Double-Stimuli-Responsive (Temperature and pH) Aqueous Biphasic Systems Comprising Ionic Liquids

: Aqueous biphasic systems (ABS) can integrate multiple unit operations and operate under continuous mode, contributing to the development of sustainable separation processes. Encouraged by the designer solvent features of ionic liquids (ILs), we herein propose their use as components of double-stimuli-responsive (temperature-and pH-driven) ABS. Resorting to choline-alkanoate-based ILs as the pH-responsive components and poly(propylene glycol) (PPG 400) as the thermo-responsive component, the ABS ternary phase diagrams are determined at various temperature (25 − 45 ° C) and pH (3 − 7) conditions. While the liquid − liquid phase diagram response to temperature obeys a lower critical solution temperature-like behavior, the response to pH correlates with the p K a of the IL anion parent acid. The simultaneous responsiveness to temperature and pH is then shown, whose results inspire the development of customizable separation techniques as proved with the simultaneous (one-step) separation of two dyes. By a proper customization of the IL chemical structure and stimuli applied, ABS may be designed to improve the performance and sustainability of separation processes.


■ INTRODUCTION
The development of sustainable processes in the chemical and biotechnology industries is crucial to achieve the goals of the UN 2030 Agenda for Sustainable Development. 1 Reductions in the total number of unit operations and resources, novel production operating modes based on continuous processing, cyclic approaches, and alternative solvents, as well as the processes miniaturization, are promising tools to improve sustainability. 2,3As industries start to be conscious of these practices, energy savings, process performance, and cost efficiency can be maximized. 2In addition to standard largescale operations, such as distillation 4 and gas drying, 5 biphasic liquid−liquid systems can be used within the concept of process intensification by integrating reactions and separation steps and easily adapting to continuous processing. 6Besides the improved technological simplicity and flexibility, liquid− liquid systems enable an easy reuse/recovery of reactants and an efficient extraction of products. 6Examples of applications involving the enantioseparation of chiral aromatic acids in liquid−liquid countercurrent processes 7 and the etherification reaction of benzyl chloride in liquid−liquid−liquid systems 8 have been reported.However, the recurrent use of organic solvents in these processes generates environmental concern and limits their biotechnological usefulness.Thus, finding alternatives that adhere to the green chemistry and sustainability guidelines while suiting biotechnological applications is an issue that remains unsettled. 9mong the proposed routes to fulfill these needs is the use of aqueous biphasic systems (ABS). 10These are composed of at least two, often nonvolatile, compounds�conventionally a polymer and a salt or two polymers�that undergo liquid− liquid demixing in aqueous medium under particular operating conditions of pH, temperature, pressure, and concentration. 11BS are endowed with a water-rich environment and an operating condition-dependent behavior, being deemed as mild extraction/separation processes.It has been shown that ABS can be used in several integrated biotech-based processes, such as in extractive bioconversions, 12,13 enzymatic reactions, 14 and biopharmaceutical manufacturing. 15Furthermore, following the addition of ionic liquids (ILs) as ABS components, 16 significant improvements have been accomplished in the efficiency, yield, and selectivity of numerous extraction and separation processes. 17Indeed, if well designed, IL-based ABS outperform their conventional polymer-based counterparts in terms of customization degree and coverable hydrophobic− hydrophilic range. 10,18−21 Despite their relevance, the integration and intensification of IL-based ABS have only recently started to gather momentum.In this domain, stimuli-responsive IL-based ABS have been proposed as viable approaches to integrate multiple process unit operations, in which isolated stimuli have been proposed. 22,23In stimuli-responsive IL-based ABS, not only the operating conditions induce transitions from mono-to biphasic regimes (and vice versa) but also the IL and the second phase-forming component can be tailored to better fit these requirements. 22,23Stimuli-responsive IL-based ABS resorting to photo/light, 24 CO 2 , 25 temperature, 26 and pH 23 have been investigated.For instance, temperature-responsive IL-based ABS have been reported as efficient extraction techniques, as shown with carotenoids 27 and proteins 22 and as integrated biocatalytic strategies. 28In turn, an integrated production−separation platform based on pH-responsive ILbased ABS has shown to overcome the harsh conditions and long times usually involved in the production 5-hydroxyme-thylfurfural�a key building block for the chemical industry. 23n this previous work, 23 response to pH was achieved by the speciation of the salt and not by the speciation of the IL as attempted in the current work�see discussion below.
