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ORIGINAL CONTRIBUTION

Thermal property changes of poly(N-isopropylacrylamide)microgel particles and block copolymers

Klaus Tauer & Daniel Gau & Susanne Schulze &Antje Vlkel & Rumiana Dimova

Received: 30 October 2008 /Revised: 8 December 2008 /Accepted: 8 December 2008 /Published online: 3 January 2009# The Author(s) 2008. This article is published with open access at Springerlink.com

Abstract Investigation of the thermo-reversible propertiesof different poly(N-isopropyl acrylamide) samples, includ-ing microgels and block copolymers, with a combination ofmethods such as electron microscopy, dynamic lightscattering, analytical ultracentrifugation, electrophoresisand ultrasound resonator technology allows comprehensivecharacterisation of the phase transition. By the combinationof methods, it was possible to show that the precipitatedpolymer phase contains at 40 C between 40 and 50 vol.%of water. Besides free bulk water, there is also bound waterthat strongly adheres to the N-isopropyl acrylamide units(about 25 vol.%). Ultrasound resonator technology, whichis a non-sizing characterisation method, revealed for themicrogel particles two more temperatures (at about 35 andbetween 40 C and 50 C depending on the chemicalnature) where characteristic changes in the ultrasoundattenuation take place. Moreover, the experimental datasuggest that the phase transition temperature is related tosurface charge density of the precipitated particles.

Keywords PNIPAMmicrogels and block copolymers .

Characterisation of thermal properties

Introduction

Since the first report of a lower critical solution temperature(LCST) of poly(N-isopropylacrylamide) (PNIPAM) in ascientific paper, 40 years ago [1], and subsequent discov-eries that by the addition of surfactants [2] or bycopolymerisation with acrylamide and cross-linkers [3]thermo-sensitive colloidal particles are accessible, thepolymer became most popular as a kind of standardmaterial. The precipitation temperature of PNIPAM indiluted solutions is about 32 C (plus or minus a fewdegrees depending on the particular conditions), which isan attractive range for biomedical applications, particularlydrug delivery [48]. Hence, quite a large number of reviewshave been published until now [3, 915]. This temperatureis frequently, also in this contribution, denoted as LCST.However, in a strict thermodynamic sense, it is a precipi-tation and dissolution temperature upon heating and cool-ing, respectively. The miscibility gap of PNIPAM has aminimum at weight fraction between 0.4 and 0.5 (slightlydepending on the molecular weight of the sample) at atemperature, the actual LCST, between 26 C and 27 C.Only in very diluted or very concentrated solutions does thephase separation take place at higher temperatures [16].Other experimental results confirm a minimum in thetemperatureconcentration phase diagram at about 50 wt.%but at temperature below 25 C [17].

The thermo-reversible behaviour of PNIPAM dependsstrongly on the architecture of the macromolecules suchas the kind of comonomers leading to random or blockcopolymers [10, 15, 1826] but also on the nature of theendgroups [2734]. Though PNIPAM is quite a long timein the focus of the research, a lot of interesting resultsappear still each year. The following examples should

Colloid Polym Sci (2009) 287:299312DOI 10.1007/s00396-008-1984-x

K. Tauer (*) :A. Vlkel :R. DimovaMPI Colloids and Interfaces,14476 Golm, Germanye-mail: [email protected]

D. Gau : S. SchulzeTF Instruments GmbH,Im Neuenheimer Feld 583,69120 Heidelberg, Germany

illustrate this. Recently, N,N-diethylacrylamide-co-N-isopropylacrylamide copolymer microgel particles havebeen described, showing a synergistic depression of thetransition temperature as the phase transition takes placeat temperature lower than that of the correspondinghomopolymers [35]. Thermo-responsive PNIPAM shellsaround hydrophobic cores that contain inorganic nano-particles are interesting inorganic polymeric compositematerials for various potential applications. The synthesisof hybrid coreshell particle with about 33 wt.% ofmagnetic nanoparticles in the core is reported in [36]. Theapplication of siloxane comonomers is a new andinnovative approach to cross-link the particles viahydrolysiscondensation reaction and an alternative routeto inorganic organic hybrid materials [37].

In summary, the LCST of PNIPAM is not a single valuecharacterising the polymer per se unambiguously but ismuch more the result of rather complex interactions takingplace over a certain temperature range and depends for agiven solvent on the polymer concentration, the molecularweight, the nature of the end groups and the comonomercontent and distribution along the chain. For chemists, thepossibility to tune the phase separation by the moleculararchitecture is a truly fascinating challenge.

Reported in this contribution are results of a comprehensivestudy of the thermo-reversible properties of chemically andmorphologically different PNIPAM samples by means ofscanning and transmission electron microscopy (SEM,TEM), dynamic light scattering (DLS), ultrasound resonatortechnology (URT), analytical ultracentrifugation (AUC) andzeta potential measurements. Particularly PNIPAM microgelswith anionic (PNIPAM-xl-A, sulfate endgroup), cationic(PNIPAM-xl-C, amidinopropane hydrochloride end group)and poly(ethylene glycol) (PNIPAM-xl-PEG, molecularweight of the PEG 2104 g/mol) endgroups and poly(styrenesulfonate)-PNIPAM block copolymers (PSS-PNIPAM) werestudied. Thermo-sensitive properties of PNIPAM-xl-PEGmicrogels and PNIPAM-PSS block copolymers are reportedfor the first time. On the one hand, the data allowconclusions regarding the influence of the state before thephase transitioneither swollen particles (PNIPAM-xl-A,PNIPAM-xl-C, PNIPAM-xl-PEG) or dissolved single poly-mer molecules (PNIPAM-PSS). On the other hand, theexperimental results prove the influence of the nature of theend groups incorporated during the polymerisation procedureor the molecular architecture. In all cases, two componentsystems are studied consisting of the particular PNIPAMsample and pure water. There is no surfactant to stabilise thecolloidal particles because its presence influences at least thesize of the precipitating particles. Moreover, it is known thatsurfactants, such as sodium dodecyl sulphate, are able tosolubilise not only organic liquids but also polymermolecules in water [32, 38, 39].

