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Swelling Deswelling Behavior of PS-PNIPAAMCopolymer Particles and PNIPAAM BrushesGrafted from Polystyrene Particles &Monoliths

Vikas Mittal, Nadejda B. Matsko, Alessandro Butte, Massimo Morbidelli*

Two sets of emulsion particles have been synthesized. In the first set, surfactant free emulsionwas used to directly synthesize PS-PNIPAAM copolymer particles. In the second set, poly-styrene particles with an ATRP initiator shell were first synthesized and subsequently graftedwith PNIPAAM brushes. Swelling/deswelling beha-vior of both sets of particles was studied with respectto temperature and time. Monoliths with two differ-ent porosities were also formed by grafting andcrosslinking of PNIPAAM chains on the aggregatedparticles and characterized. In all cases, swellingkinetics is sufficiently fast to use these supportsfor separation driven by temperature changes only.However, hindrance and cross-linking is sensiblyreducing the material performance.

Introduction

Poly(N-isopropylacrylamide) (PNIPAAM) is a well known

polymer for its thermoreversible hydrophobic and hydro-

philic behavior above and below the lower critical solution

temperature, which is close to the human body tempera-

ture (32 8C).[1–4] Owing to this property, surfaces modified

V. Mittal, A. Butte, M. MorbidelliDepartment of Chemistry and Applied Biosciences, Instituteof Chemical and Bioengineering, ETH Zurich, 8093 Zurich,SwitzerlandFax: þ41-44-632 1082; E-mail: [email protected]. B. MatskoElectron Microscopy Center ETH Zurich (EMEZ), 8093 Zurich,Switzerland

Macromol. Mater. Eng. 2008, 293, 491–502

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

with PNIPAAM chains have found wide application in

adsorption desorption of biological entities.[5–12] Though

most of the reported studies focus on flat surfaces,

spherical substrates have also been studied recently to

some extent.[5,13–17] In particular, it is believed that

PNIPAAM modified latex particles and macroporous

monoliths hold a tremendous potential of separation

processes solely controlled by temperature due to their

very large specific surface area and, thus, their use in

commercial separation processes.

Different techniques have been used to functionalize

emulsion particles with NIPAAM. Among these, free

radical copolymerization of NIPAAM and styrene to

generate PS-PNIPAAM core shell particles represents the

simplest one.[18,19] The polymerization is believed to

initially start with the polymerization of the hydrophilic

DOI: 10.1002/mame.200700409 491

V. Mittal, N. B. Matsko, A. Butte, M. Morbidelli

492

monomer, NIPAAM, and followed by copolymerization

with the hydrophobic monomer, styrene, which takes

place in the core of the forming particles. Copolymer

particles obtained by this process consisted of thick

layers of PNIPAAM on the surfaces and exhibited rough

surface morphology. Though this process is very simple,

more tunable properties of the functionalized material are

expected to be achieved using grafting techniques.[20–34] In

particular, grafting of the polymer chains from the surface

using a initiator chemically immobilized on the surface

has received widespread attention because of the genera-

tion of high density polymer brushes covalently tethered

by one end on the surface.[20,26–34] Moreover, the control

on the density of the PNIPAAM brushes was reported to

be dependant on the tuning of the initial density of the

initiator on the grafting surface.[15] This ‘grafting-from

surface’ technique becomes particularly effective when

coupled to living or pseudo-living techniques and in

particular with atom transfer radical polymerization

(ATRP).[13,29–31,33,34] However, a quantitative analysis of

the thermo-reversible behavior of PNIPAAM chains in both

PS-PNIPAAM copolymers particles and PNIPAAM grafted

PS particles is necessary. In fact, due to the complexity of

the functionalization by the ‘grafting from’ technique, it is

necessary to determine whether this is counterbalanced

by a corresponding enhancement of the material perfor-

mance.

Recently, a new technique named ‘reactive gelation’ has

been developed,[35] by which it is possible to obtain

monolithic macroporous materials by controlled aggrega-

tion of emulsion latexes, which are later re-polymerized to

fix the so-obtained macroporous particle network. By this

technique, it is possible to combine the advantages, which

arise from the precise functionalization of the emulsion

particles, with those typical of macroporous monoliths, in

which the transport of the solutes in the flow-through

phase takes place exclusively by convection. For this

reason, monoliths are becoming increasingly attractive for

chromatographic applications involving large molecules,

as proteins, which have very slow pore diffusivities. After

further functionalization of the monolith with thermo-

responsive polymers like PNIPAAM, these large molecules

can be separated solely by the use of temperature, as

initially shown by Peters et al.[36] In fact, as reported in the

literature,[6–12] proteins can adsorb onto the PNIPAAM

brushes above the LCST, i.e. on an hydrophobic surface, and

desorb below the LCST, i.e. when brushes are hydrophilic.

This process is then similar to the so-called hydrophobic

interaction chromatography but, differently from this

process, does not require the use of chemicals, as salt, to

induce the change from hydrophilic to hydrophobic

conditions. PNIPAAM modified particles can be expected

to swell and deswell very fast when subjected to

temperatures below and above the LCST owing to the



absence of any outside constraint.[17] In more constrained

environments like monoliths, a decrease in the kinetics of

swelling/deswelling process can be expected. Therefore,

study of this phenomenon on the PNIPAAM modified

monoliths is of importance in order to quantify the

efficiency of the separation material with respect to

adsorption and desorption.

