Relationship between Temperature-Induced Changes in Internal

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Relationship between Temperature-Induced Changes in Internal Microscopic Structures of Poly(N‑isopropylacrylamide) Microgels and Organic Dye Uptake Behavior Takuma Kureha,† Takaaki Sato,† and Daisuke Suzuki*,†,‡ †

Graduate School of Textile Science & Technology and ‡Division of Smart Textiles, Institute for Fiber Engineering, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, 3-15-1, Tokida, Ueda 386-8567, Japan S Supporting Information *

ABSTRACT: Temperature-induced changes in the internal structures of poly(N-isopropylacrylamide) (pNIPAm) microgels were evaluated by small-angle X-ray scattering (SAXS), and the results were used to explain organic dye uptake by the microgels. The dye uptake experiments were conducted using two organic dyes: cationic rhodamine 6G (R6G) and anionic erythrosine. In the SAXS investigation, the internal structures of the microgels were characterized in terms of the correlation length, ξ, and the distance, d*, which originated from the local packing of the isopropyl groups of two neighboring chains. With increasing temperature up to the volume phase transition temperature (VPTT) of the microgels, the correlation length, ξ, was increased and the distance, d*, was decreased. At the same time, the amounts of the dyes taken up by the pNIPAm microgels were increased, despite a decrease in the volume of the microgels. The results indicated that the pNIPAm chains were closer to each other due to the hydrophobic association of isopropyl groups, which resulted in the growth of the hydrophobic domains. Thus, the hydrophobic interactions between the dyes and pNIPAm were probably accompanied by the domain formation. With a further increase of temperature above the VPTT, the correlation length, ξ, was decreased and then not defined because the Ornstein−Zernike type contribution disappeared, and the distance, d*, was not largely changed. At the same time, the uptake amounts of the dyes per unit volume of the microgels were also not largely changed, which behaved similar to the distance, d*. It was probably due to the fact that the internal structures of the microgels were not largely changed because the isopropyl groups were in contact with each other. The view was supported by the result of the uptake study of the nonthermoresponsive microgels which did not have the hydrophobic isopropyl groups.

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INTRODUCTION Hydrogel particles (microgels) are spherical, cross-linked polymeric networks that are swollen by water. One of the most extensively studied microgels is composed of poly(Nisopropylacrylamide) (pNIPAm), which is a representative thermoresponsive polymer that has a lower critical solution temperature (LCST) of around 31 °C.1,2 Thus, pNIPAm-based microgels show a volume phase transition temperature (VPTT) around the LCST of the pNIPAm chain:3 the microgels are swollen below the VPTT, but they are deswollen above this point. It can be envisaged that the swelling/deswelling behavior of the microgels can be utilized for molecular separations and sensors. Indeed, chemical and biological separation has been achieved by controlling the interactions between the target molecules and microgels.4−13 For example, Kawaguchi et al. reported the separation of biological molecules with pNIPAm microgels.4 They investigated the temperature-dependent adsorption and desorption of human gamma globulin (HGG). It was found that the maximum protein binding to the microgels occurred at 40 °C, i.e., when the microgels were deswollen. In addition, the attached HGG could be desorbed from the microgels when the system was cooled to 25 °C, at which the microgels were swollen. It was concluded that the binding is induced by hydrophobic interactions between HGG © 2014 American Chemical Society

and isopropyl groups on the microgel. In addition to biological molecules, pNIPAm-based microgels have been used as carriers for the separation of small molecules, such as organic dyes (e.g., Orange II),5−8 heavy metal ions (e.g., Pb(II) and Cu(II)),9,10 and surfactants (e.g., cetylpyridinium chloride and sodium dodecylbenzenesulfonate).11−13 Although the application of microgels as separation carriers is promising, there are very few reports in which the temperaturedependent uptake behavior of microgels has been investigated in detail. Since pNIPAm bulk gels show critical behavior near the LCST of pNIPAm chains, it should be important to check the temperature dependence of the level of uptake of such small molecules. The volume phase transition of pNIPAm bulk gels was first reported by Tanaka et al. in 1984.14 Shibayama et al. were next reported the first extensive study on the static structure of the pNIPAm bulk gels close to the volume phase transition temperature by small-angle neutron scattering (SANS).15 It was clearly demonstrated that the SANS intensities of the pNIPAm bulk gels in q ≤ 2 nm−1 are described well by a sum of the Ornstein−Zernike (OZ) Received: May 15, 2014 Revised: July 8, 2014 Published: July 8, 2014 8717