The applied analytical techniques enable conclusionsregarding the influence of the detailed chemical nature onthe phase transition. Particularly, results are reportedregarding the reversibility of the precipitation/redissolutionprocess, the morphology of the precipitate, the chargedensity and the self-stabilisation of the particles and, to thebest of our knowledge, for the first time, the amount ofhydration water bound strongly to the PNIPAM chain. Notethat the hydration water is different from the amount ofwater present in the PNIPAM particles at T > LCST, whichis between 30 and 20 wt.% at temperatures between 35 Cand 50 C [5]. Moreover, by means of URT, othertransitions of the PNIPAM microgel particles in thetemperature range above the LCST of about 32 C havebeen detected.

Experimental information

Materials NIPAM (Acros) was recrystallised from a mix-ture of hexane/toluene (3/1, v/v), sodium styrene sulfonatefrom Sigma-Aldrich, poly(ethylene glycol) with a molecu-lar weight of 20,000 g/mol (Fluka), N,N-methylenebis(acrylamide) (MBA) from Fluka, ethyleneglycoldimetha-crylate (EGDMA) from Aldrich, potassium peroxodisulfate(Fluka), 2,2-azobis(2-amidinopropan)dihydrochlorid (V50)and 2,2-azobis (2-methyl-N-(2-hydroxyethyl) propiona-mide (VA-086) both from Wako, ceric ammoniumchloride(CAN) from Fluka which were all used as received.Polymerisation and characterisation was carried out inultrapure water (Seral purification system PURELABPlus) with a conductivity of 0.06 S cm1.

Polymerisation The PSS precursor polymer was synthes-ised with the following recipe at 70 C: 100 g of water, 20 gof sodium styrene sulfonate and 0.32 g VA-086. Afterpolymerisation, the reaction mixture was extensivelycleaned by ultrafiltration through DIAFLO membraneswith a molecular weight cutoff of 104 g/mol (type YM 10from Amicon, USA), and the polymer was isolated byfreeze drying. The PNIPAM polymers were prepared byradical polymerisation at 70 C, except PNIPAM-PEG at60 C, in all-glass reactors equipped with a jacket forheating or cooling to adjust the temperature, a condenser, anitrogen inlet and outlet, a stirrer and a bottom valve toremove the reaction mixture. The reactor lid contains anadditional opening for injecting the initiator solution afterthermal equilibration of the reaction mixture. After poly-merisation (polymerisation time 2024 h), all polymerswere cleaned by ultrafiltration through DIAFLO mem-branes with a molecular weight cutoff of 105 g/mol (typeYM 10 from Amicon) as long as the amount of originalwater was replaced ten times. Then, the polymers were

300 Colloid Polym Sci (2009) 287:299312

isolated by freeze drying. The polymerisation recipes ofpolymer samples are detailed in Table 1.

Characterisation Elemental analysis was carried out withVario micro Cube, (elementar, Hanau, Germany). Thechemical composition of PNIPAM-PEG and PNIPAM-PSS was characterised oxygen and sulfur elemental analysisdata, respectively. The molecular weight distributions of thePSS precursor polymer was analysed by analytical ultracen-trifugation according to standard procedures [40, 41]. Somesamples were investigated with electron microscopy aftersuspension preparation (either SEM with a LEO, Electro-nMicroscopy, UK or TEM with a Zeiss EM 912 Omegamicroscope operating at 100 kV). Fourier transforminfrared spectra were recorded with a Varian 1000 FT-IRspectrometer (Varian, USA) with a MIRacle ATR cell(Pike) between 600 and 4,000 1/cm. Dynamical differentialscanning calorimetry (DSC) was performed with a DSC204 (Netzsch, Germany) and glass transitions and meltingpoints data are given for heating cycles.

For all characterisations of solutions or dispersions of thepolymer samples, a stock solution/dispersion with ultrapuredistilled water was prepared. The dilution procedure isdetailed for the PSS-PNIPAM block copolymer. The blockcopolymer was dissolved in ultrapure distilled water toresult in a 0.42 wt.% solution. This concentration gives asignal during the URT measurements whilst passing theLCST that can be nicely evaluated. The stock solution wasrepeatedly diluted until measurement of the average particlesize above the LCST with routine dynamic light scatteringequipment (Nicomp 370, Santa Barbara, USA) was stillpossible. The final solids content for dynamic lightscattering was estimated to be 0.014% by weight. Thislow concentration guaranteed that the dynamic lightscattering measurements are not influenced by interactionsbetween the precipitated block copolymer particles. Thefinal solids content was in any case below 1 wt.%.

The particle size (Di, intensity weighted average particlesize) was measured by dynamic light scattering using aNICOMP particle sizer (either model 370 or 380) attemperatures between 25 C and 60 C.