The aim of this paper is to establish a quantitative scale

of comparison of the swelling deswelling properties of the

PS-PNIPAAM copolymer particles and PNIPAAM grafted

PS particles. In particular, both the extent of swelling as

well as the kinetics of swelling/deswelling is measured.

Additionally, these materials are tested against their

ability to adsorb HSA, in order to confirm the potential

of PNIPAAM grafted particles for separation processes

governed by temperature. Finally, the particles are

networked to form monoliths with different porosities

and the swelling deswelling behavior of PNIPAAM chains

in these monoliths are analyzed and compared with that of

particles.

Experimental

Materials

Styrene (S, �99.5%), divinylbenzene (DVB, �80%), a,a0-

azodiisobutyramidinedihydrochloride (AIBA, �90.0%)

and potassium peroxodisulfate (KPS, >99.0%) were pur-

chased from Fluka (Buchs, Switzerland) and were used as

supplied without further purifications. ATRP initiator end

capped with an acrylic moiety (2-(2-bromopropionyloxy)

ethyl acrylate, BPOEA) was synthesized as reported

earlier.[37]

N-isopropylacrylamide (NIPAAM, 97%) and

N,N0-methylenebis(acrylamide) (MBA, �97%) were pro-

cured from Aldrich (Buchs, Switzerland). The reagents to

run the ATRP polymerization, namely 1,1,4,7,10,10-

hexamethyltriethylenetetramine (HMTETA, 97%), cop-

per(I) bromide (CuBr, 99.99%), copper(II) bromide (CuBr2,

99.99%) and powder copper (Cu, 99%, 200 mesh), were

procured from Aldrich (Buchs, Switzerland). Ultra pure

Millipore water was employed in all experiments.

Synthesis of PS Particles and PNIPAAMGrafting by ATRP

Two different kinds of polystyrene particles were syn-

thesized and subsequently functionalized with the

ATRP initiator, denoted as material 1 and 2, respectively

(Table 1).[16]

In material 1, polystyrene seed particles were

initially prepared by surfactant free emulsion polymeriza-

tion of styrene (14 g) in the presence of 0.3 g KPS and 310 g

of water.[16]

Subsequently, surface functionalization of seed

particles was carried out by a second polymerization step,

DOI: 10.1002/mame.200700409

Swelling Deswelling Behavior of PS-PNIPAAM Copolymer Particles . . .

Table 1. Details of ATRP Initiator Modified Particles used for PNIPAAM Grafting.[16] (dTEM¼ average diameter calculated from TEMmicrographs; monomer molar ratios in the second batch: S: BPOEA¼ 3 and S: DVB¼40.)

Support Seed Second batch of monomers dTEM

nm

Material 1 100% PSa) 100% BPOEA (shot addition) 920

Material 2 100%b) Pc)(Sd) RDVBe)) SRBPOEAf) RDVB (starved addition) 530

a) Polystyrene; b)Monomer conversion; c)Copolymer; d)Styrene; e)Divinylbenzene; f)2-(2-Bromopropionyloxy)ethyl acrylate.

thus generating a thin shell of acrylic end capped ATRP

initiator (BPOEA) around them, as reported earlier.[16]

For this, 15 g of latex was heated to 70 8C at 400 rpm

and purged with alternate vacuum/nitrogen cycles.

BPOEA (0.21 g) was added to the latex followed by KPS

solution (0.0025 g of KPS in 0.5 mL of water) after 15 min.

The reaction was then allowed to run for 5 h. In material 2,

a crosslinked seed latex was synthesized as seed

similarly to the recipe of material 1, in the presence of

0.5 g of divinylbenzene. Surface functionalization was

then achieved by copolymerizing a thin shell of BPOEA

(0.21 g), DVB (0.065 g) and styrene (0.26 g) (0.01 g of

KPS in 0.5 mL of water) on the preformed seed.[16]

The

functionalized latexes were washed by repeated ultra-

centrifugation and re-suspension in millipore water cycles

prior to use.

Atom transfer radical polymerization of N-isopropyl-

acrylamide was carried out on the functionalized latex

particles (material 1 and 2) as reported earlier.[17]

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,

stirred with 4 g of latex (3.5 wt.-%) and then carefully

degassed by applying alternative vacuum and nitrogen

cycles. The reaction was kept under stirring overnight. The

latex particles were washed off any free polymer formed

in the solution by centrifugation and re-suspension in

millipore water.

