Materials Sciences and Applications, 2010, 1, 109-117
doi:10.4236/msa.2010.13019 Published Online August 2010 (http://www.SciRP.org/journal/msa)
109
Preparation of Micron-Sized Di-Functional
Magnetic Composite Polymer Particles
Hasan Ahmad1, Tania Tofaz1, Mohammad Wali Ullah Oli1, Mohammad Abdur Rahman1,
Mohammad Abdul Jalil Miah1, Klaus Tauer2
1Department of Chemistry, Rajshahi University, Rajshahi, Bangladesh; 2MPI of Colloid and Interfaces, Am Mühlenberg, Golm, Germany.
Received May 22nd, 2010; revised June 17th, 2010; accepted July 7th, 2010.
ABSTRACT
In this investigation micron-sized monodisperse magnetic composite polymer particles with amino and amide functional
groups were prepared considering their applications in biotechnology. First, polystyrene/poly (acrylic acid-acrylam-
ide-N-N-methylene-bis-acrylamide) [PS/P(AA-AAm-MBAAm)] composite polymer particles were prepared by seeded
copolymerization. The carboxyl groups present on or near the particles surface were modified by amine-nucleophile,
ethylene diamine (EDA), through pre-activation with dicyclohexyl carbodiimide as coupling agent. The aminated parti-
cles were then magnetically modified and named as aminated-Fe3O4 composite particles. Formation of such magnetic
composite particles was confirmed by scanning electron micrographs, FTIR-spectra and magnetic susceptibility meas-
urement. The produced composite particles were paramagnetic. To see the relative hydrophilic character of the parti-
cles surface the adsorption behavior of trypsin (TR) as biomolecule was studied on PS particles and aminated-Fe3O4
composite particles. The magnitude of adsorbed TR on PS particles was higher than that on aminated-Fe3O4 composite
particles.
Keywords: Aminated Fe3O4 Composite, Amino and Amide Groups, Trypsin, Hydrophilic
1. Introduction
In recent years polymer particles with well defined parti-
cle size and various reactive functional groups on the sur-
face are attracting much interest because of their applica-
tions in a broad range of fields, e.g. as binders in paints,
adhesives, paper coating, textile and as a solid support in
the biochemical and biological field as well as in cataly-
sis and calibration standards [1-5]. The functional groups
include hydroxyl [6-8], aldehyde [9-11], carboxyl [12-
16], amino [17-19], epoxy [20-22], cyano [23], sulfhydr-
yl [24] and chloromethyl groups [25-27]. A variety of
methods are available to produce monodispersed parti-
cles in the micrometer-size range. Of these, dispersion
polymerization in non-aqueous media allows successful
preparation of micron-sized polymer particles with ease
[28-33]. However, this methodology has suffered draw-
backs when functional monomers were employed [34,
35]. These limitations include polydispersity, odd-shaped
particles and in extreme case coagulation during polymer-
ization. Moreover, the presence of functional comono-
mers in dispersion copolymerization produces polymer
particles with limited number of functional groups on the
surface. To overcome these limitations dispersion pol-
ymerization followed by seeded copolymerization is exp-
ected to be suitable to prepare functional polymer parti-
cles in the micrometer-size range.
The carboxyl group is one of the most widely studied
functional groups for polymer particles intended for bio-
medical or diagnostic applications. This functional group
can undergo easy derivatization using water/oil- soluble
coupling agents. In a previous work H. Ahmad et al. rep-
orted the amination of carboxyl functionalized submic-
ron-sized copolymer latex particles using amine-nucleo-
philes [36]. The present work is an extension of this early
work, emphasizing the precipitation of magnetic nano-
particles on the surface of micron-sized aminated com-
posite polymer particles to diversify their applications in
magnetic support based separation in various fields of
biotechnology and biomedicine, like cell separation [37],
enzyme immobilization [38,39], drug delivery [40], and
protein separation [41,42]. Most of the works available in
the literature are concerned with preparation of nano to
submicron-sized magnetic latex particles [43-50] and a
few with micron-sized latex particles [51,52] bearing id-
entical functional group. To prepare magnetic latex par-
ticles numerous routes have been investigated. They in-
clude the precipitation of iron oxide submicron particles

Preparation of Micron-sized Di-functional Magnetic Composite Polymer Particles
110
in presence of water soluble polymers [53-55], polymer-
ization of suitable monomer mixtures in presence of iron
oxide particles stabilized by surfactants [56-58] and
layer-by-layer self-assembly of alternating layers of pol-
yelectrolytes and magnetic nanoparticles onto colloidal
templates [59]. In a recent work, preparation of submicr-
on-sized sulfate- and carboxyl-functionalized magnetite/
polystyrene spheres is reported for further deposition of
gold nanoparticles [60]. Despite the success of these appr-
oaches little attention has been made for the preparation
of di-functional micron-sized magnetic latex particles.