The change of the ultrasound velocity and attenuation wasmeasured with the ResoScan URT System (TF InstrumentsGmbH, Heidelberg, Germany) based onURT. This instrument

is equipped with ultrasonic transducers made of lithiumniobate single crystals with a fundamental frequency of10 MHz. It contains twin sample cells for sample andreference with a path length of 7 mm. The measurementvolume is 200 l. The temperature stability is 0.0003 Kand the resolution of the ultrasonic velocity is 0.001 ms1.The ultrasound properties of the sample solutions areevaluated in relation to a highly diluted surfactant solution(ResoStandard, TF Instruments GmbH) that practically doesnot alter the properties of pure water. All data reported areaverages of at least three repeats. The evaluated signal ofURT is either U (ultrasound velocity) or A (ultrasoundattenuation) where the delta sign refers to the differencebetween the sample solution and the standard.

The sedimentation experiments have been performed onan Optima XLI centrifuge (Beckman Coulter, Palo Alto,CA, USA) and Rayleigh interference optics at 25 C, 32 Cand 40 C in pure water and D2Owater mixture. Thedensity of the continuous phase is calculated according tothe actual dilution with D2O which is 1:10 except forsample PNIPAM-xl-B (1:30). Depending on the sedimen-tation velocity of the particles, speeds of 2,000, 3,000,50,000 and 60,000 rpm have been chosen to get an optimalresolution. The distributions of the sedimentation coeffi-cients have been evaluated with the software SEDFIT(version 10.09 beta P. Schuck 2007) [42]. The densitygradients have been performed on an Optima XLI centri-fuge (Beckman Coulter) in methanolbromoform mixtures(75:25, v/v) with absorbance optics at 500 nm and a speedof 40,000 rpm. The data have been evaluated with theprogram Newgradient (Kristian Schilling 2005, Nanolytics,Potsdam, Germany) [43].

Electrophoretic mobilities and particle size distributions attemperatures between 25 C and 50 C were determined witha Zetasizer Nano ZS (Malvern Instruments, Worcestershire,UK) operating with a 4 mW HeNe laser (632.8 nm), adetector positioned at the scattering angle of 173 and atemperature-control jacket for the cuvette. Each samplewas degassed for 15 min to remove air bubbles. Thecuvette was sealed to avoid evaporation and left for5 min to allow temperature equilibration. Five measure-ments consisting of up to 12 consecutive runs of durationof 10 s were performed for each sample and temperature.Dynamic correlation functions were fitted by a second-order

Table 1 Polymerisation recipes for PNIPAM samples with various architectures

Polymer H2O (g) NIPAM (g) Initiator/Oxidant Precursor Cross-linker

PNIPAM-xl-A 1,500 36 0.54 g KPS 1.77 g MBAPNIPAM-xl-C 1,500 33 0.486 g V50 2 g MBAPNIPAM-xl-PEG 1,455 23.7 6 g CAN in 70 g HNO3 PEG 1.2 g EGDMAPNIPAM-PSS 100 4 0.4 g CAN in 5 g HNO3 PSS

Colloid Polym Sci (2009) 287:299312 301

cumulant method to obtain the size distributions. For thezeta potential measurements, the samples (with volume0.75 ml) were loaded in folded capillary zeta potential cellswith integral gold electrodes. Three measurements consist-ing of 40 to 50 runs with duration of 10 s were performed.The mobility was converted to potential using theHelmholtzSmoluchowski relation =/0, where isthe solution viscosity, the dielectric constant of water and0 the permittivity in vacuum. The standard deviation isabout 5 mV as stated by the manufacturer.

Results and discussion

Sample characterisation

Samples PNIPAM-xl-A and PNIPAM-xl-C were prepared viacommon aqueous radical polymerisation of NIPAM in thepresence of the water-soluble MBA cross-linker with potas-sium peroxodisulfate and 2,2-Azobis(2-amidinopropan)dihydrochlorid as initiator, respectively. The resultingmicrogel particles have a quite high cross-linking densitythat allows easy re-dispergation of the purified and freeze-dried solids in water. The dispersed particles are stabilisedby the corresponding ionic end groups stemming from theinitiators and possess also at room temperature the typicalmilky white appearance of polymer latexes. Elementalanalysis and Fourier transform infrared (FT-IR) spectrosco-py give neither hints regarding the ionic endgroups nor thecross-linking units. DSC shows a glass transition temper-ature of 152 C and 154 C for PNIPAM-xl-A andPNIPAM-xl-C, respectively.

Sample PNIPAM-xl-PEG was prepared by aqueous radicalpolymerisation in the presence of the slightly water-solubleEGDMA cross-linker started with the redox initiation systemceric ion and poly(ethylene glycol) [44, 45]. The redoxreaction between Ce4+ and the methylol endgroup leadsunder proton abstraction to a carbon radical and Ce3+. Thecross-linked PNIPAM particles are stabilised by PEG chainends and actually represent PNIPAM-PEG block copolymermicrogel particles. Elemental analysis shows a ratio of about8.8 ethylene glycol units per N-isopropylacrylamide unit (Ncontent is decreased to 2.8 wt.%). FT-IR spectra confirm thepresence of PEG due to strong absorption of the ether groupsand the CH2 groups at about 1,100 and 2,900 1/cm,respectively, besides the typical absorption bands forPNIPAM. The sample can be re-dispersed in water afterultrafiltration and freeze drying without any problems.However, at similar solids content, the turbidity is at roomtemperature much lower compared to samples PNIPAM-xl-A and PNIPAM-xl-C. This behaviour points to a lowercross-linking density of the PNIPAM core, which might beexpected due to the lower water solubility of the EGDMA

cross-linker and the start of the reaction with polymeric PEGradicals. The DSC data show no glass transition temperatureof the cross-linked PNIPAM block but a melting peak at65.6 C, which is slightly higher than that observed for thestarting PEG (63.1 C).