Synthesis of PS-PNIPAAM Copolymer Particles

Copolymer particles of styrene with N-isopropylacrylamide

were also synthesized under emulsifier-free polymeriza-

tion conditions, using the recipe described in literature.[18, 19]

In a round bottom flask, a NIPAAM/S (10 wt.-% NIPAAM)

solution in millipore water was taken along with AIBA

initiator (1 wt.-% of monomers). The reaction was allowed

to run for 5 h at 70 8C. Core-shell particles were also

synthesized by adding an additional charge of NIPAAM,

MBA (crosslinker) and AIBA dissolved in water to the above

reaction mixture, after a polymerization degree of the



initial monomer charge equal to 70% was reached. The

NIPAAM used for shell is equal to 30 wt.-% of initial

styrene charge, MBA and AIBA are 1.5 and 2.5 wt.-% of the

second charge of NIPAAM, respectively. The reaction

continued for another 3 h. Particles with both crosslinked

core and shell were synthesized by starting the aqueous

polymerization of styrene and divinylbenzene (DVB/S

wt.-% of 3.5) followed by addition of different amounts of

NIPAAM (20 and 40 wt.-% of the initially added styrene)

and MBA crosslinker (MBA/NIPAAM wt.-% of 1.5) after the

initial charge of monomers was polymerized to 70%. First

polymerization step ran for 5 h, while the second was

allowed to proceed for further 3 h. The final solid fraction

in all the experiments was 5 wt.-%.

Swelling/Deswelling and Protein AdsorptionStudies on the Particles

Swelling/deswelling studies on the PS-PNIPAAM copoly-

mer particles as well as on PNIPAAM grafted particles were

conducted in Sorvall Heraeus Multifuge with temperature

control. The particles were centrifuged to measure the total

swelling at the required temperature for 60 min, the

aqueous layer was then decanted and the wet solid weight

measured. Time dependent studies were also carried out

by centrifugation of the latexes for various times at 10 8Cand 40 8C. After temperature equilibration (overnight), the

tube containing the latex were placed into the centrifuge

at the desired temperature, equilibrated and centrifuged. It

has been estimated that the time needed for temperature

equilibration inside the tubes is in the order of 1–2 min.

Adsorption studies of a sample protein were carried out

using Human Serum Albumin (HSA) on the latex particles

modified with PNIPAAM layers at 37 8C. Latex particles of

material 2 after sedimentation were re-suspended in

buffer solution (pH 5.02) and were mixed with different

amounts of protein in the same buffer solution. The

prepared vials were vigorously shaken for 72 h at 37 8C,

after which the particles were allowed to settle while

keeping the temperature constant and the supernatant

was collected and analyzed by high performance liquid

www.mme-journal.de 493


494

chromatography. The difference between the initial and

final protein concentration was used to calculate the

amount of protein adsorbed on the particles. An adsorp-

tion isotherm was then computed where the amounts of

adsorbed protein are correlated with the corresponding

equilibrium concentrations of protein in the liquid phase.

Shear Aggregation of the Particles and MonolithSynthesis by ATRP

The material 2 latex particles functionalized with BPOEA

were sonicated for 10 min (ultrasonic horn at 70%

amplitude) and shear mixed for another 10 min with an

ultra-high shear mixer (Ultra-Turax T50) to induce

aggregation. The resulting macroporous aggregates were

allowed to settle and the aqueous layer decanted to

concentrate the solid fraction. Two different monoliths

were prepared from these concentrated aggregates

(named monolith 1 and monolith 2). To produce monolith

1, the concentrated latex (1.5 g, 15 wt.-%) was transferred

to a flat bottom vial, degassed and subsequently added

with HMTETA (17 mg), CuBr (4.3 mg), CuBr2 (1.4 mg) and

Cu (2 mg). The solution was stirred for 2 min and NIPAAM

(0.4 g) and MBA (0.4 g) were added to the solution. The

viscous slurry was vigorously stirred to solubilize the

monomers. The stirrer was then removed and the vial was

degassed and kept for 12 h without shaking. At the end of

the reaction, a uniform and rigid monolithic structure was

obtained. In the production of monolith 2, the same

procedure was used but an additional 1.5 mL of water was

added to the slurry in order to see the effect of volume

changes on the properties of the monolith. The formed

monoliths were carefully removed from the vials and dried

at room temperature. After drying, the monoliths were

repeatedly washed of any unreacted monomers by placing

them in fresh water for 8–10 times.

Swelling/Deswelling Studies on the Monoliths

Swelling/deswelling studies of the PNIPAAM chains

grown from the particles in the monoliths were performed

by placing the monoliths in water at controlled tempera-

tures in the range 10–40 8C. The monoliths were then

quickly taken out of water, wiped with filter paper and

carefully weighed. The time dependant kinetics were

similarly studied at 10 8C and 40 8C.

Electron Microscopy

The surface morphology of the particles was observed

in Hitachi field emission in-lens S-900 high resolution

scanning electron microscope at accelerating voltages of



10–20 kV. Carbon/collodium coated 400 mesh copper

grids, freshly etched by charged oxygen plasma (10 s,

100 mV, 5 mbar of O2) in Balzers GEA-003-S glow-discharge

apparatus (Balzers), were placed on the droplets of particle

suspensions for 2 min, dried on filter paper followed by

sputter coating with 3 nm platinum. The SEM of monoliths

was performed by fixing small pieces of dry monoliths on

copper supports followed by sputter coating with 3 nm

platinum.

