pH of the solutions were brought up to 4.6 (degree of proton dissociation, a 50), 6.4 (a 50.50)

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and 8.0 (a 51) with the slow addition of NaOH. To remove the by-product NaCl from the systems, the solutions of pH 4.6 and 6.4 were dialyzed with running distilled water using a Medicell dialysis sack (cut-off limit 12,000–14,000 Da), the completeness of the dialysis was checked conductometrically. From the samples with pH 8, an easily sedimenting precipitate was obtained, which was collected and washed three times with distilled water. Then both the solutions and the precipitates were frozen to 270 8C, and lyophilized. Similarly to the results obtained for anionic biopolymers [17], the products were brown powders, and, under the light microscope, they were transparent, honey-colored solid substances. Infrared spectra were recorded on a Bio-Rad Digilab Division FTS-65A / 869 Fourier transform infrared spectrometer in KBr pellet between 4000 and 400 cm 21 . The spectrometer was equipped with a DTGS detector. The spectral resolution was 2 cm 21 and 128 scans were averaged. Data were processed by using the software GRAMS / 386 Ver. 3 (Galactic Corporation). Electron microscopy was performed using a Philips 301 transmission electron microscope, operating at 80 keV. A few chips of the solid iron(III)–Chit samples were suspended in a drop of water, and were applied to Formvar coated copper grids. No staining was necessary for the samples of this study, as iron(III)-containing regions are in themselves electron dense enough to provide the necessary contrast.

3. Results

3.1. pH-metric titrations When an acidic solution of Chit is titrated with NaOH, a one equivalent deprotonation step occurs between pH 5 and 7 (Fig. 1) corresponding to the dissociation of the proton from the amine group of the D-glucosamine units. From our measurements, the pKa 56.46 at a degree of proton dissociation a 50.5, which is similar to the values compiled by Muzzarelli (pKa 56.3 [33]), higher than that found for fully deacetylated Chit (pKa 55.60, I50.05 M NaCl [11]), but lower than the dissociation constant of D-glucosamine (pKa 57.52 [3]). Around pH 7–7.5 (or a | 0.75–0.80) the polymer begins to precipitate from the system (C9 in Fig. 1). During analogous titrations ([Chit] T 52 mM) in presence of ferric ions ([Fe(III)] T 51–4 mM), the electrode potential becomes sluggish at pH|2.5–3 (A in Fig. 1) indicating the onset of the slow hydrolysis of the iron(III). This sluggishness, however, disappears at pH|4 (B in Fig. 1) where a deprotonation step, corresponding to the amine nitrogen of the glucosamine units, starts and where the system becomes well-buffered. The apparent dissociation constant of Chit in presence of iron(III) and at a degree of proton dissociation a 50.5 is pKa |6.4–6.5 [practically the

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P. Sipos et al. / Journal of Inorganic Biochemistry 95 (2003) 55–63 Table 1 Viscosities of aqueous chitosan solutions at 25.0 8C, I50.15 M (NaCl) and at pH 4.6 at various levels of iron(III)-loading and at various chitosan concentrations

hred a (l g 21 )

[Chit] 21

Iron(III):Chit molar ratio b

(g l )

Fig. 1. pH-metric titration curves of the Fe(III)–chitosan system at 25 8C and at I50.15 M NaCl. Titrant: 0.05 M NaOH. Titrand: s: HCl (0.01 M), •: HCl (0.01 M)1Chit (0.002 M), m: HCl (0.01 M)1Chit (0.002 M)1Fe(III) (0.002 M). The electrode signal become sluggish at A, become stable again at B, and precipitate occurred in the solutions at C and C9.

