Saccharide‐structure effects on poly N‐isopropylacrylamide phase transition in aqueous media; Reflections on protein stability

Protein stability in aqueous solutions is important in numerous fields, particularly biotechnology and food‐science. To shed new light on the protective effect of carbohydrates on proteins, we studied saccharide‐structure effects in aqueous solutions on the coil‐to‐globule transition occurring at the lower critical solution temperature (LCST) of poly‐N‐isopropylacrylamide (PNIPA), an isomer of polyleucine, as a simple model representing certain key behaviors of proteins (e.g., denaturation/renaturation). We systematically selected sugars and polyols to relate structural and physical characteristics of these carbohydrates to their effect on PNIPA solutions. Using isothermal titration‐microcalorimetry, we showed that no significant binding of saccharides to the polymer occurs. Using micro‐DSC, we studied the decreasing polymer LCST temperature with rising carbohydrate concentration. Beyond the expected observation that steric exclusion is important, we observed previously‐unreported significant differences among the effects of isomeric aldohexoses and also among the effects of isomeric diglucoses on PNIPA LCST. We found good correlation between the sugar hydration number and its effect on LCST. We conclude that the larger and denser the hydrated cluster a carbohydrate forms, the worse a cosolvent is for the polymer, and the stronger it's lowering effect of the coil‐to‐globule transition. Such favoring of the compact globule state provides a protective effect against denaturation of globular proteins. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 2307–2318, 2008     

Preparation and characterization of pH- and temperature-responsive semi-IPN hydrogels of carboxymethyl chitosan with poly (<Emphasis Type="Italic">N</Emphasis>-isopropyl acrylamide) crosslinked by clay

A new kind of pH- and temperature-responsive semi-interpenetrating polymer network hydrogel based on linear carboxymethylchitosan (CMCS) and poly (N-isopropylacrylamide) (PNIPA) crosslinked by inorganic clay was prepared. The pH-and temperature-responsive behaviors, the deswelling kinetics, and the mechanical properties of the hydrogel were investigated. The hydrogels exhibited a volume phase transition temperature around 33 °C with no significant deviation from the conventional PNIPA hydrogels. The results of the influence of pH value on the swelling behaviors showed that the minimum swelling ratios of the hydrogels appeared near the isoelectric point (IEP) of CMCS, and when pH deviated from the IEP, the hydrogels behaved as polycations or polyanions. The novel hydrogels had much higher response rate than the conventional CMCS/PNIPA hydrogels. Moreover, the semi-IPN hydrogels crosslinked by clay could be elongated to more than 800% and the elongation could be recovered almost completely and instantaneously.     

Preparation and properties of poly(N‐isopropylacrylamide)/poly(N‐isopropylacrylamide) interpenetrating polymer networks for drug delivery

A novel poly(N‐isopropylacrylamide) (PNIPA)/PNIPA interpenetrating polymer network (IPN) was synthesized and characterized. In comparison with conventional PNIPA hydrogels, the shrinking rate of the IPN hydrogel increased when gels, swollen at 20 °C, were immersed in 50 °C water. The phase‐transition temperature of the IPN gel remained unchangeable because of the same chemical constituent in the PNIPA gel. The reswelling kinetics were slower than those of the PNIPA hydrogel because of the higher crosslinking density of the IPN hydrogel. The IPN hydrogel had better mechanical strength because of its higher crosslinking density and polymer volume fraction. The release behavior of 5‐fluorouracil (5‐Fu) from the IPN hydrogel showed that, at a lower temperature, the release of 5‐Fu was controlled by the diffusion of water molecules in the gel network. At a higher temperature, 5‐Fu inside the gel could not diffuse into the medium after a burst release caused by the release of the drug on the surface of the gel. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 1249–1254, 2004     

Synthesis and characterization of Poly(N-isopropylacrylamide)/Poly(acrylic acid) semi-IPN nanocomposite microgels