Results and Discussion

PNIPAAM functionalization of surfaces is required to

generate thermally induced hydrophobicity and hydro-

philicity on the support. The technique used to achieve this

functionalization directly affects the performance of the

surface. Although a number of techniques have been

reported in the literature, little effort has been made to

evaluate these quantitatively with respect to each other in

terms of ease of functionalization process and perfor-

mance of the obtained material. In the present study,

PNIPAAM functionalized latex particles have been synthe-

sized using two different techniques, namely copolymer-

ization and surface grafting, and their swelling/deswelling

behavior compared. The swelling/deswelling kinetics has

also been quantitatively established for the PNIPAAM

modified monoliths generated from such particles.

Table 1 shows the details of the seed latex particles

which were functionalized with a thin shell of either

BPOEA polymerized alone (material 1) or copolymerized

with styrene and divinylbenzene (material 2).[16] Figure 1a

and 1d show the SEM images of materials 1 and 2,

respectively. It should be noted that the particle surface

morphologies in these two materials are very different

from each other owing to the changes in the synthetic

process.[16] Colloidal instability of BPOEA chains in the

emulsifier free conditions and their incompatibility with

the PS particle surface are believed to induce their random

collapse on the PS particle support thus originating the

’orange-peel’ morphology in Figure 1a. On the contrary,

the use of crosslinker in the shell forming monomer feed

was observed to reduce this incompatibility and also

accelerate the fast collapse of the forming copolymer

chains on the seed particles, though rough surface

morphology is obtained (Figure 1d).[16] On both surfaces,

ATRP of NIPAAM to graft the PNIPAAM brushes was

carried out. Figure 1b and 1e respectively show the TEM

images of the latex particles of material 1 and material

2 before grafting, whereas Figure 1c and 1f show the same

particles after PNIPAAM grafting. The grafted brushes in

the particles are clearly visible indicating the successful

growth of polymer brushes by using controlled ATRP. Note

that, even if only single particles have been shown in the

DOI: 10.1002/mame.200700409


Figure 1. SEM and TEM images of the NIPAAM functionalized particles formed by surface grafting via ATRP (cf Table 1). (a), (d) SEM and(b), (e) TEM micrographs of materials 1 and 2 particles surface modified with ATRP initiator, respectively; (c), (f) high magnification TEMmicrographs of subsequent PNIPAAM brushes grown from the materials 1 and 2 particles respectively. [17]

Figure 1c and 1f to show the brushes at high magnification,

they anyway represent the overall picture of the whole of

the latex. Other details regarding the grafting procedure

can be found in ref.[16]

Table 2 reports the details of the simple free radical

emulsion polymerizations used to generate PS-PNIPAAM

copolymer particles. Material 3 was synthesized by

Table 2. Polystyrene-PNIPAAM Copolymer Particles Synthesized bycalculated from TEM micrographs (total solids 5 wt.-%).

Expt. Seed Second batch of

Material 3 100% P(Sa)RNIPAAMb)) –

Material 4 70% P(SRNIPAAM) NIPAAMR

Material 5 70% P(SRDVB) NIPAAMR

Material 6 70% P(SRDVB) NIPAAMR

a)S¼ Styrene; b)NIPAAM¼N-isopropylacrylamide; c)MBA¼N,N(-meth



copolymerization of styrene and NIPAAM without any

crosslinker. Material 4 was generated by copolymerization

of styrene and NIPAAM up to 70% conversion followed by

the addition of NIPAAM and MBA aimed to form a

crosslinked shell. Materials 5 and 6 were synthesized by

first forming crosslinked PS seed particles and letting the

polymerization to reach 70% conversion. This was then

Free Radical Emulsion Polymerization; dTEM¼ average diameter

monomers NIPAAM/S Remarks dTEM

wt.-% nm

10 no x-linking 420

MBAc) 40 x-linked shell 510

MBA 20 core-shell x-linked 470

MBA 40 core-shell x-linked 550

ylenebis(acrylamide).



496

followed by the addition of different amounts of NIPAAM

in the presence of crosslinker. Different styrene to NIPAAM

weight ratios were then obtained as indicated in Table 2.

Thus summarizing, while in materials 1 and 2, PNIPAAM

brushes have been formed by ATRP on the surface of

preformed crosslinked polystyrene particles, in the

remaining materials, 3 to 6, styrene and NIPAAM have

been copolymerized together in an uncontrolled fashion.

This is probably leading to a gradient in composition,

which in materials 3 and 4 is extended to the whole

particle, whereas in materials 5 and 6 is restricted to the

Figure 2. Microscope images of the NIPAAM functionalized particle(a), (b), (c) and (d) SEM micrographs of particles of materials 3, 4, 5



particles shell. To be noted is that in materials 4 to 6,

the PNIPAAM chains are crosslinked.

SEM micrographs of materials 3 to 6 are shown in

Figure 2. It can be observed that increasing the amount of

NIPAAM correspondingly increased the amount of this soft

polymer on the surface leading to an apparent physical

networking of the particles at the highest amount of

NIPAAM (material 4 and material 6). Very distinct

morphologies were also observed based on addition of

NIPAAM either at time t¼ 0 (Figure 2a and b) or at 70%

polymerization of styrene (Figure 2c and d). Though the

s formed by PS-PNIPAAM emulsion copolymerization (cf Table 2).and 6 respectively.