The PNIPAM-PSS block copolymer was also preparedvia aqueous radical redox polymerisation of NIPAM startedwith ceric ion and PSS precursor polymer with methylolendgroups as reductant. The molecular weight of the PSSprecursor is between 105 and 1.6106 g/mol (analyticalultracentrifugation). The nitrogen to sulphur ratio in theblock copolymer is 4.6 (elemental analysis), and hence, amolecular weight between 3.2105 and 5.1106 g/mol forthe PNIPAM block can be estimated. Then, the overallmolecular weight of the block copolymer is supposed to bebetween 4.2105 and 6.7106 g/mol. FT-IR spectraconfirm the presence of PNIPAM and PSS units. At roomtemperature, the sample dissolves easily in water and formsa transparent solution. As the block copolymer is extremelyhydrophilic, it contains practically always a few per cent ofbound water also in the dry state. The DSC trace of theblock copolymer shows a glass transition in the temperaturerange of about 130 C which belongs to the PNIPAM blockand a second peak in the range of 20 C. The latter peakarise presumably from the PSS block, as high concentratedsolutions of the homopolymer show in this range atemperature response which is attributed to the melting ofspherulites [46].

Finally, these four samples represent three classes ofPNIPAM structures: cross-linked microgel particles withdifferent endgroups (PNIPAM-xl-A, PNIPAM-xl-C), blockcopolymer microgel particles (PNIPAM-xl-PEG) and linearblock copolymers (PNIPAM-PSS).

Size and morphology of the dried sampleselectronmicroscopy

It is to be expected that the morphological changes during theprecipitation/redissolution of PNIPAM depend to largeextent on the molecular architecture of the particular sample.Evaluation of light scattering data and zeta potentialmeasurements require information on the shape of theparticles. The assumption of spherical particles, though it isfrom the thermodynamic point of view quite reasonable,must not be valid in any case. The morphology of PNIPAMaggregates can considerably deviate from solid spheresespecially in the dispersed state of non-cross-linked samples.

The morphology of PNIPAM-PAA block copolymers(2 wt.% aqueous solution) with 74 NIPAM and 110 acrylicacid units was studied by means of cryogenic TEM below(20 C) and above (45 C) the LCST as well at pH values of4.5 and 5.6 [23]. In any case, spherical objects have beenobserved. The size of the particles depends on the particular


conditions and is the largest at 20 C and pH=5.6(>300 nm in diameter with loosely packed outer regionand solid core). At pH=4.5 and 20 C as well at pH=5.6and 45 C, the images show particles with diametersbetween 10 and 30 nm. At poor solubility conditions foreither block (pH=4.5 and 45 C), solid particles of about150 nm in diameter are detected.

The electron microscopy images put together in Fig. 1show clearly solid spheres for the dried microgel particles(PNIPAM-xl-A, PNIPAM-xl-C). Moreover, the imagesprove the monodispersity of these samples. The enumera-tion of TEM pictures (only between 200 and 500 particleshave been counted as the particle size distribution is quitemonodisperse) resulted in the following average diametersfor PNIPAM-xl-A: number average diameter Dn=486.8 nmand weight average diameter Dw=487.7 nm, for PNIPA-xl-C: Dn=455.2 nm and Dw=456.3 nm.

In contrast, rather non-specifically shaped aggregates arefound on TEM images of the microgel PNIPAM-xl-PEG(cf. Fig. 2).

The shape of the PNIPAM-PSS block copolymerprecipitate is mainly spherical, however, with a quite broadsize distribution ranging from below 50 to above 200 nm(cf. images of Fig. 3).

The similarity of the structures observed for PNIPAM-PSS samples placed on the electron microscopy grid below

and above the LCST seems reasonable for this highly dilutedstarting solution and proves that spherical block copolymerparticles/micelles are formed within the biphasic region ofthe phase diagram under these particular conditions. Obvi-ously, precipitation leads to similar morphologies whetherthe phase boundary is passed by increasing the temperatureor the concentration. Additionally, smaller precipitationstructures are visible, arising very likely from moleculesthat are not aggregated onto the larger particles.

Note that in contrast to PNIPAM-xl-A and PNIPAM-xl-C,both the morphology and the size of the other two samplesare neither in the dissolved nor in the precipitated stateclearly defined. This fact restricts the data evaluation of thesesamples as discussed below.

Size and morphology of the wetted samplesdynamic lightscattering and analytical ultracentrifugation

Measurement of the hydrodynamic size of the thermo-responsive samples in dependence on temperature withroutine dynamic light scattering equipment is quite commonto characterise the LCST behaviour. Microgel particles(samples PNIPAM-xl-A, PNIPAM-xl-C, PNIPAM-xl-PEG)shrink during temperature increase, whereas soluble polymers(sample PNIPAM-PSS) precipitate and the apparent particlesize increases. This principally different dependence is

Fig. 1 Electron microscopyimages of PNIPA-xl-A (lefthand) and PNIPAM-xl-C (righthand) after ultrafiltration andredispergation; TEM (upperrow) and SEM images (lowerrow)


Fig. 2 TEM images of samplePNIPAM-xl-PEG

Fig. 3 TEM images of samplePNIPAM-PSS measured afterpreparation at room temperature(left hand) and at 40 C (righthand)


illustrated by the data put together in Fig. 4. The relativeerrors, determined from repeated heating and cooling cycles,indicate that the reproducibility for the higher cross-linkedsamples (PNIPAM-xl-A, PNIPAM-xl-C) is clearly bettercompared with the only loosely cross-linked PNIPAM-xl-PEG or the block copolymer PNIPAM-PSS.

