Quartz Crystal Microbalance Monitoring of Poly ( Vinyl Alcohol ) Sol during the Freeze – Thaw Process

A quartz crystal microbalance (QCM) working under cryo-conditions was applied to analyzing the gelation and/or phase behavior of poly(vinyl alcohol) (PVA) sol during repeated freeze–thaw processes. The development of a porous structure with the gelation of PVA sol during the freeze–thaw cycle was examined in terms of the thermal behavior of the water in the sol and the viscoelastic behavior of the sol through thermal and QCM analyses. Water was liberated from the hydrophilic PVA during the freeze–thaw process through the aggregation of PVA. The water decreased the freezing temperature and increased the melting temperature because of the development of the porous structure with gelation by the thermal treatment. The state of the water during the gelation was estimated from the phase transition temperature and enthalpy change of the water during the thermal scan by using water-saturated silica gels with a series of pore size distributions. The viscoelasticity of the PVA sol during the freeze–thaw process was measured by cryo-QCM using admittance analysis (QCM-A). The free water and/or porous structure in the PVA sol was found to increase from the viscoelastic point of view by QCM measurements showing a shift in the resonance parameters (fs, R1). A hard gel was confirmed to form by the decrease in fs and the increase in R1 with the thermal scan treatment. The cryo-QCM was found to be an effective probe for clarifying the gelation and phase behavior of PVA sol in detail with high resolution.


Introduction
The quartz crystal microbalance (QCM) is used in biological, medical and industrial fields as a molecular probe because of its fine spatiotemporal resolution of several tens of nanometers to sub-microns and its economic advantages (Janshoff, Galla, & Steinem, 2000;Marx, 2007;Iniewski, 2012;Diethelm, 2015).Modern QCMs realize a stable resonator by using a network analyzer with a sufficient power supply.This development has inspired the application of QCMs to large loading system associated with solid phases of samples and soft materials.The physical properties of soft/wet materials in a cryo-environment and/or at low temperature are also of interest.The shear-mode megahertz oscillation of the quartz in a QCM works as a probe for the rheological measurement of viscous samples.Thus, a QCM can be applied to observing the structural changes in soft materials from a viscoelastic point of view.QCMs have been applied in measuring the phase behavior of crosslinked thermo-responsive hydrogels, which are a typical soft material (Nakano, Kawabe, & Seida, 1998;Nakano, Seida, & Nakano, 2007;Seida, 2007;Seida, 2013).Viscoelasticity changes significantly depending on hydration behavior in hydrogel and hydration behavior is temperature-responsive in general.Thus we have investigated a temperature controllable QCM as a tool observing a change in hydration with high resolution based on the viscoelastic sensing function of QCM in the previous study.The use of QCM for the analysis of soft materials has several advantages.The shear-mode dynamic viscoelastic behavior of a micro-volume sample can be observed.Because a QCM has a high resonant frequency of several megahertz, the viscoelasticity of a small sample can be observed with high sensitivity.The QCM allows the hydration behavior of a thermo-responsive hydrogel to be observed in its collapse phase at higher temperature than the volume phase transition temperature (i.e., 306 K).The QCM is effective at identifying the phase behavior of a hydrogel and/or the temperature dependence of the hydration behavior at polar sites of the hydrogel, which is difficult with other more conventional techniques during the collapse phase of the gel.
The present study examined the potential of cryo-QCM for observing the gelation process of soft materials, where poly(vinyl alcohol) (PVA) sol was used as a representative soft material.In PVA, gelation occurs with a change in viscoelasticity, so it can be observed by using QCM with high-resolution.A QCM with admittance analysis (QCM-A) was applied to observing the viscoelasticity of PVA sol during its freeze-thaw process.An aqueous solution of PVA with a large degree of saponification is known to develop a strong and physically crosslinked hydrogel through repeated freeze-thaw thermal scan cycle treatments (Peppas & Stauffer, 1991) due to the formation of dense polymer aggregates and/or bundled polymers (micellar crystalline).A porous network structure is created with repeated freeze-thaw treatments of PVA sol.The morphology and viscoelastic properties of the hydrogel depend on the molecular weight, saponification, concentration of PVA, freeze-thaw temperature, time, and number of the thermal scan cycles (Peppas & Stauffer, 1991).The micellar crystallization and/or bundle formation of PVA via hydrogen bonding plays an important role in the formation of hydrogel through freeze-thaw cycles.There have been no reports of in situ mechanical measurements of PVA sol during its freeze-thaw process.The gelation process of PVA sol includes a phase change and is performed under cryo-conditions so that the mechanical measurement of the gelation itself is difficult to perform with conventional methods.QCM-A was applied to the mechanical measurement of the gelation of PVA sol through repeated freeze-thaw thermal scan cycles.The QCM response in the repeated thermal cycle process was examined from a viewpoint of hydration behavior of PVA obtained through thermal analysis of water in the PVA, referring to a pore structure-hydration behavior relationship obtained from thermal analysis of a series of water saturated porous silica gels with controlled pore size distributions.The silica gel had a hydroxyl group on its surface that was similar to that of porous PVA gel.Thus, silica gels with a series of pore size distributions were employed in the thermal analysis to investigate the influence of the pore structure on the state of water and to identify the gelation mechanism observed by the QCM in terms of the phase behavior of water in the sol.

