Communication
PNIPAAM Grafted Polymeric MonolithsSynthesized by the Reactive Gelation Processand their Swelling/Deswelling Characteristics
Vikas Mittal, Nadejda B. Matsko, Alessandro Butte, Massimo Morbidelli*
The production of macroporous monoliths functionalized with a thermo-responsive polymer(PNIPAAM) is described. The surface functionalization was achieved by copolymerization ofacrylic end capped atom transfer radical polymerization initiator (BPOEA) with divinylben-zene with or without styrene. Monoliths weregenerated by swelling them with styrene,BPOEA and divinylbenzene followed by gelationwith salt and post polymerization. Subsequentgrafting of these monoliths with PNIPAAM wasachieved by atom transfer radical polymeri-zation and their swelling deswelling character-istics quantified. The grafted monoliths providea unique chromatographic stationary phase whereadsorption/desorption can be driven by theuse of temperature only.
Introduction
The reversible hydrophobicity and hydrophilicity of
poly(N-isopropylacrylamide) (PNIPAAM) gels has been a
subject of interest for many years.[1,2] Owing to their
unique properties, these polymeric gels grown from
various surfaces have found various, very promising
applications, especially for biomedical separations.[3–6]
V. Mittal, A. Butte, M. MorbidelliDepartment of Chemistry and Applied Biosciences, Institute ofChemical and Bioengineering, ETH Zurich, 8093 Zurich, SwitzerlandFax: þ41-44-632 1082;E-mail: [email protected]. B. MatskoElectron Microscopy Center ETH Zurich (EMEZ), Institute ofChemical and Bioengineering, ETH Zurich, 8093 Zurich, Switzerland
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The growth techniques have also undergone tremendous
changes in terms of accuracy and control of molecular
characteristics.[7–11] However, the growth of these poly-
mers from spherical substrates is still not as developed as
their flat counterparts. Spherical substrates, for example
latex particles, offer a unique potential for many applica-
tions owing to their high surface area. Recently, a few
publications have focused on the use of such spherical
particles where aqueous atom transfer radical polymer-
ization (ATRP) was used to grow PNIPAAM brushes from
the emulsifier-free spherical latex particles functionalized
with a thin shell of ATRP initiator.[12–15]
Polymers are used extensively as supports for solid
phase synthesis and especially for chromatographic
separations, due to their high stability upon sanitization.
The polymer in these supports is in the form of highly
porous particles or a monolith, where the surface of the
DOI: 10.1002/mren.200700052 215
V. Mittal, N. B. Matsko, A. Butte, M. Morbidelli
216
polymer provides specific reaction or adsorption sites
based on charge, affinity, etc. As a special functionalization
aimed towards separation processes, PNIPAAM chains can
be grown from the polymer surface of the particles or the
monolith and the chromatographic separations of media,
like proteins, viruses, etc., can be achieved just by changing
the network or eluent temperature. In fact, these media are
typically adsorbing on hydrophobic surfaces [i.e., for
temperatures above the lower critical solution tempera-
ture (LCST)] and can desorb when the surface becomes
hydrophilic (i.e., below the LCST). This would help in
avoiding the use of the harsh adsorption and desorption
conditions used conventionally, which may sometimes
affect the quality of the biological media. A qualitative
adsorption and desorption behavior of tobacco mosaic
virus was successfully shown in another study on latex
particles carrying PNIPAAM brushes.[15] PNIPAAM brushes
on free particles in latex can be expected to swell and
deswell relatively fast on crossing the LCST. However, this
same behavior may be hindered in a polymeric monolith,
where the overall swelling and deswelling process of the
PNIPAAM chains is restricted by the compactness of the
structure.
The main objective of this communication is to investi-
gate the swelling and deswelling characteristics of
functionalized monoliths in order to determine whether
their application to chromatographic applications is
feasible. In particular, we first describe the monolith
formation from polymer particles carrying ATRP initiator
moieties on the surface and the subsequent generation of
ATRP brushes in the monoliths. These have been generated
by using the reactive gelation process described in the
literature for the generation of controlled networks based
on swelling of the latex particles followed by gelation and
post gel polymerization.[16] This process has the advantage
of providing a large control upon the processes of pore
formation and surface functionalization, without signifi-
cant generation of heat.[16] Note that this work represents
the first example of functionalization with polymer brushes
of a monolith produced by reactive gelation. The swelling
deswelling kinetics and hence the ability to adsorb and
desorb the biological entities of the PNIPAAM chains grown
from the monolith is then studied quantitatively.