The present work aims to describe the preparation of
monodispersed micron-sized magnetic composite partic-
les bearing amino and amide groups on or near the parti-
cles surface. The produced di-functional magnetic latexes,
named as aminated-Fe3O4 latexes, would offer advantage-
es in biomedical applications as amino functional group
can be utilized for coupling of affinity ligands or binding
of biomolecules and the remaining amide functional
group can simultaneously be used to modify the particles
surface or even can act as spacer among the immobilized
biomolecules preventing self reaction.
2. Experimental Details
2.1 Materials
Styrene (S) and acrylic acid (AA) purchased from Fluka,
Chemika, Switzerland, were distilled under reduced pre-
ssure to remove inhibitors and preserved in the refrigera-
tor until use. Acrylamide (AAm) from LOBA Chem. In-
dia and N,N'-methylene-bis-acrylamide (MBAAm) from
Sigma, Chemical Company, USA, a crosslinking agent
were used without any purification. 2,2′-Azobisisobuty-
ronitrile (AIBN) and potassium persulfate (KPS) both
from LOBA Chem. India, were recrystallized from etha-
nol and distilled water respectively and preserved in the
refrigerator before use. Poly(N-vinylpyrrolidone) (PVP)
from Fluka, Chemika, Switzerland of molecular weight
3.6 × 105 g/mol was used as a polymeric stabilizer. Tri-
caprylylmethylammonium chloride (aliquate336) from Fl-
uka, Chemika, Switzerland was used as a cationic co-
stabilizer. The biomolecule used was trypsin (TR) from E.
Merck, Germany, used as adsorbent on the surface of
polymer particles without any purification. Dicyclohexyl
carbodiimide (DCC), ethylene diamine (EDA) all from
LOBA Chem. India, were used as supplied. Ferric chlo-
ride hexahydrate (FeCl3·6H2O), ferrous sulphate (FeSO4),
sodium hydroxide (NaOH), oleic acid and other chemi-
cals were of analytical grade. Deionized water was dis-
tilled using a glass (Pyrex) distillation apparatus.
Scanning Electron Microscope, SEM (LEO Electron
Microscopy Ltd., UK); Burker Proton NMR, 250 MHz;
FTIR (8044 Simadzu, Japan); Centrifuge machine from
Kokuson Corporation, Tokyo, Japan; Helios gamma sin-
gle-beam UV-Visible spectrophotometer from Unicam,
UK; HI 9321 Microprocessor pH-meter and conductivity
meter both from HANNA instruments, Portugal were us-
ed in this study. NICOMP 380 (USA) particles sizer was
used to measure hydrodynamic diameter of the polymer
particles. Sherwood Scientific Magnetic Susceptibility Ba-
lance was used for susceptibility measurement. Thermal
analysis was carried out using thermogravimetry (TG)
analyser from Seiko Instruments Inc EXSTAR 6000 TG/
DTA 6300.
2.2 Preparation of Polystyrene (PS) Seed
Particles
Polystyrene (PS) seed particles were prepared by disper-
sion polymerization of 10 g styrene in presence of PVP
(0.432 g) and aliquate336 (0.123 g) using 0.162 g AIBN as
oil soluble initiator. The dispersion media used was
ethanol (65 mL) and the polymerization was carried out
in a 250 mL three necked round bottom flask under a
nitrogen atmosphere at 70℃ for 24 h. The conversion
was nearly 100% as measured gravimetrically.