A novel pH- and temperature-sensitive nanocomposite microgel based on linear Poly(acrylic acid) (PAAc) and Poly(N-isopropylacrylamide) (PNIPA) crosslinked by inorganic clay was synthesized by a two-step method. First, PNIPA microgel was prepared via surfactant-free emulsion polymerization by using inorganic clay as a crosslinker, and then AAc monomer was polymerized within the PNIPA microgel. The structure and morphology of the microgel were confirmed by FTIR, WXRD and TEM. The results indicated that the exfoliated clay platelets were dispersed homogeneously in the PNIPA microgels and acted as a multifunctional crosslinker, while the linear PAAc polymer chains incorporated in the PNIPA microgel network to form a semi-interpenetrating polymer network (semi-IPN) structure. The hydrodynamic diameters of the semi-IPN microgels ranged from 360 to 400 nm, which was much smaller than that of the conventional microgel prepared by using N,N′-methylenebis(acrylamide) (MBA) as a chemical crosslinker, the later was about 740 nm. The semi-IPN microgels exhibited good pH- and temperature-sensitivity, which could respond independently to both pH and temperature changes.     

Thermo-responsive polymer coated fiber-in-tube capillary microextraction and its application to on-line determination of Co, Ni and Cd by inductively coupled plasma mass spectrometry (ICP-MS)

The poly(N-isopropylacrylamide) (PNIPA) gel is a widely studied thermo-responsive material that exhibits discontinuous change in volume when the external temperature is increased. In this paper, PNIPA gel was prepared and applied as a novel polymer coating for fiber-in-tube capillary microextraction of trace Co, Ni and Cd followed by on-line ICP-MS detection. The PNIPA coating was synthesized by using ethylene triethoxysilane (ETEOS) as the cross-linking agent under acidic conditions. This siloxane incorporated PNIPA gel achieves a dramatically rapid response rate when the external temperature is changed. The micro-structure of PNIPA coating was examined by scanning electron micrograph (SEM). Various experimental parameters including pH, temperature, sample flow rate and volume, elution solution and interfering ions affecting the extraction of the target analytes have been carefully investigated and optimized. Under the optimized conditions, the limits of detection were 0.45, 4.6 and 6.9 ng L⁻¹ for Co, Ni and Cd, respectively. With a sampling frequency of 13 h⁻¹, the relative standard deviations (RSDs) for Co, Ni and Cd were 4.8, 5.1 and 6.4% (C = 1 μg L⁻¹, n = 7), respectively. The proposed method had been successfully applied to the determination of Co, Ni and Cd in human urine. To validate the proposed method, certified reference materials of NIES No. 10-b rice flour and GBW07601 (GSH-1) human hair were analyzed and the determined values were in a good agreement with the certified values. The PNIPA coated fiber-in-tube capillary can be reused for more than 150 times without decreasing the extraction efficiency.     

Semi‐interpenetrating polymer networks composed of poly(N‐isopropyl acrylamide) and polyacrylamide hydrogels

Three series of semi‐interpenetrating polymer networks, based on crosslinked poly(N‐isopropyl acrylamide) (PNIPA) and 1 wt % nonionic or ionic (cationic and anionic) linear polyacrylamide (PAAm), were synthesized to improve the mechanical properties of PNIPA gels. The effect of the incorporation of linear polymers into responsive networks on the temperature‐induced transition, swelling behavior, and mechanical properties was studied. Polymer networks with four different crosslinking densities were prepared with various molar ratios (25:1 to 100:1) of the monomer (N‐isopropyl acrylamide) to the crosslinker (methylenebisacrylamide). The hydrogels were characterized by the determination of the equilibrium degree of swelling at 25 °C, the compression modulus, and the effective crosslinking density, as well as the ultimate hydrogel properties, such as the tensile strength and elongation at break. The introduction of cationic and anionic linear hydrophilic PAAm into PNIPA networks increased the rate of swelling, whereas the presence of nonionic PAAm diminished it. Transition temperatures were significantly affected by both the crosslinking density and the presence of linear PAAm in the hydrogel networks. Although anionic PAAm had the greatest influence on increasing the transition temperature, the presence of nonionic PAAm caused the highest dimensional change. Semi‐interpenetrating polymer networks reinforced with cationic and nonionic PAAm exhibited higher tensile strengths and elongations at break than PNIPA hydrogels, whereas the presence of anionic PAAm caused a reduction in the mechanical properties. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 3987–3999, 2004     

Surface interaction of well‐defined,concentrated poly(2‐hydroxyethyl methacrylate) brushes with proteins

