Isothermal Titration Calorimetry to Probe the Coil-to-Globule Transition

Article pubs.acs.org/JPCB

Isothermal Titration Calorimetry to Probe the Coil-to-Globule Transition of Thermoresponsive Polymers Frank Termühlen,† Dirk Kuckling,‡ and Monika Schönhoff*,† †

Institute of Physical Chemistry, University of Muenster, Corrensstr. 28/30, D-48149 Muenster, Germany Chemistry Department, University of Paderborn, Warburger Str. 100, D-33098 Paderborn, Germany

‡

S Supporting Information *

ABSTRACT: Isothermal titration calorimetry (ITC) is introduced as a new way to study the effect of cosolutes on the coil-to-globule transition of thermoresponsive polymers. From isothermal titrations, critical cosolute concentrations can be identified, at which a coil-toglobule transition occurs. The concept of a temperature-dependent critical cosolute concentration is proven employing different isomers of dihydroxybenzene (DHB) and one isomer of hydroxy benzaldehyde (mHBA) in solutions of two thermoreversible polymer, namely poly(N-isopropylacrylamide) (PNiPAM) and poly(N,N-diethylacrylamide) (PDEAM). It is shown that the temperature-dependent critical cosolute concentration, determined by ITC, and the cosolute concentration-dependent critical temperature, probed via differential scanning calorimetry (DSC), yield the same phase diagram. The advantage of employing ITC is the ability to probe even critical concentrations at very low temperatures, whereas the corresponding critical temperatures are not easily accessible in DSC. In addition, kinetic information about the coilto-globule transition in different systems is obtained, and the effect of the DHB isomers on the transition temperature is found to scale as ortho > para > meta.

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INTRODUCTION The investigation of the thermoreversible properties of poly(Nisopropylacrylamide) (PNiPAM) and similar thermoresponsive polymers, like poly(N,N-diethylacrylamide) (PDEAM), in the presence of small cosolutes is exceedingly interesting in the field of drug delivery research. Generally, thermoresponsive polymers are fully dissolvable in a liquid at a specific combination of temperature and polymer mass fraction but show a miscibility gap at higher or lower temperatures corresponding to a lower critical solution temperature (LCST) or upper critical solution temperature (UCST) behavior, respectively. The critical transition temperature (Tc) typically shows a dependence on the weight fraction of the polymer, and the LCST is defined as the lowest critical temperature value. PNiPAM is the most widely studied thermoresponsive polymer of the LCST type, due in large part to its LCST of ∼32 °C in water, which is in the range of physiological temperatures.1 Several factors can influence the critical temperature of an aqueous PNiPAM solution. For example, an increasing hydrophobicity of the end groups lowers the cloud point.2 Furthermore, with increasing molecular weight the critical temperature decreases until the effect of the end groups is negligible and a plateau of Tc is reached at a critical molecular weight.3 Such a critical molecular weight was determined for PNiPAM4 as well as for PDEAM.5 Furthermore, shifts of the transition temperature of PNiPAM were reported in dependence on the type of solvent,6 the topology of the polymer,7,8 the type and fraction of comonomers,9,10 and for surface adsorbed chains.11,12 © 2017 American Chemical Society

Thermoresponsive polymers are especially interesting in terms of delivering a pharmaceutical payload in drug delivery systems. In this context the influence of dissolved drugs on the polymer transition is paramount. In terms of adding additional substances and observing their effect on Tc, it has been found that while surfactants at high concentrations increase the critical temperature significantly, even to above the boiling point of water at standard pressure, most other organic cosolutes depress Tc.13−15 Furthermore, the effect of different salts on the critical temperature of PNiPAM has been studied extensively. The strength of effect has been found to follow the Hofmeister series.16,17 More similar to drug molecular structures, prior studies have found strong effects of small aromatic cosolutes on the phase transition of PNiPAM, even at low concentrations.18−20 So far the mechanism of interaction has not been explained, and even structurally very similar cosolutes may differ in their influence on Tc by several °C.15 Several experimental techniques can be employed to study the critical transition behavior of these polymers. Distinctions must be made between the techniques based on the underlying phenomena that are being probed. Studies in the literature typically prepare samples of fixed composition and then measure them in a setup in which the temperature-dependent change of a physical quantity is observed. This may be for example the scattering intensity in turbidimetry or dynamic light scattering (DLS) experiments21,22 or the signal intensity in Received: July 27, 2017 Revised: August 23, 2017 Published: August 23, 2017 8611