DOI: 10.1002/mame.200700409


Figure 3. Swelling behavior of particles in the temperature rangeof 10 8C to 40 8C; swelling¼ amount of water swelling the PNI-PAAM chains per g of particles (excluding the water retained inthe interstitial spaces).

PNIPAAM functionalization of the particles could also be

achieved in this material, the control on the amount of the

PNIPAAM on the surface and, in particular, of its molecular

weight is not possible, due to the uncontrolled nature of

the copolymerization between styrene and NIPAAM.

Secondary nucleation leading to growth of smaller

particles was also observed to some extent especially in

the cases where higher amounts of NIPAAM were used.

In Figure 3, the swelling behavior of the particles with

respect to temperature is shown in the range 10 to 40 8C.

The effect of water in the interstitial spaces between

particles was subtracted during the swelling calculation in

order to report the true effect of swelling associated with

PNIPAAM chains only. All the particles show the sharp

reduction in the swelling behavior as soon as the lower

critical solution temperature of �32 8C is exceeded and

eventually all converge to the same swelling value. Below

this temperature, varying amounts of swelling were

observed for the different materials. Materials carrying

PNIPAAM brushes formed by controlled polymerization

(materials 1 and 2) were observed to have highest swelling

degree throughout, although they themselves are very

similar in behavior. The swelling degree was observed to

be maximum at the lowest temperature investigated, at

which the PNIPAAM brushes were swollen to nearly

3.3 times their dry weight. The copolymer particles on the

other hand showed much lower swelling degrees. Material

4, having the highest NIPAAM fraction, showed the

highest swelling among these particles with a value of

0.8 at 10 8C. Other materials were even below a swelling

degree of 0.5. This indicates that there is indeed a wide

difference in performance between the particles synthe-



sized by these two techniques. The reduced swelling in the

PS-PNIPAAM copolymer particles could have resulted

owing to the embedded and entangled PNIPAAM chains

on the surface of the particles which cannot freely float out

from the surface. Whereas in the grafted particles, the

brushes are much more efficient in swelling owing to the

well defined molecular structure of the brushes. Also no

embedding of PNIPAAM chains is suspected in this case.

One important point to note here is that the starting ratio

of NIPAAM to PS solids in the grafted latexes is much

higher than that in the copolymerized latexes. However, as

generally observed in surface grafting reactions, the

majority of the formed polymer is in solution. Kizhakke-

dathu et al. reported in a recent study that more than 80%

of the formed polymer was in solution.[13] This indicates

that only a fraction of the starting amount of NIPAAM is

probably used for grafting, therefore, bringing the effective

NIPAAM to PS solid ratio to be approximately 30% or lower

i.e. comparable to those of materials 3 to 6. In Figure 3, it is

also shown the swelling degree of a material synthesized

with the same recipe as material 1, but using half the

amount of NIPAAM (data point & at 10 8C in Figure 3). In

this material, which has an effective starting NIPAAM to

PS solids ratio lower than most of the materials produced

by direct copolymerization (materials 3 to 6), the swelling

degree has been observed to be much higher. This clearly

quantifies the superiority of the grafted brushes in terms

of swelling and thus clearly justifies the additional effort in

the synthetic process. The grafting technique also has

proved to be extremely responsive to the NIPAAM to PS

solid ratio, whereas this was not much controlled in the

case of copolymer particles. Therefore, the required

characteristics of the brushes can only be accurately

obtained in the grafting process, whereas the copolymer-

ization leads to more solid layers on the surface than the

defined brushes.

Figure 4 shows the swelling behavior of the different

materials as a function of time when kept at a constant

temperature of 10 8C. All the particles are observed to swell

fast irrespective of the technique of their synthesis. In less

than 30 min, the particles were observed to have achieved

almost complete swelling with slower changes further on.

No considerable change in swelling could be observed after

60 min. Note that most of the swelling took place in the

first few minutes. However, no experimental points were

added to Figure 4, since the handling of the sample takes

approximately the same time as the swelling time (few

minutes) and it is then very imprecise. The deswelling

kinetics were also observed to have similar behavior, as

shown in Figure 5. The particles were equilibrated at 10 8Cinitially and then quickly put at 40 8C to promote the

deswelling. A sharp deswelling was observed in less than

30 min and no further change in deswelling occurred after

60 min.



Figure 4. Swelling kinetics of the PNIPAAM functionalizedparticles at 10 8C with respect to time (details on materials 1 to6 are reported in Tables 1 and 2).

Figure 5. Deswelling kinetics of the PNIPAAM functionalizedparticles at 40 8C with respect to time (details on materials 1to 6 are reported in Tables 1 and 2).

Figure 6. Adsorbed amount of HSA per volume of polymer as afunction of the HSA concentration in the liquid phase. Squarepoints: experimental data; solid curve: best fitting of the exper-imental data using a Langmuir adsorption isotherm.