same as in absence of iron(III)]. This value did not show any dependence on iron(III) concentration. Thus the involvement of the amine nitrogen in the binding of the iron(III) in solution under the above experimental conditions can safely be ruled out. No precipitation (or even opalescence) is seen up to neutral pH and over a wide range of [Fe(III)] T / [Chit] T ratios. However, at pH |7–7.5 (i.e., where the Chit itself precipitates from the solution) a deep brown, easily sedimenting precipitate was observed. The precipitate readily dissolves in the same pH range, when the titration is performed in reverse (i.e., when the precipitated system is titrated with HCl solution), without showing any visible sign of iron(III)–hydroxide precipitate in the pH-interval of the amine nitrogen’s deprotonation. Thus the reaction appears to be perfectly reversible. Note that in Chit-free solutions, under identical experimental conditions, FeOOH(s) precipitate occurs at significantly lower pH values (4–4.5). pH-metric titrations were also performed with solutions containing ferric ions ([Fe(III)] T 51–4 mM) and the structural building blocks of the Chit, D-glucosamine or N-acetyl-D-glucosamine (2 mM). The corresponding titration curves were additive within the margin of the experimental error and no variation was seen in the precipitation pH of the FeOOH in the presence of these ligands. The pKa of D-glucosamine did not show any variation. This was found to be 7.5960.05 and 7.6260.05 in the presence and absence of the metal, respectively. Thus no pHmetrically detectable interactions were seen between the iron(III) and these two ligands.

3.2. Viscometry The viscosity data obtained from the capillary viscometric measurements are presented in Table 1. The hred

0

0.5:1

1:1

1.5:1

2:1

0.16 0.32 0.48 0.64 0.80

0.65 0.72 0.77 0.90 0.93

0.64 0.77 0.79 0.87 0.95

0.69 0.75 0.85 0.90 0.98

0.66 0.74 0.79 0.90 0.97

0.69 0.78 0.80 0.92 0.95

hlim c (l g 21 ) d 2 22 Slope (l g ) e Correlation coefficient

0.57 0.46 0.969

0.59 0.45 0.961

0.61 0.46 0.992

0.58 0.49 0.989

0.60 0.46 0.990

a hspec 5(t2t 0 ) /(t 0 3[Chit]), where t and t 0 are the flow times (or time of passage) of the sample solution and the solvent (e.g., 0.15 M NaCl in water), respectively, and [Chit] is the concentration of the chitosan expressed in g l 21 . b In the metal-to-ligand ratio, the concentration of chitosan was expressed in terms of moles of glucosamine unit per liter of solutions. c hlim or the limiting viscosity is the intercept of the hspec vs. [Chit] function. d The slope of the hspec vs. [Chit] function. e The correlation coefficient obtained from the linear regression of the hspec vs. [Chit] function.

vs. [Chit] T functions show satisfactory linearity over the concentration range investigated. The invariability of the intercept and the slope of the hred vs. [Chit] T functions indicate that there is no significant change in the molar mass and the secondary structure of the polymer, as the iron loading increases. Thus it seems that, although obviously an interaction takes place between the Chit and the hydrolysing iron(III) (e.g., there is no visible precipitation of FeOOH in presence of Chit at pH,7), it does not affect the viscosity of the solutions.

3.3. Dynamic light scattering The correlation functions from the DLS measurements were evaluated by cumulant analysis [34] which is the most often used analysis scheme for DLS measurements. The first-order autocorrelation function, g 1 (t), can be given as: ug 1 (t)u5exp[2G t1( m2 / 2!)t 2 1( m3 / 3!)t 3 1 . . . ], where G is the first cumulant (short relaxation time), which characterizes the mean, m2 the width and m3 the skewness of the distribution. For multicomponent systems the first cumulant (G 5kDlq 2 ) gives the light scattering intensity weighted diffusion coefficient, the second one ( m2 ) provides a value (often expressed as the variance) for the range of diffusion coefficients present in the sample. A large variance indicates that the sample is polydisperse. If qR,,1, the translational diffusion is the dominant dynamics, the diffusion coefficient (Dt ) can be calculated, G 5Dt q 2 where the scattering vector (q) is q5(4p n / l)sin(u / 2), and according to the Stokes–Einstein equation,