DOI: 10.1021/acs.jpcb.7b07428 J. Phys. Chem. B 2017, 121, 8611−8618

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The Journal of Physical Chemistry B liquid state NMR spectra.19 Most common, however, is to observe the heat flow in differential scanning calorimetry (DSC). DSC measurements are easy to set up, run fully automated, and can be easily processed.23 When heating a sample up to above Tc and then cooling it down again, the critical temperature in the cooling experiment will be lower than in the heating experiment. Therefore, to measure the critical temperature of a sample via DSC requires the sample to be cooled well below the critical temperature. However, as noted previously, cosolutes may cause a strong decrease of Tc, thus setting a practical limit to the critical temperatures that can be reliably measured. ITC, on the other hand, is a technique to determine binding constants and the heat of interaction of two substances and is commonly used in biochemical research.24 The benefit of ITC is that one titration experiment results in a wealth of thermodynamic parameters to describe the interaction of two substances: Assuming an appropriate binding model, enthalpies of interaction, binding constants, entropies of binding, and the stoichiometry can be determined. In the literature on thermoresponsive polymers, ITC is rarely applied. Some studies exist on the interaction of SDS with PNiPAM, PEO, and PEO-PPO-PEO block copolymers.25,14,26,27 These studies explicitly focus on the surfactant−polymer aggregation, rather than studying the influence on the polymer transition. Furthermore, Livney et al. used ITC to compare the enthalpies of interaction and binding constants of several salts with PNiPAM by fitting a binding model to the data.28 The first ITC study in an explicit drug-delivery context was performed by Waters et al. investigating enthalpies and molar ratios of binding of the chemotherapeutic drug doxorubicin to a commercial drug delivery system at different temperatures.29 Wang et al. studied the interaction of piceatannol, a natural chemopreventive drug, with PNiPAM, assuming a model of two distinct binding sites for the drug, yielding binding constants for both sites at different pH values.14,30 Shpigelman et al. further studied the interaction of PNiPAM with sugar using ITC to determine the mechanism by which sugar influences the transition temperature.31 They concluded that the effect of sugar on PNiPAM occurs via preferential exclusion. All of the aforementioned publications have in common that they employ ITC to determine thermodynamic parameters of the polymer−cosolute interaction. In contrast, we suggest here to employ ITC measurements to determine the cosoluteinduced polymer chain collapse. To this end, we propose the concept of a critical cosolute concentration. In analogy to the critical temperature that can be measured for a sample of fixed polymer and cosolute composition in a DSC setup, we demonstrate the determination of a critical cosolute concentration at a fixed temperature. The results show that DSC and ITC can be regarded as different approaches yielding the same phase diagram of thermoresponsive polymers in the presence of cosolutes. We demonstrate this concept using two thermoreversible polymers, i.e., PNiPAM and PDEAM, in combination with different cosolutes, and we discuss the advantages and limitations of the novel ITC approach.

meta, and para isomers of dihydroxybenzene (DHB) as well as 3-hydroxybenzaldehyde were used. Pyrocatechol (oDHB, 99%), resorcinol (mDHB, 99%), hydroquinone (pDHB, 99%), and 3-hydroxybenzaldehyde (mHBA, 97%) were purchased from SigmaAldrich, St. Louis, U.S.A. and used without further purification. The molecular structures are given in Scheme 1. Scheme 1. Chemical Structure of Thermoreversible Polymers PNiPAM and PDEAM, as well as Isomers of the Cosolutes DHB and HBA

