Contribution to the Study of Ferrite Nanobeads: Synthesis,

Characterization and Investigation of Horizontal Low

Gradient Magnetophoresis Behaviour
Maria Benelmekki a,*, Cristina Caparros a, Anna Montras b, Renao Gonalves a,
Senenxu Lanceros-Mendez a and Lluis Miquel Martinez c
a

Centro de Fisica, Universidade do Minho, Braga, Portugal

Sepmag Tecnologies, Parc Tecnologic del Valles, Cerdanyola del Valles (Barcelona) Spain
c
Sepmag Technologies, 191 Peachtree St. NE, 03030 Atlanta. GA, USA.
*

E-mail: benelmekki@fisica.uminho.pt

Abstract. In this work we investigate the possibilities of the use of Horizontal Low Gradient Magnetic Field (HLGMF)
(<100 T/m) for filtration, control and separation of the synthesized magnetic particles, considering, the characteristics of
the suspension, the size and the type of nanoparticles (NPs) and focusing on the process scale up. Reversible aggregation
is considered in the different steps of magnetic nanobeads synthesis. For these purpose, we synthesized Fe2O3-silica coreshell nanobeads by co-precipitation, monodispersion and silica coating. SQUID, TEM, XRD, and Zeta potential
techniques were used to characterize the synthesized nanobeads. An extensive magnetophoresis study was performed at
different magnetophoretic conditions. Different reversible aggregation times were observed at different HLGMF, at each
step of the synthesis route: Several orders of magnitude differences where observed when comparing citric acid (CA)
suspension with silicon coated beads. Reversible aggregation times are correlated with the properties of the NPs at
different steps of synthesis.
Keywords: Magnetic nanoparticles, Citric Acid, magnetophoretical properties.
PACS: 75.47.Lx; 75.50.Tt; 81.07.-b

INTRODUCTION
In recent years, substantial progress has been made in developing technologies in the field of magnetic
microspheres, magnetic nanospheres and ferrofluids. Nanospheres and microspheres containing magnetic particles
core inserted in a nonmagnetic matrix, are used in numerous biological applications [1]. They are used as carriers
which can be targeted to a particular site by using an external magnetic field. Magnetic separation of organic
compounds, proteins, nucleic acids and other biomolecules and cells, from complex reaction mixtures is becoming
the most suitable solution for large production in bioindustrial purification and extraction processes. The current
commercial NPs for In-Vitro application are coated with Polystyrene, copolymers, or silica [2,3,4]. Due to the low
surface/volume ratio of the microspheres, and the unclear magnetic behaviour of the particles, commercial magnetic
micro and nano beads have low efficiency for large volume production and potential industrial application. These
limitations hinder their use as efficient and low cost carriers for bioindustrial purification and extraction processes
[2,5]. In order to be effective for large scale magnetic separation processes, low cost and efficient commercial beads
are necessary. The efficiency of magnetic beads may be improved by maximizing their surface/volume ratio and by
a better understanding of the behaviour of the magnetic particle core, allowing better control of the separation
process.
In this work, the essential steps involved in the life cycle of magnetic particles, from the synthesis process to
their subsequent recovery and removal using external magnetic fields (magnetophoresis) have been
comprehensively studied. The aim of this study is to increase our knowledge and improve our understanding of the
effect of different parameters on the behaviour of magnetic beads during their use in potential applications.
AIP CREDIT LINE TO BE INSERTED ON THE FIRST PAGE OF EACH PAPER

CP1311, 8th International Conference on the Scientific and Clinical Applications of Magnetic Carriers
edited by U. Hfeli, W. Schtt, and M. Zborowski
2010 American Institute of Physics 978-0-7354-0866-1/10/$30.00