For the PNIPAM-PSS block copolymer, the difference inthe heating and cooling curve indicates hysteresis. It is justa single point below the LCST during cooling, but it wasrepeatedly observed. Caution is recommended regardingthe temperature during the measurements, as the there is nopossibility to check the temperature for the particular lightscattering equipment. However, the occurrence of hystere-sis is not surprising as it is also observed for the coilglobule transition of single PNIPAM homopolymer chainsas intensely studied by more sophisticated light scatteringtechniques with much higher sensitivity [47]. Contrary, thecross-linked particles do not show hysteresis but ratherpulsating type of behaviour during temperature cycles. Thedifferent behaviour is reasonable, as the morphologicalchanges during the redissolution of the PNIPAM-PSSparticles require chain disintegration or disentanglementinside the multi-chain particles. The spring-like pulsation ofthe microgel particles requires only minor reorientation ofmacromolecules, but only the diffusion of water moleculesin or out of the network.

The extent of swelling can be quantitatively expressed invarious ways bymeans of particle sizes determined in the non-swollen (dry) or swollen (wet) state. The average diametersdetermined by enumerating TEM images correspond to the

size of the dried particles (Ddry), whereas the DLS values arethat of the swollen or wet particles (Dwet or Di). Thus, theswelling ratio (Hv) as defined by Eq. 1 or the volumefraction of water (w) are easily accessible, but reliablevalues can be obtained in this way only for the highly cross-linked monodisperse samples PNIPAM-xl-A and PNIPAM-xl-C. The overall volume of water per unit mass of polymer(vw1) requires additionally the knowledge of the density ofthe dry polymer (dry; Eqn. 2 and cf. below).

Hv DwetDdry

3 1

1 fw1

vw1 D3wet D3dryrdry D3dry

: 2

Ultracentrifugation relies on a given relation betweenvolume and mass of the moving particles as illustrated bythe fundamental equation of this technique, which can bewritten in the form as given with Eq. 3:

VsedCacc

S 118

1h0

D2mov rwp r0: 3

In Eq. 3, Vsed means the sedimentation velocity in m/s,Cacc the centrifugational acceleration in m/s

2, S thesedimentation coefficient in Svedberg (1S=1013 s), 0 theviscosity of the continuous phase (water) in kg/(s m), Dmovthe diameter of the moving particle in nm, rwp the density of

Fig. 4 Change of the averageparticle size with temperaturemeasured with dynamic lightscattering (filled symbols; inten-sity-weighted average diameter,Di) and AUC (open symbols);data were obtained under iso-thermal condition during AUCand after about 20-min equili-bration time for the DLSmeasurements


the polymer and 0 the density of the continuous phase. Thesedimentation coefficient is experimentally accessible asthe centrifugational acceleration is known and the sedimen-tation velocity is measured. Equation 3 suggests that forparticles with unknown density and size, like the PNIPAMparticles studied, from AUC runs in two solvents withdifferent density (H2O, D2O) the unknowns (D, rwp ) can bedetermined. The data summarised in Table 2 show that themethod, which is successful for solid hydrophobic particles,fails for the PNIPAM particles.

For the microgels, the average particle size increaseswith temperature, which is the opposite of the expectedbehaviour and also contradictory to the DLS results (cf.Fig. 1). Only for the PNIPAM-PSS did block copolymerAUC show increasing size with temperature, but the valuesare much smaller than that from DLS. The density (rwp )shows no general trend in either direction. The straightfor-ward explanation for these results is that various amounts ofboth solvent molecules (H2O and D2O) adhere strongly andmove along with the polymer, and hence, the constancy ofthe volumemass relation is violated. Note that thedependence of Tp on the composition of the continuousphase (H2O, D2O) is, if so ever, of minor importance, as thehighest temperature (40 C) is also above the LCST ofPNIPAM in D2O that is about 2 greater as in H2O [48, 49].

However, a further evaluation of the sedimentation data ispossible with the assumption that the hydrodynamic diameter

from DLS corresponds to the size of the moving particles inthe centrifugational field. Applying this assumption to Eq. 1,the density difference r rwp r0

can be calculated.

values calculated in this way show quite significantdifferences between the samples reflecting the moleculararchitecture in a reasonable manner (cf. Fig. 5a).

The values as displayed in Fig. 5a suggest that thedensity of the moving objects is not constant withtemperature, which again is a hint to bound water movingalong with the polymers. Another interesting result is thedifferent temperature dependence of observed for thecross-linked samples (PNIPAM-xl-PEG, PNIPAM-xl-A,PNIPAM-xl-C) and the linear block copolymer PNIPAM-PSS. For the microgels, increases with increasingtemperature as expected during deswelling. Furthermore,these data show that the PNIPAM-xl-PEG particles aremuch less cross-linked than the other two samples asalready concluded from the TEM images. In contrast, decreases for PNIPAM-PSS with increasing temperature,hinting that increasing amounts of water are bound in thephysical PNIPAM network that is gradually built up.

