Self-Association of a Thermosensitive Amphiphilic Block Copolymer

Dec 17, 2012 - The self-association behavior of a thermosensitive amphiphilic block copolymer, poly(N-isopropylacrylamide)-b-poly(N-vinyl-2-pyrrolidon...
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Self-Association of a Thermosensitive Amphiphilic Block Copolymer Poly(N‑isopropylacrylamide)‑b‑poly(N‑vinyl-2-pyrrolidone) in Aqueous Solution upon Heating Takahiro Sato,†,* Kohei Tanaka,† Akiko Toyokura,† Rika Mori,† Rintaro Takahashi,† Ken Terao,† and Shin-ichi Yusa‡ †

Department of Macromolecular Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2280, Japan

‡

S Supporting Information *

ABSTRACT: The self-association behavior of a thermosensitive amphiphilic block copolymer, poly(N-isopropylacrylamide)-b-poly(Nvinyl-2-pyrrolidone) (PNIPAM-b-PNVP) in water upon heating was investigated by static and dynamic light scattering (SLS and DLS), small-angle X-ray scattering (SAXS), and pulsed field gradient NMR (PFG-NMR). Combining SLS and DLS with SAXS and PFG-NMR, we conclude that above 40 °C, the unimer or a few arms star micelle of the copolymer coexists with large spherical particles of uniform density, which should be regarded as concentrated-phase droplets produced by a liquid−liquid phase separation. The spherical particles of PNIPAM-bPNVP samples with the PNIPAM content less than 0.32 became smaller with increasing temperature. This temperature dependence was explained in terms of the packing parameter for amphiphiles.

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INTRODUCTION Amphiphilic block copolymers are macromolecular surfactants or lipids, and they may self-associate into various types of micelles in solution, just like low-molar mass counterparts. The morphology and size of the micelles are determined not only by the ratio of block chain lengths but also by interaction parameters among both blocks and solvent.1−11 If one block of a copolymer is thermosensitive, i.e., changes from hydrophilic to hydrophobic by changing temperature, we expect a thermally induced micellization of the copolymer in aqueous media. With the advent of novel controlled polymerization techniques, it has become possible to prepare various thermosensitive block copolymers. Poly(N-isopropylacrylamide) (PNIPAM)12 and poly(2-isopropyl-2-oxazoline)13,14 are typical thermosensitive blocks, and poly(ethylene glycol),15−19 polypeptides,20 poly(N-alkyl acrylamide),21 and poly(acrylic acid).22 are hydrophilic blocks. Yusa et al.23 recently synthesized a block copolymer of PNIPAM and poly(N-vinyl-2-pyrrolidone) (PNVP) (see Scheme 1) by organotellurium-mediated controlled radical polymerization. PNIPAM is known to be soluble in water at room temperature but become insoluble above the lower critical solution temperature (LCST). On the other hand, PNVP is soluble in water in a wide temperature range. Therefore, the block copolymer PNIPAM-b-PNVP is a watersoluble polymer at room temperature, and become amphiphilic upon heating. In fact, Yusa et al. reported the formation of large © 2012 American Chemical Society

Scheme 1. Chemical Structure of Poly(Nisopropylacrylamide)-b-poly(N-vinyl-2-pyrrolidone) (PNIPAM-b-PNVP)

aggregates with a hydrodynamic radius as large as 100 nm in aqueous PNIPAM-b-PNVP upon heating. However, the reported hydrodynamic radii were larger than those expected for the normal star (or spherical) micelle, and the detailed morphology of the large aggregates was not elucidated in the previous work. In this study, we investigated the morphology of the large aggregates of PNIPAM-b-PNVP in hot water by static and dynamic light scattering (SLS and DLS), small-angle X-ray scattering (SAXS), and pulsed field gradient NMR (PFGNMR). It was found that the thermosensitive block copolymer solution containing largely different size scattering components, was difficult to analyze by SLS and DLS because the light Received: October 7, 2012 Revised: December 5, 2012 Published: December 17, 2012 226

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where K is the optical constant, c is the mass concentration of the total copolymer, k is the magnitude of the scattering vector, and wi, Mw,i, ⟨S2⟩z,i, and A′2,i are the weight fraction (in the total copolymer), weight-average molar mass, the z-average square radius of gyration, and the apparent second virial coefficient of the component i, respectively. Furthermore, the first cumulant Γi of the component i, calculated by the summation of A(τ)/τ over the τ range of the component i, is related to the hydrodynamic radius RH,i of the component i by

scattering power of the smaller size scattering component is much weaker than that of the larger one. The combination with SAXS data was crucially important to characterize the selfassociation of the block copolymer.