Experimental Part
Materials
Styrene (STY, �99.5%), divinylbenzene (DVB, �80%), sodium
dodecyl sulfate (SDS, >98%) and radical initiator (potassium
peroxodisulfate, KPS, >99%) were purchased from Fluka (Buchs,
Switzerland) and were used as supplied without further purifica-
tions. ATRP initiator end capped with an acrylic moiety
[2-(2-bromopropionyloxy) ethyl acrylate, BPOEA] was synthesized
as reported previously.[17] N-isopropylacrylamide (NIPAAM, 97%)
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and other reagents to run the ATRP polymerization, namely
1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA, 97%),
copper (I) bromide (CuBr, 99.99%), copper (II) bromide (CuBr2,
99.99%) and copper powder (Cu, 99%, 200 mesh), were procured
from Aldrich (Buchs, Switzerland) and used as received. Ultrapure
Millipore water was employed in all experiments.
Synthesis of Crosslinked Polystyrene Latex and
Surface Functionalization
The crosslinked polystyrene latex was prepared in a Mettler
Toledo reactor (LabMax), as reported previously, using Millipore
water (240 g), styrene (48 g), divinylbenzene (12 g) and SDS (3 g)
followed by the addition of 0.3 g of KPS in 10 mL water after a
temperature of 70 8C was reached.[14] The final solid weight
percent of the latex was 20 wt.-% and the average hydrodynamic
diameter of the particles was found by laser light scattering to be
160 nm. For the surface functionalization, the above synthesized
crosslinked polystyrene seed latex (2.6 g) was heated to 70 8C at
400 rpm and purged with alternate vacuum/nitrogen cycles.
BPOEA (0.21 g) and DVB (0.065g) were added, either alone or with
styrene (0.26 g), to the heated latex as a single shot followed by a
KPS solution (0.01 g of KPS in 0.5 mL of water) after 15 min. The
reaction was allowed to run for 5 h. In an another trial to
investigate the effect of the mode of addition of the monomer feed
on the resulting particle morphology, the KPS solution was first
added to the heated crosslinked polystyrene latex, followed by the
addition of a monomer feed of BPOEA and DVB with and without
styrene in starved conditions. Finally, the functionalized latexes
were washed by repeated ultracentrifugation and resuspension in
Millipore water cycles.
Reactive Gelation
The gelation process consists of latex swelling, gelation and post
gel polymerization.[16] For the swelling process, the washed latex
was added to a flat bottom glass vial. A monomer mixture
consisting of styrene, divinylbenzene and BPOEA was then added
(swelling degree of 10 wt.-% of the solid fraction, DVB weight
fraction 10 wt.-%, styrene to BPOEA weight ratio of 1). An oil
soluble radical initiator, AIBN (1 wt.-% of the monomer weight),
was also added, together with monomer feed. The latex was
degassed and allowed to swell under stirring for 4 h. After
swelling, a solution of NaCl (0.25 mol � L�1) was added to the latex
under vigorous stirring. The dry solid fraction of the final monolith
to be achieved was adjusted always to 10 wt.-% by adjusting the
amount of NaCl solution. The stirrer was then removed and the gel
was left at room temperature for further 8–10 h. The polymeriza-
tion of the swollen gel was initiated subsequently by placing the
vial in an oil bath maintained at 70 8C. The reaction was allowed to
continue for 24 h. The porous monolith was then removed from
the vial and dried in air at room temperature.
ATRP
ATRP of N-isopropylacrylamide was first carried out on the latex
particles functionalized with BPOEA according to the procedure
DOI: 10.1002/mren.200700052
PNIPAAM Grafted Polymeric Monoliths Synthesized by the Reactive Gelation Process . . .
reported in the literature,[12,15] in order to confirm the generation
of brushes from these particles. The crosslinked polystyrene
particles functionalized by forming a shell of styrene, BPOEA and
DVB (added in starved conditions) were used for this study.