The interaction of concentrated polymer brushes with proteins was chromatographically investigated. By the use of surface‐initiated atom transfer radical polymerization, a low‐polydispersity poly(2‐hydroxyethyl methacrylate) (PHEMA) was densely grafted onto the inner surfaces of silica monoliths with mesopores of about 50 and 80 nm in mean size. The graft density reached 0.4–0.5 chains/nm². The 80‐nm‐mesopore monolithic column with the concentrated PHEMA brush was characterized through the elution of low‐polydispersity pullulans with different molecular weights, clearly showing two modes of size exclusion, that is, one by the mesopores and the other by the brush phase. The latter mode gave a sharp separation with a critical molecular weight (size‐exclusion limit) of about 1000. This molecular size of pullulan was comparable to the distance between the nearest‐neighbor graft points. The elution behaviors of five proteins of different sizes (bovine serum thyroglobulin, bovine serum immunoglobulin G, bovine serum albumin, horse heart myoglobin, and bovine serum aprotinin) were studied with this PHEMA‐grafted column. The smallest protein, aprotinin, with a pullulan‐reduced molecular weight slightly larger than the critical value of 1000, was eluted much behind the corresponding pullulan, and this indicated that it barely got into the brush layer, suffering from a strong affinity interaction within the brush. On the other hand, the other four larger proteins were eluted at the same elution volumes as the equivalent pullulans, and this meant that they were perfectly excluded from the brush layer and separated only in the size‐exclusion mode by the mesopores without an affinity interaction with the brush surface. This excellent inertness of the concentrated brush in the interaction with the large proteins should afford the system long‐term stability against biofouling. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 4795–4803, 2007     

FTIR study on molecular structure of poly(N-isopropylacrylamide) in mixed solvent of methanol and water

The intrachain and interchain hydrogen bonding of poly(N-isopropylacrylamide) (PNIPA) and intermolecular hydrogen bonding between PNIPA chains and the solvent molecules in the mixed solvent of methanol and water have been quantitatively investigated by using Fourier transform infrared (FTIR) spectroscopy at 25 °C. In this spectroscopic system with curve fitting program, we found that in the C-H stretching region, both the N-isopropyl group and the backbone underwent conformational change upon the solvent composition. An analysis of the amide I band suggested that the amide groups of PNIPA were mainly involved in intermolecular hydrogen bonding with water molecules, and the polymer chains were flexible and disordered in the mixed solvent when the methanol volume fraction (χ_v) was lower than 15%. While χ_v was in the range of 15-65%, about 30% of these intermolecular hydrogen bonding between the polymer and water were replaced by intrachain and interchain hydrogen bonding, consequently, PNIPA shrinked as aggregates. If χ_v was above 65%, the interchain hydrogen bonding became predominant due to the solubility characteristics of amphiphilic methanol, and the PNIPA system was homogeneous solution again. We believe that the reentrant transition is related to the weaker interaction between PNIPA molecules and methanol-water complexes, (H₂O)_m(CH₃OH)_n (m/n = 5/1, 5/2, 5/3, 5/4, 5/5) as compared to that between PNIPA and free water or free methanol.     

A new temperature-sensitive polymer: Poly(ethoxypropylacrylamide)

In this study, a new temperature sensitive polymer was obtained by the solution polymerization of ethoxypropylacrylamide. The monomer, N-(3-ethoxypropyl)-acrylamide was synthesized by the nucleophilic substitution reaction of 3-ethoxy-propylamine and acryloyl chloride. The solution polymerization was performed in ethanol at 70 °C, by using azobisizobutyronitrile as the initiator. Poly(N-(3-ethoxypropyl)acrylamide), PEPA, exhibited a reversible phase transition by the temperature. The effects of polymer and salt concentrations on the lower critical solution temperature, (LCST) behaviour were investigated. LCST was found to be strongly dependent on the polymer concentration. The dynamic light scattering (DLS) measurements confirmed the formation of aggregates by the association of nucleated polymer chains at the temperatures higher than LCST. However an unusual behaviour, a marked decrease in the hydrodynamic diameter by the increasing PEPA concentration was observed below the LCST. The effect of salt concentration on the critical flocculation temperature of PEPA was reasonably similar to poly(isopropylacrylamide), PNIPA. In the ethanol-water media, the reversible phase transition behaviour was observed up the ethanol concentration of 30% v/v. This study indicated that PEPA was a new alternative thermally reversible material for PNIPA. With respect to the well-defined temperature-sensitive polymers like PNIPA, polymer concentration dependent LCST of PEPA can provide significant advantages in the applications like drug targeting, affinity separation and immobilization of bioactive agents.     

