Coacervate

Article Cite This: Macromolecules XXXX, XXX, XXX−XXX

Scattering Neutrons along the Polyelectrolyte Complex/Coacervate Continuum Hadi M. Fares,† Yara E. Ghoussoub,† Jose D. Delgado,† Jingcheng Fu,† Volker S. Urban,‡ and Joseph B. Schlenoff*,† †

Department of Chemistry and Biochemistry, The Florida State University, Tallahassee, Florida 32306-4390, United States Center for Structural Molecular Biology, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States

‡

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S Supporting Information *

ABSTRACT: The coil size of narrow molecular weight distribution deuterated poly(styrenesulfonate), PSS, within a polyelectrolyte complex doped with KBr was tracked across the continuum from solid to coacervate to solution using small-angle neutron scattering. While PSS alone in solution exhibited the familiar and pronounced “polyelectrolyte effect” of coil shrinkage with increasing [KBr], the radius of gyration Rg of the PSS in the complex remained surprisingly constant up to 1.4 M KBr, which is close to the transition between complex and coacervate behavior. Thereafter, Rg decreased with increasing KBr, remaining slightly larger than Rg for PSS in KBr alone. Upturns in the scattering at low angle, seen for complexes in lower [KBr], are consistent with porosity, observed macroscopically as whitening of the bulk complexa universal property of polyelectrolyte complexes. Reasons for this porosity, imaged by scanning electron microscopy, are discussed. At high q ranges, a correlation peak between deuterated coils of PSS was observed.

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where Pol+Pol− is a pair of oppositely charged polyelectrolyte repeat units and CA is a salt. The conformations of polyelectrolytes in solution and in their complexed form have been explored through a number of scattering techniques. Light scattering provided in-depth information on the shape and interactions of single polyanions in solution. 15 Traditional or modified light scattering techniques were also employed to gain insight into the mechanism of polycation−protein complexation.16,17 Smallangle X-ray scattering (SAXS) was used to study polyelectrolytes in their free18 or bound form.19,20 Though these scattering techniques provide information on single polymer chains in solution or on the internal construction of complexes, they fall short of elucidating the molecular structure of single polymers within their materials. Small-angle neutron scattering (SANS) is a powerful tool that delivers this additional information.21 Taking advantage of selective isotopic labeling, SANS allows the monitoring of the chain in any surrounding.22 It has been employed to observe polyelectrolytes in salt-free solutions,23,24 salt-containing solutions,25 and polyelectrolyte− protein complexes and coacervates.26−29 So far, the use of SANS in the investigation of synthetic polycation−polyanion complexes has been limited to studying the polymers in strongly interacting solid polyelectrolyte complexes30 and in coacervates of weakly interacting polyelectrolytes.31

INTRODUCTION Polyelectrolytes are an intriguing class of macromolecules. Beyond classical rules that dictate the behavior of neutral polymers,1 the presence of charge adds a dimension of complexity to their dynamics and structure.2 Both intra- and interpolymer interactions are controlled by solution ion concentration, type, and valency. The strength of (charged) segment−segment repulsions determines molecular dimensions, while a host of possible attractive interactions between polyelectrolytes and other macromolecules, surfaces, or colloids/nanoparticles leads to interesting materials with various properties and morphologies. Polyelectrolyte complexes (PECs) are produced by mixing oppositely charged polymers in solution3 or by depositing them on surfaces in an alternating fashion.4 Salt plays an integral part in controlling the charge compensation within these products.5 The outcome is a wide range of morphologies from ultrathin films and capsules,6 to gels7 and precipitates8 which, using the proper processing technique, can be molded into virtually any shape.9 Beyond the rather simple idea of mixing positively and negatively charged entities lies a multitude of parameters underpinning this phenomenon.10−12 Salt and water concentration play a central role in the mechanical properties of PECs: salt water plasticizes or dopes PEC (eq 1), transforming the properties of the material from a rubbery solid to a viscous liquid along the so-called polyelectrolyte complex/coacervate “continuum”13,14 Pol+Pol−s + C+aq + A −aq ⇌ Pol+A −s + Pol−C+s © XXXX American Chemical Society