2.3 Preparation of Micron-Sized Composite
Polymer Particles
Micron-sized PS/P(AA-AAm-MBAAm) composite pol-
ymer particles were prepared by seeded copolymeriza-
tion of AA, AAm and MBAAm in presence PS seed par-
ticles. Prior to the seeded copolymerization PS seed par-
ticles were transferred into water and magnetically stirred
with AA, AAm and MBAAm comonomers at room
temperature for 12 h in order to minimize the formation
of soluble copolymer. The reaction flask was then trans-
ferred to a preheated thermostat water bath maintained at
70℃. The polymerization reaction was carried out under
a nitrogen atmosphere for 12 h using KPS as an initiator.
Polymerization conditions are shown in Table 1.
2.4 Measurement of Hydrodynamic Particle Size
PS/P(AA-AAm-MBAAm) composite polymer particles
were washed by serum replacement to remove any unrea-
cted monomer and initiator fragments. A dilute dispers-
ion of approximately 0.01% polymer solid was prepared,
Table 1. Preparation of PS/P(AA-AAm-MBAAm)a) compo-
site polymer particlesb)
Ingredients
PS dispersionc) (g) 29.5130
AA (g) 0.8881
AAm (g) 0.8759
MBAAm (g) 0.036
KPS (g) 0.29
Water (g) 60.0
a)Core/shell ratio: 1/0.6; b)70˚C, N2, 120 rpm, 24 h; c)Polymer solid, 4.89
g/L

Preparation of Micron-sized Di-functional Magnetic Composite Polymer Particles111
pH was adjusted using acid/alkali solution and intensity
weighted average hydrodynamic size was measured by
NICOMP particle size. Each sample was measured tw-
ice and the average value is reported. The deviation be-
tween successive measurements was less than ± 5%.
2.5 Amination of PS/P (AA-AA-MBAAm)
Composite Particles
PS/P(AA-AA-MBAAm) latex particles were transferred
from water to ethanol medium by ultra-centrifugal was-
hing at 12,000 rpm and the solid content of the latex was
adjusted to about 2%. EDA was covalently bonded to the
carboxyl groups of composite particles by the following
method [36].
Pre-activation method: 50 g of latex dispersion was mi-
xed with 2.1166 g coupling agent (DCC) in a reagent
bottle, magnetically stirred at 25˚C for 1 h. The activated
carboxyl group was then reacted with 0.6154 g EDA at
25˚C for 3 h and then again at 0˚C for 24 h.
2.6 Preparation of Aminated-Fe3O4 Composite
Particles
2.5 g of modified aminated composite particles were
taken in a round bottom flask containing 70 mL water. The
dispersion was cooled (0-5˚C) in an ice-water bath under a
nitrogen gas bubbling. 0.5 g (3 mmol) of FeCl3·6H2O and
0.2558 g (2.7 mmol) of FeSO4 were dissolved in 25 mL
water. Then the iron solution was added to the flask con-
taining the aminated composite dispersion. A light brown
color mixture was formed. The ice bath was removed and
the flask was immersed in a preheated water bath at 85˚C.
Immediately, 2.9123 mL of 5 M aqueous NaOH solution
was added. The reaction mixture gradua- lly turned to
black. The mixture was kept stirring at 85˚C for 2 h.
Oleic acid (0.01456 g, 2.26 mmol) was slowly added
towards the end of the process to stabilize the ami-
nated-Fe3O4 dispersion. Then the dispersion was cooled
to room temperature. The resultant magnetic particles
were centrifuged and thoroughly washed with deionized
distilled water, followed by 0.1 M HCl and again by de-
ionized distilled water.
2.7 Measurement of Magnetic Susceptibility
Aminated-Fe3O4 composite particles were washed, sepa-
rated from their dispersion by centrifugation and dried in
oven at 80˚C for 2 h. The dried powders were placed in a
pre-weighed sample tube and measured the magnetic
susceptibility (g) using the following equation:


9
0
10

m
RRLC
g

where, C is calibration constant of the balance, L is
length of the sample, R0 and R are reading of the empty
and sample tubes and M is weight of the sample (dry
basis) in C.G.S unit.
2.8 Thermogravimetric Analysis (TGA) of
Aminated-Fe3O4 Composite Polymer
Particles
Thermal properties of the dried aminated-Fe3O4 powder
was measured by heating samples under flowing nitrogen
atmosphere from 40˚C to 600˚C at a heating rate of 20˚C
/min and the weight loss was recorded.