NIPAAM (0.21 g, 1.9 mmol), HMTETA (11.3 mg, 49 mmol), CuBr
(2.37 mg, 16 mmol), CuBr2 (0.81 mg, 3.6 mmol) and Cu powder
(1.46 mg, 23 mmol) were carefully measured and stirred with 0.4 g
of the functionalized latex. This was then carefully degassed by
applying alternative vacuum and nitrogen cycles. The reaction
was carried out at room temperature and kept under stirring
overnight. The so-obtained latex particles carrying the PNIPAAM
brushes were washed of any free polymer formed in the solution
by centrifugation and resuspension in Millipore water.
Growth of PNIPAAM chains from the monoliths was achieved
by placing the monoliths in an aqueous solution of required
amounts (based on the dry weight of monolith) of NIPAAM,
HMTETA, CuBr, CuBr2 and Cu powder followed by degassing and
purging with nitrogen. The monoliths were kept immersed in the
monomer solution overnight at room temperature and were
subsequently placed in Millipore water 5–7 times to wash off any
unreacted monomer.
Electron Microscopy, Laser Light Scattering and
Swelling Deswelling Studies
The surface morphology of the particles was observed using
scanning electron microscopy (SEM), with an Hitachi field
emission in-lens S-900 high resolution scanning electron micro-
scope, following the procedure reported previously.[14] SEM
imaging of monoliths was performed by fixing small pieces of
dry monoliths on copper supports followed by sputter coating
with 3 nm platinum. Multiangle dynamic laser light scattering
(DLS, Brookhaven) was used to evaluate the size of the polystyrene
latex particles. Volume average mean diameter of the particles
was taken. Very dilute particle emulsions in distilled water were
measured after equilibration for a sufficient time. The size
determination of particles with PNIPAAM chains at different
temperatures was conducted after the specimen has been
equilibrated to the set temperature for 45 min. Swelling/
deswelling studies of the PNIPAAM chains grown from the
monoliths was performed by placing the monoliths (cut into cubes
of 0.5 cm edge) dipped in water in the baths maintained at
controlled temperatures. The monoliths were then quickly taken
out of water, wiped with filter paper and carefully weighed.
Results and Discussion
Chromatographic supports grafted with PNIPAAM chains
have a great potential for adsorption and desorption
processes driven by temperature only. This may not only
simplify the whole separation process, but also ensure
better handling of the sensitive biological media. In order
to realize this potential, free latex particles coated with a
thin shell of a functional monomer carrying an ATRP
initiator were first tested for PNIPAAM grafting.
Emulsion polymerization in the automated reactor led
to narrowly sized crosslinked polystyrene latex particles
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with an average hydrodynamic diameter of 160 nm.
Figure 1a is a high magnification image of these particles.
The particles are not perfectly spherical, as observed in an
earlier study for crosslinked particles produced by sur-
factant-free polymerization.[14] As styrene and divinyl-
benzene were both entirely added at the beginning of the
polymerization, a gradient in the crosslinking degree can
be expected owing to the fact that divinylbenzene reacts
faster than styrene in copolymerization conditions.[18] There-
fore, under these conditions, the surface of the particles can
be expected to be softer (less crosslinked) than the core.
Functionalization of the surface of the crosslinked
polystyrene particles was achieved by co-polymerizing
an acrylic end capped ATRP initiator (BPOEA) and divinyl-
benzene, with or without styrene. These shell forming
monomers were fed either as a single shot or under starved
conditions in order to analyze the effect of these process
changes on the resulting particle surface and size.[14]
Figure 1b–e show SEM micrographs of these particles. With
the exception of the final particle size, there is no other
visible difference in the morphology of the functionalized
particles. However, when similar trials were carried out in
emulsifier-free conditions, a number of differences in the
particles surface morphology (surface smoothness, rough-
ness, etc), and secondary nucleation were observed.[14] The
presence of emulsifier in the present conditions is believed
to provide colloidal stability as well as a better compat-
ibility between the copolymer chains and the seed
particles. An average particle size of 190 nm was observed
when no styrene was used, whereas a size of 210 nm
resulted when styrene was present in the monomer feed.