As the density of water decreases with temperature, thedensity of the moving particles (rwp ) must not possess thesame dependence as . Note that the rwp values calculatedin this way differ from those obtained by the standardevaluation of the AUC data in H2O and D2O (Table 2).Surprisingly, the PNIPAM samples split into two groups,

Table 2 Apparent diameter and density of the PNIPAM particles determined by AUC in H2O and D2O at different temperatures

Sample Dmov (nm) 25 C rwp (g/cm

3) 25 C Dmov (nm) 32 C rwp (g/cm

3) 32 C Dmov (nm) 40 C rwp (g/cm

3) 40 C

PNIPAM-xl-PEG 2.1 1.200 2.4 1.139 51.5 1.107PNIPAM-PSS 4 1.320 3.4 1.246 19 1.929PNIPAM-xl-A 251 1.187 291 1.167 305 1.152PNIPAM-xl-C 249 1.194 295 1.168 344 1.172

Fig. 5 Dependence of the den-sity difference and the density ofthe moving particles in depen-dence on temperature calculatedfrom the sedimentation coeffi-cients according to Eq. 1


showing a distinctly different dependence of the rwp values ontemperature (Fig. 5b). The rwp values for the cross-linkedhomopolymers (PNIPAM-xl-A and PNIPAM-xl-C) and thePNIPAM block copolymers (PNIPAM-xl-PEG and PNIPAM-PSS) increase and decrease with increasing temperature,respectively. The homopolymers show the expected typicalbehaviour of PNIPAM, that is, deswelling and consequentlyincreasing density with rising temperature. The contrarybehaviour is observed for the block copolymers. The shrink-age of the PNIPAM core during heating and the exclusion ofwater are obviously overcompensated by the incorporationand binding of water molecules in the hydrophilic corona.

In order to further evaluate the values, the drydensity of PNIPAM (dry) must be known. The density ofthe microgel particles PNIPAM-xl-A and PNIPAM-xl-Cwas determined by ultracentrifugation in a density gradi-ent built up in a methanol bromoform mixture. This studysuggests a density for these particles between 1.35 and1.42 gcm3, which is slightly higher than 1.269 gcm3

used in [50] for calculating the PNIPAMwater interactionby means of a lattice-fluid hydrogen-bond theory. Eitherof these values for the density of the dry PNIPAM isclearly larger than the values calculated from the values (cf. data of Fig. 5). The same holds also for theblock copolymer samples, as the density of PEG and PSSare between 1.2 and 1.3 gcm3 [51, 52]. The density ofthe moving particles can be expressed by Eq. 4 leading toEq. 5 for the volume of water bound per unit mass ofmonomer.

rwp mp

vp vbw 4

vw2 rdry rwprwp rdry

: 5

In order to calculate the amount of bound water as mol(nbw) or mass (mw2) ratio, the density of the bound watermust be known. As this value in dependence on temperatureis not available, the bulk density of water can be used at leastto get an idea about nbw and mw2. The nbw and mw2 values asdepicted in Fig. 6a, b are exactly in the same order ofmagnitude as those calculated in [50] and experimentallydetermined in [5]. These values correspond to the watermolecules bound via hydrogen bonds to the NIPAM units.

The data of Fig. 7 compare the amount of overall waterin the microgel particles (vw1) with the amount of boundwater, which adheres so strongly that it moves along withthe polymer in the centrifugational field (vw2).

The data of Fig. 7 suggest that the cationic microgelparticles swell to slightly larger extent than the anionicparticles. In general, this might be due to several reasons, asthe degree of swelling is mainly influenced by the degree ofcross-linking, the particle size and the interfacial tension.As the particles size is almost identical (cf. Fig. 4c, d),differences in the cross-linking density and the interfacialtension remain as possible reasons. Moreover, there isample experimental evidence that the structure of microgelparticles is heterogeneous regarding the cross-linkingdensity, that is, the cross-links are non-homogeneouslydistributed and their number decreases towards the shell[53, 54].

The amount of water in either type of microgel particlesdecreases over the whole temperature range, whereby thedrop is the steepest in the vicinity of the LCST. At 40 C,the overall water volume fraction in the particles (w) isstill about 42% and 51% for PNIPASM-xl-A and PNIPAM-xl-C, respectively. A similar value has been found forPNIPAM microgels with carboxylic end groups [55]. Thevw2 values at 60 C correspond to w of even 26%(PNIPAM-xl-A) and 43% (PNIPASM-xl-C). Moreover,

Fig. 6 Molar (a) and mass (b)ratio of the water bound toPNIPAM in the highly cross-linked microgel particles in de-pendence on temperature; nbw isexpressed as mole water permole repeating unit


the experimental data prove that PNIPAM microgelscontain two types of water: free bulk water that easilydrains the particles and bound water that strongly adheres tothe polymer. The amount of the latter is practicallyindependent of the nature of the ionic groups in themicrogel and corresponds at 40 C to a volume fractionof about 25%. The fact that vw1 is over the whole range oftemperature larger than vw2 proves that the combination ofmolecular characteristics determined with very differentmethods (electron microscopy, dynamic light scattering,analytical ultracentrifugation) leads to reliable conclusions.It must be mentioned that these results are in qualitativeagreement with previous results obtained for coil-to-globuletransition of a single PNIPAM chain with laser lightscattering [47]. In addition, the evaluation of neutronscattering data for cross-linked PNIPAM particles revealedthat at 25 C and in the shrunken state (50 C), the polymervolume fraction is about 0.1 and 0.5, respectively, dependingon particular size and cross-linking density [54, 56].