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Dt 5kT / 6phR H , the hydrodynamic radius (R H ) can be obtained. (In these equations: k is the Boltzmann’s constant, T is the temperature, h is the viscosity of the medium, n is the refraction index of the solution, l is the wavelength of the scattered light and u is the scattering angle). The reciprocal of q is the dimension of length, which sets essentially a length scale on which the diffusion is probed. For qR..1 the DLS only probes the internal modes, i.e., the rotational and configurational degree of freedom [35]. Any measurements aimed at the size determination the quadratic q-dependence of G in G 5kDlq 2 must therefore be checked. When other dynamic processes, due to the internal modes, also contribute to the intensity fluctuations [36,37] the exponent of q in the equation G 5kDlq 2 will be larger than 2. For sufficiently large qR..1 the internal modes will be completely dominant and the linewidth G will be proportional to the third power of the scattering vector, q. In some unusual cases fractal colloidal aggregates gave an anomalous fractional linewidth exponent, G |q a with 2,a ,3. This unusual phenomena has been studied intensively by Martin and coworkers [36–39] and the detailed theoretical analysis of the dynamic scaling has been published. The exponent a can experimentally be determined from the measurement of the scattering angle dependence of G defined as the initial decay of correlation function. This theory correlates with the various structural phenomena of these systems, such as fractal character and polydispersity. The iron(III)–Chit systems appeared to be clear solutions over the studied range of pH and iron(III) to Chit ratio at 0.15 M ionic strength. However, a weak light scattering appeared in these solutions when they were illuminated by a laser beam. Conditions for DLS measurements with the given apparatus proved to be suitable for solutions containing at least 4 mM Chit. The DLS measurement of 4 mM Fe(III) and 4 mM Chit with average intensity of counted photons 34.3 KCps resulted in Z average hydrodynamic radius of 246 nm using automatic mode of data collection and third-order cumulant analysis. Since the correlation function is generated from the fluctuation of scattered light intensity in the sample, and the iron oxide particles are strong scatterers due to their high refractive index, the contribution of the Chit polyelectrolyte was also checked. The average intensity of counted photons for a 4 mM Chit solution was very low (3.6 KCps), therefore the contribution of free chitosan to intensity fluctuation in Fe(III) chitosan solution is less than |10%. A Z average hydrodynamic radius 525 nm was determined for the pure chitosan solution. Considering the molecular mass of |60 kDa from the limiting viscosity value measured in the present work, this value seems to be quite large but can be explained by the polydispersity of the Chit used here. The scattered light intensity shows a quadratic dependence on the volume of scatterer according to the classical Rayleigh theory [40], the contribution of larger molecules is more pronounced in a polydisperse

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Fig. 2. IR spectra of chitosan obtained from solutions with pH of (a) 4.6, (b) 5.4, (c) 6.4, (d) 7.2, (e) 8.0.

sample in general. Different commercial Chit samples were characterized by means of static light scattering among the other methods in a recent paper [30]. Some hundreds nm of radius of gyration (R G ) were given for the original sample with molar mass similar to ours, while fractions separated by gel permeation chromatography had only some tens nm of R G .

3.4. IR spectra The IR spectrum of iron(III)-free Chit is characterized by broad and overlapping vibrational bands [1,6] and displays characteristic variations with the pH of the solution from which the sample was taken prior to lyophilization (Fig. 2). The peaks have been resolved via Fourier-deconvolution [41–43] by narrowing the width of the bands. The result of such deconvolution for the fully protonated (pH 4.6) and fully deprotonated (pH 8) form of Chit is shown in Fig. 3. The majority of the resolved peaks shows no significant change upon variation of the pH. However, the strong band around 1730 cm 21 in the range of the C=O stretching vibration which is present in the pH 4.6 sample, fully disappears in samples of pH$6.4. Another two bands (around 1630 and 1515 cm 21 ) also lose intensity with the

Fig. 3. Fourier deconvolution of the spectrum of chitosan obtained from solutions of pH 4.6 (A) and 8.0 (B). Dashed lines: original spectra, full lines: deconvoluted spectra.