PVA Sample
PVA (molecular weight: 2000, saponification: 98%) purchased from Wako Pure Chemicals Co. Ltd.(Japan) was used as received.A 15 wt% aqueous PVA solution was prepared by dissolving the PVA in pure water at 371 K under vigorous stirring.The prepared viscous solution was gently stirred for 1 day to remove bubbles that were produced during the preparation stage.The sol showed gelation when it was kept at 253 K for 1 h in a freezer followed by thawing at room temperature.

Silica Gel
Silica gels with a series of pore size distributions d p were supplied from Fuji Silysia Chemical Ltd.The N 2 adsorption/desorption method was used to characterize the porous structure of the silica gels at 77 K.The Brunauer-Emmett-Teller (BET) method was used to determine the specific surface area S BET from the N 2 desorption isotherm of each silica gel.The Barrett-Joyner-Halenda (BJH) method was used to determine the pore size distribution and pore volume V p by using the N 2 desorption isotherms (Gregg & Sing, 1982).Figure 1(a) shows the pore size distribution of each silica gel.The silica gels used in this study had relatively narrow pore size distributions: sharp with a single peak.Figure 1(b) shows the specific surface area as a function of the pore diameter.The water absorption capacity of each silica gel was determined based on the mass balance between water-saturated and dry samples.The water-saturated silica gels were prepared by immersing the series of silica gels in a large amount of distilled water for 48 h with ultrasonication for 10 min in the initial stage of immersion.The water content of the series of the silica gels was a simple linear function of the pore volume of the samples as shown in Figure 1(c).Table 1 summarizes the structural characteristics of the series of silica gels.

Thermal Analysis
The thermal analysis of the series of water-saturated silica gels was performed with a differential scanning calorimeter (DSC) to understand the phase behavior of pore water in the silica gels during a freeze-thaw operation as a function of the pore size in the silica gel.The freezing and thawing temperatures T f , T m and freezing and melting enthalpies of water ΔH f , ΔH m in each water-saturated silica gel were determined from DSC cooling and heating charts, respectively.The water-saturated silica gels were packed in an aluminum DSC sample cell, and cooling and heating charts were recorded with a DSC200 (Seiko Corporation Instrument).The scanning temperature range and rate in the DSC measurement were 293-243 K and 1 K/min, respectively.Thermal analysis was also performed on the 15 wt% PVA sol sample.The DSC measurements were performed under the same analytical conditions as the case of silica gels.The PVA sample was cooled and heated for five cycles with data acquisition to identify changes in the water behavior (hydration structure) of the PVA.