While the use of a single stimulus has been the investigated strategy to produce stimuli-responsive ABS, the use of several stimuli simultaneously may create new opportunities.Doublestimuli-responsive systems provide a higher degree of customization of the target application and thus can be more easily adapted to industrial applications.−32 To the best of our knowledge, the development of IL-based ABS with double responsiveness to temperature and pH was not previously reported.
The basis for the development of temperature-dependent ABS relies on the possibility of these (at least) ternary mixtures to the present one of the two following behaviors: 33 (i) lower critical solution temperature (LCST), where the ABS formation ability is increased by increasing the temperature or (ii) upper critical solution temperature (UCST), where the ABS formation ability is decreased by increasing the temperature.In conventional ABS, it is well-established that polymer + polymer ABS may present a UCST-like behavior, 34,35 while polymer + salt ABS, whose formation is ruled by a salting-out effect, usually show an LCST-like behavior. 36,37In IL + polymer ABS, either UCST-like or LCST-like behaviors are typically induced depending on the IL−polymer pair. 38,39egarding pH-responsive ABS, the use of salts and ILs, whose constituent ions must follow speciation as a function of the pH, is mandatory. 23,40erein, we take full advantage of the multiplicity of interactions and stimuli-induced phase transitions occurring in IL-based ABS, particularly by using ILs and polymers able to simultaneously respond to pH and temperature.Doublestimuli-responsive (temperature and pH) ABS comprising ILs are here proposed for the first time, while showcasing their potential as customizable separation platforms.To accomplish the goal of developing double-stimuli-responsive ABS, ILs able to respond to the pH and a polymer able to respond to temperature have been chosen as phase-forming components.The ternary phase diagrams of the ABS were initially determined at different temperatures and pH values, allowing to represent the effect of the stimuli in the ABS liquid−liquid phase behavior.For the synthesis, choline bicarbonate (purity 80%), purchased from Sigma, propionic acid (99% purity) from Acros Organics, and glycolic acid (99% purity) and lactic acid (88−92% purity), both from Sigma-Aldrich, were used.For the washing steps, ethyl acetate (analytical purity) purchased from Fischer Chemical was used.For water content determinations, the analyte Hydranal -Coulomat AG was obtained from Riedel-de Haen.The purity was confirmed by proton and carbon nuclear magnetic resonance ( 1 H and 13 C NMR) using deuterium oxide (D 2 O) (99.9 atom % D) containing 0.05 wt % of 3-(trimethylsilyl)propionic-2,2,3,3-d 4 acid, sodium salt (TSP) from Sigma-Aldrich.All information on the IL synthesis procedure, as well as purity confirmation by NMR, is available in the Supporting Information (Table S1).Poly(propylene glycol) with an average molecular weight of 400 g mol −1 (PPG 400) was obtained from Sigma-Aldrich.The Brilliant Blue FCF (E133) (>98% purity) was obtained from Vahine, and Sudan III (>99% purity) was acquired at Merck.

Materials
Determination of the Liquid−Liquid Phase Diagrams.The phase diagrams of the ternary systems composed of each IL, PPG 400, and water were determined for three temperatures (25, 35, and 45  °C) and pH values within the range of 3−7 (according to the pK a of the respective acid or the pH of the initial IL aqueous solution) in all possible combinations.The temperature was maintained at the desired value using a window bath with a precision of ±0.01 °C (ME-18 V Visco-Thermostat, Julabo).The pH of aqueous solutions of ILs was adjusted by adding the same acid as used in the IL synthesis, using a Metrohm 827 pH lab pH meter with an uncertainty of ±0.1.