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equation, I(q) = Ioz(0)/(1 + ξ2q2), and the Guinier equation, IG(q) = IG(0) exp(−Rgq2/3), where q is the magnitude of the scattering vector, ξ is the correlation length, Ioz(0) is the OZ intensity extrapolated to the zero scattering vector, RG is the radius of gyration, and IG(0) is the asymptotic Guinier intensity at zero scattering vector. They found that the correlation length, ξ, showed critical behavior near the VPTT of pNIPAm bulk gels. Similar behavior was observed for other pNIPAm systems, such as chains16 and cryo-gels,17,18 by other research groups. Nonetheless, there seem to be conflicting views on the thermoresponsive behavior of the correlation length, ξ, for pNIPAm microgels.19−24 For example, in one of the frequently cited paper, the mesh size of the network is linearly related to the hydrodynamic diameter of the microgels, showing no critical behavior.22 Thus, the occurrence of the critical behavior in the microgel system is still unclear. Recently, we focused on the use of small-angle X-ray scattering (SAXS) in an effort to clarify the microscopic features of the microgels.25 The maximum q-value obtained in the pioneering SANS studies on pNIPAm- and pNIPAm-based materials was often limited to ca. 2−3 nm−1 despite its excellent low-q resolution.15,19−24,26−28 In our case, the scattering profiles of the microgels were analyzed in a wide q-range (0.07 ≤ q/nm−1 ≤ 20), and this allowed us to observe the microscopic features of the microgels.25 This study primarily aimed to determine the internal microscopic structure of pNIPAm microgels across their volume phase transition by SAXS. Moreover, the dye uptake behavior of the pNIPAm microgels as a function of temperature was investigated to clarify the influence of the microscopic structural changes in pNIPAm microgels on the separation of small molecules. First, monodisperse pNIPAm microgels were synthesized by aqueous free-radical precipitation polymerization. Next, the pNIPAm microgels were used to investigate the uptake of organic dyes as a function of temperature. In these experiments, two types of organic dyes, cationic rhodamine 6G (R6G) and anionic erythrosine, were chose in the uptake study (Figure 1). The amounts of the dyes taken up

chloride (NaCl, 99.5%), and ethanol (99.5%) were purchased from Wako Pure Chemical Industries and were used as received. Water for all reactions, solution preparation, and polymer purification was distilled and then ion-exchanged (EYELA, SA-2100E1). Synthesis of pNIPAm Microgels. Thermoresponsive pNIPAm microgels were prepared via aqueous free-radical precipitation polymerization using the water-soluble anionic initiator KPS. Polymerization was performed in a 300 mL three-neck, round-bottom flask equipped with a mechanical stirrer condenser and nitrogen gas inlet. The initial total monomer concentration was held constant at 150 mM. A mixture of NIPAm (3.361 g, 99 mol %), BIS (0.046 g, 1 mol %), and SDS (5.8 mg, 0.1 mM) were dissolved in 195 mL of water. The monomer/surfactant solution was heated to 70 °C under a stream of nitrogen with constant stirring at 250 rpm. This solution was allowed to stabilize for a period of at least 30 min prior to initiation. Free radical polymerization was then initiated with KPS (0.109 g) dissolved in 5 mL of water. The stirring solution was allowed to react for a period of 4 h. After the completion of polymerization, the microgel dispersion was cooled to room temperature. The microgels were purified by centrifugation/redispersion with water (two times) using a relative centrifugal force (RCF) of 50000g and by dialysis for 5 days with daily changes of water. Synthesis of Poly(AAm-co-MAc) Microgels. Nonthermoresponsive poly(AAm-co-MAc) microgels were prepared via free-radical precipitation polymerization in ethanol using the oil-soluble initiator AIBN according to a previous report.29 Polymerization was performed in a 300 mL three-neck, round-bottom flask equipped with a mechanical stirrer condenser and nitrogen gas inlet. The initial total monomer concentration was maintained constant at 500 mM. A mixture of AAm (2.666 g, 75 mol %), MAc (0.861 g, 20 mol %), and BIS (0.385 g, 5 mol %) was dissolved in 200 mL of ethanol. The monomer solution was heated to 70 °C under a stream of nitrogen with constant stirring at 250 rpm. This solution was allowed to stabilize for a period of at least 30 min prior to initiation. Free radical copolymerization was then initiated with AIBN (0.033 g) dissolved in 5 mL of ethanol. The stirring solution was allowed to react for a period of 4 h. After the completion of polymerization, the microgel dispersion was cooled to room temperature. The microgels were purified by centrifugation/redispersion with water five times using an RCF of 50000g and by dialysis for 5 days with daily changes of water. Characterization of Microgels. The hydrodynamic diameters of the microgels were determined by dynamic light scattering (DLS, Malvern Instruments Ltd., ZetasizerNanoS). Data were an average of 15 measurements with a 30 s acquisition time of the intensity autocorrelation. DLS experiments were conducted at a microgel concentration of ∼0.001 wt %. NaCl was used to adjust the total salt concentration to 1 mM. When the DLS measurements were performed in the presence of the dyes, concentrations of the dyes were 0.02 mM in each case. The samples were allowed to thermally equilibrate at the desired temperature for 10 min prior to measurement. A time-dependent scattering intensity was detected at the total scattering angle of 173°, which corresponds to a scattering vector of 0.0264 nm−1 in aqueous media (refractive index n = 1.33). The hydrodynamic diameters of the microgels were calculated from the measured diffusion coefficients using the Stokes−Einstein equation (Zetasizer software v6.12). The optical transmittance of the microgel dispersions was measured on a UV−vis spectrophotometer (JASCO, V-630iRM). The temperature dependence of the optical transmittance was investigated at a heating rate of 0.5 °C/min from 20 to 50 °C with constant stirring at 800 rpm. A wavelength of 600 nm was selected to detect optical transmittance. The electrophoretic mobility (EPM) of the microgels was measured with a ZetasizerNanoZS instrument (Malvern, Zetasizer software ver. 4.20). The samples were allowed to thermally equilibrate at the desired temperature for 10 min prior to measurement. The EPM of the microgels was measured at a microgel concentration of ∼0.001 wt % NaCl was used to adjust the total salt concentration to 1 mM. Dye Uptake by Microgels. Stock solutions of R6G (1 mM) and erythrosine (1 mM) in water were prepared. The pNIPAm or