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EXPERIMENTAL SECTION

Samples. Five PNIPAM-b-PNVP copolymer samples were used in this study. All samples were prepared by the organotellurium-mediated controlled radical polymerization described in a previous paper.23 Number-average degrees of polymerization of the PNIPAM precursors N0,n(PNIPAM) and of the copolymer chains N0,n, as well as the mole fractions x of the NIPAM monomer unit in the copolymer chain [=N0,n(PNIPAM)/N0,n] were estimated by 1H NMR. Ratios of the weight- to number-average molecular weights Mw/Mn of the copolymer samples were determined by size exclusion chromatography (SEC), using dimethylformamide as the eluent and a calibration curve constructed with standard polystyrene samples. Details of NMR and SEC measurements were also described previously.23 The results summarizes in Table 1, along with the number-average degree of

RH, i = lim

c , θ→ 0

N0,n(PNIPAM)a

N0,na

N0,n(PNVP)a

Mn/ 104 a

Mw/ Mnb

Mw/ 104 c

0.67 0.32 0.31 0.28 0.21 0.14

110 110 100 95 100 60

163 345 321 335 473 429

53 234 218 240 373 369

1.83 3.85 3.58 3.74 5.27 4.77

1.09 1.11 1.16 1.18 1.12 1.25

1.99 4.27 4.15 4.41 5.90 5.96

a

Estimated by 1H NMR. bEstimated by SEC. cEstimated from the results in the fifth and sixth columns. The results for x = 0.67 and 0.32 were reported previously.23

polymerization of the PNVP block N0,n(PNVP) and Mn and Mw of the total copolymer chain. Size exclusion chromatograms for a PNIPAMb-PNVP block copolymer sample (x = 0.31) and its precursor PNIPAM are given in theSupporting Information. Light Scattering. Static light scattering (SLS) measurements were carried out using a Fica 50 light scattering photometer (vertically polarized incident light of 436 and 546 nm) or an ALV/DLS/SLS5000 light scattering system (vertically polarized incident light of 532 nm) with no analyzer. The light scattering systems were calibrated using benzene or toluene as the reference material to determine the excess Rayleigh ratio Rθ of the solution over that of the solvent at the scattering angle θ: The correction of the dispersity with respect to the copolymer composition to the molar mass was estimated to be less than 0.1%, from ∂n/∂c of the PNVP homopolymer (0.186 cm3/g at 436 nm and 0.180 cm3/g at 546 nm) and of the PNIPAM homopolymer (0.167 cm3/g at 436 nm and 0.162 cm3/g at 546 nm) as well as the dispersity in the composition of the PNIPAM precursors and copolymer samples. In what follows, we have neglected this correction.24 The refractive index increment of PNIPAM-b-PNVP in water exhibited no appreciable temperature dependence. Dynamic light scattering (DLS) measurements were made using the ALV/DLS/SLS-5000 light scattering system equipped by an ALV5000 multiple τ digital correlator. Intensity autocorrelation functions g(2)(t) obtained were analyzed by a CONTIN program to get the relaxation time spectrum A(τ). The spectrum A(τ) of PNIPAM-b-PNVP solutions showed a bimodal or trimodal distribution. In such a case, Rθ was divided into two or three components using A(τ). The excess Rayleigh ratio Rθ,i for the component i in dilute solution is expressed in the form25

I(g ) = exp(− Dsζ ),

⎛ 1 1 ⎞ ζ ≡ (γgδ)2 ⎜Δ − δ − τ ⎟ ⎝ 3 2 ⎠

(3)

for a monodisperse polymer solution where γ is the gyromagnetic ratio (= 2.67 × 108 rad/sT).

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RESULTS AND DISCUSSION Light Scattering Components below 35 °C. Figure 1 shows autocorrelation functions g(2)(t) and relaxation time spectra A(τ) obtained by DLS for a PNIPAM-b-PNVP solution with c = 1.0 × 10−2 g/cm3 quickly heated to 25−35 °C. Here, the abscissa is the delay time t and the relaxation time τ multiplied by (kBT/6πηS)k2, and the scattering angle was fixed to 90°. There are three components with different relaxation times in the relaxation spectra. The fast, middle, and slow

1

1 + 3 ⟨S2⟩z , i k 2 Kc = + 2A′2, i c R θ ,i wM i w, i

(2)

where kBT is the Boltzmann constant multiplied by the absolute temperature and ηS is the solvent viscosity coefficient. Each test solution was prepared by mixing a PNIPAM-b-PNVP sample and pure water at room temperature, and optically cleaned by filtration through a 0.2-μm pore-size membrane filter at room temperature. The light scattering cell containing each test solution such prepared was immersed into the xylene bath of the light scattering instrument set at a given temperature. Scattering intensities of all the solutions examined (except for the solution shown in Figure 9) became constant within 30 min after the cell was transferred to the xylene bath from the room temperature. To examine the heating rate dependence of the aggregation, a few copolymer solutions were placed in an air-bath also set at a given temperature. The rate of the temperature raise of the test solution was 0.3 °C/min, which was much slower than that of the above conventional heating method (ca. 10 °C/min). Small-Angle X-ray Scattering. Small angle X-ray scattering (SAXS) measurements were conducted on aqueous solutions of a PNIPAM-b-PNVP sample with x = 0.31, using the BL-10C beamline in KEK-PF (Ibaraki, Japan). The wavelength, the camera length, and the accumulation time were chosen to be 0.15 nm, 1000−2000 mm, and 300 s, respectively. A test solution at room temperature was rapidly heated to the desired temperature by setting the capillary containing the test solution in the heating block of the sample holder. The intensity of the scattered X-ray was measured using an imaging plate detector, which reached asymptotic values within 30 min. Pulsed Field Gradient NMR (PFG-NMR). NMR measurements were performed for a D2O solution of the PNIPAM-b-PNVP sample with x = 0.31 at concentration c = 0.01 g/cm3, on a VARIANNMR600 spectrometer operating at a proton NMR frequency of 600 MHz using a pulsed field gradient coil. Test solutions in precision coaxial tube inserts of 2 mm o.d. were set in the spectrometer, and the DBPPSTE (dosy bipolar pulsed pair stimulated echo) sequence was applied,26 where the width of the pulsed gradient δ and the gradient recover time τ were chosen to be 2 and 0.5 ms, respectively. The diffusion time Δ was chosen to be 50 ms, and 25 echoes were acquired at different values of the gradient amplitude, g, up to 64 G/cm. The variation of integrated peak intensity I(g) with g is determined by the self-diffusion coefficient Ds of the component, and can be written in the form