All ITC measurements were performed on a nano ITC (TA Instruments, U.S.A.). Samples were prepared in ultrapure water (Millipore, resistivity >18 MΩ cm). The sample cell volume was 953 μL, and titration was performed from a syringe with a volume of 250 μL. The reference cell contained ultrapure water. The sample cell was filled with an aqueous solution of the polymer with an initial weight fraction of 0.4 or 0.2 wt %. The syringe was filled with the cosolute solution and sealed with an air bubble to prevent premature mixing. Each titration step was defined by the injection volume (typically 5 μL), cosolute concentration (adjusted for each experiment), and equilibration time after the injection (typically 1800 s for PNiPAM and 400 s for PDEAM). Stirring speeds were routinely increased above the recommended value of 250 rpm up to a value of 350 rpm, to speed up the mixing and permit shorter equilibration times, albeit at the cost of the baseline quality. Blank titrations concerning dilution of the polymer solution and the concentration of the titrant generally showed negligible heat flow values (see Figure S1 in Supporting Information) and were thus not subtracted from the data to prevent the introduction of additional noise. Data evaluation was performed with the program NanoAnalyze (TA Instruments), which computes the concentrations of both substances for each titration step. In the following we only evaluate the concentration of the titrant and the heat flow. DSC experiments were performed on a micro DSC (micro DSC III, Setaram, France). The DSC setup consisted of two cells, one filled with pure water as the reference and the other filled with the sample. Volumes were always 320 μL, the temperature was cycled from 5 to 50 °C and back at a rate of 1 °C/min, and each time the sample was left to equilibrate at 5 °C for 2 h. Four heating and cooling cycles were recorded, and critical temperatures were determined as the average over the last three cycles.

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MATERIALS AND METHODS PNiPAM (Mn = 96 300, Đ = 4.0) was synthesized by free radical polymerization.10 PDEAM (Mn = 55 000, Đ = 1.16) was purchased from PolymerSource, Canada. As cosolutes, ortho,

RESULTS AND DISCUSSION Titration Raw Data. It is well established in the literature that the critical temperature of a thermoresponsive polymer in a

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take place upon mixing. As the coil-to-globule transition is linked to endothermic enthalpy changes, the reverse process must be linked to exothermic contributions. We thus conclude that the small exothermic contributions we observe in region b stem from local globule-to-coil transition processes, following an initial local coil-to-globule transition. While the maximum of the heat flow in Figure 1a is almost constant for a few titration steps around region c, the integrated heat per injection in Figure 1b shows a sharp peak. Region d then denotes injections that show a decreasing maximum heat flow again and corresponds to a decrease of the heat per injection in Figure 1b. In region e the heat flow decays back to the initial plateau of region a. Introduction of a Critical Concentration. All previous ITC studies on thermoresponsive polymers have always evaluated the titration in terms of molar ratios (i.e., cosolute per monomeric unit of the polymer) in order to determine binding enthalpies. Here, however, we want to describe the titration in terms of absolute cosolute concentration to determine a critical concentration, ccrit, for the coil-to-globule transition. To do so we need to investigate how far the titration results are dependent on the polymer concentration. As an example, Figure 2 shows the titration of 50 mM mHBA into PDEAM solutions of 0.1, 0.2, and 0.4 wt %. Another

mixture with cosolutes may be influenced by the cosolute concentration.19,32,33 Here, we want to conversely determine a critical cosolute concentration in dependence on the temperature. Figure 1 shows the raw data of a titration of 1 M mDHB solution into an aqueous PNiPAM solution of 0.4 wt % initial

Figure 1. (a) Raw data of the titration of 1 M mDHB solution into 0.4 wt % PNiPAM in 5 μL steps with an equilibration time of 1800 s at 5 °C. (b) Integrated heat per injection for the same experiment.