Concentration Dependence of Elution Volumes in size Exclusion Chromatography of Polymer Molecules. II. Poly(Methyl Methacrylate) in Ideal Solvent

Abstract

The concentration effects occurring in size exclusion chromatography of polymer molecules have been investigated for the case of elution of macromolecules in an ideal solvent. It is shown that increases in the elution volumes with polymer concentration are detectable with the high efficiency columns employed. The concentration dependence of elution volumes is much lower than that previously determined with good solvents. The main factor contributing to the shift of elution volumes seems to be the viscosity of the polymer solutions, but another effect appears also to be present. This latter effect possibly suggest that in semi-concentrated solutions of polymer molecules in ideal solvents a continuous contraction of the polymer chains occurs, below their unperturbed dimensions, similar to that happening when the temperature of ideal solutions is decreased. More investigation on this point is needed. the ideal solutions is increased. Further investigations will be carried out to clarify this point.

In conclusion, the concentration dependence of polymer elution volumes in SEC has been found to be detectable also in a thermodynamically ideal solvent, though it is much lower than that observed in good solvents. In such a system, the main cause of the shift of elution volumes appears to be the viscosity of the polymer solutions, but a small additional contribution is also seen to be present. At the moment, it seems not possible to evaluate with certainty the origin of this latter contribution, especially in the absence of data on the macromolecular conformations in semi-concentrated solutions in ideal solvents.     

Size‐selective purification of hepatitis B virus‐like particle in flow‐through chromatography: Types of ion exchange adsorbent and grafted polymer architecture

Hepatitis B virus‐like particles expressed in Escherichia coli were purified using anion exchange adsorbents grafted with polymer poly(oligo(ethylene glycol) methacrylate) in flow‐through chromatography mode. The virus‐like particles were selectively excluded, while the relatively smaller sized host cell proteins were absorbed. The exclusion of virus‐like particles was governed by the accessibility of binding sites (the size of adsorbents and the charge of grafted dextran chains) as well as the architecture (branch‐chain length) of the grafted polymer. The branch‐chain length of grafted polymer was altered by changing the type of monomers used. The larger adsorbent (90 μm) had an approximately twofold increase in the flow‐through recovery, as compared to the smaller adsorbent (30 μm). Generally, polymer‐grafted adsorbents improved the exclusion of the virus‐like particles. Overall, the middle branch‐chain length polymer grafted on larger adsorbent showed optimal performance at 92% flow‐through recovery with a purification factor of 1.53. A comparative study between the adsorbent with dextran grafts and the polymer‐grafted adsorbent showed that a better exclusion of virus‐like particles was achieved with the absorbent grafted with inert polymer. The grafted polymer was also shown to reduce strong interaction between binding sites and virus‐like particles, which preserved the particles’ structure.     

Isomeric sugar effects on thermal phase transition of aqueous PNIPA solutions,probed by ATR-FTIR spectroscopy; insights to protein protection by sugars

To illuminate the impacts of sugar concentration and stereochemistry on protein protection we used attenuated-total-reflectance Fourier-transform infrared-spectroscopy (ATR-FTIR) to study the effects of four aldohexoses on poly-N-isopropylacrylamide (PNIPA) phase transition. Protein stability in aqueous solutions is essential in numerous fields, predominantly biotechnology and food science. Saccharides protect proteins against thermal denaturation, but the mechanisms are still debatable. We therefore studied the effect of sugar concentration and stereochemistry on the LCST phase transition of PNIPA as a model for protein cold renaturation, using ATR-FTIR. The transition temperature of PNIPA, as observed by both the shift in amide II peak and its area, revealed the following order: galactose > glucose > mannose > talose, i.e., galactose is the most kosmotropic of the four isomers and talose is the least. We concluded that the soluting-out effect exerted on the polymer by these sugars positively correlates with the sugar hydration number governed by sugar stereochemistry.     