For the binodal curve determination, the cloud point titration method was employed. 22,23To a PPG 400 aqueous solution at 60 wt %, an aqueous solution of ILs (at concentrations ranging from 60 to 70 wt % depending on the IL) was added drop by drop and under constant agitation until a cloudy mixture was identified.This point indicates that a biphasic regime is reached.Afterward, the addition of pure water drop by drop and under constant agitation was performed until the solution turned limpid.At this stage, a monophasic region is reached.Following the addition of the IL solution or water, the ternary mixture compositions were determined through weight quantification (within ±10 −4 g).The experimental binodal curves were adjusted by the original equation proposed by Merchuk et al. 43 and its modified versions presented in the Supporting Information (eqs S1−S5).
Determination of the Stimulus-Responsive Region Extent.The extent of the stimulus-responsive region corresponds to the Euclidian distance between two mixture compositions of the binodal curves determined at the upper and lower extremes of the condition (temperature or pH) under appraisal (see eq S6 in the Supporting Information).The two mixture compositions used for the determination of the extent of each stimulus-responsive region correspond to [IL] = [PPG] and were calculated by the intersection of a line that passes through the graph origin with the respective binodal curve, considering the weight percent data.Figure S1 in the Supporting Information provides a schematic representation on how these determinations have been made.
Separation of Dyes Using Stimuli-Responsive ABS.For the evaluation of dyes separation in stimuli-responsive ABS, Sudan III was previously dissolved in PPG 400, while E133 was added directly to the system.For partition studies involving temperature, the mixture point used was composed of 7 wt % [Ch][C 2 O 2 ] + 20 wt % PPG 400 (at pH 9).Temperature responsiveness was proved by varying the temperature from 25 (monophasic) to 50 °C (biphasic).For partition studies regarding the pH, the mixture composition adopted was 8 wt % [Ch][C 2 O 2 ] + 32 wt % PPG 400 (at 25 °C).pH responsiveness was confirmed by a pH variation from pH 9 (biphasic) to 5 (monophasic) through the addition of acetic acid.Biphasic mixtures were left to reach the equilibrium overnight, after which the phases were separated, weighed, and collected for quantification purposes.
The dye content in each phase was assessed by UV−visible spectroscopy with the absorbance of the phases measured at a wavelength of 633 nm for E133 and 348 nm for Sudan III using a SYNERGY|HT microplate reader.Blank systems without dyes were also prepared to address the interference of the ABS phase-forming compounds.
Both dyes (one-step) separate to opposite ABS phases.Accordingly, the extraction efficiency of E133 (EE E133 , %) was determined according to eq 1 where Abs E133 IL and Abs E133 PPG are the absorbance of E133 at 633 nm in the IL-rich and in the PPG-rich phase, respectively.w IL and w PPG are the weight of the IL-rich and the PPG-rich phase, respectively.
where Abs Sudan III PPG and Abs Sudan III IL are the absorbance of Sudan III at 348 nm in the PPG-rich and the IL-rich phase.w PPG is the weight of the PPG-rich phase, while w IL is the weight of the IL-rich phase.

■ RESULTS AND DISCUSSION
Four choline-based ILs sharing the same cation, choline, while varying the organic acid-derived anion have been here studied for the development of double-stimuli-responsive ABS, namely Figure 1 with the respective chemical structures.These were chosen due to the possibility of modifying the anion speciation in solution by altering the pH, at amenable conditions, thus enabling an easy response to this stimulus.These ILs were paired with the polymer PPG 400, which is a thermoresponsive polymer in aqueous solution, to create ABS.In addition to their valuable features as stimuli-responsive chemicals, decision-making in selecting PPG 400 and choline-based ILs was also supported by their acknowledged potential as phase-forming components in the ABS field.Currently, it is well-established that PPG 400 is able to form ABS with choline-based ILs, e.g., those comprising anions derived from Good's buffers, 44 amino acids, 45 and other organic acids. 46By addressing the simultaneous effect of temperature and pH on the ABS formation, this study further contributes to enlarge the range of responsive systems available for process design.Moreover, ABS composed of PPG 400 and choline-based ILs have been shown to be a biocompatible extraction/separation platform, 47 further highlighting the potential of these systems.