weight fraction at 5 °C. The time between 5 μL titration steps was set to 1800 s to allow for adequate equilibration of the sample after each injection, while still allowing for measurements to be completed within 1 day. The main finding consists of endothermic heat flow contributions, mainly occurring in a certain cosolute concentration range, yielding a peak as shown in Figure 1b. This behavior has no similarity to systems where the heat flow is dominated by binding between two compounds, typically yielding a sigmoidal curve shape of the heat per injection. We thus attribute the peak to the endothermic collapse of the polymer, induced at a certain critical cosolute concentration. In more detail, several regimes can be identified in the graph (Figure 1): The first injection deviates from the following ones due to an air bubble sealing the syringe against premature mixing of the components and must thus be ignored. In the first few injections, labeled a in the graph, a constant and noticeable endothermic enthalpy is detected for each injection, which is significantly higher than the pure dilution enthalpies (compare to the reference values in the Supporting Information, Figure S1). Consequently, the cosolute shows a significant degree of interaction with the polymer, even at very low concentrations. In regime b, we see an increase in the endothermic heat rate after each injection and additionally observe small exothermic contributions directly following the more pronounced endothermic peaks. As we show further below, this endothermic peak is due to the coil-to-globule transition enthalpy. The titration in ITC experiments is stepwise and leads to a strong, local concentration increase, which is then homogenized over the entire sample via the stirrer. The local concentration at the tip of the syringe will be initially higher than the global concentration after mixing. In the case where the local cosolute concentration is high enough to depress the critical temperature to or below the experimental temperature, we must expect to observe the endothermic contributions of the polymer coil-to-globule transition locally at the injection position. If, however, the global concentration after mixing is too low to depress the critical temperature to or below the experimental temperature, a local globule-to-coil-transition will

Figure 2. Calorimetric titration data for 0.1, 0.2, and 0.4 wt % PDEAM solutions titrated at 20 °C with 50 mM mHBA in ultrapure water;( a) in dependence on the cosolute concentration and (b) in dependence on the molar ratio.

example (PNiPAM and mDHB, showing the weight fraction range from 0.1 to 0.4 wt % at 25 °C) is given in the Supporting Information, Figure S3. The lower resolution of Figure S3 is a consequence of a lower number of titration steps due to the slower transition kinetics of PNiPAM, see Figure 4. We will therefore discuss the PDEAM data in Figure 2 here, but we note that PNiPAM data are qualitatively the same in terms of the polymer concentration dependence. In Figure 2a the integrated heat flow per injection is plotted against the cosolute concentration and against the molar ratio in Figure 2b. Note that by the term molar ratio we always refer to the ratio of cosolute per monomeric unit of the polymer. We observe a very good agreement in Figure 2a, whereas titration experiments evaluated in terms of molar ratios clearly differ from each other. Thus, the cosolute concentration, rather than the molar ratio, is the proper parameter to describe the enthalpy changes. Note that this confirms our interpretation above, stating that the peak of the heat flow observed here is not due to binding enthalpies of cosolute and polymer but due to the collapse of the polymer. In addition, the heat flow itself increases with the polymer concentration, yielding higher heat flow at higher polymer concentrations. This heat flow of the 8613