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increasing pH, such bands can be found in the range of the stretching bands of the C–O bonds, but it also contains the bending bands of X–H type groups, like N–H or O–H. In the wavenumber range of C–C and C–O stretching, two bands (at 1090 and 1065 cm 21 ) loses, while one (at 1120 cm 21 ) gains intensity with the increasing pH. There are two potential protonation sites of Chit. The amino side-chains are the most basic in character while the nitrogen atom of the acetamido group is a strong acid. It seems likely from the IR spectra that, in the solid state, the amide moiety in the acetamido group is bound to hydrogen via a hydrogen bond. The samples prepared from the two most acidic solution show a band around 1730 cm 21 , which disappears at higher pH values, indicating that at lower pH the amide is ‘‘protonated’’, and which breaks up the conjugated system of the amide group resulting in a ‘‘normal’’ C=O group. The bands appearing at 1630 and 1515 cm 21 gradually disappear with the increasing pH, and are characteristic to the ammonium side chains. They can be assigned to the das NH 31 and ds NH 31 bending modes, respectively. The two bands around 1665 and 1550 cm 21 exhibit decreasing intensities with the increasing pH, and are likely to correspond to the das NH 2 and ds NH 2 in-plane deformation modes of the deprotonated amino group. The gradual intensity changes between 1000 and 1100 cm 21 are caused by the decreasing amount of charge carried by the side chains contributing to the skeletal vibrations of the polymeric chain. This is because the electric transition dipole moment is proportional to the charge localized on the atoms which motion is included in the given normal mode. The IR spectra of iron(III)–Chit samples at various metal-to-ligand ratios obtained from solutions at pH 4.6 and 8 are shown in Figs. 4 and 5, respectively. The deconvolution of the spectra of the pH 4.6 samples revealed that neither the composition nor the relative intensities of the bands changes significantly with the increasing metal-to-ligand ratio. There are, however, minor differences between the spectra of the iron-free and ironcontaining samples. In the spectra of the iron(III) containing samples, the relative intensities of the bands

Fig. 4. IR spectra of Fe(III)–chitosan compounds obtained at pH 4.6, at metal-to-ligand ratios of 3 (a), 2 (b), 1 (c) and 0 (d).

Fig. 5. IR spectra of Fe(III)–chitosan compounds obtained at pH 8.0 at metal-to-ligand ratios of 3 (a), 2 (b), 1 (c) and 0 (d).

assigned to the ammonium side chain decreased and their maxima is somewhat shifted towards the higher wavenumbers. The band at 1730 cm 21 is also lacking, which might indicate that the hydrogen bonding interaction within the acetamido group is broken down in presence of iron(III). The deconvolution of the spectra obtained for pH 8 samples resulted in a very similar picture. The metal-toligand ratio affects the spectra only to a minor extent, and the band at 1665 cm 21 corresponding to the non-protonated amino group is shifted towards the higher wavenumbers as iron(III) is added to the Chit. The shifts observed both for the pH 4.6 and for the pH 8 samples can be appropriately interpreted without invoking a direct amino coordination to the iron(III). It is well known that the FeOOH itself has IR spectrum [44], with broad vibrational bands around |1620, |1500 and |1340 cm 21 , respectively. When the IR-spectrum of FeOOH is numerically subtracted from that of the iron(III)–Chit ‘‘complex’’, a spectrum that is practically identical to that of the iron-free Chit can be obtained (Fig. 6).

3.5. Electron microscopy A typical electron micrograph of an iron(III)–Chit compound is shown in Fig. 7. The spherical electron dense

Fig. 6. (a) IR spectrum of the Fe(III)–chitosan compound at a metal-toligand ratio of 3:1 and at pH 8. (b) IR spectrum of FeOOH. (a2b) The difference between the spectrum (a) and (b). (c) IR spectrum of chitosan obtained from a solution of pH 8.

P. Sipos et al. / Journal of Inorganic Biochemistry 95 (2003) 55–63

Fig. 7. Transmission electron micrograph of an Fe(III)–chitosan compound obtained from a solution of pH 4.6, at a metal-to-ligand ratio of 1:1, showing electron dense spheres of FeOOH, with particle sizes ranging from approximately 5 to 10 nm. The scale bar represents 100 nm.

particles are likely to be FeOOH balls. It is noteworthy that the diameter of these spheres is rather uniform, falling between 5 and 10 nm [18].