2.9 Adsorption of Trypsin (TR) on PS Seed and
Aminated-Fe3O4 Composite Polymer
Particles
A mixture of 20 mL was prepared from purified polymer
dispersion (polymer solid 0.15 g) and TR (200 mg/g).
The pH value of the mixture was immediately adjusted at
the isoelectric point (pH 10.0) using a buffer solution. In
each measurement the dispersion-TR mixture was al-
lowed to stand at 25˚C for 45 min. Polymer particles
were then separated by centrifugation at 10,000 rpm for
10 min. In order to remove wafting particles completely,
the supernatant was centrifuged once more. The concen-
tration of TR in the supernatant was determined by a
UV-visible spectrophotometer at the wavelength of 280
nm. The magnitude of TR adsorbed was calculated by
subtracting the concentration of TR in the medium from
that of the initial concentration. Calibration curve was
used for this purpose.
3. Results and Discussion
Figures 1(a) and (b) shows the SEM photographs of PS
and PS/P(AA-AAm-MBAAm) composite polymer parti-
cles. The average diameters and coefficient of variations
are 1.47 µm and 5.87% for PS seed and 1.51 µm and
2.43% for PS/P(AA-AAm-MBAAm) composite particles.
The average size of PS/P(AA-AAm-MBAAm) composite
particles increased slightly after seeded copolymerization.
Both the PS seed particles and the composite polymer par-
ticles are fairly monodispersed and spherical, having a sm-
ooth homogeneous surface. Submicron-sized P(AA-AAm-
MBAAm) copolymer particles are not observed. This
suggests that seeded copolymerization of AA, AAm and
MBAAm proceeds smoothly on PS seed particles, with
a) b)
2.5 µm
2.5 µm
a) b)
2.5 µm2.5 µm
2.5 µm2.5 µm
(a) (b)
2.5 μm2.5 μm
Figure 1. SEM photographs of (a) PS seed; (b) PS/P(AA-
AAm-MBAAm) composite polymer particles

Preparation of Micron-sized Di-functional Magnetic Composite Polymer Particles
112
out any secondary nucleation.
Figure 2 shows the FTIR spectra of PS seed particles
and PS/P(AA-AAm-MBAAm) composite polymer parti-
cles. Both polymer particles were washed and dried at
70˚C prior to the recording of FTIR spectra. In the spec-
trum of PS/P(AA-AAm-MBAAm) composite particles a
broad O-H stretching of carboxyl group appeared at
2400-3500 cm-1. The signal due to N-H stretching of
primary amide expected to appear at 3200-3500 cm-1 is
possibly been overlapped with O-H stretching signal.
The inclusion of moisture may also affect the appearance
of N-H stretching. Distinct but weak signals due to C=O
stretching vibration for -COOH and -CONH2 groups in
composite particles are appeared at around 1718 and
1690 cm-1, respectively. The combination band due to
phenyl ring of PS largely affected the appearance of
those stretching signals. The weak stretching signals of
-COOH and -CONH2 groups are attributed to the smaller
total surface area of micron-sized composite particles.
Figure 3 shows the 1H NMR spectrum of PS/P(AA-
AAm-MBAAm) composite polymer particles. The che-
mical shifts of the aliphatic groups -CH2 and -CH ap-
peared at 1.4 ppm and 1.86 ppm, respectively. The in-
tense signal due to aromatic protons from PS ring is ob-
served at 6.6 ppm. The characteristic NMR signals due to
acidic (-COOH) and amidic (-CONH2) protons are ex-
pected to be at 10.0-12.0 ppm and 5.0-9.0 ppm, respec-
tively. But it is known that the chemical shifts of these
groups are variable, depend not only on the chemical
environment, but also on concentration, temperature, and
solvent [61]. In the magnified spectra of composite parti-
cles, signals appeared at 8.1 ppm and 4.68 ppm, indicated
the presence of -COOH and -CONH2 groups. The de-
viation of both signals to upfield is due to possible hydr-
ogen bonding between amide and carboxyl groups, hence
b) PS/P(AA-AAm-MBAAm
40003000 20001500
a) PS seed
b) PS/P(AA-AAm-MBAAm
40003000 2000150040003000 20001500
a) PS seed
Wave Number
(
cm
-
1
)
% T
Figure 2. FTIR spectra of a) PS and b) PS/P(AA-AAm-
MBAAm) composite polymer particles
the protons are shielded. The weak signal intensity of
-COOH and -CONH2 groups are attributed to the poor
solubility of cross-linked hydrophilic composite particles
in non-polar CDCl3. Additionally, the presence of -COOH
and -CONH2 was also confirmed by elemental and func-
tional group analyses.
