Gelation, Phase Separation, and Fibril Formation in Aqueous

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Gelation, Phase Separation, and Fibril Formation in Aqueous Hydroxypropylmethylcellulose Solutions Timothy P. Lodge,*,†,‡ Amanda L. Maxwell,† Joseph R. Lott,† Peter W. Schmidt,‡ John W. McAllister,† Svetlana Morozova,† Frank S. Bates,*,‡ Yongfu Li,§ and Robert L. Sammler∥ †

Department of Chemistry and ‡Department of Chemical Engineering & Materials Science, University of Minnesota, Minneapolis, Minnesota 55455, United States § Analytical Sciences and ∥Material Science and Engineering, The Dow Chemical Company, Midland, Michigan 48674, United States S Supporting Information *

ABSTRACT: The thermoresponsive behavior of a hydroxypropylmethylcellulose (HPMC) sample in aqueous solutions has been studied by a powerful combination of characterization tools, including rheology, turbidimetry, cryogenic transmission electron microscopy (cryoTEM), light scattering, small-angle neutron scattering (SANS), and small-angle X-ray scattering (SAXS). Consistent with prior literature, solutions with concentrations ranging from 0.3 to 3 wt % exhibit a sharp drop in the dynamic viscoelastic moduli G′ and G″ upon heating near 57 °C. The drop in moduli is accompanied by an abrupt increase in turbidity. All the evidence is consistent with this corresponding to liquid−liquid phase separation, leading to polymer-rich droplets in a polymer-depleted matrix. Upon further heating, the moduli increase, and G′ exceeds G″, corresponding to gelation. CryoTEM in dilute solutions reveals that HPMC forms fibrils at the same temperature range where the moduli increase. SANS and SAXS confirm the appearance of fibrils over a range of concentration, and that their average diameter is ca. 18 nm; thus gelation is attributable to formation of a samplespanning network of fibrils. These results are compared in detail with the closely related and well-studied methylcellulose (MC). The HPMC fibrils are generally shorter, more flexible, and contain more water than with MC, and the resulting gel at high temperatures has a much lower modulus. In addition to the differences in fibril structure, the key distinction between HPMC and MC is that the former undergoes liquid−liquid phase separation prior to forming fibrils and associated gelation, whereas the latter forms fibrils first. These results and their interpretation are compared with the prior literature, in light of the relatively recent discovery of the propensity of MC and HPMC to self-assemble into fibrils on heating.

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INTRODUCTION Cellulose ethers represent a large family of chemically modified polysaccharides, which have been in commercial use for many decades.1,2 Two of the most prevalent are methylcellulose (MC) and hydroxypropylmethylcellulose (HPMC), depicted in Figure 1. Applications range from food additives and pharmaceutical excipients to construction materials; in most cases, they are used in an aqueous milieu. While the technological relevance of these materials is clear, there has recently been renewed interest in gaining a more fundamental understanding of the properties of MC and HPMC solutions.

Of particular interest is the occurrence, or nonoccurrence, of either gelation or liquid−liquid phase separation, or both, on heating dilute and semidilute solutions. In the case of the most common grades of MC, which typically have an average degree of substitution (DS) of methoxygroups of about 1.7−2.1 out of three hydroxyls per anhydroglucose unit, it has long been established that semiflexible MC chains are molecularly dissolved at low temperatures, but undergo gelation, and turn turbid, upon heating. There has been ongoing controversy about the nature of the gelation process, and in particular whether it precedes, is accompanied by, or follows, liquid− liquid phase separation.3−17 This controversy has largely been resolved in recent years, however, by virtue of application of a combination of powerful experimental tools, including cryogenic transmission electron microscopy (cryoTEM) and small-angle neutron scattering (SANS).18−20 The crucial new

Figure 1. Idealized chemical structures of cellulose ethers. For methylcellulose (MC) R = H or CH3; for hydroxypropylmethylcellulose (HPMC), R = H, CH3, or CH2CH(OH)CH3. © XXXX American Chemical Society