Zeta potential and charge density

PNIPAM samples with charges increase and decrease theirelectrostatic surface potential during the heating and cool-ing cycles, respectively. The samples of this study differdistinctly in their surface properties as exemplarily illus-trated by the zeta potential () at temperature above theLCST (cf. Fig. 8). The zeta potential spans a range ofalmost 100 mV which is clearly related to the chemicalnature of the samples. It is known that for soft particles, thezeta potential is less meaningful than the Donnan potentialthat is built up in the polyelectrolyte layer, and hence, aspecial data evaluation should be used [57]. However, forthe sake of comparison between the different samplesemployed in this study, the applied relation to convert

mobility in zeta potential seems to be sufficient. Allsamples, including the nominally electrically neutral PNI-PAM-xl-PEG, possess a zeta potential. The slightly nega-tive of PNIPAM-xl-PEG is explainable with theassumption of a contact potential as described by Coehnsrule [58]. Accordingly, an electric potential exists betweentwo materials with different permittivity in a way that thematerial with the lower dielectric constant carries thenegative charge.

In dependence of the magnitude of the temperature-induced size change, the zeta potential of the particles changesas well. The data of Fig. 9 illustrate the changes independence on the particle size and temperature. Figure 9brelates the zeta potential to the particle surface and can beconsidered as measure, of course not exactly but as anestimate, of a surface potential. This value increases duringheating as the average particle size decreases. From a colloidchemical point of view, this is good, as it counteractsinstability which is usually enhanced at higher temperatures.

Expectedly, the size dependence is, especially for thePNIPAM-PSS sample, extremely steep (Fig. 9a). This isbecause firstly, the size changes only little above the LCST,and secondly, the stabilizing polyelectrolyte chains carry ahuge number of charges compared with the other samples.

URTa powerful tool to investigate thermal phasetransitions of polymers

URT technology allows to evaluate either the ultrasoundvelocity or the ultrasound attenuation. The ultrasoundvelocity in fluids depends on the compressibility and thedensity of the medium. The attenuation is more complexand influenced by much more parameters (especiallyviscosity as well as vibration and structure relaxationprocesses) [59], and hence, it is charged with larger scatter.Therefore, it is more convenient to consider the difference

Fig. 8 Zeta potential () of the different PNIPAM samples at 40 C

Fig. 7 Overall (vw1) and bound water (vw2) in the highly cross-linkedmicrogel particles in dependence on temperature


of the ultrasound velocity especially when diluted samplesare investigated.

URT principally differs from light scattering or otheroptical techniques, as it does not rely on alteration of theturbidity but on the propagation of sound waves that issensitive to both changes in the physical state of thepolymer and the solvent molecules. This analytical tech-nique is considered to belong to the methods with anextremely high resolution in measurements of physicalparameters of solutions and colloidal suspensions [59].Thus, the method seems to be ideally suited to investigatethe critical solution behaviour of polymers. Very recently,the apparent activation free energies of the precipitation andredissolution of PNIPAM-PSS block polymers have beendetermined for the first time by means of transient-thermalstudies with URT [60].

Figure 10 elucidates by means of runs with samplePNIPAM-xl-C the kind of data that are obtained with URT.

The curves of A and U exhibit completely differentshapes and contain also different information. The Utemperature curve is characterised by just one point ofinflection (U1 at T1) which gives the LCST of the PNIPAMblock (cf. also [60]). At this temperature, the coilglobuletransition of the PNIPAM block takes place and theformerly homogeneous solution changes to a more or lessturbid dispersion. On the contrary, the AT curves showat least three characteristic points (A1, A2, A3) during bothheating and cooling. The point of inflection (A1) occursagain in the range of the LCST, whereas T2 and T3 at A2(sharp maximum in the AT curve) and A3 (shallowminimum in the AT curve), respectively, appear only inthe curves of the ultrasound attenuation at temperaturesabove the LCST. Moreover, A2 and A3 are only observedfor the microgel particles (cf. Fig. 11) and not for thePNIPAM-PSS block copolymer.

The great difference in A between the samples can beobviously related rather to the morphology of the samplesthan to the nature of the charge or the charge density. Theorder of A reflects the density of the PNIPAM core, as itis the lowest for the non-cross-linked block copolymer andthe highest for the heavily cross-linked microgel particles.Moreover, these data allow the conclusion that the reasonfor the transitions at T2 and T3 is connected withtransitions in the cross-linked PNIPAM core, whereas T1is clearly caused by the change in the solubility of thePNIPAM moieties.

In contrast to the AT curves (Fig. 11), the UTdependencies show for all curves a similar feature that is asteep minimum at the LCST in the dU/dT curves(Fig. 12).

