PNIPAAM Grafted Polymeric Monoliths Synthesized by the Reactive Gelation Process and their Swelling/Deswelling Characteristics

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

Macromol. React. Eng. 2008, 2, 215–221

� 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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%)



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



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

www.mre-journal.de 217


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

� 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/mren.200700052


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.



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



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.

220Macromol. React. Eng. 2008, 2, 215–221


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


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



Keywords: ATRP; electron microscopy; emulsion polymerization;monolith; polystyrene (PS)

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PNIPAAM Grafted Polymeric Monoliths Synthesized by the Reactive Gelation Process and their Swelling/Deswelling Characteristics