Figure 1. Chemical structures of rhodamine 6G (R6G) (left) and erythrosine (right).

by the pNIPAm microgels were determined by UV−vis spectroscopy. The temperature-induced changes in the internal microscopic structures of pNIPAm microgels were investigated by SAXS. The relation between the temperature dependence of the microscopic structures of the pNIPAm microgels and the dye uptake behavior is discussed.

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EXPERIMENTAL DETAILS

Materials. N-Isopropylacrylamide (NIPAm, purity 98%), N,N′methylenebis(acrylamide) (BIS, 97%), sodium dodecyl sulfate (SDS, 95%), potassium peroxodisulfate (KPS, 95%), acrylamide (AAm, 95%), methacrylic acid (MAc, 99%), 2,2′-azobis(isobutyronitrile) (AIBN, 98%), rhodamine 6G (R6G, 98%), erythrosine (95%), sodium 8718

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Figure 2. (a) Temperature dependence of hydrodynamic diameters (solid diamonds) and the first derivatives of the temperature transition curves (solid line) for the pNIPAm microgels. (b) Temperature dependence of electrophoretic mobilities for the pNIPAm microgels as measured by laser Doppler velocimetry. (c) Temperature dependence of hydrodynamic diameters of the pNIPAm microgels in the absence (solid diamonds) and presence of the dyes: R6G (open squares) and erythrosine (open circles). poly(AAm-co-MAc) microgel dispersions were poured into a vial. The final concentration of microgels was 0.2 g/mL in all experiments. The vial was allowed to thermally equilibrate at the desired temperature for 1 h with constant stirring at 300 rpm in an incubator (CN-25C, Mitsubishi Electric Engineering Co., Ltd.). After the solution had stabilized in the incubator, the appropriate dye stock solution was injected into the vial. The final concentration of dye was 0.02 mM in all experiments. After 5 min of exposure, the mixtures were divided into the three centrifuge tubes (SC-0200, INA-OPTIKA CO., LTD). A period of 5 min was adequate for the uptake study because the uptake amounts of the dyes did not change after times of 5, 10, 30, and 60 min. The mixtures were centrifuged at an RCF of 20000g in order to pack all the microgels at the bottom of the tubes. The three supernatants were carefully removed from the centrifuge tubes without disturbing the microgel pellet at the bottom of the tubes, and the absorbance of the supernatants was measured using a UV−vis spectrophotometer. The overall process is illustrated in SI Scheme 1. Small-Angle X-ray Scattering (SAXS). SAXS measurements were conducted on the pNIPAm microgels at 20, 25, 30, 33, 35, and 40 °C. The SAXS intensities of the pNIPAm microgels in water were measured by SAXSess camera (Anton Paar, Graz) equipped with a block collimator and a sealed-tube anode X-ray generator (GE Inspection Technologies, Germany). The X-ray generator apparatus was operated at 40 kV and 50 mA. A Göbel mirror and block collimator provided a focused monochromatic X-ray beam of Cu Kα radiation (λ = 0.1542 nm) with a well-defined shape. 2D scattered intensity distribution recorded by an imaging-plate (IP) detector was read out by a Cyclone storage phosphor system (PerkinElmer). The 2D data were integrated into an one-dimensional scattering intensities I(q) as a function of the magnitude of the scattering vector. All I(q) data were normalized to the same incident primary beam intensity for the transmission calibration and were corrected for the background scattering from the capillary and water. The absolute scale calibration was made by using water intensity as a secondary standard.30 The sample temperature was controlled with a thermostated sample holder unit (TCS 120, Anton Paar). A model-independent collimation correction (desmearing) procedure was conducted on the basis of a Lake algorithm. All fitting analyses were performed on the demeaned, absolute scattering intensities.