4. Discussion and conclusions Chit contains amino- and amide nitrogen, alcoholic- and etheric oxygen donor atoms, which are potential binding sites for iron(III) in solutions [3]. Both our pH-metric titrations (in solution) and IR spectroscopic measurements (on solid samples) indicate that there is no direct involvement of the glucosamine’s amino group in the iron(III) binding. Since the ferric ion is of typical ‘‘hard’’ character in complex forming reactions, this observation is not particularly surprising. Regarding the possible role of donor groups containing oxygen atoms, the situation is much less obvious. Several water soluble iron(III)–alkoxylate complexes have been described in the literature [4,45–47]. The iron(III)-induced deprotonation of the alcoholic hydroxide moieties of sugar type ligands is quite common in strongly alkaline aqueous systems [4,48,49] and with some monosaccharides it can occur even in solutions of pH 7–10 [50–52]. Regarding iron(III)–Chit systems, given that the solutions are acidic, it seems unlikely that alcoholic hydroxide moieties could become deprotonated during the interaction, however, this can not be unambiguously stated from the experimental data available at this stage. The invariability of the viscometric behavior of Chit upon iron(III) addition also seems to support that the Chit and the iron(III), (or more accurately, its hydrolysis products, FeOOH), coexist in solution without any specific coordination chemical interaction (or site binding). This invariability also indicates the lack of cross-linking. However, the constancy of the limiting viscosity, hlim , with the

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iron loading is somewhat surprising since, even in the case of simple steric stabilization, hlim (or, in other words, the molar mass of the polymer) should significantly increase. From the transmission electron microscopic measurements it seems that the dominant form of the iron(III) in presence of Chit at pH|4.6 is a 5–10 nm diameter sphere. A core like this contains around 3000–6000 ferric atoms. It is easy to show that about 10–20% of the ferric atoms are situated on the surface of such spheres, while 80–90% resides in the bulk and therefore is not accessible for the various solution species. Thus, in molar ratio terms, only a small fraction of the Chit polymer is required to cover the surface of the FeOOH spheres . This way the dominant form of the Chit in such solutions is free (not bound to the spheres), hence the invariability of the hlim . A similar behavior was observed by Jones et al. [20] in systems containing k-carrageenan and cellulose-sulfate at pH values around 7. The DLS measurements indicate that the size of the dominant hydrodynamic units is about 200–250 nm in the solutions containing iron(III) and Chit. From this it can be inferred that that the FeOOH spheres with 5–10 nm diameter, which were identified on the transmission electron microscopic pictures, are covered with a loose layer of adsorbed Chit molecules providing steric stabilization for nanoparticles formed spontaneously under the given experimental conditions. Steric stabilization of colloidal particles by polymer adsorption is well-known both in academic and in industrial fields [53]. There is a close relationship between the adsorption and dispersion stability. The theoretical models of steric stabilization predict that the stabilization effect increases with the thickness of the adsorbed layer, which increases with increasing molecular mass of polymers, and therefore the larger macromolecules, above a critical molecular mass (|10 kDa in general) are more effective in dispersion stabilization. The polymer concentration required to cover the surface of particles, and so to stabilize the dilute dispersions is low in the order of 10 mg l 21 magnitude. To prove our in situ steric stabilization hypothesis, DLS measurement with solutions containing 2 mM Fe(III)–4 mM Chit was also performed. The average intensity of counted photons was found to be 14.7 KCps, e.g., the intensity of the scattered light was roughly halved when the iron(III) concentration was halved in the system. However, the Z average hydrodynamic radius was calculated to be 252 nm, which is practically identical to the value found for the systems with higher iron(III) concentrations. These results support well the in situ formation of more or less uniform stabilizing chitosan layer around the FeOOH nanoparticles during hydrolysis. The iron(III)–Chit system is a composite colloidal system, which does not obey certain ideal conditions (e.g., monodisperse compact particles in sols or random coils of monodisperse macromolecules) assumed in the theoretical background of this method. In the evaluation method used,