A novel sodium carboxymethylcellulose/poly(N‐isopropylacrylamide)/Clay semi‐IPN nanocomposite hydrogel with improved response rate and mechanical properties

A novel semi‐IPN nanocomposite hydrogel (CMC/PNIPA/Clay hydrogel) based on linear sodium carboxymethylcellulose (CMC) and poly(N‐isopropylacrylamide) (PNIPA) crosslinked by inorganic clay was prepared. The structure and morphology of these hydrogels were investigated and their swelling and deswelling kinetics were studied in detail. TEM images showed that the clay was substantially exfoliated to form nano‐dimension platelets dispersed homogeneously in the hydrogels and acted as a multifunctional crosslinker. The CMC/PNIPA/Clay hydrogels swell faster than the corresponding PNIPA/Clay hydrogels at pH 7.4, whereas they swell slower than the PNIPA/Clay hydrogels at pH 1.2. The CMC/PNIPA/Clay nanocomposite hydrogels showed much higher deswelling rates, which was ascribed to more passway formed in these hydrogels for water to diffuse in and out. The deswelling process of the hydrogels could be approximately described by the first‐order kinetic equation and the deswelling rate decreased with increasing clay content. The mechanical properties of the CMC/PNIPA/Clay nanocomposite hydrogels were analyzed based on the theory of rubber elasticity. It was found that with increasing clay content, the effective crosslink chain density, v_e, increased whereas the molecular weight of the chains between crosslinks M_c decreased. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 1546–1555, 2008     

Preparation and characterization of sodium carboxymethylcellulose/poly(N-isopropylacrylamide)/clay semi-IPN nanocomposite hydrogels

A new kind of pH-/temperature-responsive semi-interpenetrating polymer network hydrogels based on linear sodium carboxymethylcellulose (CMC) and poly(N-isopropylacrylamide) (PNIPA) cross-linked by inorganic clay (CMC/PNIPA/Clay hydrogel) was prepared. The temperature- and pH-responsive behaviors, the mechanical properties of these hydrogels were investigated. The CMC/PNIPA/Clay hydrogels exhibited a volume phase transition temperature around 32 °C with no significant deviation from the conventional PNIPA hydrogels. The swelling ratio of the CMC/PNIPA/Clay hydrogels gradually decreased with increasing the contents of clay. The influence of pH value on swelling behaviors showed that there is a maximum swelling ratio at pH 5.9. Moreover, the CMC/PNIPA/Clay hydrogels exhibited excellent mechanical properties with high tensile stress and elongation at break in excess of 1200%.     

Monte Carlo simulation of size exclusion chromatography for branched polymers formed through free‐radical polymerization with chain transfer to polymer

The elution curves of size exclusion chromatography (SEC) for branched polymers formed through free‐radical polymerization that involves chain transfer to polymer were theoretically investigated by using a Monte Carlo method. We considered two types of measured molecular weight distribution (MWD), (1) the calibrated MWD relative to standard linear polymers, and (2) the MWD obtained by using a light scattering photometer (LS) in which the weight‐average molecular weight of polymers within the elution volume is determined directly. It was found that the calibrated MWD clearly underestimates the high molecular weight tail, and the measured distributions are narrower than the true MWD. On the other hand, the present simulation results showed that the LS method gives reasonable estimates of the true MWDs. The mean square radius of gyration of the polymer molecules having the same molecular weight was also investigated. The radii of gyration showed clear deviation from the Zimm‐Stockmayer equation^[1] because of the non‐random nature of branched structure and the difference in the primary chain length distribution.     

Elution behavior of polyethylene in polar mobile phases on a non-polar sorbent

Linear polyethylene standards in the range of 1-500 kg/mol, dissolved in 1,2,4-trichlorobenzene, were injected into a column packed with oligo(dimethylsiloxane) modified silica gel. Fifteen polar solvents (cyclohexanone, cyclohexylacetate, cyclohexanol, nonylalcohol, dimethylformamide, dimethyl sulfoxide, ethylene- and diethylene glycol monobutyl ether, benzylalcohol, hexylacetate, bis(2-ethyl-hexyl)phthalate, N,N-dimethylacetamide, propylene carbonate, dipropylene glycol and N-methyl-pyrrolidone) were evaluated as mobile phases. Depending on the type of mobile phase evaluated, different elution behaviors are observed for polyethylene: (1) polyethylene was eluted in the size exclusion mode, (2) polyethylene was eluted together with the sample solvent peak at constant elution volume, (3) polyethylene was partially or fully retained on the column. The retained polymer was easily removed from the column by injecting a small volume of trichlorobenzene. The use of ethylene glycol monobutyl ether as the mobile phase enabled separation of the polyethylene from polypropylene. In this case polypropylene is eluted in the size exclusion mode, while polyethylene is eluted at a constant elution volume or remains in the column.     