498Macromol. Mater. Eng. 2008, 293, 491–502


The grafted particles were quantitatively much better in

their extent of swelling than the copolymer particles. As an

additional study, the adsorption capacity of these particles

was also quantified with respect to a model protein to

confirm their importance for separation processes. Figure 6

shows the quantitative protein (HSA) adsorption studies at

37 8C on the PNIPAAM grafted particles (material 2). No

adsorption has been observed for HSA below the LCST. The

adsorption reached a plateau value roughly at a value of

80 mg of HSA per mL of support. This value indicates a very

good capacity of the PNIPAAM functionalized particles,[38]

which is comparable to the value measured for commer-

cial beads for adsorption.[39] It should also be noted that,

due to the relatively large size of the particles used in the

experiment (�530 nm), the specific surface area of

material 2 is only about 10 m2 � g�1. On the other hand,

a 5 to 10 fold-increase of the surface area could be obtained

by reducing the diameter of the primary particles by the

same factor, thus reaching the typical surface area for

conventional monolithic stationary phase, which has been

reported to range in between 60 and 210 m2 � g�1.[40] For

this reason, it is expected that by decreasing the particle

size, a considerable improvement of the material capacity

can be obtained. Overall, this result clearly confirms the

potential of these materials for preparative separations of

the biological media lonely driven by temperature.

As the use of small particles would be impractical in

packed chromatographic columns, monoliths represent a

convenient support for adsorption. Therefore the swelling

deswelling behavior of the monoliths and hence their

efficiency to adsorb and desorb needs to be analyzed and

compared with that of particles. To generate monoliths in

the emulsified systems, the reactive gelation process has

DOI: 10.1002/mame.200700409


Figure 7. SEM micrograph of the high shear aggregated particlesof material 2 used for synthesis of monoliths.

Figure 8. (a) and (b) SEM micrographs of monoliths 1 and 2respectively. The two monoliths were synthesized by using sameamount of aggregates, but different solid percents of the latexcontaining the aggregates.

been used.[35] Due to the large size of the particles formed

in the emulsifier-free polymerization, these are typically

colloidally stable and destabilization cannot be obtained

even when using large amounts of salt. Therefore, a

different approach based on high shear mixing was

employed to generate the monoliths. When the latex

particles of material 2 (functionalized with BPOEA, but

without PNIPAAM brushes) were subjected to high shear

forces in a sonicator and shear mixer, a network of physical

aggregates resulted owing to the destabilization and

collapse of the latex. Figure 7 shows the SEM image of

these aggregates. The aggregates are clearly visible to

be porous thus providing the ideal material to generate

monoliths. Stable monoliths were obtained when these

aqueous aggregates were subjected to PNIPAAM grafting

reactions using ATRP. A high amount of water soluble

crosslinker was used in order to provide strength to these

structures. Two monoliths with different solid fractions

were studied. The solid fractions were altered in order to

estimate the effect of availability of free volume to the

PNIPAAM chains in the monolith on their swelling

deswelling properties. Figure 8a and b shows the SEM

images of the monoliths. Figure 8b shows the monolith

with half the solid fraction as the monolith in Figure 8a. In

both SEM images, the primary particles used as support for

the functionalization can be identified, as well as the large

micron-size pores in between them which can be used for

the convective transport of the solutes to the polymer

brushes. Owing to low solid fraction, monolith 2 in

Figure 8b was observed to be more porous and, thus, more

space may be available to the PNIPAAM chains to expand

during the swelling studies.



In Figure 9, the swelling curves of the monoliths as a

function of temperature are shown. Though the curves

have similar shape as those observed for the particles

(cf Figure 3), the amount of swelling decreased to a large

extent. High extent of crosslinking is believed to be the

main reason for such a decrease. The restriction to expand

in the monolith environment also is expected to contribute



Figure 9. Effect of temperature on the equilibrium swelling valueof PNIPAAM chains in the monoliths.

Figure 11. Deswelling of PNIPAAM chains in the monoliths at atemperature of 40 8C with respect to time.

500

to the reduced swelling of the chains. The effect of

restriction to swell is clearly visible when the behavior of

monoliths is compared. Monolith 2, having the higher

porosity, is far more swollen than monolith 1 owing to

higher free volume available in the structure. In spite of

the effect of space restrictions in the monolith structure,

the swelling behavior appears to be very similar to that

observed for particles of materials 1 and 2, which have also

been functionalized with PNIPAAM using ATRP. On the

other hand, the effect of space restrictions and crosslinking

can possibly be the reason for the different swelling

kinetics observed for the monoliths, as shown in Figure 10

and Figure 11. The monoliths achieved high extents of

swelling in 90 min though even further the swelling

slowly increased even after 2 h (Figure 10). Similar

behavior was also observed for the deswelling kinetics

(Figure 11). This indicates that both the time and extent of

Figure 10. Swelling kinetics of PNIPAAM chains in the monolithsas a function of time at a temperature of 10 8C.



reversible hydrophilic and hydrophobic behavior was

reduced in the monoliths as compared to the free particles.

However, it also confirmed that this thermally induced

behavior can also be generated in the networks. Therefore,

it can be safely said that these monoliths have tremendous

potential in the separation process technology as they can

induce additional functionality in the process thus

improving the whole process in terms of simplicity and

economics. Also, it is important to notice that the covalent

bonding among the particles is guaranteed by the cross-

linking of the PNIPAAM brushes only. Accordingly, the

particles act as nodes of a network structure which can

swell, and in general, behave as a sponge. It has been in

fact observed that monoliths have a spongy consistence

when wet and turned hard solid when dry, therefore, these

reversible sponges can also be suitable for use in fluid

filters & cleaners as well as in surface cleaning media.