below the VPTT and were deswollen above the VPTT (e.g., Dh ≈ 490 nm at 25 °C and Dh ≈ 200 nm at 40 °C). Above the VPTT, the hydrodynamic diameter did not show significant variation. Moreover, the hydrodynamic diameters, Dh, in the presence of the dyes were not largely changed, compared with that in the absence of the dyes below the VPTT as shown in Figure 2c. Therefore, the dyes did not interfere with the VPT behavior of the pNIPAm microgels. However, Dh, in the presence of the R6G was dramatically increased above the VPTT (Dh ≈ 1 μm at 33 °C). It was due to the fact that the pNIPAm microgels were aggregated. The aggregation behavior of the microgels was also seen in the case of R6G uptake study (details will be described below). In contrast, the hydrodynamic diameter of the poly(AAm-coMAc) microgels was independent of temperature (e.g., Dh ≈ 1.23 μm at 25 °C and Dh ≈ 1.24 μm at 40 °C). The EPMs of the pNIPAm microgels as a function of temperature are shown in Figure 2b. The charge per microgel may have a constant, and as a consequence, smaller (deswollen) microgels had a higher charge density than swollen microgels.32 Thus, the EPMs of the pNIPAm microgels were nearly zero in their swollen states, and they increased sharply in the deswollen states. For poly(AAmco-MAc) microgels, the EPM was independent of temperature (e.g., −3.1 × 10−8 m2 V−1 s−1 at 25 °C and −3.6 × 10−8 m2 V−1 s−1 at 40 °C). The uptake of the cationic dye (R6G) by the pNIPAm or poly(AAm-co-MAc) microgels as a function of temperature was investigated. First of all, we investigated the effects of temperature and R6G concentration on the visible absorption spectrum of R6G in aqueous solutions in the absence of the microgels. The temperature range studied was from 20 to 40 °C, and the concentration range of R6G was from 0.001 to 0.02 mM, which covers the experimental conditions for the dye uptake. Significant differences in the absorbance were not observed under these conditions (see Figure 1 in Supporting Information). In addition, the effect of the presence of R6G on the phase transition behavior of the pNIPAm microgels was investigated by monitoring the temperature dependence of transmittance (see Figures 2 and 3 in Supporting Information). At each R6G concentration, the transmittance of the pNIPAm microgels showed a marked change at ∼33 °C. The results indicate that the transition behavior of the pNIPAm microgels was not perturbed by the presence of R6G. Note that we did not focus on the dyes uptake behavior upon cooling though there is hysteresis behavior in this system (see Supporting Information Figure 3b). The amount of R6G taken up by the

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RESULTS The temperature sensitivity of microgels is most easily demonstrated by DLS. The hydrodynamic diameters, Dh, of the pNIPAm microgels as a function of temperature are shown in Figure 2a along with the first derivatives of the estimated volumes of the microgels for the transition curves. The first derivative provides a convenient measure of the VPTT.31 From the DLS measurements, the VPTT of the microgels was determined to be 33 °C. The pNIPAm microgels were swollen 8719

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Figure 3. Temperature dependence of R6G uptake by the pNIPAm microgels. (a) Uptake amount of R6G per unit gram of microgels (circle) and correlation length, ξ, of the microgels below the volume phase transition temperature (triangle). (b) Uptake amount of R6G per unit volume of microgels (circle) and inverse of the distance, d* (diamond). Each point represents an average of three replicate uptake experiments, and the error bars denote a standard deviation.