Received: November 14, 2017 Revised: February 12, 2018

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DOI: 10.1021/acs.biomac.7b01611 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules discovery is that MC chains assemble into long fibrils on heating, with a remarkably uniform diameter in the range of 15 nm. Gelation represents formation of a sample-spanning network of these fibrils; both linear and nonlinear rheological properties of these gels are consistent with models for fibrous networks. 21−23 The diameter of the fibrils is largely independent of molecular weight, concentration, and temperature of formation. Turbidimetric and rheological experiments, carefully conduced under equivalent heating rates, show that generally the onset of turbidity is contemporaneous with gelation.21 The turbidity can be attributed to micron-scale heterogeneities in the network, and therefore may or may not always be indicative of liquid−liquid phase separation. HPMC shows qualitatively different behavior from MC, even though the degree of hydroxypropyl substitution (MS) is relatively low, often about 1 for every 4 or 5 anhydroglucose units. For example, whereas the dynamic elastic moduli G′ and G″ for a typical MC solution shows three regimes on heating (a pregel regime, where both moduli decrease gently; the gelation regime, over which G′ sharply increases by orders of magnitude and exceeds G″; the hot gel regime, where G′ saturates), HPMC is known to exhibit a sharp drop in G″ at a certain temperature, often designated T1.6,8,17,18,24,25 Upon continued heating, G′ ultimately increases sharply, consistent with gelation, but HPMC never achieves a hot gel modulus anywhere close to that characteristic of MC. The interpretation of these phenomena has remained uncertain, although a variety of explanations have been advanced. For example, three recent papers consider the behavior of HPMC in some detail, and two compare the results directly with MC, but the interpretations of the results differ. Bodvik and co-workers attribute the drop in viscosity for HPMC to “extensive aggregation of polymers” and “formation of compact objects”, and the ensuing increase in moduli to the formation of fibrils, but which are “more linear” and “less entangled” than in the case of MC.18 For Fairclough and co-workers, gelation is caused by phase separation (attributed to spinodal decomposition) for both MC and HPMC, with the rheological differences stemming from the fact that in MC the two processes are contemporaneous, whereas for HPMC phase separation comes first.17 By contrast, Shahin et al. consider HMPC to form coexisting permanent and transient networks, and that the drop in viscosity is due to “collapse of the transient network” due to “microphase separation”.26 These studies all build on extensive prior experimental work, especially in terms of rheology and turbidimetry, and other interpretations have been advanced.27−32 In order to develop a more consistent picture, we here report an unprecedented combination of experiments on a single representative HPMC sample, as a function of concentration and temperature. Turbidimetry, rheology, light scattering, cryoTEM, and small-angle scattering (SAXS and SANS) results are presented and discussed. Based on this comprehensive data set, we propose an overall picture for HPMC behavior, and compare our interpretation with the previous literature. The importance of fibril formation to the HPMC gelation process is confirmed, as in the pioneering work of Bodvik et al.18 It is worth noting from the outset that, in the main, similar experiments (especially rheology and turbidimetry) by different groups yield more-or-less similar results, and so the two main goals of this paper are to present new experimental information, and to propose an internally consistent interpretation. It should also be recalled that different groups have examined HPMC

samples that may differ in important molecular characteristics, including DS, MS, and molecular weight (M), which further complicates establishing a universal interpretation.

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EXPERIMENTAL SECTION

Materials. The water-soluble HPMC sample was graciously provided by The Dow Chemical Company. This material was made by a heterophase commercial process and sold under the trade name of METHOCEL brand cellulosic ethers. Its degrees of side-chain substitution (DS(−OCH3) = 1.98 mol [−OCH3]/mol [AGU]; DS(−OCH2CHOHCH3) = 0.27 mol [−OCH2CHOHCH3]/mol [AGU]) were measured by the Zeisel method.33 Its nominal solution viscosity measured at standard conditions (2 wt %, 20 °C) is about 4000 mPa·s. The monomodal molecular weight distribution (weightaverage Mw = 370 kg/mol; dispersity Đ = 3.6) was characterized by size-exclusion chromatography using the method of Li, and the reported molecular weights were calibrated absolutely.34 HPMC powder was dried in a vacuum oven at 40−60 °C overnight prior to use and stored in a desiccator. Polymer was added to half the necessary amount of ultrapure water at ∼75 °C and allowed to stir for about 10 min to obtain a well-dispersed mixture. Sufficient room-temperature water was added to give the final desired concentration, and the solution was then stirred for another 10 min at room temperature followed by 10 min in an ice bath. The cold solutions were degassed with brief exposure to vacuum with stirring and then stored under static vacuum in a refrigerator for at least 24 h to allow for hydration and clearing of bubbles. Methods. Turbidimetry. Optical transmittance measurements were conducted using a home-built apparatus. Solutions were contained in 10 mm o.d. cylindrical glass ampules and placed in a temperaturecontrolled copper block (Omega CN3251). A 10 mW HeNe laser beam (vacuum wavelength λ = 633 nm) passed through a neutral density filter and the sample, and then was focused onto a photodiode detector using a lens. Labview software (National Instruments) was used to automate the temperature control and data collection processes. Heating rates were controlled over the range of 0.1−5 °C/min while cooling rates were controllable only at rates T2, where T2 corresponds to the minimum in G′, see Figure 3a). There is therefore an inevitable built-in F