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The Journal of Physical Chemistry B transition is large compared to the heat flow resulting from interactions or binding, which is seen in regime a in Figure 1a. Since we attempt to determine a critical concentration in analogy to the critical temperature in DSC, we propose to determine ccrit in a similar way: In DSC, the onset and peak temperatures are the most common parameters used to describe the critical temperature of thermoresponsive polymers. In the following, the concentration of the injection with the highest integrated heat flow is defined as the critical concentration, similar to the peak temperature in DSC. The precision of such a determination is therefore dependent on the cosolute concentration in the titrand solution. Using mDHB and PDEAM as an example, Figure S2 compares titrations with three different titrand concentrations and demonstrates the higher precision of ccrit when using a low concentration. In Table S1 typical values of ccrit are summarized in combination with the respective uncertainty due to the discrete nature of the titration steps. It demonstrates that this uncertainty is rather low, when suitable titration parameters are chosen. An important question is in how far ccrit might depend on the polymer concentration. In Figure 2a the critical concentration values are 9.1, 9.3, and 9.3 mM for initial polymer weight fractions of 0.1, 0.2, and 0.4 wt % PDEAM, respectively. The small differences arise from the discrete nature of the titration experiment, as in the titration of 0.1 wt % the maximum is reached one injection earlier. We could thus show that the weight fraction change from 0.4 to 0.1 wt % PDEAM does not noticeably change the critical concentration. The same is valid for PNiPAM solutions (data not shown). All further measurements in this study were started with an initial polymer weight fraction of 0.2 wt %, and the dilution within a titration amounts to only approximately 25%. Therefore, we assume that the maxima in our titration experiments are independent from changes in the polymer weight fraction occurring due to dilution during a titration. Similar experiments have been conducted for different cosolutes and different temperatures, qualitatively showing the same behavior. We thus assume the independence of the critical concentration, defined as the heat flow peak maximum, on polymer concentration to be valid for all samples measured in this study. This justifies to evaluate the critical concentrations, rather than a critical molar ratio typically relevant in binding studies. It is interesting to note that in previous work we probed the dependence of the critical temperature of PNiPAM in mixtures with aromatic cosolutes on the concentration of PNiPAM at much higher weight fractions, ranging from 1 to 5 wt % and found that here the critical temperature in fact depends on the polymer weight fraction.19 We interpreted this as a direct interaction of cosolute and polymer inducing the transition, rather than a decrease in the solvent quality upon cosolute addition. This effect was relevant at polymer concentrations exceeding 1 wt %.19 In contrast, the lower initial polymer weight fractions in the present study do not show this previously observed weight fraction dependence. We could show that the critical concentration of the cosolutes in PNiPAM and PDEAM solutions is independent of the polymer concentration within the range of 0.4−0.1 wt %. Therefore, we must assume that the stoichiometry of cosolute to polymer is of minor importance in this concentration range or shows no effect at all in determining the critical temperature of a mixture. We note that the concept of a critical concentration inducing aggregation as well as a critical temperature bears some analogy

to other polymer systems: Pluronics are known to have a critical micellization concentration (cmc) and were shown to undergo micellization even above a certain critical micellization temperature (cmt).34,35 There, the concentration refers to that of the polymers themselves, while in our case it refers to the concentration of the cosolute. Interpreting ITC Data in Terms of Binding Models. While we interpret the observed enthalpies as coil-to-globule transition enthalpies, previous publications on PNiPAM− cosolute systems have interpreted similar ITC enthalpies as binding enthalpies, extracting thermodynamic data of binding.28,30,29 It is thus instructive to attempt the same interpretation and apply a binding model to our data. For this purpose, the titration data must be fitted with appropriate binding models in dependence on the molar ratio.28,30,29 We already plotted the present data in this way in Figure 2b, yielding a large deviation for different polymer concentrations. We nevertheless used a two-site binding model, similar to Wang et al. to fit our data.30 The resulting fit parameters are shown in Table S2 in the Supporting Information. The parameters for different initial polymer weight fraction differ remarkably, yielding contradictory results, and questionable concentration-dependent binding constants. It is thus evident that the enthalpy data are not consistent with the assumption that they stem from a binding process. Interpreting the enthalpy values as transition enthalpies, however, Heskins and Guillet already described in their 1968 paper how the molar heat of transition depends on the polymer concentration of PNiPAM,1 by describing the transition process in a Flory−Huggins model. This concentration dependence is the reason for the fact that the heat flow in Figure 2a does not quantitatively scale with the polymer concentration as expected, and only a qualitative trend of increasing heat flow with concentration is found. Therefore, we restrict our analysis to the extraction of ccrit, not further considering absolute enthalpy values. In conclusion, our data are consistent with a phase transition but not with a binding model. As far as the above-mentioned publications go, Wang et al.30 titrated piceatanol into a phosphate buffered PNiPAM solution at 25 °C. The qualitative trend of their heat flow data is remarkably similar to our experiments, in which we determine a critical concentration. We might thus speculate that they too observed the transition of the polymer, rather than binding of the cosolute to the polymer. Comparing PNiPAM and PDEAM Raw Data. In the following we want to compare the raw titration data of PNiPAM and PDEAM. The injections with the highest integral are compared for either polymer for a titration of 250 mM mDHB into 0.2 wt % polymer, see Figure 3. For comparison, the injection with the highest integral of an identical titration into 0.2 wt % PDEAM is shown as well. The enthalpy values are calculated per mole of the polymer within the sample cell, to allow for comparison of the two experiments, because the maximum integrals of injection occur at different polymer weight fractions despite the same initial concentrations. While the maximum heat rate for PNiPAM is only approximately −6 J/(s mol), the maximum heat rate for PDEAM is more than twice as large, i.e., −14 J/(s mol). The integrated heat values per titration step, however, are rather similar at −746 J/mol for PNiPAM and −836 J/mol for PDEAM. This is because the heat flow in the PNiPAM titration persists for much longer than for PDEAM. Two regimes in the PNiPAM titration can be clearly defined, as a regime of slow 8614