Figure 4. Temperature dependence of erythrosine uptake by the pNIPAm microgels. (a) Uptake amount of erythrosine per unit gram of microgels (circle) and correlation length, ξ, of the microgels below the volume phase transition temperature (triangle). (b) Uptake amount of erythrosine per unit volume of microgels (circle) and inverse of the distance, d* (diamond). Each point represents an average of three replicate uptake experiments, and the error bars denote a standard deviation.

spectrum of erythrosine in aqueous solutions were assessed in the absence of the pNIPAm microgels (see Figure 4 in Supporting Information). In addition, the effect of the presence of erythrosine on the phase transition behavior of the pNIPAm microgels was also checked by monitoring the temperature dependence of transmittance (see Figure 5 in Supporting Information). In a similar way to the case of R6G, the transition behavior of the pNIPAm microgels was not perturbed by the presence of erythrosine. The amount of erythrosine taken up as a function of temperature is shown in Figure 4a. The amount of erythrosine taken up by the microgels increased as the temperature was increased from 20 to 33 °C, in a similar manner to that found in the R6G uptake study. However, in contrast to the case of R6G shown in Figure 3a, the amount of erythrosine taken up showed a maximum at 33 °C, i.e., at the VPTT of the pNIPAm microgels. Then, it decreased with a further increase of the temperature to 34 °C but was not largely changed at higher temperatures. The amount of erythrosine taken up per unit volume of the pNIPAm microgels as a function of temperature is shown in Figure 4b. The maximum

pNIPAm microgels as a function of temperature is shown in Figure 3a. It can be seen that as the temperature was increased, the amount of R6G taken up increased at temperatures below 33 °C (i.e., the VPTT of the pNIPAm microgels). The amount of R6G taken up by the pNIPAm microgels did not significantly change with temperature above the VPTT. Since the volume of pNIPAm changes as a function of temperature as shown in Figure 2a, the dye uptake amount was normalized by the volume of the pNIPAm microgels at each temperature (Figure 3b). It is clear that a large amount of R6G per unit volume of pNIPAm was removed by the microgels on increasing temperature. Note that the microgels were aggregated when the experiments were conducted above the VPTT. The aggregation behavior of the microgels was also seen from the DLS measurements (Figure 2c). It may influence the accuracy of the data above the VPTT shown in Figure 3a,b. The uptake behavior of erythrosine by the pNIPAm microgels as a function of temperature was investigated. In the same manner as described above, the effects of temperature and erythrosine concentration on the visible absorption 8720

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Figure 5. Temperature dependence of the dye uptake by the poly(AAm-co-MAc) microgels. (a) Uptake amount of dyes per unit gram of the microgels. (b) Uptake amount of dyes per unit volume of microgels. Each point represents an average of three replicate uptake experiments, and the error bars denote a standard deviation.

scattering intensities in q < 0.2 nm−1, which can be described by the Guinier equation, seems to have arisen from solidlike density fluctuation due to the inhomogeneities of chemical cross-links in the microgels.18,25

at 33 °C was no longer visible in the normalized quantity, and the amount of erythrosine taken up per unit volume of the pNIPAm microgels increased with temperature, which is similar to the trend observed for the uptake of R6G. In contrast to the result of R6G study represented in Figure 3, the microgels were not aggregated above the VPTT. Figures 3a and 4a also showed the correlation length, ξ, below the VPTT and the distance, d*, of the microgels. The details will be described at greater length below. On the one hand, when the nonthermoresponsive poly(AAm-co-MAc) microgels were used, the amount of both R6G and erythrosine taken up was almost independent of temperature (Figure 5). Nonetheless, there was a large difference in the uptake amount: the uptake amount of R6G was approximately 10 times higher than that of erythrosine, which is probably due to electrostatic attraction between the cationic R6G and the anionic microgels. SAXS was employed to reveal the internal structures of the pNIPAm microgels according to methods described in a previous study.25 The representative scattering profiles of the pNIPAm microgels obtained at 20, 25, 30, 33 (VPTT of the pNIPAm microgels), 35, and 40 °C are shown in Figure 6. For a quantitative description of the measured I(q) of the pNIPAm microgels, a sum of five contributions was required. The forward intensity obeyed a Porod law, which originated from the interface of the microgel.33−35

I(q) ∝ q−4

⎡ R 2q2 ⎤ IG(q) = IG(0) exp⎢ − G ⎥ 3 ⎦ ⎣

(2)

where RG is the radius of gyration and IG(0) is an asymptotic Guinier intensity at q → 0. The contribution from density fluctuations of the network in the microgel was found in low-to-intermediate-q range and was described by the Ornstein−Zernike equation.15,36