Semi‐online nanoflow liquid chromatography/matrix‐assisted laser desorption ionization mass spectrometry of synthetic polymers using an octadecylsilyl‐modified monolithic silica capillary column

We have designed a semi‐online liquid chromatography/matrix‐assisted laser desorption/ionization mass spectrometry (LC/MALDI‐MS) system to introduce eluent from a octadecylsilyl (ODS) group modified monolithic silica capillary chromatographic column directly onto a sample plate for MALDI‐MS analysis. Our novel semi‐online system is useful for rapidly and sensitively examining the performance of a monolithic capillary column. An additional advantage is the small elution volume of a monolithic capillary column, which allows delicate eluents, such as 1,1,1,3,3,3,‐hexafluoroisopropyl alcohol (HFIP), to be used to achieve cost‐effective analysis. Using the semi‐online LC/MALDI‐MS system, chromatographic separation of polymers by the monolithic column with different eluents was studied. Separation of poly(methyl methacrylate) and Nylon 6/6 showed that the column functioned via size‐exclusion separation when tetrahydrofuran or HFIP eluent was used. On the other hand, the separation behavior of Nylon 11 indicated a reversed‐phase mode owing to the interaction of the polymer with the modified ODS group in the column. Using tetrahydrofuran/methanol (1:1, v/v) as the eluent, the LC/MALDI‐MS spectra of poly(lactic acid), which contains both linear and cyclic polymer structures, showed that the column could separate the hydrophobic cyclic polymer and elute it out relatively slowly. The monolithic column functions basically via size‐exclusion separation; the reversed‐phase separation by interaction with the ODS functions may have less influence on column separation. The semi‐online monolithic capillary LC/MALDI‐MS method we have developed should provide a means of effectively analyzing synthetic polymers. Copyright © 2010 John Wiley & Sons, Ltd.     

High performance liquid chromatography characterization of macromolecules

A new method, temperature gradient interaction chromatography(TGIC) is employed for the characterization of macromolecules. Fine and reproducible control of interaction between polymer chains and the alkyl chain bonded silica packing material can be achieved by varying the temperature of the column. This method provides a far superior resolution to the conventional size exclusion chromatography. In addition, this method has a high sample loading capacity to be effective for preparative purpose. Furthermore, this method can be used to characterize binary polymer mixtures, where one component of a polymer mixture is separated by the size exclusion mechanism and the other is by the interaction mechanism simultaneously from single isocratic elution.     

Relations between the separation coefficient,longitudinal displacement and peak broadening in size exclusion chromatography of macromolecules

The separation of a polymer by size exclusion chromatography is described as a series of interactions, i.e. consecutive establishments of equilibria between polymer fractions in the mobile and stationary phases followed by displacements of mobile phase containing the polymer. The elution curve is derived as the longitudinal concentration profile in the column observed in one position in space during the time of the analysis. The mean value of elution volume of a particular polymer species turns out to be the interstitial volume of the separation system divided by the mean fraction of polymer in the mobile phase. The number of the displacement-equilibrium steps can be estimated from the limiting values of the variance of the spreading function.     

Positron annihilation lifetime study of continuous volume phase transition of poly(N-isopropylacrylamide) gel induced in methanol–water mixed solvent

The macroscopic volume shrinkage and swelling of poly(N-isopropylacryl-amide) (PNIPA) gel induced by the compositional change in the methanol–water mixed solvent is correlated to the change in the nanoscopic free volume size and numerical concentration formed in the PNIPA gels. The free volume size and numerical concentration are estimated from the longest component appearing in the positron annihilation lifetime curves. The apparent free volume fraction calculated by the free volume size and numerical concentration, and dispersion of the free volume deduced by the size distribution are utilized to analyze the origin and location of the free volumes. The free volume parameters obtained by analysis of the positron annihilation data show various nanoscopic phases occuring within the PNIPA gels during the volume change, implying the variation of the strength of the interactions among the solvent molecules and the polymer chains of the PNIPA. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36: 1141–1151, 1998