Figure 4 shows the variation of hydrodynamic diame-
ter of washed PS/P(AA-AAm-MBAAm) composite poly-
mer particles with different pH values at 20˚C. Hydrody-
namic diameter increased with the increase of pH value.
This behavior indicates that at lower pH value the com-
posite polymer particles are relatively hydrophobic as the
majority of carboxyl groups derived from AA comono-
mer is protonated. Whereas with increasing pH value
more and more carboxyl groups are deprotonated, which
facilitated the swelling of composite polymer particles
through hydrogen bonding with water.
Figure 5 shows the SEM photographs of aminated
PS/P(AA-AAm-MBAAm) composite, magnetite (Fe3O4),
Figure 3. 1H NMR spectrum of composite polymer particles
Figure 4. Variation of average hydrodynamic diameter of
washed PS/P(AA-AAm-MBAAm) composite polymer parti-
cles with pH values measured at 20˚C

Preparation of Micron-sized Di-functional Magnetic Composite Polymer Particles113
and aminated-Fe3O4 composite particles. All the particles
were washed by serum replacement prior to the observa-
tion by SEM. It is to be mentioned that after three wash-
ings the supernatant obtained from the aminated-Fe3O4
composite particles was clear indicating no leaching of
Fe3O4 particle. The observed magnetite (Fe3O4) particles
were prepared by co-precipitation of Fe2+ (aq) and Fe3+
(aq) in presence of NaOH and aminated-Fe3O4 composite
particles were prepared by the impregnation of iron ions
and precipitation of Fe3O4 on aminated composite poly-
mer particles. The mean diameter of aminated-Fe3O4
composite particles is 1.60 µm which is greater than the
mean diameter of aminated composite polymer particles
(1.54 µm). It is also observed that after amination the
average size of composite particles slightly increased as
compared to that before amination (1.51 µm). Similar inc-
rease in average size after amination was also observed
in our previous work [36]. The surface of the aminated
composite polymer particles is smooth but the surface of
the aminated-Fe3O4 composite particles is not smooth
with many small Fe3O4 particles attached to the surface
of aminated composite particles. Additionally, the preci-
pitation of Fe3O4 particles on aminated composite surface
was confirmed by the measurement of magnetic suscep-
tibility and the value was recorded as positive (2.4768 ×
10-4). This result suggests that the prepared aminated-
Fe3O4 composite particles are paramagnetic. Prior to this
measurement the dispersion was centrifuged and dried at
low temperature (80˚C) to prevent any crystal deforma-
tion of the impregnated Fe3O4 particles.