DOI: 10.1021/acs.biomac.7b01611 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules spatial heterogeneity to fibril growth, which is presumably deleterious to the formation of a strong fiber gel, and may also contribute to the decreased stability. In a recent report, it was also shown that grafting short poly(ethylene glycol) chains to MC suppressed fibril formation.39 The experiments do not have sufficient resolution to address the question of how fibrils may be interconnected, i.e., exactly what constitutes a cross-link, so any difference in cross-linking efficiency between HPMC and MC, while conceivable, would be hard to discern. This scenario differs from prior reports, in some cases in substantial ways, and also agrees with aspects of previous interpretations. Neither Fairclough et al.17 nor Shahin et al.26 considered the presence or role of fibrils (and, of course, nor did any other work prior to 2010), which the cryoTEM and SANS/SAXS results confirm is an essential element. The phenomena occurring at T1, according to Shahin et al., reflects “microphase separation” and the “collapse of a transient network”.26 We are not in agreement with this terminology. First, the phase separation is clearly macroscopic, or there would not be such strong turbidity in the absence of gelation. Furthermore, the term “microphase separation” as usually applied to polymer systems implies a preferred length scale, which would be manifest as a peak in SANS and SAXS; no such peak is evident. Second, the “transient network” to which Shahin et al. allude is nothing more than a viscoelastic polymer solution; their rheological data below T1 show terminal behavior and a longest relaxation time, but no plateau in G′.26 We are in agreement with Fairclough et al. that T1 corresponds to liquid−liquid phase separation, but we do not see evidence for spinodal decomposition.17 Their interpretation relies on optical microscopy, which reveals an apparently bicontinuous structure at relatively long times when heated above T1. However, bicontinuity is not synonymous with spinodal decomposition. For example, the growth of fibrils from phase-separated droplets would ultimately lead to a samplespanning network, which is also by definition bicontinuous. Perhaps the most important argument against spinodal decomposition is that it is most likely to apply to the earliest stages of phase separation. It is difficult to reconcile how a process that creates a bicontinuous structure would lead to a sharp drop in the dynamic moduli; bicontinuity should, if anything, enhance the moduli, and especially G′. Conversely, the formation of polymer-rich droplets (by nucleation and growth) provides a straightforward explanation for the decline in the moduli. Parenthetically, several previous studies have invoked the process of spinodal decomposition in MC and HPMC solutions, but the relevant experiments invoked a temperature jump into the gelation region,15,29,40 rather than the kind of temperature ramp experiments emphasized here. Some prior reports have identified the temperature at which the moduli reach a minimum on heating as T2, and discuss its possible significance. In the scenario we propose, however, this represents a competition between two process involving nucleation and growth (liquid−liquid phase separation, and fibril formation). Consequently, one may anticipate that the precise value of T2 will depend on details of the experiment, such as heating rate, sample size, etc., and will not necessarily be simply related to a thermodynamic transition. This is consistent with our observation of nonreproducibility of the moduli, on heating above T1, even for the same solution (see Figure S2 for an example). Light scattering measurements on very dilute solutions of this HPMC sample do reveal one interesting feature that, as yet,

is not fully resolved. Figure 9 shows the weight-average molar mass, z-average radius of gyration, and second virial coefficient,

Figure 9. Molar mass, radius of gyration, and second virial coefficient obtained from Berry plots (see Supporting Information).

A2, respectively, as obtained from Berry plots (see Supporting Information for examples), from 25 to 52 °C (i.e., just below T1). Within the scatter, Mw and Rg are independent of T, whereas A2 decreases with increasing T, consistent with LCST behavior. However, in typical polymer solutions approaching liquid−liquid phase boundary, one would expect A2 to change sign (i.e., at the theta point), prior to phase separation. In this case, however, A2 remains positive. One possible interpretation is that, although nominally dilute, the solutions considered contain some form of aggregates that interfere with the simple interpretation of A2. Alternatively, this observation is not inconsistent with the previous speculation that the growth of fibrils at temperatures where A2 is positive may reflect an underlying nematic order parameter. The phase diagram associated with solutions of rigid and semiflexible polymers is characterized by a wide two-phase window that separates an isotropic solution at low concentrations and a concentrated nematic phase; this window narrows to a channel at intermediate concentrations as χ decreases, including when χ < 1/2. The value of χ associated with the 2-phase boundary at low concentrations is sensitive to chain stiffness (i.e., the axial ratio within the context of the Flory theory), and weakly dependent on concentration (see Figure 11 in ref 21). Significantly, the persistence length of MC and HPMC is consistent with phase separation when χ < 1/2 at the concentrations where gelation occurs. Curiously, MC nucleates fibrils without phase separation while HPMC appears to phase separate prior to fibril formation. Although it is not yet clear whether this reflects thermodynamic or kinetic (nucleation) effects, the detailed fibril structure, which we have deduced involves more water in the case of HPMC, likely plays an important role in tipping the balance between these two types of transitions. Note that light scattering measurements could not be made reliably above 53 °C for HPMC, due to large-scale intensity G