Ioz(q) =

Ioz(0) 1 + q 2ξ 2

(3)

where ξ is the correlation length and Ioz(0) is an asymptotic OZ intensity when q → 0. ξ represents the length scale of the spatial correlation of density fluctuations caused by a polymer chain. The scattering curves below the VPTT are shown in Figure 6, and it appears that the contribution of the OZ scattering extended over the low-to-intermediate-q range. In particular, the contribution of the OZ scattering at 33 °C (VPTT of the microgels) was larger than that at 25 and 30 °C. Then, the contribution of OZ scattering became smaller drastically at 35 °C, and the contribution disappeared completely at 40 °C. In the high-q regime, two interference peaks were observed (e.g., q ≈ 3.6 nm−1 and q ≈ 15.6 nm−1 at 25 °C). These were formally fitted by pseudo-Voigt equations.25

(1)

When the microgels were swollen at 20, 25, and 30 °C, the Porod behavior was not clearly observed in the scattering intensities of the microgels. It was probably due to the fact that the interface of the swollen microgels was rather vague. On the other hand, the Porod behavior became clearly visible in q < 1 nm−1 with increasing temperature when the VPTT was approached. This result indicates that the interface of the microgels was well-defined, and the overlapping contribution from the polymer network fluctuation disappeared by deswelling of the microgels. In addition, the Guinier equation was necessary to fit the data in low-q regime. The raised

IV(q) =

IV(0) 1 + (q − q*)2 ξ*2

(4)

where q* denotes the peak position and ξ* is related to the size of the organized domains. The low-q peak position, q*, was gradually shifted from 3.6 nm−1 at 25 °C to 5.6 nm−1 at 40 °C, and this shift was accompanied by an increase in the peak intensity. The low-q peak was attributed to interchain correlations or the domain formation of pNIPAm. On the 8721

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Figure 6. Small-angle X-ray scattering (SAXS) intensities, I(q), of the pNIPAm microgels at 20, 25, 30, 33, 35, and 40 °C. The gray open circles represent the measured intensities. The solid black curves are the best fit curves based on the sum of eq 1 (solid red curve), eq 2 (dashed blue curve), eq 3 (solid purple curve), and eq 4 (solid and dashed green curve). The gray region highlights the Ornstein−Zernike type contribution. Upper insets show the variation of the residuals with q.

other hand, the position of the high-q peak centered at q ≈ 15 nm−1 remained largely unchanged within the accuracy of the data. The high-q peak is able to be ascribed to the local structure of pNIPAm such as intrachain correlations.17,18,25 The characteristic distance, d*, corresponding to the low-q peak position, q*, can be approximated by Bragg’s equation, d* = 2π/q*. The correlation length, ξ, and the distance, d*, as a function of temperature are shown in Table 1 and plotted in Figures 3 and 4. The correlation length, ξ, of the pNIPAm microgels increased with temperature up to 33 °C (VPTT). On the other hand, the distance, d*, decreased, but it became nearly constant above 35 °C. The best fit was obtained using a weighted least-squares method in SAXS analysis. A residual of the fit was divided by the standard deviation at each scattering vector, q (Figure 6).

Table 1. Summary of the DLS and SAXS Results Obtained at the Different Temperatures T/°C

Dh/nm

ξ/nm

q*a/nm−1

d*b/nm

20 25 30 33 35 40

520 490 419 246 206 196

2.4 2.5 3.5 6.1 0.5

3.5 3.6 4.1 5.4 5.6 5.6

1.8 1.7 1.5 1.2 1.1 1.1

a

q* denotes the peak position. bd* is estimated by Bragg’s equation, d* = 2π/q*.

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DISCUSSION To discuss the effect of electrostatic interactions between dyes and microgels, two types of organic dyes were selected for the uptake study (i.e., cationic R6G and anionic erythrosine). The 8722