Received: April 2, 2018 Revised: June 5, 2018

(1) A

DOI: 10.1021/acs.macromol.8b00699 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Investigating the PEC structure over the full range of the complex/coacervate continuum provides insight into the response of polymer chains to external variables. At one end, the effect of Pol+Pol− dissociation is observed in solid complexes as they are doped with salt and become progressively softer. At the other end, continued breaking of interactions produces coacervates, and eventually isolated chains.13,32 Since the introduction of the term “coacervate”,33 this phenomenon of “unmixing” in liquids has gained attention as a model for early precellular life.34 More recent studies have implicated coacervates as membraneless cellular organelles,35,36 in the production of bioadhesives,37,38 and it is showing promise in manufacturing new multifunctional materials.39−42 In this study, the dimensions of polyelectrolytes in PECs were investigated, going from solids to coacervates to liquids by changing the concentration of salt. KBr alone allowed transit through the phase diagram of the poly(diallyldimethylammonium) PDADMA/poly(styrenesulfonate) PSS complex, without changing other parameters such as the concentration of polymers or temperature during the measurements.13 The continuum in morphology has traditionally been broken down into a solidlike “complex” phase, PECOX, and a more liquidlike coacervate material, PECOV. The boundary between the two morphologies is somewhat arbitrary but has been taken as the point where the elastic modulus G′ equals the loss modulus, G′′.13 Passage from complex to coacervates is accompanied by strong increases in volume (for a fixed amount of PEC). Two hypotheses were advanced to explain how the polymer chains might behave as the network morphs from solid to liquid (Scheme 1A−C). In the first hypothesis, salt and water uniformly swell the PEC and the polymer chains expand into the new volume created by doping (Scheme 1B). In the second scenario, polymers remain compacted as the extra volume is excluded to a more polymer-poor phase (Scheme 1C). The same low connectivity between polymers is maintained in both cases. The goal of this work was to establish which hypothesis, Scheme 1B or 1C, was most consistent with the observed response of coil dimensions to salt concentration. The outcome will have important implications on understanding the largerscale structure of PECs, since Scheme 1B is a more uniform distribution of polymer whereas Scheme 1C requires a certain amount of microphase separation to accommodate the extra salt and water while maintaining the compactness and some association of polyelectrolytes.

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Scheme 1. (A) Proposed Structures of the Polyelectrolyte Complex Made from the Polycation/Polyanion Pair PDADMA/PSS and of the Same Complex as It Transitions into the Coacervate Phase with the Addition of KBr as Salt; Chains in the Network Are Hypothesized To Either (B) Expand while Remaining Connected at Certain Polymer/ Polymer Points with Salt Either Closely Associated with the Polyelectrolyte or in the Surrounding Solution or (C) Phase Separate into Denser Regions To Maintain Some Polymer Connectivity, while Remaining More Tightly Coiled, with Excess Salt and Water Occupying an Adjacent Microphasea

a

The square on the right shows how the charged repeat units are compensated by small ions or, to a lower extent, the other polyelectrolyte.

in 60 mL of dichloroethane and then precipitated by adding the solution slowly to 200 mL of methanol under vigorous stirring. The reprecipitated PS was collected after decanting the liquid mixture and dried under vacuum for 12 h to yield a fine white powder which was sorted through a 106 μm sonic sifter sieve (Advantech Manufacturing). The reaction was started by mixing 1.0 g of the H- or D-PS powder with 50 mL of H2SO4 or D2SO4, respectively, in a roundbottom flask which was immersed in a silicone oil bath preheated to 90 °C. Because H-PS was reacted in larger quantities (4.2 g), the sulfuric acid volume increased proportionally. The mixture was vigorously stirred for 4 h after which the reaction was stopped by placing the round-bottom flask in an ice bath. The resulting dark brown liquid was then poured over around 500 mL of ice-cold water. This was followed by dialysis of the mixture using SnakeSkin dialysis tubing (3500 MWCO, Thermo Scientific) for around 6 days, with changes of the outer water solution every 6−8 h, until the external pH reached 3−4. The content of the tubes was then neutralized with 0.1 M NaOH (BDH) to obtain sodium poly(styrenesulfonate) (H-PSSNa or DPSSNa). The volume of the solution was reduced using a rotary evaporator at 65 °C, and the resulting mixture was lyophilized to obtain a white solid with the yield of different batches ranging between 90 and 100%. Size Exclusion Chromatography (SEC). SEC columns in series, from Tosoh Bioscience, were used to determine the molecular weight of the H- and D-PSS: two polymethacrylate columns (particle size = 17 μm, length (L) = 30 cm, internal diameter (i.d.) = 7.5 mm, TSKgel G5000PW) and one hydroxylated polymethacrylate column (particle size = 13 μm, L = 30 cm, i.d. = 7.8 mm, TSKgel GMPWxl), protected with a guard column (L = 7.5 cm, i.d. = 7.5 mm, TSKgel PWH Guard column). H-PSSNa and D-PSSNa was prepared at 3.95 and 4.47 mg mL−1, respectively, in 0.3 M NaNO3. 50 μL samples were injected at a flow rate of 0.7 mL min−1 after filtering through a 0.1 μm Supor membrane filter (Pall Corp.). The eluent, also 0.3 M NaNO3, was