Figure 6 shows the FTIR spectra of magnetite (Fe3O4)
and aminated-Fe3O4 composite particles. In the FTIR sp-
ectra of Fe3O4, the characteristic stretching vibrations due
to Fe-O bonds of Fe3O4 particles appeared at 582.5 and
439.7 cm-1 respectively, as also reported elsewhere [62,
0.2 µm
a)a)
2.5 µ m2.5 µm
a)b)
0.2 µm0.2 µm0.2 µm
a)a)
2.5 µ m2.5 µm2.5 µm2.5 µ m
a)b) (a) (b)
2.5 μm 0.2 μm
b)
2.5 µm
c)
2.5 µm
b)
2.5 µm
c)
2.5 µm
b)
2.5 µm
c)
2.5 µm
(c)
2.5 μm
Figure 5. SEM photographs of (a) aminated PS/P(AA-AAm-
MBAAm) composite polymer; (b) magnetite (Fe3O4); (c) ami-
nated-Fe 3O4 composite particles
63]. The broad band at 3330.8 cm-1 can be assigned to
the stretching vibration of surface water molecules or the
envelope of hydrogen bonded surface OH groups. In am-
inated-Fe3O4 composite particles a combined strong sig-
nal for N-H and -OH stretching derived from amide, car-
boxyl and surface water molecules is appeared at 3404.1
cm-1. The stretching signal due to Fe-O is also observed
in aminated-Fe3O4 composite particles at around 439 cm-1
and the signal appeared at 540 cm-1 broadened due to the
out of plane C-H bending vibration for substituted phenyl
ring of PS. The above results also confirmed that the
Fe3O4 particles have successfully been precipitated on
the aminated composite polymer particles. TGA was us-
ed to determine the total inorganic material i.e. iron oxide
content in aminated-Fe3O4 composite particles. TGA
curve is illustrated in Figure 7. It is expected that as
theaminated-Fe3O4 composite particles are heated from
ambient temperature to 600˚C, the organic part of the
50010001500200030004000
a) Fe
3
O
4
particles
b) Aminated- Fe
3
O
4
composite
50010001500200030004000
a) Fe
3
O
4
particles
b) Aminated- Fe
3
O
4
composite
50010001500200030004000 50010001500200030004000
a) Fe
3
O
4
particles
b) Aminated- Fe
3
O
4
composite
Wave Number
(
cm
-
1
)
% T
Figure 6. FTIR spectra of a) magnetite (Fe3O4); b) amina-
ted-Fe3O4 composite polymer particles
0
20
40
60
80
100
120
0100200 300 400500 600
Temperature (°C)
Weight loss (%)
443.9 °C
8.8 %
0
20
40
60
80
100
120
0100200 300 400500 600
Temperature (°C)
Weight loss (%)
443.9 °C
8.8 %
Figure 7. TGA thermogram of aminated-Fe3O4 composite
polymer particles

Preparation of Micron-sized Di-functional Magnetic Composite Polymer Particles
114
composite would be burned and the percentage of the
remaining part after calcination would give the iron ox-
ide content. As such TGA thermogram indicates that
about 8.8% (w/w) magnetic Fe3O4 nano-particles are pre-
cipitated on the aminated composite polymer particles.
Considering the larger average size of the composite par-
ticles, the percentage of magnetic iron oxide is pretty
high to modify the particles surface [64].
Figure 8 shows the adsorption behaviors of TR on the
PS seed and aminated-Fe3O4 composite particles. It is
well known that magnitude of adsorption is largely dep-
endent on the degree of hydrophobic interaction between
the particle surface and biomolecules and also on the to-
tal particle surface area. Since the sizes of the seed and
aminated-Fe3O4 composite particles are not the same as
measured from the SEM photographs, so the adsorption
behavior of TR has been presented per unit area of the
particles. The magnitude of adsorption on the aminated-
Fe3O4 composite particles is lower than that on PS seed
particles. As already mentioned, in order to use polymer
particles, as a carrier for biomolecules, the particle sur-
face should be sufficiently hydrophilic with limited am-
ount of hydrophobicity. It is well known that the strong
hydrophobic interaction of biomolecules with solid sur-
face results in the loss of native conformation as well as
the activity of adsorbed biomolecule.
4. Conclusions
A synthetic rout for the preparation of monodisperse mi-
cron-sized magnetic composite polymer particles with
amine and amide functional groups on or near the surface
has been developed. The process involved the prepara-
tion of micron-sized PS latex particle by dispersion poly-
merization followed by seeded copolymerization with
AA, AAm and MBAAm, surface modification with EDA
and subsequent precipitation of iron ions to form Fe3O4.
0.00
0.07
0.14
0.21
0.28
0.35
PS
Magnitude of adsorbed TR
(mg m
-2
)
Aminated-Fe
3
O
4
composite
0.00
0.07
0.14
0.21
0.28
0.35
PS
Magnitude of adsorbed TR
(mg m
-2
)
Aminated-Fe
3
O
4
composite
Figure 8. Magnitude of trypsin (TR) adsorbed on PS seed
and aminated-Fe3O4 composite particles measured under
the constant concentration against the total solid content at
25˚C. pH: 10.0; particles: 0.15 g; TR: 200 mg/g of particles
The presence of amino and amide groups improved hy-
drophilicity and hence could prevent nonspecific adsorp-
tion of proteins.
5. Acknowledgements
This work was supported by the financial grant from
MOSICT, Dhaka (Economic code: 3-2605-3993-5921).
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