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concentrations in DSC measurements, which we described previously.19 In addition, the broad transitions at lower temperatures show a clearly asymmetric behavior. This is again a motivation to define ccrit in terms of a peak value rather than onset values, as described above. PNiPAM Critical Concentrations of DHB Isomers. Figure 5a shows the dependence of the critical concentration

Figure 3. Comparison of injections showing the highest integral of titration experiments of 250 mM mDHB at 25 °C into aqueous solutions of 0.2 wt % PDEAM and PNiPAM, respectively. Enthalpy values are plotted per mole of the corresponding monomer. The time scale is referenced to the beginning of the injection.

change is following a regime of more rapid heat flow, whereas the former does not seem to occur for PDEAM, see Figure 3. We can assume that the slow change stems from continuing reorganization of the polymer−water interaction and consequent restructuring of the polymer aggregates. Because this behavior cannot be found for PDEAM, we can assume that any potential reorganization processes for PDEAM occurs much faster. This is expected, because PDEAM is incapable of forming interchain or intrachain hydrogen bonds, as it only includes a hydrogen bond acceptor functionality. As a result of the slow PNiPAM kinetics, equilibration times in PNiPAM experiments were set far longer than those in similar PDEAM titrations (see Materials and Methods). Because the second regime of a slowly decaying heat flow is only observed in the region of increased heat flow (regime c in Figure 1), all other injections could be followed by a shorter equilibration time. This example demonstrates that, in contrast to DSC experiments, ITC studies allow kinetic information about the process of chain collapse to be extracted. Temperature Dependence. When increasing the cosolute concentration in a DSC experiment, the critical temperature is shifted downward. Likewise, we assume that ccrit is shifted to higher values for lower temperatures. As an example, a temperature series of the injection of oDHB into PDEAM solutions in a temperature range between 5 and 30 °C is shown in Figure 4. As expected, at lower temperatures a higher critical cosolute concentration is found. In addition, the peaks at lower temperatures, or conversely higher critical concentrations, are broader than those at high temperatures. This is in analogy to the broadening of the transitions at higher cosolute

Figure 5. Temperature dependence of the critical concentration of DHB isomers in (a) PNiPAM and (b) PDEAM solutions. Lines are empirical fits with an exponential function in panel a and with a linear function in panel b.