Recently, another kind of monolithic structure functio-

nalized with PNIPAAM has been reported.[41] Two macro-

scopic differences can be readily identified in their

structure as compared to the monoliths described in this

work. A monolith consisting of PS emulsion particles has

been first formed using the reactive gelation technique.

This structure has been then functionalized by ATRP, using

the same recipe as for materials 1 and 2. Accordingly, this

structure appears very rigid and did not appear as a

sponge, due to the very rigid backbone of PS particles

obtained by reactive gelation. The second major difference

with the monoliths discussed herein is that in monoliths in

ref.[41], the PNIPAAM brushes are not cross-linked and,

therefore, are able to behave as material 1 and 2. It has

been shown that the rigid monoliths produced by reactive

gelation are able to swell to values which are comparable

to those of material 1 and 2 (swelling ratio of 1.5), but still

smaller. Accordingly, it can be said that the cross-linking of

DOI: 10.1002/mame.200700409


the PNIPAAM chains is the main responsible for the large

decrease in the swelling ratio observed for the ‘‘spongy’’

monoliths (monolith 1 and 2), although the space

constraints could still play a non-negligible role in limiting

the swelling ratio. In addition to this, a similar kinetics of

swelling as compared to materials 1 and 2 was observed

for the rigid monolith, for which most of the swelling took

place in about 30 min, and it was completed in 90 min. This

result further demonstrates that the tailoring of PNIPAAM

functionalized monolithic supports is possible according to

the requirements of the separation processes.

Conclusion

In this work, the potential performance of several supports

functionalized with the thermo-responsive polymer

PNIPAAM was systematically evaluated. In particular,

PS-PNIPAAM copolymer particles and PNIPAAM grafted PS

particles were first synthesized and their swelling/

deswelling behavior compared quantitatively with respect

to temperature and time. PNIPAAM grafted particles

showed a swelling degree of more than 3 times, whereas

the copolymer particles were swollen to less than 1 at the

same conditions. The well defined brush morphology is

responsible for the much better response of the grafted

particles. In particular, it is believed that the copolymer

structure does not allow PNIPAAM to fully exhibit its

potential in switching between hydrophilic and hydro-

phobic behavior, nor does the presence of crosslinking. This

also indicates that much better control on the process can

only be achieved if a grafting route involving ATRP is

selected to functionalize the particles. The rate of swelling

and deswelling of these particles was unaffected by the

technique of functionalization. The HSA protein adsorp-

tion studies on the PNIPAAM grafted particles also

confirmed the large potential that such supports may

have for chromatographic separation processes. The

capacity of these particles to adsorb the protein molecules

per unit surface area was in fact comparable to (if not

larger) the capacities of the commercially available

separation supports.

A second type of support was also analyzed. In fact,

emulsion particles are impractical for commercial use in

chromatographic columns, since it is unpractical to pack

so small particles and their use would then result in

extremely large pressure drops. A convenient support is

then represented by macroporous monoliths, since these

offer large surfaces for adsorption, limited pressure drops

and an excellent transport of the bio-molecules to the

adsorption sites. ’Aggregate grafting’ process was selected

to synthesize these monoliths, thus avoiding the use of salt

used for gelation and monolith generation. Monoliths

obtained from these aggregated particles after PNIPAAM



functionalization were stable and strong. The same

behavior of thermo-reversible hydrophobicity and hydro-

philicity of the PNIPAAM chains was observed in the

monoliths, though the extent and rate of swelling is

hindered owing to the large degree of crosslinking among

PNIPAAM brushes and space constraints to the chains. This

technique is demonstrating that spongy structures can be

achieved using PNIPAAM brushes. On the other hand,

another study on the functionalization of highly rigid

supports prepared via reactive gelation has demonstrated

that similar behavior to that of the single particles carrying

PNIPAAM brushes can be obtained, both in terms of

swelling degree and kinetics.[41] Therefore, this study

demonstrates that the PNIPAAM functionalization of the

particles can be achieved in macroporous structures, as

monoliths, thus indicating that these networks hold

tremendous potential to be used as chromatographic

separation media solely controlled by temperature.

Received: December 17, 2007; Revised: April 7, 2008; Accepted:April 7, 2008; DOI: 10.1002/mame.200700409

Keywords: ATRP; electron microscopy; monolith; PNIPAAM;polystyrene

[1] R. Yoshida, K. Uchida, Y. Kaneko, K. Sakai, A. Kikuchi, Y.Sakurai, T. Okano, Nature 1995, 374, 240.

[2] Y. H. Bae, T. Okano, S. W. Kim, J. Polym. Sci., Part B: Polym. Phys.1990, 28, 923.

[3] M. Heskins, J. E. Guillet, J. Macromol. Sci. Chem. 1968, A2, 1441.[4] H. Ringsdorf, E. Sackmann, J. Simon, F. M. Winnik, Biochim.