DOI: 10.1021/acs.biomac.7b01611 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

properties is largely due to the persistence of fibrils on cooling, as in the case of MC.

fluctuations and irreproducibility. Note also that the measurement at each T was made on a different set of solutions, heated directly to the target T from room temperature immediately prior to measurement. This protocol was adopted based on our previous study with MC solutions, where it was discovered that the slow growth of fibrils could interfere with the measurements. However, in contrast to HPMC, in the MC case, the values of A2 thus obtained changed sign at about 47 °C. One further interesting question, which remains to be fully resolved, is the precise nature of the chain packing within the fibrils. Probably the most surprising aspect of fibril formation in MC and HPMC is the fact that the polymer samples in question are extremely heterogeneous, both in molar mass and in monomer substitution pattern sequence, and thus on general principles self-assembly into regular structures is not anticipated. McAllister et al. tentatively proposed an approximately helical chain conformation for MC; in this model, the welldefined fibril diameter arises naturally as twice the persistence length of a single MC (or HPMC) chain.22 Ginzburg et al. proposed a related model whereby the chains formed toroidal bundles that could associate end-to-end.41 Kong et al. simulated wormlike chains with attraction, and found a tendency to form loops for certain combinations of stiffness and attraction strength, in at least qualitative agreement with the proposed helical conformation.42 More detailed atomistic simulations by Huang et al. showed that isolated MC chains can form loops in dilute solution;43,44 end-to-end stacking of such loops might be the origin of fibrils, as considered by Ginzburg et al.41 The tendency of MC to wind into helices, and then stack end-toend, was also examined by Li et al., who uncovered an interesting conformational fluctuation that may assist multiple chains in avoiding “misfolded” conformations, and thereby to promote the formation of long fibrils.45 In the future, it will be interesting to see if simulations can capture how the relatively small structural perturbation of the sparse hydroxypropyl groups exert such a strong influence on fibril formation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b01611. Further rheological properties, light scattering data, and details of SAXS/SANS analysis (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Timothy P. Lodge: 0000-0001-5916-8834 Frank S. Bates: 0000-0003-3977-1278 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported primarily by the National Science Foundation, through the University of Minnesota MRSEC (DMR-1420013), and also by a business unit (Dow Pharma and Food Solutions) of The Dow Chemical Company. Helpful discussions with Kevin Dorfman, Robert Schmitt, Tirtha Chatterjee, and Valeriy Ginzburg are appreciated. We acknowledge the support of Oak Ridge National Laboratory (ORNL) and U.S. Department of Energy in providing the neutron research facilities used in this work. Portions of this work were performed at both Sector 12-ID-B and the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) located at Sector 5 of the Advanced Photon Source (APS). DND-CAT is supported by E.I. DuPont de Nemours & Co., The Dow Chemical Company and Northwestern University. Use of the APS, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRSEC program.

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CONCLUSIONS The thermoresponsive properties of aqueous solutions of a hydroxypropylmethylcellulose (HPMC) sample have been examined by turbidimetry, rheology, cryoTEM, SANS, SAXS, and light scattering, for a range of concentrations. The combined results support a consistent picture of HPMC phase separation and gelation, which differs in important ways from prior reports. Upon heating a homogeneous solution, the sharp drop in moduli at a particular temperature, traditionally designated T1, is attributed to liquid−liquid phase separation, with polymer-rich droplets dispersed in a polymer-depleted matrix. This phase separation is accompanied by an abrupt increase in turbidity, and an increase in low wavevector SANS and SAXS. Upon further heating, the moduli begin to increase, due to the formation of fibrils, leading to weak gels at elevated temperature. The structure and quantity of the fibrils are revealed by real-space imaging via cryoTEM, and by quantitative analysis of a distinct feature in both SAXS and SANS patterns. The fibrils are thicker (diameter ca. 18 nm), shorter, and less stiff than their methylcellulose (MC) counterparts. These attributes, combined with the fact that fibril growth begins from a spatially inhomogeneous material, explains the significantly lower hot-gel modulus of HPMC compared to MC. Upon subsequent cooling, the sample recovers optical clarity, and the fibrils dissolve, by about 40 °C. SANS results indicate that the hysteresis in the rheological

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DOI: 10.1021/acs.biomac.7b01611 Biomacromolecules XXXX, XXX, XXX−XXX