EXPERIMENTAL SECTION

Materials. Protiated polystyrene, H-PS (Mw = 53 300 g mol−1, Mn = 51 000 g mol−1, Mw/Mn = 1.05), and deuterated polystyrene, D-PS (Mw = 56 700 g mol−1, Mn = 52 000 g mol−1, Mw/Mn = 1.09) were obtained from Polymer Source Inc. Poly(diallyldimethylammonium) solution (PDADMA, 20 wt % in H2O, Mw = 200 000−350 000 g mol−1) was from Aldrich. This polycation was presumed to have a wide molecular weight distribution of Mw/Mn ∼ 2. Sulfuric acid (H2SO4, 98 wt %) was obtained from Merck, and sulfuric acid-d2 (D2SO4, 98 wt % in D2O, 99.5 atom % D) was from Acros Organics. Sodium chloride (NaCl, Fisher), potassium bromide (KBr, SigmaAldrich), methanol (Sigma-Aldrich), and 1,2-dichloroethane (Fisher Scientific) were all used as received. Deuterium oxide (D2O, 99.9% D), obtained from Cambridge Isotope Laboratories, Inc., and deionized water (Barnstead 18 MΩ E-pure) were used to prepare aqueous solutions. Polystyrene Sulfonation. H-PS and D-PS were sulfonated according to a procedure described previously. 1.0 g PS was dissolved B

DOI: 10.1021/acs.macromol.8b00699 Macromolecules XXXX, XXX, XXX−XXX

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side polished silicon 100 wafers (Okmetic, Inc.). The thin film deposited on the wafer after water evaporation was analyzed with Fourier transform infrared spectroscopy (FTIR, Thermo Nicolet Avatar 360 equipped with a DTGS detector). The decrease of the HPSS band (1130 cm−1) in the sample relative to the background (taken as 100% H-PSS) was measured to be around 20%. An increase in the D-PSS peak (at 1080 cm−1) was also observed (Figure S2B). For scanning electron microscopy (SEM), an FEI Nova nanoSEM 400, with an ETD detector, was used at 15 kV and a working distance of 5 mm. Samples were broken into small pieces, and not cut using a blade, to prevent the “smudging” of the pores’ contours. They were coated with a 4 nm layer of iridium using a Cressington HR 208 sputter coater. To prepare the coacervate samples, 0.5 g of the PEC powder was weighed under Ar and dissolved in 2.5 M KBr prepared in 25:75 by mole D2O:H2O solvent. After full dissolution, which took 24−36 h, specific volumes of 25:75 D2O:H2O were slowly added to the solutions for a total volume of 5 mL (Table 1), and the mixture was

filtered through a 0.2 μm filter with a poly(ether sulfone) membrane (VWR) by vacuum filtration. Two detectors, both from Wyatt Technology, were used: a DAWN-EOS multiangle light scattering (MALS) setup and an Optilab rEX refractometer. The analysis was preceded by a measurement of the refractive index increment (dn/dc) of the polymer solutions in 0.3 M NaNO3 (dn/dc = 0.1749). Light Scattering. Static Light Scattering. The chain conformation and solvent quality were probed in a series of H-PSS solutions at concentrations that ranged between 0.1 and 0.9 mg mL−1 in KBr (0.1, 0.5, 1.0, 2.0, and 2.5 M). After measuring the dn/dc, the optical constant K was calculated: K=

2π 2n0 2

2

( ddnc )

NAλ 4

(2)

where n0 is the solvent refractive index, NA the Avogadro constant, and λ the wavelength of the scattered light (λ = 690 nm). The optical constant was used in the general expression of static light scattering which associates the polymer scattering, represented by the excess Rayleigh ratio ΔRθ that excludes solvent scattering, with its mass average molar mass M̅ w and radius of gyration Rg:

⎡ ⎛ 16π 2n 2 sin 2 ⎡ 1 ⎤⎢ 0 Kc ⎜ =⎢ + 2A 2 c + ...⎥⎢1 + ⎜ ⎜ ΔR θ ⎣ M̅ w ⎦⎢ 3λ 2 ⎝ ⎣

Table 1. Expected and Measured KBr Concentrations in the Prepared Coacervate Samples and Volumes of KBr and Water Added to 0.5 g of Dried PEC To Reach Them

( θ2 ) ⎞⎟R 2⎤⎥ ⎟⎟ ⎠

g

⎥ ⎥⎦

(3)

where A2 is the second virial coefficient, θ the angle of the detector with respect to the nonscattered light, and λ the wavelength of the scattered light (690 nm). Scattering was measured at different angles, and a Zimm plot relating Kc/ΔRθ to sin2(θ/2) (+ k′c, where k′ is a constant included to provide a clear separation of the data points) was constructed for each salt concentration. SEC and static light scattering experiments were performed at 25.0 °C, and the data analysis used ASTRA 6.1.2.84 software (Wyatt Technology). Dynamic Light Scattering. A compact goniometer system (ALV/ CGS-3) equipped with a He−Ne laser (λ = 632.8 nm) with a vertically polarized light was used to compare the hydrodynamic radii (Rh) of Hand D-PSS in KBr at room temperature (23.0 °C). Solutions were passed through a 0.1 μm filter into a clean 10 mm diameter borosilicate glass tube which was placed in the cylindrical reservoir containing toluene (used for refractive index matching). Polymer concentrations were 0.5−0.6 mg mL−1 with the KBr concentration fixed at 0.1 M. Three acquisition periods of 10 s each were averaged to obtain the autocorrelation function which was fit to obtain the Rh values deduced from the diffusion coefficient, D, using the Stokes− Einstein equation Rh =

kBT 6πηD

expected [KBr] (M)a

actual [KBr]b (M)

2.5 M KBr in 25:75 D2O:H2O (mL)

25:75 D2O:H2O (mL)

1.4 1.5 1.6 1.7 1.8 2.0

1.44 1.53 1.61 1.72 1.80 2.00c

3.0 3.2 3.4 3.6 3.8 4.2

2.0 1.8 1.6 1.4 1.2 0.8

a c

From Wang et al.13 bObtained by conductivity measurements. Estimated concentration.

vortexed then annealed in a water bath at 60 °C for 3 h to reach equilibrium. This thermal annealing treatment has been shown to accelerate the approach to equilibrium.13 They were then carefully taken out of the water bath and slowly allowed to return to room temperature, yielding clear phase-separated mixtures. The details of the preparation technique, known as the “backwards method”,13 are shown in Table 1. The KBr concentration in the coacervate was measured by diluting 50 μL of the dilute phase (upper phase) in 15 mL of water. After 24 h, following the complete release of salt, conductivity measurements were performed in a thermostated cell equipped with a stir bar at 25 °C. A four-probe conductivity electrode (Orion 3 Star, Thermo Scientific) was used for this purpose. A calibration curve was established to convert μS cm−1 values to KBr concentrations. The 2 M sample, falling on one extreme of the complex continuum spectrum where no phase separation is observed (fully dissolved PEC), was estimated from the KBr concentration added. To prepare the solid samples, the remaining PEC powder was fused together by doping in 2 M NaCl and annealing at 70 °C for 2 h resulting in a transparent PEC globule. The latter was further compacted by centrifugation in a Beckman Optima XL-100 K ultracentrifuge at 155000g for 4 h at 20 °C in polycarbonate thick wall tubes. Removal of the salt was done by washing the PEC in water. They were then squeezed to remove excess water, air-dried overnight, cut into ca. 1 cm pieces, and pressed at 50 °C in a melt-press (Carver) to a thickness of 0.8 mm. They were finally cut into 1.5 × 1.0 cm2 slabs which were immersed in different KBr solutions prepared in 25:75 D2O:H2O ranging from 0.1 to 1.0 M. The surrounding solution was replaced with a fresh one after 24 h. The measurements were taken in the same solutions. The above treatment, for PEC spanning solids to liquids, was also performed on samples used for background measurements, in an identical manner. Neutron Scattering. All samples were analyzed in quartz cuvettes (1 mm path length, VWR). Neutron scattering was performed on