Table 1. Molecular Characteristics of PNPIAM-b-PNVP Samples Used xa

kBT k 2 6πηS Γi

(1) 227

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Figure 1. Intensity autocorrelation functions g(2)(t) and relaxation time spectra A(τ) for an aqueous solution (c = 0.01 g/cm3) of a PNIPAM-bPNVP sample with x = 0.31 at temperatures from 25 to 35 °C, obtained by DLS at θ = 90°.

relaxation components have (kBT/6πηS)k2τ of 1, 10, and 100 nm orders, respectively, and the temperature dependence of the relaxation strength for each component are shown in Figure 2. At 25 and 32.5 °C, the fast and middle components are the major one, respectively, and above 34 °C, only the slow component is detected by DLS.

Combining DLS and SLS data, we estimated the fast and middle components of Kc/Rθ at temperatures from 25 to 32.5 °C as well as the slow component at 34 and 35 °C, as functions of c (Experimental Section). Figure 3 shows the concentration dependences of Kc/R0,i (Kc/Rθ,i extrapolated to θ = 0) for the three components in the form of the Berry plot. By extrapolating to the zero c, we have determined wiMw,i for 228

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fast component are almost independent of the temperature, but wmiddleMw,middle and RH,middle for the middle component are slightly increasing and decreasing functions of the temperature. The large values of RH,middle indicate that the middle component is aggregates of the PNIPAM-b-PNVP copolymer. Similar aggregates were reported to exist in aqueous solutions of a thermosensitive poly(vinylether) even a temperature much lower than the LCST.27 If the middle component is a random aggregate of the linear copolymer chain, RH,middle should be smaller than RH of the linear copolymer with the same molar mass (the monomer molar masses of NIPAM and NVP are almost equal). Kubota et al.28 reported the following power-law relation between RH and Mw for PNIPAM homopolymer in water at 20 °C:

Figure 2. Temperature dependences of relaxation strengths of the fast, middle, and slow components for an aqueous solution (c = 0.01 g/ cm3) of a PNIPAM-b-PNVP sample with x = 0.31 at temperatures from 25 to 35 °C.

RH/nm = 0.0160 × M w 0.54

(4)

Neglecting the conformational difference between the PNIPAM homopolymer and PNIAPM-b-PNVP copolymer below the LCST, we roughly estimated the lower limit of Mw of the copolymer aggregate to be 5 × 106 g/mol. Combining this result with wmiddleMw,middle shown in Figure 4a, the upper limit of wmiddle is estimated to be 1% at 25 °C and 4% at 32.5 °C. Since RH of the random aggregate should be smaller than that of the linear chain with the same Mw,29 the actual wmiddle is expected to be considerably smaller than the upper limits. Although the aggregation of the copolymer chain below the LCST is an interesting matter, we do not argue this problem here because the component of such a tiny amount may not be important to most solution properties. The small values of wmiddle mean that the results of wfastMw,fast shown in Figure 4a can be equated to Mw,fast in a good approximation. The relation between this approximate Mw,fast and RH,fast is in a good agreement with eq 4 presented by Kubota et al. for the PNIPAM homopolymer in 20 °C water. Thus, the fast component is assigned to the single PNIPAM-bPNVP chain taking a random coiled conformation. When wfast ≫ wmiddle and RH,fast ≪ RH,middle, the slope of the plot in Figure 3 for the fast component provides the second virial coefficient A2,fast in the aggregate-free solution in a good approximation.25 The estimated A2,fast was 6.8 × 10−4 cm3g−2 mol irrespective of the temperature, indicating that water at 32.5 °C is still a good solvent for PNIPAM-b-PNVP. This corresponds to the temperature insensitiveness of RH,fast up to 32.5 °C. In Figure 4, wslowMw,slow values for the slow component are much larger than those of the fast and middle components, and