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study reported here, we used small- and wide-angle X-ray scattering (SWAXS) to achieve a wide q-range coverage of 0.07 ≤ q/nm−1 ≤ 20. The correlation length, ξ, was precisely quantified because the two interference peaks in high-q regime were fitted by pseudo-Voigt equations. The correlation length, ξ, was increased with rising temperature to 33 °C (VPTT). In addition, the scattering from density fluctuations of the network in the microgel disappeared above the VPTT. As a result, the correlation length, ξ, was not able to be defined as shown in Table 1. Thus, the correlation length, ξ, of the pNIPAm microgels exhibits the divergent behavior, which is similar to that of pNIPAm bulk gels and chains.15,16,18 At temperature below the VPTT, the uptake amounts of the dyes were increased at the same time with an increase of the correlation length, ξ (Figures 3a and 4a). After the uptake amounts of the dyes were normalized by the volume of the pNIPAm microgels at each temperature (Figures 3b and 4b), the uptake amounts of the dyes per unit volume were sharply increased at temperature below the VPTT, and then they were not largely changed above the VPTT. Ahmed et al. reported that the hydrophobic association of isopropyl groups of two neighboring chains and/or hydrogen bonds between amide groups occur with increasing temperature, which was revealed by UV resonance Raman experiments.39 Furthermore, Chalal et al. reported that the distance, d*, which was observed for pNIPAm-based cryogel, originates from the local packing of isopropyl groups of two neighboring chains and/or hydrogen bonds between amide groups.17,18 Therefore, the pNIPAm main chains were closer to each other with increasing temperature due to the hydrophobic association of isopropyl groups, which resulted in the deswelling of the pNIPAm microgels below the VPTT. Above the VPTT, the hydrodynamic diameter of the pNIPAm microgels did not show significant variation because the isopropyl groups were in contact with each other. The temperature-induced structural changes of the pNIPAm microgels across the VPTT associated with the temperature dependence of the distance, d*, and the amounts of the dyes taken up. The amounts of the dyes taken up, which were normalized by the volume of the pNIPAm microgels, were inversely proportional to the distance, d*, approximately (Figures 3b and 4b). The hydrophobic interactions between the dyes and pNIPAm microgels were probably accompanied by the domain formation. To reveal the behavior, SAXS measurements on the pNIPAm microgels with the dyes may be helpful although careful SAXS measurements will be necessary to be performed in the exactly the identical experimental conditions (see Supporting Information Figure 7). We will further investigate the temperature dependence of the correlation length, ξ, and the distance, d*, of the pNIPAm microgels in detail, and this will be reported elsewhere. Thus, the dyes were adsorbed through hydrophobic interactions with the isopropyl groups in the pNIPAm microgels, and the amounts of the dyes taken up increased as the hydrophobic domains in the pNIPAm microgels grew. This uptake behavior supports the result that the hydrophobic interaction is an important factor when hydrophobic molecules, such as organic dyes, are separated by pure pNIPAm microgels. However, in the R6G uptake study, the pNIPAm microgels aggregated above the VPTT. Hence, it was questionable whether the uptake amount of R6G was directly related to the changes in internal structure, which was evaluated by SAXS, because the effect of the aggregates of the pNIPAm microgels

pNIPAm microgels were negatively charged, which was confirmed by the EPM data shown in Figure 2b, due to the presence of sulfate groups from the initiator KPS.32 Below the VPTT (33 °C), the amount of the R6G taken up was always greater than that of the erythrosine, which is due to the electrostatic attraction between the cationic R6G and the anionic pNIPAm microgels. Note that the uptake amount of cationic R6G was much larger than that of erythrosine when the poly(AAm-co-MAc) microgels with a higher negative charge were used (Figure 5). On the other hand, above the VPTT, where the pNIPAm microgels were deswollen, the uptake of R6G was twice larger than that of erythrosine. The reason for the difference cannot simply be explained because the pNIPAm microgels were aggregated only in the case of R6G uptake study. The colloidal stability of the deswollen microgels was provided by the electrostatic repulsion as is the case with the hard particles, such as polystyrene and silica.32 We infer that the decreased dispersion stability was due to electrostatic attractive forces between the cationic R6G and the anionic pNIPAm microgels and the resulting surface charge neutralization. In contrast, the electrostatic attractive forces between the anionic erythrosine and the anionic pNIPAm microgels did not occur, and the microgels did not aggregate. Similar aggregation behavior was reported by Snowden et al. for the adsorption of lead ions onto anionic pNIPAm-based microgels.9 The influence of the structural changes in the pNIPAm microgels on the temperature dependence of the uptake of the dyes will be discussed in the following. Recently, Parasuraman and Serpe reported that poly(NIPAm-co-acrylic acid) microgels efficiently adsorb the azo dye Orange II (anionic dye).5 Their results suggested that greater uptake was possibly due to the increase in the size of the microgels as the mole percent of acrylic acid increased, where the microgels were in swollen states. Therefore, it was expected that the pNIPAm microgels with a larger volume would take up larger amounts of the dyes. However, it can be seen in Figures 3a and 4a that the uptake amounts of the dyes increased as the temperature was increased from 20 to 33 °C (VPTT), despite a decrease in the size (volume) of the pNIPAm microgels, as shown in Figure 2a. The uptake studies were carried out in the equivalent mass of the microgels. In the case of the nonthermosensitive poly(AAm-co-MAc) microgels, the uptake amounts of the dyes were not temperature dependent (Figure 5). It is clear that the VPT behavior of the pNIPAm microgels influences the amounts of the dyes taken up. Additionally, despite the fact that the volumes of the poly(AAm-co-MAc) microgels, which did not have hydrophobic isopropyl groups, were larger than those of pNIPAm microgels, the uptake amounts of the dyes per unit volume by the poly(AAm-co-MAc) microgels were much lower than those for the pNIPAm microgels (Figure 5b). Thus, we infer that the hydrophobic interaction is an important factor. Note that the R6G was not removed from the microgels during the centrifugation process, as shown in Supporting Information Figure 6 . Small-angle scattering of X-rays (SAXS) data related to the variation of the internal microscopic structures of the pNIPAm microgels. As described in the Introduction, there seem to be conflicting views on the thermoresponsive behavior of the correlation length, ξ, for pNIPAm microgels.19−24 Stieger et al. mentioned that the incoherent background is large, and the statistical error of the experimental data is rather large in the qrange of their SANS experiments; thus the correlation length, ξ, could not be extracted reliably by SANS data analysis.38 In the 8723