To appraise the response to temperature and pH, the ternary phase diagrams of ABS were determined within a temperature range from 25 to 45 °C and a pH range from 3 to 7. The temperature or pH dependency of the ABS can thus be appraised by the respective binodal curves, comparing the largest biphasic/monophasic area while fixing the other condition, that is pH or temperature, respectively.Figure 2 shows the responsiveness of ABS to both stimuli for representative conditions.Experimental binodal data (Tables S2−S15) and their fitting and correlation parameters (Tables S16−S29), as well as all of the remaining possible representations (Figures S2−S9), are provided in the Supporting Information.
Figure 2a shows the effect of temperature on the ABS formation ability, by fixing the pH at a value of 5 as a representative condition.It should be highlighted that the same trend was found for all ILs under investigation regardless of the pH applied (cf.Figures S2−S5 in the Supporting Information).In general, there is an increase in the facility of two-phase formation by an increase in temperature.Based on this tendency, it is possible to conclude that all ternary mixtures bearing choline-based ILs + PPG 400 + H 2 O present an LCST-like behavior, following the LCST of PPG 400− water binary mixtures. 48,49igure 2b depicts the pH response at a fixed temperature of 35 °C.Based on the binodal curves presented, decreasing the pH generally translates into a downgrade in the ability to form two phases.The same trend is found for all of the temperatures studied; however, it is more evident at lower temperature, i.e., 25 °C, with a partial overlapping being observed for the phase diagrams at different pH values obtained at 45 °C (cf.Figures S6−S9 in the Supporting Information).The pH response is directly related to the pK a of the IL anion corresponding acid, as shown in Table 1, and the percentage of neutral versus negatively charged species as a function of the pH.Representations of the speciation curves of the acids are given in the Supporting Information (Figures S10−S13).Below the pK a , the largest fraction of the ILs is in their neutral state�the corresponding acid�while above the pK a , negatively charged ions are more prevalent, i.e., a higher percentage of ILs is in solution.Overall, the reduction of pH toward values closer to the pK a , thus leading to neutral species of the corresponding acid, reduces the ABS formation ability.Moreover, below the pK a of the respective acid, in concentration regions for which anionic species become minimal, the appearance of ABS is not observed.Accordingly, ILs bearing anions derived from acids of lower pK a , i.e., lactic and glycolic acids versus propanoic and acetic acids (cf.Table 1), have the capacity to form ABS at lower pH.When IL anions are present in solution, their interactions with water are more likely to occur than PPG 400−water and PPG 400−IL interactions.Since the ABS formation mechanism is driven by the IL anions and their ability to form hydration complexes, 46 the decrease of the pH toward values closer to the pK a of the corresponding acid reduces the concentration of anions present and the ABS formation ability, explaining the trend observed for all ILs under study.
Overall, the response to both stimuli of the proposed IL + polymer ABS is guided by an interplay of interactions between the three constituents, 50 herein consisting of PPG 400−water, IL−water, and PPG 400−IL interactions.Particularly, hydrogen bonding interactions and hydrophobic effects are significantly affected by temperature or pH.An increase in temperature favors the occurrence of hydrophobic effects, while decreasing hydrogen bond interactions leads to the observation of the previously mentioned LCST-like behavior.On the other hand, the response to pH is mainly driven by ion−dipole interactions between the IL anions and water.