Figure 3. Concentration dependences of the fast, middle, and slow components of (Kc/R0)1/2 for aqueous solutions of a PNIPAM-bPNVP sample with x = 0.31 at temperatures from 25 to 35 °C. For the slow component, (Kc/R0,slow)1/2 is multiplied by ten in the plot.

each component i (cf. eq 1). Similarly, the fast, middle, and slow components of the first cumulant Γ estimated from A(τ) data (Experimental Section) were extrapolated to c = 0 to obtain the hydrodynamic radius RH,i of each component i. The results of wiMw,i and RH,i of the three components are plotted against the temperature in Figure 4 by triangles, unfilled circles, and filled circles, respectively. Both wfastMw,fast and RH,fast for the

Figure 4. Temperature dependences of wiMw,i and RH,i for the fast, middle, and slow components of a PNIPAM-b-PNVP sample with x = 0.31 in water. 229

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Figure 5. (a) Temperature dependence of wslowMw,slow and (b) the z-average radius of gyration ⟨S2⟩z,slow1/2 plotted against wslowMw,slow for PNIPAM-bPNVP samples in water at 40−60 °C (c = 10−4 g/cm3). Solid curves in panel b, theoretical values for the bilayer vesicle with a = 1.4 nm (x = 0.67), 0.3 nm (x = 0.32), 0.15 nm (x = 0.28), 0.09 nm (x = 0.21), and 0.032 nm (x = 0.14), along with κ = 10−3 nm5/3 mol/g, mi/m = 0.3, and cc = 0.8 g/ cm3.

RH,slow values take more than 100 nm above 34 °C, indicating that the slow component is a large assembly of the copolymer chains. In what follows, the morphology of the copolymer assembly for the slow component is investigated by SLS and SAXS in more detail. Components Existing in the Copolymer Solution above 35 °C. Since the scattering intensity was very strong above 35 °C, we decreased the copolymer concentration to c = 10−4 g/cm3 to measure SLS. Figure 5a shows the temperature dependence of wslowMw,slow for the slow component in aqueous solutions of five PNIPAM-b-PNVP samples upon quickly heating from the room temperature. With increasing temperature, wslowMw,slow first decreases and then stays constant at higher temperatures for all samples. The radius of gyration ⟨S2⟩z,slow for the slow component is plotted against wslowMw,slow for the PNIPAM-b-PNVP samples at 40−60 °C in Figure 5b. When compared at same wslowMw,slow, ⟨S2⟩z,slow1/2 is smaller for higher x, because the PNIPAM block takes a more compact conformation than the PNVP block in the associate. Most of data points for samples of x = 0.67, 0.32, and 0.28 seem to follow lines with the slope of ca. 0.5. Since relaxation time spectra A(τ) were essentially unimodal above 35 °C, we assumed that wslow = 1. Then, the square root Mw,slow dependence of ⟨S2⟩z,slow1/2 in Figure 5b implies that the slow component may be a bilayer vesicle. Solid curves in the figure indicate theoretical results of the vesicle model for all the samples, calculated by equations given in the Supporting Information.30 To obtain good fittings, we have chosen the thickness a of the hydrophobic shell (cf. Figure S2 in Supporting Information) = 1.4 nm (x = 0.67), 0.3 nm (x = 0.32), 0.15 nm (x = 0.28), 0.09 nm (x = 0.21), and 0.032 nm (x = 0.14). Although the fittings seem to be fairly good, the values of a chosen are too small. To investigate the origin of the unphysical fitting, we have made SAXS measurements on aqueous PNIPAM-b-PNVP solutions upon heating. Figure 6 shows SAXS profiles for aqueous solutions of the PNIPAM-b-PNVP sample (x = 0.31) with the copolymer concentration 0.01 g/cm3 at 25 °C and with the concentration 0.002 g/cm3 at 40 and 60 °C, along with SLS profiles for the same solutions (k < 0.03 nm−1), where k is the magnitude of the scattering vector. Values of Rθ/Kc for the SAXS profile was adjusted to smoothly connect the SLS data. As already mentioned, the SLS profile is strongly dependent on the temperature, but the SAXS profile in the high k region is insensitive to the temperature. Although the scattering profiles

Figure 6. SAXS and SLS profiles for aqueous solutions of the PNIPAM-b-PNVP sample (x = 0.31) with the copolymer concentration 0.01 g/cm3 at 25 °C and with the concentration 0.002 g/cm3 at 40 and 60 °C. The value of Rθ/Kc at 40 and 60 °C are multiplied by 100.5 and 10, respectively for the clarity of view.

of 40 and 60 °C are shifted vertically in the figure for the viewing clarity, if they are not shifted, the SAXS profiles of the three temperatures are almost overlap each other at k > 0.4 nm−1. This strongly indicates that the aqueous copolymer solution contains a component with a comparable size to the fast component at 25 °C other than the large slow component, although A(τ) obtained by DLS (Figure 1, parts g and h) did not indicate the existence of such a small component above 34 °C, due to very strong light scattering power of the slow component. At high k measurable by SAXS, P(k) of the slow component diminishes so much that the scattering from the small component becomes detectable. Since Rθ/Kc in the SLS k region is affected by the intermolecular interference effect, we use the following scattering function including the effect for a solution containing two scattering components 1 (the small component) and 2 (the large component)31 230