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was included.5,6 However, this factor should be relevant in the erythrosine uptake study.

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ASSOCIATED CONTENT

S Supporting Information *

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Information on effects of temperature and concentration on the absorption spectrum of dyes aqueous solutions measured on a UV−vis spectrophotometer. This material is available free of charge via the Internet at http://pubs.acs.org.

CONCLUSIONS The relation between the temperature-induced changes in the internal microscopic structures of pNIPAm microgels and the dye uptake behavior was investigated. The uptake study was performed using two organic dyes: cationic R6G and anionic erythrosine. The dye uptake behavior of the pNIPAm microgels was clarified by comparing the microscopic structural information on the pNIPAm microgels and the dyes uptake of the nonthermosensitive poly(AAm-co-MAc) microgels. The internal structures of the pNIPAm microgels were investigated by SAXS in the extended q-range of 0.07 ≤ q/nm−1 ≤ 20. In the SAXS study, we found that the scattering intensity of the pNIPAm microgels showed five different structural features. The forward intensity obeyed the Porod behavior, which originated from the interface of microgels. In addition, the Guinier equation was necessary to fit the data in low-q regime without causing systematic deviations. The fluctuations of the gel network, which were described by the Ornstein−Zernike equation, were characterized in terms of the correlation length, ξ. The appearance of two interference peaks was observed in q ≈ 3−6 nm−1 and q ≈ 15 nm−1, and these were formally fitted by pseudo-Voigt equations. Since the correlation length, ξ, of the pNIPAm microgels was increased with rising temperature to 33 °C (VPTT), and then it decreased above the VPTT. The volume phase transition behavior of the pNIPAm microgels was similar to that of pNIPAm bulk gels and chains. The characteristic distance, d*, was able to be estimated by the Bragg’s equation, d* = 2π/q*, where q* is the low-q peak position centered at q ≈ 3−6 nm−1. The distance, d*, originated from the local packing of the isopropyl groups of two neighboring chains. The value of d* decreased as the temperature was increased from 20 to 33 °C, but it became constant above the VPTT. The temperature dependence results signify that the hydrophobic domains grew because of the hydrophobic interaction between the isopropyl groups in the pNIPAm microgels on increasing the temperature up to the VPTT. The temperature-induced changes in the internal microscopic structures of the pNIPAm microgels were able to explain the separation behavior of the dyes. The uptake amounts of the dyes, which were normalized by the volume of the pNIPAm microgels, were inversely proportional to that of the distance, d*. The result suggests that hydrophobic interactions occurred between the hydrophobic domains in the pNIPAm microgels and dyes, and the uptake amounts of the dyes increased as the hydrophobic domains in the pNIPAm microgels grew. This view was supported by the result of the dyes uptake study of the nonthermosensitive poly(AAm-coMAc) microgels, which did not have the hydrophobic isopropyl groups. We believe that an understanding of the thermoresponsive behavior of pNIPAm microgels will help encourage their actual use as separation carriers for small molecules. In addition, since the uptake and adsorption of small molecules by microgels are important phenomena, especially for novel functional microgels, such as oscillating microgels40−42 and organic/inorganic hybrid microgels that are used for catalysts, 43−45 the fundamental aspects clarified in this study will also be useful for other applications of microgels.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (D.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS D.S. acknowledges Grant-in-Aid for Young Scientists (A) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (22685024).

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REFERENCES

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