121

MATERIAL AND METHODS

Synthesis of Ferrite Nanobeads
Nanobeads were prepared by co-precipitation of Fe3O4 from an aqueous mixture of FeSO4 and FeCl3. NH4OH
was used as a precipitation agent. For these purpose, FeSO47H2O (0.5 mM) and FeCl36H2O (1 mM) were mixed
and heated to 80 C. In order to precipitated the iron hydroxides, the pH value was raised and maintained to pH=3
for 30 min. The solution was rigorously stirred at a constant temperature during all the process. Then, the pH value
was increased to 10.5 - 11.
Citric acid (CA) was used to stabilize the magnetic nanoparticles, according to the method proposed by Campelj
et al. [6]. Approximately 2 g of nanoparticles were mixed with 65 mL of an aqueous solution of 2% CA. The pH
value of the mixture was adjusted to pH = 5 and, then, was raised to pH = 10 using concentrated ammonia. The
adsorption step of the CA was maintained at 80 C and rigorously stirred for 60 min. The prepared suspension was
washed with distilled water and magnetically separated several times to remove the excess of CA. Finally the NPs
were dried in order to proceed to the following step.
The CA-modified NPs were coated with silica, using tetraethyl orthosilicate (TEOS). 10 mL of NPs were mixed
in a 20 mL ethanol solution (35% v/v) containing 3% (v/v) TEOS. The pH value of the solution was maintained at
11 for 90 min.

Characterization Methods
A LEO 906E electron microscope operating at 100 keV, was used for transmission electron microscopy. The
samples were prepared by deposition of a droplet of particle solution on a copper grid coated with carbon and
allowed to dry. Dynamic light scattering (DLS) and potential were performed with a Zetasizer Nano ZS (Malvern
instruments), provided by a He/Ne laser of 633 nm wavelength. The Fe2O3- water dried sample was characterized by
X-ray diffraction with a Bruker D8 Discover diffractometer using Cu K incident radiation. Hysteresis loop of the
Fe2O3 aqueous solution at room temperature curves were measured with a superconducting quantum interference
device (SQUID) magnetometer (Quantum Design MPMS5XL). The experimental results were corrected for the
holder contribution.

Magnetophoresis Experiments
The magnetophoresis setups employed in our experiment are the SEPMAG LAB 1x25 ml 2042 and 2042 Plus
systems, commercially available from SEPMAG company [7]. The system consists of a cylindrical cavity
containing a high permanent magnetic field with a uniform horizontal gradient pointing toward the walls of the
cylindrical vessel. All magnetic NPs are working at saturation regime. The magnetophoresis experiment is
performed at Low Gradient Magnetic Field (LGMF) (<100 T/m), by placing a bottle of radius 1.5 cm containing 25
ml of aqueous solution inside the SEPMAG cylindrical cavity. The initial yellow-brown dispersion becomes
transparent progressively, reaching a transparent final state with all particles close to the walls of the bottle. Opacity
measurements were performed using the external light source SEPMAG CBL Q250 ml, also available in SEPMAG
company [7]. As reported by De la Cuevas and co-workers [8], the magnetophoresis process is driven by a
cooperative phenomenon consisting of reversible aggregation of the particles, followed by the moving of the
aggregates to the walls of the bottle. Typical magnetophoresis curves consist of a plateau corresponding to the
reversible aggregation time followed by a progressive decay of the suspension opacity until 100% transparency is
reached.

RESULTS AND DISCUSSION

Transmission electron microscopy images of the dried Fe2O3-water suspension show clusters of Fe2O3-silica
core-shell. The aggregation of clusters occurs during the drying sample process for TEM imaging. Single clusters
have about a 150 nm diameter size (FIGURE 1). Theses results are in agreement with DLS measurements. X- ray
diffraction was performed on dry Fe2O3 samples. Because of the modest amount of available sample, the peaks of
the diffractogram are not very well defined. They can be indexed either to maghemite or magnetite. Magnetic

122

measurements of Fe2O3-silica core-shell sample are shown in FIGURE 2. The sample is saturated at 4 kOe with a
saturation magnetization value Ms of 44 emu/g.