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Rθ = {w1M w,1P1(k) + w2M w,2P2(k) + 2cw1w2M w,1 Kc

solution of another thermosensitive block copolymer consisting of a poly(2-isopropyl-2-oxazoline) (PIPOZ) block and a poly(2-ethyl-2-oxazoline) (PEOZ) block (PIPOZ-b-PEOZ) at a temperature between the LCSTs of PIPOZ and PEOZ. They fitted SAXS profiles for PIPOZ-b-PEOZ by assuming that the solution contains the star micelle (i = 1) and polydisperse spherical particles of uniform density (i = 2). Here, we fit the SAXS profiles for PNIPAM-b-PNVP at 40 and 60 °C using the same model. The star micelle and polydisperse spherical particles of uniform density determine the shape of the scattering function in low k and high k regions, respectively. The particle scattering functions of the star micelle P1(k) and of the polydisperse spheres of uniform density P2(k) are also given in the Supporting Information. Both P1(k) and P2(k) contain several adjustable parameters. By choosing the parameter values listed in Table 2, we can the data at 40 and 60 °C in Figure 6, as shown by solid curves. The product w1Mw,1 mainly determines the height of the second shoulder at the higher k, and the radius of gyration of the coronal chain ⟨S2⟩corona1/2 and the radius of the hydrophobic core Rcore are main factors determining the decay of the second shoulder. We have examined many parameter sets for the fitting, but did not find any good fit by other sets of the parameters. At 40 °C, the aggregation number of the component 1 (Mw,1 divided by the molecular weight of the copolymer chain) is ca. unity, which means that this component is a tadpole with a PNIPAM hydrophobic head and a PNVP tail chain. The hydrophobic head is assumed to be spherical, and the PNIPAM mass concentration inside the spherical head is calculated to be 0.30 g/cm3 from Mw,1 and Rcore listed in Table 2. On the other hand, the aggregation number of the component 1 at 60 °C is ca. 3, and the PNIPAM mass concentration inside the spherical hydrophobic core of the star micelle is calculated to be also 0.30 g/cm3 from Mw,1 and Rcore. The height of the first shoulder at the lower k is mostly determined by the product w2Mw,2, and the decay of the first shoulder is sensitive to radii of the spherical particles of uniform inside the density or the copolymer mass concentration csphere in spheres if Mw,2 is fixed (see eq S15 in the Supporting Information). We had to choose a large number of w2Mw,2, and almost uniquely for a given Mw,2. The was able to select csphere in listed in Table 2 indicate that the spheres fitting results of csphere in contain a lot of water. Yusa et al.23 did not observe the NMR peak broadening of the methylene protons of the PNVP pyrrolidone ring in water at 60 °C, which may be due to that is not high enough to slow down the rotational motion of csphere in the pyrrolidone ring. The value of ⟨S2⟩z,sphere1/2 (= 160 nm) is consistent with a cryogenic transmittance electron microscopic image shown in the Supporting Information (cf. Figure S3). We also tried to fit the data assuming a disk or bilayer vesicle as the component 2, but did not obtain a good fit, because P2(k) of the disk or bilayer vesicle decays more slowly in a high k region than that of the sphere of uniform density. The apparently successful fitting of the ⟨S2⟩z,slow data shown in Figure 5b may arise from the wrong assumption that wslow = 1. Although not determined for the solutions in Figure 5b, we can expect that wslow decreases with decreasing temperature, or decreasing the hydrophobicity of the PNIPAM block, as demonstrated in Table 2. Therefore, the double logarithmic plot of ⟨S2⟩z,slow1/2 vs Mw,slow should give a slope smaller than 0.5 expected for the vesicle. The slope for the sphere of uniform density is 1/3.

M w,2P1(k)P2(k)(A11 + A 22 − 2A12 )} /{[1 + 2w1cM w,1P1(k)A11][1 + 2w2cM w,2P2(k)A 22 ] − 4w1w2c 2M w,1M w,2P1(k)P2(k)A12 2 }

(5)

where wi, Mw,i, and Pi(k) have the same meaning as in eq 1 for the component i = 1 and 2, and Aij is the second virial coefficient between i and j (=1 and 2). First, we fit Rθ/Kc data at 25 °C, where the solution contains the single copolymer chain (i = 1) and a random aggregate component32 (i = 2). Equations for P1(k) and P2(k) are given in the Supporting Information (eqs S1−S3). The calculation of Rθ/Kc needs parameters, w2 (=1 − w1), Mw,1, Mw,2, ⟨S2⟩1 (the square radius of gyration of the single copolymer chain), M0 (the unimer molar mass) and b (the unimer bond length), as well as the three second virial coefficients. We have selected the single copolymer chain as the unimer in the component 2, and P2(k) is insensitive to the choice of f (the functionality). When values listed in Table 2 are chosen as those parameters, the Table 2. Parameters Chosen for the Fitting of SAXS Profiles temperature, °C 25