Biophys. Acta 1993, 1153, 335.[5] J. N. Kizhakkedathu, R. Norris-Jones, D. E. Brooks, Macromole-

cules 2004, 37, 734.[6] D. Cunliffe, C. H. Alarcon, V. Peters, J. R. Smith, C. Alexander,

Langmuir 2003, 19, 2888.[7] T. O. Collier, J. M. Anderson, A. Kikuchi, T. Okano, J. Biomed.

Mater. Res. 2002, 59, 136.[8] K. Hosoya, Y. Watabe, T. Kubo, N. Hoshino, N. Tanaka, T. Sano,

K. Kaya, J. Chromat. A 2004, 1030, 237.[9] D. M. Jones, R. R. Smith, W. T. S. Huck, C. Alexander, Adv.

Mater. 2002, 14, 1130.[10] S. Okajima, T. Yamaguchi, Y. Sakai, S. Nakao, Biotech. Bioeng.

2005, 91, 237.[11] T. Okano, N. Yamada, M. Okuhara, H. Sakai, Y. Sakurai,

Biomaterials 1995, 16, 297.[12] G. Chen, Y. Ito, Y. Imanishi, Biotech. Bioeng. 1997, 53, 339.[13] J. N. Kizhakkedathu, K. R. Kumar, D. Goodman, D. E. Brooks,

Polymer 2004, 45, 7471.[14] J. N. Kizhakkedathu, A. Takacs-Cox, D. E. Brooks, Macromole-

cules 2002, 35, 4247.[15] J. N. Kizhakkedathu, D. E. Brooks, Macromolecules 2003, 36,

591.[16] V. Mittal, N. B. Matsko, A. Butte, M. Morbidelli, Polymer 2007,

48, 2806.[17] V. Mittal, N. B. Matsko, A. Butte, M. Morbidelli, Eur. Polym. J.

2007, 43, 4868.



502

[18] D. Duracher, R. Veyret, A. Elaissari, C. Pichot, Polym. Int. 2004,53, 618.

[19] D. Duracher, F. Sauzedde, A. Elaissari, A. Perrin, C. Pichot,Colloid Polym. Sci. 1998, 276, 219.

[20] T. Sun, G. Wang, L. Feng, B. Liu, Y. Ma, L. Jiang, D. Zhu, Angew.Chem. Int. Ed. 2004, 43, 357.

[21] M. K. Chaudhury, G. M. Whitsides, Science 1992, 256, 1539.[22] Y. Nagasaki, K. Kataoka, Trends Polym. Sci. 1996, 4, 59.[23] P. Mansky, Y. Liu, E. Huang, T. P. Russell, C. J. Hawker, Science

1997, 275, 1458.[24] G. Fytas, S. H. Anastasizdis, R. Seghrouchni, D. Vlassopoulos,

J. Li, B. J. Factor, W. Theobald, C. Toprakcioglu, Science 1996,274, 2041.

[25] Y. Mir, P. Auroy, L. Auvray, Phys. Rev. Lett. 1995, 75, 2863.[26] O. Prucker, J. Ruhe, Macromolecules 1998, 31, 592.[27] O. Prucker, J. Ruhe, Macromolecules 1998, 3, 602.[28] B. Zhao, W. J. Brittain, J. Am. Chem. Soc. 1999, 121, 3557.[29] K. Matyajaszewski, P. J. Miller, N. Shukla, B. Immaraporn,

A. Gelman, B. B. Luokala, T. M. Siclovan, G. Kickelbick,T. Vallant, H. Hoffmann, T. Pakula, Macromolecules 1999,32, 8716.



[30] B. Zhao, W. J. Brittain, Macromolecules 2000, 33, 8813.[31] J. Kim, M. L. Bruening, G. L. Baker, J. Am. Chem. Soc. 2000, 122,

7616.[32] Y. Z. You, C. Y. Hong, C. Y. Pan, P. H. Wang, Adv.Mater. 2004, 16,

1953.[33] C. Perruchot, M. A. Khan, A. Kamitsi, S. P. Armes, T. von Werne,

T. E. Patten, Langmuir 2001, 17, 4479.[34] M. Ejaz, S. Yamamoto, K. Ohno, Y. Tsujii, T. Fukuda, Macro-

molecules 1998, 31, 5934.[35] N. Marti, F. Quattrini, A. Butte, M. Morbidelli, Macromol.

Mater. Eng. 2005, 290, 221.[36] E. C. Peters, F. Svec, J. M. J. Frechet, Adv. Mat. 1997, 9, 630.[37] K. Matyajaszewski, S. G. Gaynor, A. Kulfan, M. Podwika,

Macromolecules 1997, 30, 5192.[38] J. Janzen, Y. Le, J. N. Kizhakkedathu, D. E. Brooks, J. Biomater.

Sci. Polym. Edn. 2004, 15, 1121.[39] G. Bayramoglu, A. U. Senel, M. Y. Arica, Polym. Int. 2006, 55,

40.[40] F. Svec, J. Sep. Sci. 2004, 27, 747.[41] V. Mittal, N. B. Matsko, A. Butte, M. Morbidelli, accepted for

publication in Macromol. React. Eng.

DOI: 10.1002/mame.200700409

swelling deswelling behavior of ps-pnipaam copolymer particles and pnipaam brushes grafted from...

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