Principle of QCM-A
Based on the mathematical equivalency between the mechanical model of the QCM oscillation and LCR electric circuit model under forced oscillation (Figure 2), the viscoelastic property of the viscoelastic soft samples on the QCM were evaluated by admittance analysis of the QCM oscillation.Details of the admittance analysis have been reported elsewhere (Muramatsu, Tamiya & Karube, 1988).The resonance frequency f s at the maximum conductance G max and the resonance resistance R 1 (i.e., inverse of the G max ) of the QCM oscillation were obtained from the admittance analysis through a frequency scan around the resonance frequency.f s depends on a substantial loading mass and/or elasticity of the load on the QCM (Sauerbrey 1959).R 1 is used as an index for the viscosity of a load.In a QCM, f s and R 1 shift when the viscoelastic property of the sample changes.f s also shifts for a viscous sample due to the influence of viscosity on the oscillation of QCM, so f s is not a simple function of the loading mass in the case of a viscous sample (Kanazawa & Gordon, 1985).Martin, Granstaff, and Frye (1991) reported the independence of f 2 (i.e., the frequency at half of G max and the minimum susceptance B min ) from the influence of the load viscosity on the QCM.Thus, the frequency f 2 was also measured in this study to evaluate the mass effect that is free from the influence of the sample viscosity.

QCM Measurement
The viscoelasticity of the PVA sol during its freeze-thaw process was measured by QCM-A.A 5 MHz QCM with a gold electrode (φ10 mm diameter) purchased from Mitadenpa Co. (Japan) was used in this study.Figure 3 shows a schematic diagram of the QCM system used in this study.The temperature dependence of the frequencies f s and f 2 and the resonance resistance R 1 of the bare QCM were measured first.Then, 1 µL of the PVA sol sample was dropped onto the surface of the QCM within an area of 1 mm 2 .The QCM was installed in a thin cryo-cell with a Peltier device at the bottom of the cell.To reduce the loss of the aliquot PVA sol sample by drying and to avoid condensation from moisture in the atmosphere on the QCM, the PVA sol sample was covered with a thin polymer film made of poly(propylene), and the entire QCM electrode was covered with a silicon sheet.The film had a reverse dimple with a φ1 mm diameter and 1 mm depth at its center to hold the sample sol.The influence of the thin film on the QCM oscillation was examined in advance and was found to be negligible compared to the changes in f and R 1 induced by the sample on the QCM.The sample temperature was measured by using a φ1 mm thermocouple placed near the edge of the quartz of the QCM.The sample temperature was controlled by using the Peltier device with a proportional-integral-derivative (PID) controller.The sample was cooled from 283 K to 243 K and then heated to 283 K in a stepwise manner.This temperature swing was repeated three times.The cooling and heating rates were 1 K/min.The admittance of the QCM oscillation during the thermal scan cycle was analyzed at each temperature to collect the f s , f 2 , and R 1 data with a network analyzer.
Figure 3. Schematic diagram of the QCM system used in this study