40

i=1 random coil chain w1 = 1 Mw,1 = 4.15 × 104 b ⟨S2⟩11/2 = 6.5 nm tadpole w1 = 0.77 Mw,1 = 4.2 × 104 b

60

⟨S2⟩corona1/2 = 4.0 nm Rcore = 2.6 nm star micelle w1 = 0.6 Mw,1 = 1.3 × 105 b ⟨S2⟩corona1/2 = 3.7 nm Rcore = 3.7 nm

a

i=2

Aija

randomly branching chain

A11 = 6.8a

w2 = 6 × 10−5, Mw,2 = 3.2 × 108 b f = 3, M0 = 4.15 × 104 b

A22 = 1.0a A12 = 1.0a

b = 1.5 nm polydisperse sphere of A11 = 0 uniform density 9b w2 = 0.23, Mw,2 = 4.2 × 10 A22 = 0.036a A12 = 0 Mw,2/Mn,2 = 2.5, c insphere = 0.35c ⟨S2⟩z,sphere1/2 = 160 nm

polydisperse sphere of uniform density w2 = 0.4, Mw,2 = 8.7 × 107 b Mw,2/Mn,2 = 2.5, c insphere = 0.40c ⟨S2⟩z,sphere1/2 = 42 nm

A11 = 0 A22 = −0.02a A12 = 0

In units of 10−4 cm3g−2 mol. bIn units of g/mol. cIn units of g/cm3.

calculated values indicated by the solid curve nicely fit the experimental data points for 25 °C in Figure 6. The value of ⟨S2⟩11/2 is consistent with RH for the fast component shown in Figure 4b, and A11 agrees with A2,fast determined by light scattering (Figure 3). The very small number of w2 is also consistent with the estimate of the upper limit of wmiddle from wmiddleMw,middle and RH,middle. (As mentioned in the Supporting Information, the value of b is affected by the excluded volume effect.) Recently, SAXS profiles similar to those of 40 and 60 °C in Figure 6 were reported by Takahashi et al.13 for an aqueous 231

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power, but if the condition w1Ds,1 ≫ w2Ds,2 and w1M1Ds,1 ≪ w2M2Ds,2 is fulfilled, the decay of I(g) is not affected by this slow component 2. This remarkable difference in the decay rate between g(1)(t) and I(g) clearly demonstrates the existence of the smaller component 1 other than the larger slow component 2 in the copolymer solution at 35 °C. Takahashi et al.13 reported that spherical particles of uniform density in aqueous PIPOZ-b-PEOZ at a temperature between the LCSTs of the two blocks further coalesce into macroscopic concentrated phase, which was observed by eye or a microscope. On the other hand, spherical particles of uniform density in aqueous PNIPAM-b-PNVP (c = 0.055 − 0.53 g/cm3) did not coalesce into droplets observable by an optical microscope. The PNVP block, which may cover the surface of the spherical particles, may be more hydrophilic than the PEOZ block to provide colloidal stability. Temperature Dependence of the Size of the Slow Component above 40 °C. When a solution containing the smaller component 1 and the larger component 2 is dilute enough, the intercept and initial slope of the Guinier plot give us the weight-average molar mass Mw and z-average square radius of gyration ⟨S2 ⟩ z (averaged over all scattering components), respectively, and they are written as

If the component 2 is a multilayered vesicle, P2(k) should resemble that of the sphere of uniform density with the same radius, but should have a peak at k fulfilling the Bragg condition, k = 2π/d, where d is the bilayer thickness. Since the thickness d may be nearly equal to 4(Rcore + ⟨S2⟩corona1/2), the peak should appear around k ∼ 0.2 nm−1. Actually, no peak is observed in the SAXS profiles in such a k region at 40 and 60 °C (see Figure 6). This implies that there is no definite bilayer structure inside the sphere of the component 2. Although DLS failed to detect the component 1, w1 determined from SAXS is considerably large. The existence of the smaller component 1 can be also checked by PFG-NMR. When the solution contains the smaller component 1 and larger component 2, the cumulant expansions of I(g) obtained by PFG-NMR and g(1)(t) ≡ [g(2)(t) − 1]1/2 obtained by DLS give the following equations.25 ln I(g ) ≈ −(w1Ds,1 − w2Ds,2)ζ

ln g(1)(t ) ≈ −

w1M1Ds,1(1 + 2A11M1c) + w2M 2Ds,2

× k 2t

w1M1 + w2M 2

(6a)

(1 − υ ̅ c) (6b)

where Ds,i is the self-diffusion coefficient of the component i and υ̅ is the polymer partial specific volume, and we assume that M1 ≪ M2 and Ds,1 ≫ Ds,2. If w1M1 ≫ w2M2, the plots of ln 2 I(g) vs ζ and of ln g(1)(t) vs (1 + 2A11M1c)(1 − υc)k ̅ t should provide the same slope −Ds,1. On the other hand, if w2 is comparable to w1 and w1M1Ds,1 ≪ w2M2Ds,2, the former plot decays with the rate w1Ds,1,but the latter with the rate Ds,2/(1 + 2A11M1c). Figure 7 compares PFG-NMR and DLS results for aqueous solutions (c = 0.01 g/cm3) of PNIPAM-b-PNVP sample (x =