FIGURE 1. Typical TEM images of clusters of Fe2O3-silica core-shell

Magnetophoresis Behaviour
FIGURE 3 (Left) shows magnetophoresis curves of the obtained nanobeads at different gradients, 60 T/m, 30
T/m, and 15 T/m. The total time separation depends on the magnetic force applied inside the SEPMAG system. For
15 T/m gradient, the aggregation time takes about 100s. This time decreases one order of magnitude (~10 s) when
60 T/m gradient is applied. No relevant differences between the aggregation times corresponding to 60 T/m and 30
T/m applied magnetic gradients are observed. As explained previously, magnetophoresis separation is induced by
reversible aggregation of NPs [8], this reversible aggregation depends mainly on the total interaction potential
between the particles: van der Waals interaction, stabilizing electrostatic energy and magnetic interaction [9].

FIGURE 2. Magnetization curve of magnetic silica nanoclusters at room temperature

123

By applying 60 T/m, at pH 10.5, the aggregation time increases from less than 1 s for non-modified NPs to 300
s for CA-modified NPs (FIGURE 3 (right)). The potential of the non-modified NPs, and the CA-modified NPs is 20 mV and -43 mV, respectively. This result suggests the important influence of the electrostatic stabilization in the
magnetophoresis process. High electrostatic stabilization guarantees a very stable suspension, but induces a large
separation time.
In the case of nanobeads, the aggregation time takes 10s and the potential is -30 mV. The theoretical limit of
stability in a colloidal system is |30| mV. Colloidal systems show good stability if its potential is higher than 30
mV or lower than -30 mV [10]. This result indicates that the synthesized nanobeads in the present work are at the
limit of stability in colloidal systems, and will be subject to improvement in future works.

FIGURE 3. Magnetophoresis curves. Obtained nanobeads at different gradients (Left). Reversible aggregation times on NPs
at each step of the synthesis route (Right).

Effect of pH on the Reversible Aggregation Process

As shown in FIGURE 4, CA has three dissociable protons, associated with its three carboxylate groups. It has
been claimed that CA act as an adsorbate tribasic acid on metal oxides (adsorbent) [11].

FIGURE 4. Schematic representation for the synthesis of NPs, using CA to produce aqueous stable suspension

The deprotonation of CA depends on the pH of the suspension. At pH5.5, CA appears mainly with two
deprotonated species (AH2-), and then is adsorbed onto the NPs surface positively charged [6]. At pH ~ 10, CA
appears at their fully deprotonated state (A3-). As reported by Hajdu and co-workers [12], the CA may be adsorbed

124

on the surface of the NPs by coordinating via one or two of the carboxylate functionalities depending on the steric
necessity and the curvature of the surface. When the CA affinity limit is reached, the CA adsorption increases
further and a rearrangement of CA binding towards a tighter packed surface structure can occur. Only one
carboxylate group of CA will be bound to the NPs surface. In these conditions, there will be at least one carboxylic
acid group exposed to the solvent that should be responsible for the surface charge. Furthermore, the presence of a
terminal carboxylic group provides a possible way of extending the bond formation [13]. This chemisorption
process provides the electrostatic stability of the suspension.
Different samples of CA-modified NPs, with different pH in the range 2-10 were prepared separately. Samples
with pH<3 were not considered for this study because NPs degradation and cluster formation are observed. This
behaviour could be attributed to the suppressed dissociation of CA carboxylic groups in this acidic region. As shown
in FIGURE 5, the DLS measurements show homogeneous aggregates of the CA-modified NPs of about 40 45
nm, these aggregates could be induced by the chemisorption process of CA onto NPs [11,12]. The aggregates' size is
not significantly affected by pH changes in the considered range.