(4)

with kB the Boltzmann constant, T the temperature, and η the solvent viscosity (0.997 cP).43 Sample Preparation for Neutron Analysis. PDADMA/PSS polyelectrolyte complexes were prepared by mixing ca. 200 mL solutions of 0.125 M polymer (with respect to their monomer units) in 0.25 M NaCl added simultaneously into a beaker while stirring.9 To ensure the polyanion was completely dry before solution preparation, lyophilized PSS powder and NaCl were weighed in a glovebox under argon. Two batches were prepared: complexes used for background measurements contained 100% H-PSS while those used for samples contained 20:80 D:H-PSS by mole. The mixtures were stirred for around 30 min on a hot plate set to around 50 °C in order to produce the maximum yield of the complex. The PEC precipitates were then decanted and washed repeatedly with DI water to remove the salt. When the conductivity of the water decreased below 8 μS, the PECs were taken out of the beaker, squeezed to remove excess water, and dried in an oven at 120 °C for around 24 h. The dry complex was then ground into powder and stored under Ar. To test the composition of the complex, a small amount of powder was fully dissolved in 2.5 M KBr.13 Around 0.5 mL of the resulting solution was spread on doubleC

DOI: 10.1021/acs.macromol.8b00699 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules beamline CG-3 (Bio-SANS), part of the Center for Structural Molecular Biology at the High Flux Isotope Reactor − Oak Ridge National Laboratory. The wavelength of the incident beam of neutrons was 6 Å. The instrument is equipped with two 2-D 3He positionsensitive detectors: a 1 × 1 m2 main detector and an additional 1 × 0.8 m2 wide-angle detector allowing measurements to be performed on a wide Q-range (0.003 < Q < 0.8 Å−1), with Q = (4π/λ)(sin(θ/2), without changing the position of the main detector (15 m). The sample holder temperature was set at 23 °C for all measurements. Scattering profiles were azimuthally averaged to yield intensity plots (I(Q) vs Q). Reduction of data included subtracting scattering from the empty cuvette, scaling the intensity to that of a porous silica standard (Porasil B) to obtain absolute intensity, and correcting for the detector dark current and sample thickness. Backgrounds data, combined from at least two runs and a total of 3 h measurements, were subtracted from sample data, measured for the same duration. Data processing, correction, and background scaling and subtraction were performed on MantidPlot (Ver. 3.11). Fitting of the obtained data was performed using the KCL package (K.C. Littrell, Oak Ridge National Laboratory) for Igor Pro (Ver. 7).

the effect of the surrounding environment on the chain conformation is obtained. The concentration of deuterated PSS within PECs was maintained below the overlap concentration (C*) to minimize the effects of interchain correlations.30,47 C* was calculated to be 0.83 M from an Rg of about 100 Å30 using C* =

Rg3

(6)

with N = 500 and NA Avogadro’s number. A mole ratio of 20:80 D:H-PSS was used, similar to a previous study using the same system (polymer structures are shown in Scheme 1 and Figure S2A).30 Using [D-PSS] = 0.2 × [total PSS], the highest [D-PSS] employed here was about 0.5 M. FTIR analysis verified the compositions of the complex used for sample and background measurements (Figure S2B). To ensure that the coherent scattering resulted exclusively from deuterated polymer, other components in the environment had to scatter uniformly. To achieve this contrast matching, the exact atomic composition was required, which relied on the phase (composition) diagram established for PDADMA/PSS in KBr. 13 Different compositions were extracted from the phase diagram13 to calculate the molar ratios of PEC:water:KBr. The resulting formula of the nondeuterated PEC in D2O:H2O C16H23O3NS(KBr)x(H2O)y(D2O)z was plugged into the NIST online calculator48 (using density = 1.1 g cm−3, sample thickness = 0.1 cm, and neutron wavelength = 6 Å) to predict the expected scattering length densities (SLDs) for each composition (shown in Figure S3). Though the intersections of SLDs the fraction of D2O:H2O at which the compositions scattering is similarranged between 22:78 and 28:72 D2O:H2O, the ratio of 25:75 D2O:H2O was used as the average mixture ratio to prepare all the samples. Figure 1 shows the relationship between the PEC water content and the salt concentration, adapted from Wang et al.,13 over the [KBr] employed in the neutron scattering experiment. The transition from complex (low [KBr]) to coacervate (high [KBr]) is somewhat arbitrarily defined as the point where the loss modulus starts to exceed the storage modulus, which