M w = w1M1 + w2M 2 ,

⟨S2⟩z =

w1M1⟨S2⟩1 + w2M 2⟨S2⟩2 w1M1 + w2M 2 (7)

where wi, Mi, and ⟨S ⟩i (i = 1 and 2) are the weight fraction, molar mass, and square radius of gyration of component i, respectively. If w1M1 ≪ w2M2 and also w1M1⟨S2⟩1 ≪ w2M2⟨S2⟩2, the above equations can be approximately written in the form 2

M w ≈ w2M 2 ,

⟨S2⟩z ≈ ⟨S2⟩2

(7′)

Therefore, ⟨S ⟩z for such a solution can be approximated to that of the larger component 2, but Mw cannot be approximated to that of the component unless w2 ∼ 1. In what follows, using this approximation, we discuss the temperature dependence of ⟨S2⟩21/2 for the larger component (i.e., the slow component in Figure 1) above 40 °C, but not of M2. We made SLS measurements on dilute aqueous solutions of PNIPAM-b-PNVP samples with different x upon quickly heating above 40 °C from the room temperature. The copolymer concentration was as low as 10−4 g/cm3, which is much lower than that at the above- mentioned SAXS and PFGNMR measurements. Therefore, the virial term in the scattering intensity may be negligible to obtain ⟨S2⟩21/2 for the slow component, corresponding to polydisperse spheres of uniform density, from SLS. Figure 8 shows the temperature dependence of ⟨S2⟩21/2 for polydisperse spheres of uniform density in aqueous solutions (c = 1 × 10−4 g/cm3) of PNIPAM-b-PNVP samples with different x upon quickly heating above 40 °C from the room temperature (unfilled symbols). For the samples of x = 0.21, 0.28, and 0.32, ⟨S2⟩21/2 definitely decrease with increasing temperature, while the temperature dependence of ⟨S2⟩21/2 is much weaker for the samples of x = 0.14 and 0.67. As already indicated in Table 2, ⟨S2⟩z,sphere1/2 for the aqueous solution (c = 2 × 10−3 g/cm3) of the sample with x = 0.31 also decreases with increasing temperature from 40 to 60 °C. When the copolymer solutions with x = 0.32 and 0.67 are slowly heated to 60 °C (see Experimental Section), ⟨S2⟩21/2 2

Figure 7. Comparison between PFG-NMR and DLS results for aqueous solutions (c = 1 × 10−2 g/cm3) of PNIPAM-b-PNVP sample with x = 0.31 at 25 °C and upon quickly heating to 35 °C.

0.31) at 25 °C and upon quickly heating to 35 °C. The function I(g) decays almost the same rate as g(1)(t) at 25 °C. It can be seen from Table 2 that w1M1 is considerably larger than w2M2 at 25 °C, so that we can expect that both I(g) and g(1)(t) provide approximately the same decay rate Ds,1. On the other hand, the decay of g(1)(t) is much slower than that of I(g) at 35 °C. As already mentioned, the slow decay rate of g(1)(t) corresponds to the slow component with very strong scattering 232

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Israelachvili et al. characterized the shape of the amphiphilic molecule in terms of the packing parameter λ to discuss various morphologies of micelles formed by the amphiphile. Viewing the hydrophobic part of the amphiphile as a circular cone or frustum of circular cone with the volume υ, the area a, and the length l (cf. Figure 10a), λ is defined by

λ ≡ υ/al

(8)

According to Israelachvili et al., the amphiphile with λ ∼ 1/3 and 1/2 forms spherical and cylindrical micelles, respectively, and that with 1/2 < λ < 1 tends to form a vesicle. From a geometrical consideration on the outer monolayer of the vesicle at 1/2 < λ < 1, the outer radius R of the hydrophobic shell is related to λ by33,34

Figure 8. Temperature dependence of the radius of gyration ⟨S2⟩21/2 for polydisperse spheres in aqueous solutions (c = 1 × 10−4 g/cm3) of PNIPAM-b-PNVP samples with different x above 40 °C.

R=

reach to the values indicated by filled square and filled circle in Figure 8, respectively. While ⟨S2⟩21/2 for the sample of x = 0.67 is essentially independent of the heating rate, ⟨S2⟩21/2 for the sample of x = 0.32 heated slowly is much larger than that at quickly heating (the unfilled square) at 60 °C, and almost equal to ⟨S2⟩21/2 for the solution quickly heated to 48 °C. This indicates that, during the slowly heating process, the polydisperse spheres of uniform density grow in the solution up to around 48 °C but it is frozen to prevent from reaching to the thermodynamically more stable smaller spheres above that temperature. Since ⟨S2⟩21/2 for the sample of x = 0.67 is essentially independent of the temperature above ca. 45 °C, the heating rate dependence of ⟨S2⟩21/2 was not observed. After an aqueous copolymer solution (x = 0.28, c = 2 × 10−4 g/cm3) with ⟨S2⟩21/2 = 28 nm at 60 °C is quenched to 46 °C, ⟨S2⟩21/2 increases with time, as shown in Figure 9. This clearly demonstrates that the thermodynamically stable size of the polydisperse spheres of uniform density is a decreasing function of the temperature.