Thermal Behavior of Pore Water in the Silica Gels
Figure 4 indicates the relationship between the pore size of the silica gels and the change in enthalpy with freezing ΔH f obtained from the cooling charts in the DSC analysis.The water in the silica gels showed supercooled freezing with a single exothermic peak.The freezing temperature T f decreased monotonically with increasing pore size.ΔH f in Figure 4 was obtained from DSC time charts because the temperature drift occurred at the freezing point due to the exothermic heat process of the freezing.ΔH f increased with the pore size and reached near the value for pure water in the silica gel with a pore size of more than 30 nm.This indicates the existence of a large amount of non-freezable bound water in the silica gel with small pores of less than 16 nm.This was because of the increase in the surface area relative to the pore volume with the decreasing pore size.The ratio between the specific surface area S BET and water content in sample A was three times greater than that in sample C, as indicated in Table 1.T f for pure water obtained by the same method was lower (253 K) than the temperature observed in the silica gels with large pores.Increasing the pore size of the silica gels was found to decrease T f and increase ΔH f .(Watase, Nishinari, & Hatakeyama, 1988;Seida & Nakano, 1996).The melting enthalpy of freezable bound water ΔH m2 was calculated from the peak area below 273 K.The melting enthalpy of free water ΔH m1 was determined from the area above 273 K.As shown in Figure 5(a), the peak due to melting of the bound water shifted toward a higher temperature with increasing pore size.The endothermic peaks below 273 K indicate the existence of freezable bound water, as mentioned above.T m1 was defined as the melting temperature of free water.T m2 was defined as the melting temperature of bound water that could be observed in the DSC analysis.T m1 was determined from the inflection point around 273 K in the case of the multi-peak sample.T m2 was determined by a conventional method as indicated in Fig. 5(a).The endothermic peak attributed to the freezable bound water disappeared in Sample A. Figure 5(b) indicates the melting temperatures T m1 , T m2 and the melting enthalpy of water ΔH m (= ΔH m1 + ΔH m2 ) as a function of the silica gel pore size.T m2 and ΔH m were found to decrease with the pore size of silica gel.The results indicate the existence of some amount of non-freezable bound water in the silica gels with small pores.In the silica gels with pore sizes larger than 30 nm, the total ΔH m reached close to the melting enthalpy of pure water.This indicates that free water was dominant in the pores larger than 30 nm.The fraction of water that was free from the influence of polar sites on the pore surface was large in the large pores, so T m2 and ΔH m1 were found to increase with the pore size.The melting temperature T m2 of freezable bound water and the melting enthalpy ΔH m of freezable water can be associated with the pore structure in the case of silica gels.Slight decrease of T m1 occurred with the thermal cycle treatment due to a overlapping of the melting peak of bound water in the definition of T m1 .The DSC heating charts are a better way to clearly represent the state of water in the present system.

Thermal Behavior of PVA Sol
PVA sol did not significantly affect the oscillation of the QCM.The change in oscillation due to the application of the thin film was within several hertz in the considered temperature range.
Figures 7 (a), (b), and (c) indicate temperature dependences of f s , f 2 , and R 1 , respectively, in the QCM analysis of the PVA sol during the freeze-thaw thermal scan cycle.During the cooling process, f s decreased and R 1 (=1/ G) increased with decreasing temperature.In contrast, f 2 slightly decreased, which indicates that the viscosity R 1 change has a significant influence on f s (Kanazawa & Gordon, 1985).The increase in R 1 during the cooling process was due to the increased density of water with temperature.A drastic shift in f s , f 2 , and R 1 occurred at the freezing temperature of the PVA sol after some supercooling.A large change in the effective load (i.e., penetration depth of the shear wave of the QCM) induced by the phase transition of the water from a liquid to solid (i.e., freezing) caused the behaviors of f s and R 1 .The transition temperature (i.e., point at which the red-shift of f s occurred) shifted down closer to the freezing temperature of pure water with the thermal scan cycle.free water of the PVA sol during the thawing process.The changes below 273 K would reflect the melting of freezable bound water in the PVA. Figure 7 represents the first measurements of the soft/wet material during its freeze-thaw process with QCM.