The dU/dT curves as depicted in Fig. 12 reveal ahysteresis between the heating and cooling curves that also

Fig. 10 Change of U and A during temperature scans between25 C and 85 C for sample PNIPAM-xl-C with heating rate of300 mK/min; the arrows point to characteristic changes that have beenconsidered in the further data evaluation (cf. text)

Fig. 9 Zeta potential in depen-dence on the hydrodynamicparticle size (Di) (a) and zetapotential (absolute value) divid-ed by the squared hydrodynamicparticle size in dependence ontemperature (b); the differencein the particle size for thePNIPAM-PSS in comparison tothe data of Fig. 4 is due to thefact that for the block copoly-mer, the particle size above Tpdepends on the concentration[59], and for the measurementsconsidered here, the concentra-tion is about eight times higher(0.12 instead of 0.014 wt.%)


strongly depends on the morphology of the sample. For thehighly cross-linked samples, the hysteresis, expressed asdifference of the temperature between heating and coolingextremum, is below 0.3 but increases for the loosely cross-linked PNIPAM-xl-PEG sample to 0.55 and to 0.99 forthe block copolymer. Note that the magnitude of thehysteresis depends on the heating and cooling rate asdetailed in [60]. Moreover, the graphs of Fig. 12 show thatalso the width of the transition range depends on the cross-linking density of the samples. The peak is broad for the

microgel particles PNIPAM-xl-A and PNIPAM-xl-C andalmost spike-like for the block copolymer.

Figure 13 includes a summary of the transition temper-atures detected with A and U for all samples. Thiscollection elucidates nicely the influence of the both thecomposition of the sample and the nature of the particularultrasound property used for the analysis.

Besides the chemical nature of the samples and thedegree of cross-linking, the charge density of the particles/molecules can be related to the transition temperatures as

Fig. 12 Temperature deriva-tives of UT curves for thefour PNIPAM samples duringheating and cooling in the tem-perature range between 25 Cand 45 C; each graph showsthree repeats; heating/coolingrate 300 mK/min

Fig. 11 Change of the differ-ence of the ultrasound attenua-tion (A) during heating andcooling in the temperature rangebetween 25 C and 85 C for allsamples (a) and magnificationof the AT curves for thecharged microgel particlesPNIPAM-xl-A and PNIPAM-xl-C (b) (the order of legendscorresponds to the order of thecurves); the curves are averagesover three or five consecutiveruns; heating/cooling rate300 mK/min


well. Figure 14 shows that with increasing zeta potential,the first transition temperature (T1), that is, the LCST,increases for both the precipitation during heating and thedissolution during cooling. This result seems, at least forthe precipitation, absolutely reasonable, as covalentlyattached charges counteract the aggregation of the PNIPAMmoieties. This counterforce should be the stronger thehigher the charge density. Therefore, the value of T1,determined from the difference of the ultrasound velocity,varies for the precipitation of 2 between the electricallyneutral PNIPAM-xl-PEG and the highly charged PNIPAM-PSS block copolymers. However, there must be still othereffects acting, as the largest difference of about 2.38 ismeasured for T1 during cooling in the A curves. Thereason might that the PNIPAM-PSS block copolymermolecules must completely disentangle during the dissolu-tion, whereas the microgel particles only expand.

The URT technology is indeed a powerful tool toinvestigate phase transitions. The data presented here show

not only one characteristic temperature (T1) but also anothertwo characteristic transitions for microgel particles at highertemperatures if the attenuation of the ultrasound is evaluated.Unfortunately, these changes cannot unambiguously bedirectly connected with structural changes either inside thecross-linked cores of the microgel particles or the hydrophilicshells or the water structure in either of these phases.

Conclusions

The results of this comprehensive study of four chemicallydifferent PNIPAM samples clearly show that the combina-tion of methods leads to new insights in the thermo-reversible properties of this polymer. The combinationTEM, DLS and AUC allows for microgel particles todistinguish between the amount of tightly bound water andthe quantity of water that only swells the microgels. Theevaluation of data obtained with DLS, zeta potential

Fig. 13 Transition temperatures for the different PNIPAM samples determined from AT curves (a) and UT curves (b); the plain grey andcrossed grey symbols represent heating and cooling runs, respectively, the lines in a are just for guiding the eye

Fig. 14 Dependence of theLCST (T1) on the absolutevalue of the zeta potential: a andb show T1 values for heatingand cooling, respectively; thevalues of the zeta potential at50 C


measurements and URT permits the identification of othercharacteristic transition temperatures above the LCSTwhich are only hardly, if so ever, accessible by opticalmethods alone. By the evaluation of the ultrasoundattenuation, two more transitions at temperatures abovethe LCST have been observed for PNIPAM microgelparticles, but not for the PNIPAM-PSS block copolymer.The joint evaluation of this data with measurements of theelectrophoretic mobility revealed a strong relation betweenthe zeta potential of the precipitated structures and thetransition temperatures for both the precipitation (duringheating) and the redissolution (during cooling).

Acknowledgement The authors thank Mrs. Ursula Lubahn and Mrs.Sylvia Pirok for preparative and analytical assistance and Mrs. RonaPitschke and Mrs. Heike Runge for the electron microscopy images.Financial support of the Max Planck Society and the TF InstrumentsGmbH is gratefully acknowledged. The interest of Theodor Funk in theprogress of this work and many discussions with him are appreciated.

Open Access This article is distributed under the terms of theCreative Commons Attribution Noncommercial License which permitsany noncommercial use, distribution, and reproduction in any medium,provided the original author(s) and source are credited.

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Thermal property changes of poly(N-isopropylacrylamide) microgel particles and block copolymersAbstractIntroductionExperimental informationResults and discussionSample characterisationSize and morphology of the dried sampleselectron microscopySize and morphology of the wetted samplesdynamic light scattering and analytical ultracentrifugationZeta potential and charge densityURTa powerful tool to investigate thermal phase transitions of polymers

ConclusionsReferences

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