of DHB isomers in PNiPAM solutions on the temperature, in the range 32−5 °C. As shown above, the critical concentration generally increases with decreasing temperature. The advantage of ITC as compared to DSC results is that we can determine ccrit at far lower temperatures than we can determine critical temperatures in DSC. Whereas critical concentrations of oDHB, mDHB, and pDHB are very similar at 32 °C, ccrit values at lower temperatures differ remarkably. At 5 °C, ccrit of oDHB in PNiPAM is 297 mM, whereas ccrit of mDHB is only 153 mM, a difference of almost a factor of two. Critical concentrations for pDHB were only determined from 32 to 15 °C, because the solubility of pDHB was not sufficient to observe critical concentrations at lower temperatures: The final concentration of the cosolute in the mixture is only roughly 1/4 of the syringe concentration. To probe the demixing transition it must occur within the concentration range of the experiment, i.e., ccrit must be lower than 1/4 of the titrant concentration. This limits the range of substances and temperatures to be studied using ITC to those with high solubility and/or strong critical temperature depressing effect. A clearly different behavior can be observed for all three cosolutes, with oDHB always requiring a higher concentration at a given temperature to reach the transition than pDHB, which always requires a higher concentration than mDHB. The influence of the substituent positions on the transition follows the order ortho < para < meta. Domjan et al. previously studied the influence of DHB isomers on hydrogels of PNiPAM at two concentrations and found a similar order, proclaiming the order ortho ∼ para < meta.36 Since via using ITC we can investigate the transition at much higher concentrations, we are here able to resolve a difference between the ortho and para form. PDEAM Critical Concentrations of DHB Isomers. Similarly, we investigated the phase transition of PDEAM in solution with DHB isomers in the temperature range between 5 and 30 °C and show this in Figure 5b. While the critical concentration range for the probed temperatures in the PNiPAM system ranges from 3.8 to 297 mM, the critical cosolute concentrations with PDEAM vary only between 2.4 and 35 mM. Thus, the transition inducing effect of DHB

Figure 4. Titration of oDHB (in increasing concentrations) into 0.2 wt % PDEAM at temperatures between 30 and 5 °C. 8615


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the importance of hydrogen bonding in the interaction strength of either polymer with the aromatic cosolutes. Coherent Phase Diagram Resulting from ITC and DSC Experiments. If ITC is indeed the analogue to DSC in that it determines the temperature dependent critical cosolute concentration, rather than the cosolute concentration dependent critical temperature, both techniques should agree in their respective critical values and yield the same phase diagram. Figure 7 shows the temperature-dependent critical cosolute

isomers is much stronger for PDEAM than for PNiPAM. However, the same order of isomers is relevant for the effect of cosolute on either polymer. The critical concentrations at a given temperature in PDEAM solutions also follow the trend oDHB > pDHB > mDHB, just like for PNiPAM. In contrast to PNiPAM, however, PDEAM shows a linear correlation between the temperature and the critical concentration, indicating a constant Tc shift per added cosolute molecule, regardless of the cosolute concentration. This might, however, be an effect of the small concentration range, allowing a linear regression, which would apply to the PNiPAM ccrit values at high temperatures as well. The cosolute effects on the critical temperature of PDEAM can be quantified from the slopes as −0.93 °C/mM for oDHB, −2.38 °C/mM for mDHB, and −1.27 °C/mM for pDHB. The effect of mDHB per molecule is more than twice that of oDHB. Comparing PNiPAM and PDEAM Critical Concentrations. Figure 6 displays the ratio r of the critical concentrations

Figure 7. Comparison of ccrit(T) from ITC and Tcrit(c) from DSC experiments in aqueous solutions of PNiPAM with oDHB. ITC measurements performed in H2O and DSC measurements in D2O.

concentration (ccrit(T)) from ITC and the cosolute concentration-dependent critical temperature (Tcrit(c)) from DSC peak temperatures in aqueous solutions of PNiPAM with oDHB in one diagram. To account for the large concentration range, the concentration axis is scaled in a logarithmic fashion. ITC measurements were performed with H2O as solvent, while DSC samples were prepared in D2O. As has been published previously, PNiPAM critical temperatures in D2O are approximately one degree higher than in H2O.39 Except for this isotope effect, however, the coil-to-globule transition probed via both techniques is identical and shows a very good agreement over the entire concentration range. We can therefore conclude that both methods can probe the same process but in different ways. Note that for this comparison we have chosen oDHB, because it has the smallest effect on the critical temperature and thus allows the largest concentration range to be studied by DSC and to be compared to ITC results. oDHB is thus a suitable compound to prove the independence of the phase diagram on the experimental method. The advantage of an ITC determination of phase transitions, however, is more pronounced for cosolutes which cause larger Tc shifts, since by ITC the transition can be studied at lower temperatures (and thus higher concentrations), which are not accessible in DSC. The method of determining critical cosolute concentrations, however, depends on the assumption that the critical temperature of the polymer does not change significantly due to the dilution in the titration experiment. We were able to verify this in this study (see Figure 2 and subsequent discussion), but we note that this assumption must be verified if the method is to be applied to different polymers or other polymer weight fractions of the same polymer.