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RESULTS AND DISCUSSION PDADMA/PSS Complex/Coacervate. The PDADMA/ PSS pair is an ideal system to study the full range of morphologies spanning complexes to coacervates. With essentially pH-independent charges, the PEC composition (water, salt, and polymers weight ratios) can be manipulated with salt concentration alone.13 Potassium bromide, falling in the middle of the Hofmeister series,44 is a convenient salt for this purpose: with its capacity to moderate the “medium strength” interactions between PDADMA and PSS, small increments in [KBr] provide substantial changes in PEC morphology and properties.13 The path to different morphologies along the continuum relies on dissociating the Pol+Pol− polymer pair by doping with salt (eq 1). Preparing equilibrated samples (associating the polyelectrolyte complex) follows the opposite of eq 1: Pol+A −aq + Pol−C+aq ⇌ Pol+Pol−s + A −aq + C+aq

N /NA

(5)

This complexation generally has a strong entropic component, driven by the release of salt and water.45 For the specific system employed here complexation is almost athermal.45 Under the preparation conditions described, a 1:1 PEC is produced, with the stoichiometry validated by NMR and radiolabeling.9,46 Neutron scattering takes advantage of the markedly different interactions of the incoming beam with hydrogen and deuterium within the sample. To that end, protiated and deuterated narrow molecular weight polystyrene polymers (HPS and D-PS), having similar degrees of polymerization, were sulfonated to obtain their charged counterparts, H-PSS and DPSS. The resulting polyelectrolytes had similar molecular weights with H-PSS Mw = 104 200 g mol−1 (Mw/Mn = 1.01; degree of polymerization, N = 501) and D-PSS Mw = 111 300 g mol−1 (Mw/Mn = 1.02; N = 515) determined by gel permeation chromatography with light scattering detection (Figure S1, Supporting Information). Their similar hydrodynamic radii in 0.1 M KBr measured by dynamic light scattering provided additional validation that their mass and behavior in solution were almost identical (Figure S1). By probing the polyanion with neutron scattering, the chain conformation in the associated state and other complex morphology details can be deduced.30 With information about the complex structure and composition, a picture of

Figure 1. Water per PDADMA/PSS pair (mole ratio) in the series of PEC solids, coacervates, and liquid. The inset shows the amount of water at low salt concentrations, in the solids. The data are adapted from ref 13. D

DOI: 10.1021/acs.macromol.8b00699 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules occurs at about 1.3 M KBr.13 Complete dissolution, where almost all PDADMA/PSS ion pairs are broken, occurs more precisely at [KBr] = 1.80 M. Images of solid complex and coacervate samples, equilibrated in KBr and the mixture of D2O:H2O, are shown in Figure 2.

was previously seen to persist,30 resulting in light scattering which gives rise to the white appearance. By contrast, the coacervate samples (Figure 2 and Figure S4) were clear, which implies a more homogeneous structure. To obtain this morphology, the samples were equilibrated at 60 °C for 3 h and then left to cool slowly in the beaker containing the bath water. The slow cooling step was critical in producing the clear coacervate, resulting from a slow return to its room temperature (rt) equilibrium. In fact, with more concentrated polymer samples (less concentrated KBr), this cooling process could be clearly seen, as the translucent material slowly turned transparentusually from the edges, which implies a temperature gradient. While the movement of polymer chains is very slow, the annealing conditions used here should provide the equilibrium conformation of chains. For example, at 1.4 M NaCl, with Dpolymer = 2 × 1015 cm2 s−1 at rt,10 the diffusion length is around 190 nm during the room temperature annealing period of around 24 h. An additional diffusion length of about 200 nm is estimated during the 3 h 60 °C annealing phase. These distances, calculated for materials in the more rigid complex (as opposed to coacervate) phase, are far greater than the chain Rg, implying the coils should be relaxed following the equilibration treatment. Scattering Results. Figure 3 shows the neutron scattering profiles for PEC samples ranging from solid at 0.1 M KBr to liquid at 2.0 M KBr, passing through a series of increasingly fluid coacervate samples from 1.4 to 1.8 M KBr. The jump

Figure 2. Scheme showing D-PSS chains included in the PDADMA/ H-PSS complex with example photographs comparing the white, opaque solid PEC sample and a phase-separated clear coacervate sample (at 1.7 M KBr).