Discussion
In the case of the aliquot of pure water on the QCM, the QCM behavior during the freezing and thawing processes of the water was reproducible, and the supercooled freezing temperature and melting temperature did not change with repeated temperature scan cycle measurements.The freezing and melting temperatures were determined from the points of drastic shifts in f s and R 1 during the QCM measurement.The trend of heating chart for f s and f 2 of ice from pure water was reproducible as a simple decreasing function of the temperature with a small peak just before the drastic blue-shift due to the ice melting (Seida 2016).
The size and/or amount of samples used in both the DSC analysis and QCM measurements were much larger than the size of the micellar crystalline aggregates of PVA (Hassan & Peppas, 2000) that are produced in the initial stage of the gelation of PVA sol and the pore sizes created in the PVA by the freeze-thaw cycle treatment.From this point of view, knowledge on the gelation mechanism for bulk PVA sol is applicable to this system (Watase & Nishinari, 1985;Peppas & Stauffer, 1991).
The pore size and thermal properties (hydration behavior) of water in the pores correlated well for the silica gels, as indicated above.Increasing the pore size decreased the freezing temperature T f and increased both the melting temperature of freezable bound water T m2 and melting enthalpy of freezable bound water ΔH m1 in the silica gels.
The freezing water excluded dissolved solutes from its ice crystal, which enhanced the bundle formation and/or micellar crystalline aggregates of PVA and resulted in the porous gel.The elasticity decreased near the melting temperature in the freezing phase of the PVA sample, as expected from the f 2 trend for the heating process shown in Figure 7 (b).This would also enable the polymer molecule to be excluded from the ice crystal and enhance the aggregation of PVA.The fractional change in hydration of the PVA also occurred with the progress of the aggregation and/or gelation during the freeze-thaw treatment, as indicated by the trend of ΔH according to the DSC analysis (Figure 6).Thus, the results shown in Figure 6 correspond to the development of a porous structure and gelation with repeated thermal scan cycles.
For the sol of 15 wt% PVA with 80% saponification and a small molecular weight, no obvious temperature shift with the freeze-thaw thermal cycle was observed (Seida, 2015).Bulk gelation does not occur in PVA sol with a small degree of saponification (~80%).Watase and Nishinari (1985) reported that acetate groups can inhibit the formation of gel in PVA.PVA with a small degree of saponification hardly shows bulk gelation even if crystalline PVA aggregates may be produced by the freeze-thaw thermal treatment.Thus, the change in the resonance parameters (i.e., f s and R 1 ) during the freeze-thaw thermal scan cycle of the PVA sol, as observed in Figure 7, was attributed to the gelation of the PVA sol.
Rapid freezing produces an internal strain in the freezing phase and/or the interfacial strain between the ice and electrode surface of the QCM.Relaxing the strain would take a long time compared to the scan rate of the temperature in this study.Thus, the strain would affect the f and R 1 trends in the freezing phase of the temperature scan measurement.The scan rate of the temperature is also a potential factor for determining the f and R 1 behaviors in the freezing phase.The scan rate of the temperature in the present study was determined based on the conventional scan rate used in the DSC analysis.
The freezing temperature of the sample observed with the QCM decreased with the progress of gelation; this was associated with the increases in viscoelasticity and/or R 1 .The shift in the f s and R 1 charts for the heating process were also associated with the increased viscoelastic properties with the thermal scan cycles.The increased melting temperature and temperature range of the melting peak of the freezable bound water on the PVA would indicate an increase in free water due to the development of the porous structure.The drastic shift of f s was due to a large drastic change in the effective load (i.e., the product of the penetration depth δ of the shear wave of the QCM and the density of the load ρ (Kanazawa and Gordon, 1985)).The drastic change in f 2 indicated a drastic change in the penetration depth of the shear wave of QCM in the sample, which means a change in the effective load on the QCM.The steep increase in R 1 during the heating process indicated increased damping of the shear wave energy along with the increased penetration depth of the shear wave.The red-shift of f s and f 2 with the freezing of the sample may seem to be quite different compared to the generally accepted relation between the loading mass and frequency shift (Sauerbrey, 1956).The large blue-shift of f s occurred with increases in the water (liquid) phase and penetration depth of the shear wave.The increased effective load with small viscosity observed by the QCM decreased f s , f 2 , and R 1 .Because the shear oscillation frequency of a QCM is very high, the freezing phase would behave like viscous body.In the case of a load with a much larger viscosity, f s and f 2 increase in spite of the large R 1 (Seida, 2016).According to the pore structure-hydration behavior relationship obtained from the results in Figure 5, the decrease of red-shift temperature and the increase of blue-shift temperature of f s and f 2 occur in the QCM response along with the increase of free water and/or formation of pore structure in the PVA.R 1 becomes large with the increase of free water in the PVA.
The findings obtained from the thermal analysis of the silica gels and the PVA sol with repeated freeze-thaw thermal scan cycles indicate that a QCM can be used to probe the structural formation of a PVA porous network during the gelation of PVA sol.