FIGURE 5. DLS measurements (Left), and potential (Right) of the CA-modified NPs at different pH values

Charge distribution depends strongly on the pH of the suspension, according to the FIGURE 5 (right). Acidic
pH's show considerable changes in the potential due to the suppressed dissociation of CA carboxylic groups that
should be responsible for the changes of NPs surface charge. At a pH of 6.75 and 4.62 a new peak appears in the
potential curves at about -10 mV indicating that the suspension is becoming electrostatically heterogeneous. At basic
pH (8.43), the potential curve shows a narrow peak at about -43 mV indicating the stability of the suspension.
As previously mentioned, a typical magnetophoresis curve of CA-modified NPs at basic pH, presents a unique
plateau corresponding to reversible aggregation time, followed by progressive decreasing of the suspension opacity.
As shown in FIGURE 6, at pH 6.72 and 4.62, magnetophoresis curves show a first Plateau followed by a
progressive decay of the suspension opacity until ~50%, and a second plateau followed by a progressive decaying of
the opacity until 100% of the suspension transparency is reached. These results are in agreement with potential
measurement and suggest that the separation process occurs in two steps by the separation of a first group of NPs
with a lower potential followed by the separation of NPs with higher potential. At lower potential, the
attractive magnetic interactions overcome the electrostatic repulsion, and the time separation is reduced.

125

FIGURE 6. Magnetophoresis behaviour at 60 T/m of CA-modified NPs at different pH's.

CONCLUSION
This paper reports on the effects of the stabilizing electrostatic energy on magnetophoresis process at HLGMF
(<100 T/m). The experiments demonstrate that potential affects drastically the separation time of several orders of
magnitude. Total separation time shifts from ~ 1.5 min for non-modified NPs (-20 mV), to ~330 min for CAmodified NPs (-43 mV). Small potential allows the attractive magnetic interaction to overcome the electrostatic
repulsion, and then, separation time is accelerated
Modification of potential of NPs could be achieved inducing pH changes of the magnetic suspension.
Destabilization and deterioration of the magnetic suspension due to the changes of electrostatic charges on the
surface of NPs could be monitored during magnetophoresis process.

ACKNOWLEDGMENTS
The authors thank the Portuguese Foundation of science and technology (FCT) for financial support under
grants: NANO/Nmed-SD/0156/2007 and PTCD/CTM69316/2006.

REFERENCES
Z. M. Sayed, S. D. Telang and C. N. Ramchand, BioMagnetic Res Technol 1 (2003).
K.S. Wilson, L.A. Harris, J.D. Goff, J.S. Riffle and J.P. Dailey, European Cells and Materials 3, 206-209 (2002).
P. Tartaj, M. Del Puerto Morales, S. Veintemillas-Verdaguer, T. Gonzalez-Carreno, C. J. Serna, J. Phys. D, Appl. Phys.
36, R182-R197 (2003).
4.
K. S. Wilson, J. D. Goff, J. S. Riffle1, L. A. Harris and T. G. St Pierre, Polym. Adv. Technol. 16, 200211 (2005).
5.
B. Chu, International Patent N WO99/12000-PCT/US98/21266 (1999).
6.
S. Campelj, D. Makovec, and M. Drofenik, J. Phys.: Condens. Matter. 20, 204101 (2008).
7.
Sepmag technologies, www.sepmag.eu
8.
G. De Las Cuevas, J. Faraudo, J. Camacho, J. Phys. Chem. C 112, 945-950 (2008).
9.
J. Faraudo, J. Camacho; Colloid Polym. Sci. 2888:207-215 (2010).
10. E. Taboada, E. Rodriguez, A. Roig, J. Oro, A. Roch, and R.N. Muller, Langmuir 23, 4583-4588 (2007).
11 . N. Kallay and E. Matijevic, Langmuir 1, 195-201 (1985).
12 . A. Hadju, E. Illes, E. Tombacz, and I. Borbath: Colloids Surf. A: Physicochem. Eng. Aspects 347, 104 (2009).
13. A. Goodarzi, Y. Sahoo, M.T. Swihart, P.N. Prasad; Mat. Res. Soc. Symp. Proc.789, 6.6.1-6.6.6 (2004).
1.
2.
3.

126

Copyright of AIP Conference Proceedings is the property of American Institute of Physics and its content may
not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written
permission. However, users may print, download, or email articles for individual use.