3+

3(4λ − 1) 6(1 − λ)

l

(9)

As shown in Figure 10b, the radius of the vesicle monolayer is an increasing function of λ according to eq 9. At elevating temperature, the PNIPAM block chain is dehydrated to shrink. If the shrinkage is isotropic, λ may decrease with increasing temperature. Therefore, the increase of temperature shifts the abscissa of the graph in Figure 10b from the right to left side. Since eq 9 was derived from the consideration of the outer monolayer of the vesicle, we can apply the same argument to spherical particles covered by the amphiphilic monolayer. Equation 9 or Figure 10b thus explains the temperature dependence of spherical particle size shown in Figures 8 and 9.

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CONCLUSION We have investigated the thermally induced self-association behavior in water of a thermosensitive block copolymer PNIPAM-b-PNVP with different compositions x (the content of the PNIPAM) from 0.14 to 0.67. By combining SLS and DLS with SAXS and PFG-NMR, we have concluded that above 35 °C, the unimer or a few arms star micelle of the copolymer coexists with large spheres of uniform density. This conclusion might miss to be obtained without using the SAXS and PFGNMR data. We should be careful at analyzing light scattering data for solutions containing components with largely different sizes (or scattering powers). Similar coexistence of smaller star micelle and large spherical particles of uniform density was observed in another thermosensitive block copolymer of poly(2-isopropyl-2- oxazoline) and poly(2-ethyl-2-oxazoline) (PIPOZ-b-PEOZ) in water at a temperature between the LCSTs of the two blocks,13 and it may be regarded as a kind of a liquid−liquid phase separation phenomenon. In fact, a macroscopic phase separation was observed in the aqueous PIPOZ-b-PEOZ. On the other hand, the large spheres of uniform density in aqueous PNIPAM-bPNVP solution did not grow to macroscopic concentrated phase, because the PNVP block chain is more hydrophilic than the PEOZ block chain, and may stabilize the colloidal droplets. Although no direct observations, the large spheres in aqueous PNIPAM-b-PNVP solutions heated must be concentratedphase droplets in the liquid−liquid phase separation, because the SAXS results indicate that the copolymer concentration inside the sphere is uniform and as high as 0.35−0.4 g/cm3. The hydrophilicity of the PNVP block in the copolymer chain may not be strong enough to form micelles, and the copolymer may behave like a homopolymer in a poor solvent.

Figure 9. Time evolution of the radius of gyration ⟨S2⟩21/2 for polydisperse spheres in aqueous solutions (c = 2 × 10−4 g/cm3) of a PNIPAM-b-PNVP sample (x = 0.28) quenched from 60 to 46 °C.

The hydrophilic PNVP block chains may cover the surface of spheres as the monolayer in aqueous PNIPAM-b-PNVP above 40 °C to stabilize the colloidal particles. Therefore, the size of the spherical particles of uniform density may be determined by this monolayer, and we can argue their optimum size using Israelachvili et al.’s packing parameter,33,34 originally proposed to discuss the micellar morphology for low-molar mass amphiphiles. 233

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The size of the concentrated-phase droplets decreases with increasing temperature (except for x = 0.67). This temperature dependence may be explained by preferred curvature of the monolayer at the surface of the droplets by using the packing parameter originally proposed by Israelachvili et al.33,34 Zhou and Chu35,36 have already reported a similar phase separation phenomenon in aqueous solutions of a poly(oxyethylene)-b-poly(oxypropylene)-b-poly(oxyethylene) thermosensitive triblock copolymer. They observed anomalously strong light scattering and large hydrodynamic radius in an intermediate temperature region between the unimer and normal micelle regions, and ascribed the anomaly to a tiny amount of large concentrated-phase droplets, which were formed by a small amount of hydrophobic copolymer components with low LCST in the sample. In our copolymer system, however, the weight fraction of the droplets (the slow component) is as high as 23−40% (see Table 2), so that the droplets are not formed by an impurity or a minor component of the copolymer samples. The composition heterogeneity of the copolymer sample is not necessary for a liquid−liquid phase separation to occur.

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ASSOCIATED CONTENT

S Supporting Information *

Size exclusion chromatograms, calculation of the radius of gyration, equations for the particle scattering functions, and cryogenic transmission electron microscopy. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was partly supported by a Grant-in-Aid for Scientific Research (No. 23350055) from the Japan Society for the Promotion of Science as well as a Grant-in-Aid for Scientific Research on Priority Area “Soft Matter Physics” of the Ministry of Education, Culture, Sports, Science and Technology, Japan. SAXS experiments were performed under the approval of the Photon Factory Program Advisory Committee (Proposal Nos. 2010G080 and 2011G557).

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REFERENCES

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