Conclusion
In the present study, the gelation process of PVA gel during repeated freeze-thaw thermal cycles was evaluated based on the thermal behavior of pore water and the viscoelastic behavior of the PVA sol obtained by the cryo-QCM developed in this study.The relationship between pore structure and hydration behavior of pore water was clarified based on the freezing temperature T f , melting temperature T m , and freezing and melting enthalpies ΔH f , ΔH m of the pore water obtained from the thermal analysis using water saturated porous silica gels with a series of pore size distributions.The non-freezable and freezable bound water decreased with increasing pore size up to a diameter of 16 nm in the silica gels.T f decreased and T m2 increased with the increase of pore size because of the increased amount of free water in the silica gel.The development of a porous structure in the PVA sol during the thermal scan cycle treatment was observed based on the parameters T f , T m , and ΔH m in the thermal analysis.The QCM response of the PVA sol in the repeated freeze-thaw thermal scan cycle was obtained fairly well.The QCM response was interpreted from the viewpoint of hydration behavior referring to the pore structure-hydration behavior relationship observed in the silica gels.The results obtained in this study indicate the viability of the cryo-QCM for clarifying the gelation as well as hydration behaviors of PVA sol during freeze-thaw process in detail from a viscoelastic point of view.
Figure 1.(a) The pore size distribution, (b) the relationship between the pore size and the specific surface area and (c) the relationship between the pore volume and the water content for the series of silica gels used in the present study

Figure 2 .
Figure 2. Model comparison between the mechanical oscillation model of QCM and the LCR electric circuit model Figure 2. Model comparison between the mechanical oscillation model of QCM and the LCR electric circuit model

Figure 3 .
Figure 3. Schematic diagram of the QCM system used in this study

Figure 4 .
Figure 4. T f and ΔH f of water in each silica gel obtained from the cooling charts of DSC.□: weight basis enthalpy, ■: water content basis enthalpy, ×: freezing temperature T f

Figure 6
Figure6(a) presents the DSC heating charts of the PVA sol obtained in the freeze-thaw measurements.An endothermic melting peak appeared from around 253 K, which indicates the existence of freezable bound water in the PVA sol.A slight shift in the endothermic peak was observed with the thermal scan cycle.Figure6(b)summarizes T f , T m , and ΔH m obtained from each DSC chart.The supercooled freezing temperature T f of the PVA sol shifted toward a lower temperature with the thermal scan cycle.In contrast, the melting temperature T m determined based on the conventional slope method, as described above, decreased very slightly with the thermal scan cycle.This was due to the tail overlapping of the endothermic melting peak of freezable bound water with the melting peak of free water.Figure6(b)shows ΔH m at each freeze-thaw cycle normalized by ΔH m of the first freeze-thaw cycle ΔH m,1st .The increase in ΔH m /ΔH m,1st indicates the increase in free and freezable bound water with each repetition of the thermal scan cycle treatment.Thus, the melting temperature of the freezable bound water increased because of the decrease in non-freezing bound water with the gelation of PVA.Based on the thermal behavior of water observed in the silica gels, the decrease in T f of the PVA sol with the thermal scan cycle indicates an increase in free water.The partial densification and/or aggregation of PVA as a result of repeated thermal scan cycle treatment produces a pore structure along with gelation of the sol(Peppas & Stauffer, 1991, Watase, Nishinari, & Nanbu, 1983).The formation of bundled polymers (micellar crystalline aggregates) via hydrogen bonding among hydroxyl groups in PVA results in pore formation with gelation.Based on the knowledge obtained from thermal analysis of the silica gels, the results in Figure6indicates the formation and development of a porous structure in the gelling PVA sol by the freeze-thaw thermal scan cycle.

Figure 7 .
Figure 7. QCM behaviors of the sample during the repeated freeze-thaw temperature scan cycle.(a) f s , (b) f 2 and (c) R 1

Table 1 .
Characterized structural parameters of the silica gels