The solid complexes were white and opaque in [KBr] < 1 M, a feature attributed to the presence of pores. Even after compacting with ultracentrifugation, the porosity of the sample

Figure 3. Small-angle neutron scattering profiles in polyelectrolyte complex samples including 0.1−1.0 M KBr-doped solids (KBr concentration shown on graph), 1.4−1.8 M KBr gel-like complex coacervates, and 2.0 M KBr solution. Markers show data after correction and background subtraction. Lines show fits according to the Debye function for Gaussian polymers. The solid black line shows the slope of Q−1.7. The arrows show the approximate maxima for the high-Q peak in the solid samples. Profiles have been shifted vertically for clarity. E

DOI: 10.1021/acs.macromol.8b00699 Macromolecules XXXX, XXX, XXX−XXX

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Figure 4. (A) As the concentration of salt decreases in the PEC, the amount of water follows the same trend, shrinking the solid. However, as some water is trapped in the PEC before reaching the solution, pores appear within it and scatter light, giving it a white appearance. The pores in the lower panel are representative and not meant to reproduce the actual size or its distribution. (B) Scanning electron micrograph of pores in the fully hydrated 20:80 D:H-PSS/PDADMA. The scale bar is 20 μm.

quartz cuvettes. A crucial step followed: they were sealed and again placed in a water bath at 60 °C followed by slowly cooling to room temperature. This seems to have allowed the complex to return to its equilibrium state in the concentrated phase and to have homogenized the macrostructure of the PECOV. In theory, if the background has been perfectly contrast matched, there should be no variations in SLD and no observable density fluctuations. However, as is standard practice, an H2O/D2O ratio has been selected here to match the SLDs of as many of the compositions as possible. Magnification of the calculated SLDs in Figure S3 shows slight mismatch for some systemsenough to provide contrast and reveal density fluctuations. It may be a matter of chance that certain PECOX/PECOV systems are better matched than others at certain salt concentrations. There is significant evidence that certain types of coacervate show heterogeneous microscale compositions, even when apparently equilibrated. For example, protein−polyelectrolyte coacervates exhibit significant scattering at low Q corresponding to length scales greater than 100 nm.29 Cryo-TEM (transmission electron microscopy) images of coacervates made from polyelectrolytes show interesting bicontinuous phase separation on the 100 nm length scale.51 The origin of porosity within solid complexes is apparent from Figure 1: a significant amount of water is lost going from high to low [KBr]. The PEC sample is too thick for water to be expelled back into solution and thus ends up in pores of micrometer size (as seen in scanning electron micrographs in Figure 4B).49 For example, going from 1.0 to 0.2 M KBr the water content decreases from 25 to 10 H2O per Pol+Pol− pair, which corresponds to an approximate volume reduction of 50%. Instead of all water being expelled to the bulk solution, some of it appears in pores within the PEC, causing inhomogeneities and scattering (Figure 4A). The fact that the surface tension between complexes/coacervates and the salt bath is very low51−53 removes the (Laplace) pressure that would cause small pores to merge into larger ones and eventually lead to a homogeneous phase. In addition, the mobility of the polymers significantly decreases with lower [salt], further trapping microscopic pores within the macroscopic sample.10 The resulting material has an equilibrium composition over length scales smaller than the distance between pores but is out of equilibrium at larger length scales. Of course, the pores are full of liquid and salt and perhaps a

between 1.0 and 1.4 M KBr corresponds to the transition into the coacervate phase.13 Samples here are too viscous to be handled through the preparation technique used in this study. However, from the results discussed below, significant changes in polymer conformation are not expected between 1.0 and 1.4 M KBr. Scattering profiles allow the exploration of these systems at different length scales represented by different Q ranges on the plots of the low-Q Guinier region (Q < 0.01 Å−1 corresponding to a scale above 2π/Q = 600 Å), an intermediate-Q Guinier region (0.01 < Q < 0.1 Å−1, comprising the scattering from the polymer chain: 600−60 Å), and the Porod region (Q > 0.1 Å−1, corresponding to a range