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an a priori fact is the square dependence on scattering vector (G 5kDlq 2 ). This is true for the regime qR,,1, which seems to be fulfilled, if the size of the FeOOH nanoparticles, 5–10 nm, and the region of scattering vector 0.02 to 0.01 nm 21 in the range of 1208#Q #608 are considered. However, the size of the dynamic units in the iron(III) Chit systems was much larger, than the size of nanoparticles determined with transmission electron microscopy. In some cases (e.g., fractal scatterers, polydisperse samples) an anomalous fractional exponent, G |q a with 2,a ,3 have been published [36–39,54]. The exponent a was determined experimentally for the selected colloidal solutions containing 2 and 4 mM Fe(III), respectively, besides 4 mM chitosan and 0.15 M NaCl at pH 4.6. The cumulant analysis of the correlation functions measured at different angles between 60 and 1208 resulted in G values (characteristic of exponential decay of correlation function) which did not obey the quadratic dependence on scattering vector (G 5kDlq 2 ). The exponents calculated as the slope of log G vs. log q functions (Fig. 8) were found to be 2.63 and 2.35 for Fe(III) Chit solutions containing 2 and 4 mM Fe(III), respectively, instead of 2, which would be expected for an ideal system. One consequence of this unusual angular dependence is that only the apparent values of hydrodynamic radius (R H ) can be calculated. This changes systematically with the scattering angle (the larger the u the smaller the R H . Larger variation in the apparent R H values (from 345 to 233 nm) was observed at 2 mM iron(III) concentration than at 4 mM where the apparent R H values changed between 279 and 241 nm. It can be stated that the behavior of iron(III) Chit solutions is far from the ideal, which is probably due to the polydisperse and fractal character of Chit coated iron oxide nanoparticles. Therefore the effect of excess (free, non-adsorbed) Chit present in the solutions has to be also considered. This is why the behavior of the sample with higher iron(III) concentrations (i.e., less free chitosan content) shows significantly smaller deviation from ideal.

It is interesting to compare the solution chemical behavior and the morphology of the iron(III) ‘‘complexes’’ of various anionic polysaccharides studied previously with those of Chit. First it seems plausible that the polysaccharides, when iron(III) (or FeOOH) is bound to them, determine the morphology of the inorganic particles formed. This has been illustrated in our previous [17,19] and present electron microscopic and viscosimetric measurements. The different functional groups (e.g., different ionization and coordination properties) on the two kinds of polysaccharides provide the different nucleation sites for the growth of the FeOOH particles. The carboxylate and sulfonate donor groups efficiently sequester iron(III), resulting in two effects. One is the unfolding of the polymeric chain, the other is the nanometric level regulation of the assembly of the FeOOH precipitate formed. In our model, the (practically) infinite array of donor groups on the anionic polymers act as a chain of nucleation sites from which further growth of an iron(III) hydroxide nanophase takes place. Ultimately, this process results in elongated, ‘‘rod-like’’ structures, consisting of long linear sections of the unfolded polymeric backbone covered with a few-nanometers-thick FeOOH layer [17]. The donor groups on the Chit are less effective nucleation sites than those of the anionic polymers, and therefore their effect on the self assembly of the FeOOH nanoparticles is much less pronounced. In the process of the iron(III)–Chit interactions, the deprotonation processes of the two components proceed independently in aqueous solution. As the pH increases, the hydrolysis of iron(III) takes place first at the usual range of pH 3–4, resulting in spherical FeOOH particles with a diameter of 5–10 nm [55]. Because of this it seems unlikely that Chit is incorporated in the FeOOH spheres. In the absence of complex forming agents, these nanoparticles agglomerate, and form a macroscopic precipitate (usually ferrihydrite). When the FeOOH is formed in a network of the polymeric Chit matrix, such coagulation does not happen. Thus the solution structure of the iron(III)–Chit system can be best described as FeOOH spheres with 5–10 nm diameter, sterically stabilized by the polysaccharide, which adsorbs on the surface of the nanoparticles.

Acknowledgements

Fig. 8. Dependence of the first cumulant (G, s 21 ) on the scattering vector (q, nm 21 ) for solutions containing Chit (4 mM) and iron(III) (s: 2 mM, •: 4 mM), at I50.15 M NaCl, pH 4.6.

¨ ¨ Technical assistance by G. Kormoczi (IR spectra), R. ´ Szekely-Nagy (capillary viscometry), G. Machula (rotation viscometry) and P. Fallon (electron microscopy) is gratefully acknowledged. This work was financially supported by the Department of Industry, Trade and Commerce of the Commonwealth of Australia and by the Hungarian Research Foundation (OTKA) grants T-029554 and T043551. P.S. also acknowledges the financial support of the ´ Bolyai Janos Fellowship.

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