We therefore can count on a particle functionalization
technique that responds positively to any monomer feed
ratios, thus allowing us to change the amount of ATRP
initiator, and therefore its surface density, according to the
requirement of the process.
Grafting of PNIPAAM chains from the free particles was
performed first to confirm the growth of brushes. The
thoroughly washed latex particles functionalized with a
thin shell of styrene, BPOEA and divinylbenzene (added in
delayed mode) of Figure 1e were selected as a trial. The
latex was washed rigorously after the PNIPAAM grafting
and dilute solution of this latex was analyzed with laser
light scattering at different temperatures. Figure 2 shows
the plot of thickness of grafted layer as a function of
temperature. The size of the grafted layer decreases
with temperature and almost completely collapses as soon
as the gel temperature exceeds the lower critical solution
temperature of �32 8C. This result confirms the successful
growth of PNIPAAM brushes from the free particles which
as the next step was replicated on the particles joined
together in the network (gel).
Two different starting latexes were employed for the
monolith generation. The first latex was the crosslinked
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V. Mittal, N. B. Matsko, A. Butte, M. Morbidelli
Figure 1. SEM micrographs of: a) seed PS particles; b) seed particles functionalized with a thin layer of surface polymerized BPOEA and DVB(added as a shot); c) seed particles functionalized with a thin layer of surface polymerized BPOEA and DVB (added in starved fashion); d) seedparticles functionalized with a thin layer of surface polymerized STY, BPOEA and DVB (added as a shot); and, e) seed particles functionalizedwith a thin layer of surface polymerized STY, BPOEA and DVB (added in starved fashion).
218Macromol. React. Eng. 2008, 2, 215–221
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PNIPAAM Grafted Polymeric Monoliths Synthesized by the Reactive Gelation Process . . .
Figure 3. SEM pictures of polymeric monoliths generated by reactivemonolith produced from the particles functionalized with a thin layeand, d) high, magnification images of the monolith synthesizedfunctionalization (named as Monolith 2).
Figure 2. A plot of the thickness profile of the PNIPAAM layeraround the functionalized PSTY particles as a function oftemperature, measured by laser light scattering.
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polystyrene particles carrying a thin shell of polymerized
styrene, BPOEA and divinylbenzene (particles of Figure 1e;
network named as Monolith 1). The second latex used for
monolith generation was the thoroughly washed parent
crosslinked polystyrene seed particles themselves (parti-
cles of Figure 1a; network named as Monolith 2). In both
cases, the latex particles were swollen with an additional
load of styrene, BPOEA and divinylbenzene, which was
followed by gelation and post gel polymerization. The use
of these two different latexes allowed us to analyze the
effect of initially present ATRP initiator on the particle
surface apart from the one added during swelling, on
the final grafting of PNIPAAM chains and hence their
characteristics. Figure 3 shows the high and low magni-
fication images of Monolith 1 and 2. Both the monoliths
have porous structure and the primary particles are still
visible, though these seem to be partly fused together in
Monolith 2. The particles in Monolith 1 are well separated
gelation process: a) low, and, b) high, magnification images of ther of polymerized STY, BPOEA and DVB (named as Monolith 1); c) low,from the original crosslinked PSTY particles without subsequent
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V. Mittal, N. B. Matsko, A. Butte, M. Morbidelli
Figure 4. a) Swelling characteristics of Monolith 1 and 2 withrespect to temperature (equilibrated for 1 h at every tempera-ture), where swelling is the amount of water swelling thePNIPAAM brushes per g of particles (excluding the water retainedin the interstitial spaces); b) time dependant swelling of themonoliths as a function of time when placed at 10 8C instan-taneously after equilibrating at 40 8C; and, c) time dependantdeswelling of the monoliths as a function of time when placed at40 8C instantaneously after equilibrating at 10 8C. The lines serveonly as visual guides.
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from each other; this is due to the fact that these particles
have been coated with an additional layer of crosslinker
and other monomers during the functionalization step.