Table 1.pK a of the Acids Used for the IL Synthesis and Their Relationship with the pH Dependency of IL-Based ABS ab a N.D.: not determined due to the impossibility of reaching this pH at ternary compositions able to form ABS. b Red cells: no ABS formation; green cells: ABS formation.pK a values were retrieved from Chemaxon. 51o get full mileage out of the ILs' "designer solvent" features, it is important to shed light on the impact of the IL anion in ABS formation and response to both stimuli.In Figure 3, it is shown the comparison on their ability to form ABS for a representative set of conditions: 35 °C and pH 6.The remaining possible representations are provided in Figures S14−S18, Supporting Information.Although some slight variations are found depending on the set of pH/temperature conditions, the ability to form ABS can be ranked as follows: Since all ILs present the same cation, the differences in ABS formation are derived from the anion.Particularly, the number of carbons in the alkyl chain, which dictate the hydrophilic− hydrophobic character of the IL, and the number of hydroxyl groups that governs the capacity of the IL for hydration play a relevant role in ABS formation.Here, it is possible to study (i) the influence of hydroxyl groups on the formation of two phases by comparing the behavior of    significant impact on the IL + PPG ABS under investigation.Since this effect seems to be more pronounced for for most sets of temperature and pH conditions tested (see also Figures S14−S18 in the Supporting Information), an influence of a hydrophilic−hydrophobic balance in the behavior of the systems herein reported can be anticipated.
Table 2 provides the temperature-and pH-responsive region extent for the different IL-based ABS studied, which was determined using the distance between the binodal curves at the respective extreme conditions.Among the ILs studied, As previously mentioned, this is due to the lower pK a of the parent acids and wider range of workable pH conditions (cf.Table 1).For temperature, comparable responsive regions are found for all of the ILs under investigation despite the pH condition.Indeed, the response to temperature is dominated by the PPG 400, which is common for all systems.
Figure 4 depicts the double-responsiveness behavior of the studied systems and, together with Table 2, allows enlightening on how the conjugation of both stimuli works and which stimulus is the most influential for ABS formation.In general, increasing the temperature turns the pH effect significantly less pronounced.On the other hand, there is a constant and pronounced effect of temperature on the binodal curves, which intensifies at pH ≤ 5.It is thus clear that temperature has a higher impact in the formation of ABS than the pH, with the response to the pH being weaker with the increase in temperature.
The potential of the developed double-stimuli-responsive ILbased ABS was evaluated with a proof-of-concept applica-tion�the simultaneous separation of two dyes: Sudan III and Brilliant Blue FCF (E133).The use of dyes as model molecules provides a clear visual perception on the phenomenon of temperature and pH responsiveness, that is, the transition from mono-to biphasic systems upon temperature/pH variations.To evaluate the partition of both dyes, studies were performed using [Ch][C 2 O 2 ] as the representative IL under the following conditions: (i) for the temperature-responsiveness assessment, the mixture composition adopted was 7 wt % [Ch][C 2 O 2 ] + 20 wt % PPG 400 at pH 9 with a temperature variation from 25 to 50 °C; (ii) for the pH-responsiveness assessment, the mixture composition adopted was 8 wt % [Ch][C 2 O 2 ] + 32 wt % PPG 400 at 25 °C together with a variation of the pH from 9 to 5. All numerical values of the partition parameters� extraction efficiencies (EE Sudan III and EE E133 , %)�are detailed in the Supporting Information (cf.Table S30).
Figure 5 shows the concept of double-stimuli-responsive ABS through the application of temperature and pH.As visually perceived and further confirmed by the determination of the partition parameters, the two dyes selectively partition to opposite phases in one step, by the application of both stimuli.Sudan III partitions to the (more hydrophobic) polymer-rich phase, while E133 partitions to the (less hydrophobic) IL-rich phase.The hydrophobicity/hydrophilicity of these dyes explains the observed partition tendencies: Sudan III is considered an oil-soluble compound, given its octanol/water partition coefficient (log K ow ) of 7.63, 52 while E133 is a hydrophilic dye with a log K ow of −4.94. 53here is a transition from a monophasic to a biphasic mixture rising the temperature from 25 to 50 °C, further allowing the complete (one-step) separation of both dyes, with 100 and 98% of Sudan III and E133 partitioning to opposite phases, respectively.Under the conditions tested, the polymerrich and IL-rich phases correspond to the bottom and top phases, respectively.In the case of pH responsiveness, the enhanced ability to separate Sudan III from E133 (EE Sudan III = 96% and EE E133 = 94% for the polymer-rich and IL-rich phase, respectively) is maintained at pH 9.These results also reveal that, under the conditions adopted, an inversion of the phases takes place because of variations in the phases' densities and in line with previous observations with [Ch][C 2 O 2 ] + polymer ABS. 54Such a feature allows further increasing the tunable degree of IL-based ABS sensitive to stimuli.