Figure 6. Ratio r = ccrit(DHB in PNIPAM)/ccrit(DHB in PDEAM) in dependence on temperature.

of each of the three DHB isomers in combination with PNIPAM divided by the respective value in PDEAM against the temperature. r = ccrit(DHB in PNiPAM)/ ccrit(DHB in PDEAM)

With decreasing temperature the difference between PNiPAM and PDEAM becomes significantly more pronounced, as r increases to r ≫1. While ccrit of oDHB at 30 °C is only roughly twice as high in PNiPAM as compared to PDEAM, this factor increases to about eight in the critical concentration at 5 °C. A similar behavior can be observed for mDHB and pDHB, although pDHB data is only available to a temperature of 15 °C, due to solubility constrains in lower temperature measurements of pDHB with PNiPAM. The slopes are very similar for all isomers. As such, the influence of DHB cosolutes on the critical temperature of PNiPAM and PDEAM must occur by a similar mechanism, which is more effective in PDEAM than in PNiPAM. The main difference between PDEAM and PNiPAM is the ability to form hydrogen bonds, either with itself or with the solvent. PDEAM is incapable of forming hydrogen bonds with itself and can form fewer hydrogen bonds to water than PNiPAM. In the literature about PNiPAM, it is well established that the critical temperature is determined by the balance of entropic and enthalpic contributions. The large entropy gain of the release of hydration water from the polymer side groups is the driving force for the coil-to-globule transition, despite the unfavorable enthalpy of transition.37,1,38 The fact that the ratio r decreases with increasing temperature is a strong hint toward

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CONCLUSIONS We were able to show that titration calorimetry of thermoresponsive polymer solutions, determining the cosolute critical concentration at a given temperature, is a comple8616


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mentary method to commonly employed DSC experiments yielding the critical temperature in dependence on the cosolute concentration. The main advantage of ITC over DSC experiments is the possibility to probe even critical cosolute concentrations at very low temperatures, whereas the corresponding critical temperatures in DSC are not accessible. By introducing this concept we further critically discussed, at least for the particular systems used in this study, the interpretation of titration data in ITC in the frame of binding models. Such an attempt to determine thermodynamic parameters like binding ratios, enthalpy, or entropy changes is not applicable, since the observed heat flow is given by transition enthalpies rather than binding enthalpies. ITC measurements further give valuable insights into the kinetics of the interaction and following polymer rearrangement. By comparing raw titration data of PNiPAM and PDEAM injections, we were able to show that PNiPAM requires far longer to reach equilibrium conditions after cosolute addition than PDEAM does, and we concluded that this must be linked to the slow rearrangement of PNiPAM chains in a strong interchain and intrachain hydrogen bonding network. In the future, based on the concept of a critical concentration introduced here, ITC measurements may play a larger role in investigating the influence of active molecules on the coil-toglobule transition of thermoreversible polymers. Advantages over DSC measurements are particularly relevant when high concentrations of actives are required or for active molecules with a large influence on Tc.

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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.jpcb.7b07428. Tabulated values of critical concentration and thermodynamic fit data. Graphs of ITC titrations of additional systems and reference systems. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +492518323419. Fax +49-2518329138. ORCID

Monika Schönhoff: 0000-0002-5299-783X Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS LCST, lower critical solution temperature; Tc, critical temperature; DSC, differential scanning calorimetry; ITC, isothermal titration calorimetry

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REFERENCES

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Isothermal Titration Calorimetry to Probe the Coil-to-Globule Transition

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