The presence of a crosslinked surface unfavors the particle
interpenetration during gelation. On the other hand, the
crosslinked polystyrene particles in Monolith 2 have softer
shells, as noted above, which swelled more during the
swelling step and allowed a partial fusion of the particle
surfaces. This is in spite of the presence of BPOEA in
the swelling feed, which, as noted in a previous work, is
less compatible with polystyrene.[14] Nevertheless, smooth
particle surfaces are still observable in the monolith, with
a complete absence of secondary nucleation. Moreover,
due to the higher degree of interpenetration of the
particles in Monolith 2, this resulted in a very rigid final
structure. In both cases, a relatively large porosity is
observed. Pores in the order of 1 mm can be distinguished,
which are in the usual range for polymeric monoliths.[19]
The generated monoliths were porous enough to graft
PNIPAAM on the networked particles by ATRP. Figure 4a
shows the swelling properties of grafted PNIPAAM brushes
on the monoliths. Both the curves show sharp deswelling
on exceeding the lower critical solution temperature.
Below the LCST, Monolith 1 is slightly higher in swelling
extent as compared to Monolith 2. This effect could be
explained considering that in Monolith 1 the emulsion
particles were already functionalized with the ATRP
initiator and, thus, the grafting density in this monolith
is higher. Both the curves converge to the same value
above the LCST. It should be noted that PNIPAAM chains on
free particles were observed to swell to a much higher
extent than in an earlier study.[15] The reduced swelling
extent in the monoliths could be due to possible cons-
traints to the PNIPAAM layers in the network. In Figure 4a,
the monoliths were equilibrated for an hour at every tem-
perature. However, it is equally necessary to analyze the
rate of response of the monoliths to temperature changes,
nin order to quantify the efficiency of these monoliths as
supports for chromatographic separations. In fact, it is
believed that the transition from the hydrophobic to
hydrophilic state should take place in a time which is
smaller than or comparable to the characteristic time of
the purification process. Figure 4b and c show the kinetics
of monolith swelling and deswelling when the monoliths
were brought from 10 to 40 8C, and vice versa. It is clear
from the plots that the swelling and deswelling of the
PNIPAAM chains was not instantaneous in these mono-
liths, whereas this was qualitatively observed to be very
fast in the case of free particles.[15] However, the brushes
were swollen to a high degree in less than 30 min and after
90 min there was no further change in the swelling at all.
Deswelling behavior was similar but faster, so that
deswelling was almost complete in less than 30 min,
with subsequent minor changes when kept further at
DOI: 10.1002/mren.200700052
PNIPAAM Grafted Polymeric Monoliths Synthesized by the Reactive Gelation Process . . .
40 8C. This indicates that the grafted PNIPAAM chains have
the ability to swell and deswell even when grown in
constrained environment and confirms the ability of the
generated monoliths to be used as a special support for
the chromatographic separations totally driven by temp-
erature.
Conclusion
Following our previous work, where free latex particles
have been used as a support to grow PNIPAAM brushes
with a controlled polymerization technique (ATRP),[15] it
has been shown in this work that the same result can be
obtained in a constrained environment, for example in
macroporous monoliths. The monolithic structure has
been obtained using the ‘reactive gelation’ technique.[16]
This technique not only allows a precise control of the
macroporous structure – as evident from the microscope
images shown in this work – but also, most importantly,
allows precise control of the surface functionalization of
the latex and the resulting monoliths. The resulting
structures show similar behavior to free particles once
they are functionalized with PNIPAAM brushes. The
reduced swelling capabilities and the slower kinetics in
swelling and deswelling can probably be ascribed to the
constrained environment of the monolith, as opposed to
free particles. Therefore, it is believed that this material
shows an immense potential in chromatographic separa-
tions of biomolecules driven by temperature changes only.
Received: December 17, 2007; Revised: March 17, 2008; Accepted:March 17, 2008; DOI: 10.1002/mren.200700052
Macromol. React. Eng. 2008, 2, 215–221
� 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Keywords: ATRP; electron microscopy; emulsion polymerization;monolith; polystyrene (PS)
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