■ CONCLUSIONS
In this work, IL-based ABS showing a double response to external stimuli, namely, temperature and pH, are proposed.Following the acquisition of the binodal data of ABS comprising choline-alkanoate-based ILs and PPG 400 under distinct temperature/pH conditions, it was shown that temperature response shows an LCST-like behavior, whereas the pH response is dependent on the anion parent organic acid pK a and the fraction of neutral versus negatively charged species present in the aqueous medium.Furthermore, it was demonstrated that double-stimuli-responsive ABS can separate the dyes Sudan III and E133 in one step, by applying any of the stimulus, enlightening on their performance as sustainable separation techniques.
Overall, double-stimuli-responsive IL-based ABS can be developed and customized to the needs of different applications.The ability to simultaneously respond to two stimuli, in this case temperature and pH, makes ABS more easily adjusted to the optimal conditions, selectivity requirements, and equipment available.On the one hand, doublestimuli-responsive ABS have an extra degree of freedom in the development of separation processes when compared to more conventional temperature-responsive ABS since more than one stimulus can be used to finely tune the partition of molecules.On the other hand, these systems hold high potential for process integration and intensification, such as in extraction− purification and reaction−separation processes.Since different process operations may require different optimal conditions of temperature and/or pH, a more fit-for-purpose and efficient process integration can be achieved.By applying an external stimulus or a combination of two stimuli, transitions between monophasic and biphasic regimes can be promoted, contributing to more sustainable processes.In this regard, more than one operation step can be simultaneously performed in "one-pot" and under continuous mode, further saving resources and increasing process efficiency.It is expected that the double-stimuli responsiveness of the systems here presented would enhance the flexibility of applications while opening new perspectives for chemical, pharmaceutical, and biotechnological companies.

Figure 1 .
Figure 1.Chemical structures, names, and abbreviations of the ILs investigated.
[Ch][C 3 O 2 ] versus [Ch][C 3 O 3 ] and [Ch][C 2 O 2 ] versus [Ch][C 2 O 3 ] and (ii) the influence of the anion alkyl chain length by comparing the behavior of [Ch][C 2 O 2 ] versus [Ch][C 3 O 2 ] and [Ch][C 2 O 3 ] versus [Ch][C 3 O 3 ].While the addition of a hydroxyl group in the anion enhances the range of concentrations in which a twophase system is observed, the alkyl chain elongation has a less

Figure 4 .
Figure 4. Double responsiveness to temperature and pH of ABS composed of PPG 400 + IL + H 2 O.The red points on the graphics correspond to the points that define the binodal curves of the system, being [IL] = [polymer].

Figure 5 .
Figure 5. Application of double-stimuli-responsive IL-based ABS in the separation of dyes: (a) description of the double-stimuliresponsive IL-based ABS concept and (b) extraction efficiencies of both dyes (EE Dye , %) obtained using ABS composed of [Ch][C 2 O 2 ] and PPG 400 upon application of temperature and pH stimuli.The blue bars represent the EE E133 to the IL-rich phase, while the red bars represent the EE Sudan III to the PPG 400-rich phase.

Table 2 .
pH-and Temperature-Responsive Regions Accounting for the Distance between the Binodal Curves for the IL + PPG 400 + H 2 O Systems pH-responsive region (wt %)temperature-responsive region (wt %)

■ ASSOCIATED CONTENT * sı Supporting Information The
Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssuschemeng.3c00059.IL synthesis protocol, NMR spectra of ILs, binodal data in weight percent, correlation parameters of the experimental binodal data, binodal curve representations, speciation curves of acids, and extraction efficiencies of dyes (PDF)