Enhanced partitioning of tryptophan in aqueous biphasic systems formed by benzyltrialkylammonium based ionic liquids: Evaluation of thermophysical and phase behavior

Accepted Manuscript Enhanced partitioning of tryptophan in aqueous biphasic systems formed by benzyltrialkylammonium based ionic liquids: Evaluation of thermophysical and phase behavior

V.P. Priyanka, Anusha Basaiahgari, Ramesh L. Gardas PII: DOI: Reference:

S0167-7322(17)33022-2 doi:10.1016/j.molliq.2017.09.111 MOLLIQ 7948

To appear in:

Journal of Molecular Liquids

Received date: Revised date: Accepted date:

7 July 2017 27 August 2017 27 September 2017

Please cite this article as: V.P. Priyanka, Anusha Basaiahgari, Ramesh L. Gardas , Enhanced partitioning of tryptophan in aqueous biphasic systems formed by benzyltrialkylammonium based ionic liquids: Evaluation of thermophysical and phase behavior. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Molliq(2017), doi:10.1016/j.molliq.2017.09.111

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ACCEPTED MANUSCRIPT Enhanced partitioning of Tryptophan in Aqueous Biphasic Systems formed by Benzyltrialkylammonium based Ionic Liquids: Evaluation of thermophysical and phase behavior V.P. Priyanka1, Anusha Basaiahgari1, and Ramesh L. Gardas*

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Department of Chemistry, Indian Institute of Technology Madras, Chennai - 600 036, INDIA

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*Corresponding author: Phone: +91 44 2257 4248; Fax: +91 44 2257 4202

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E-mail: [email protected] ; Web: http://www.iitm.ac.in/info/fac/gardas 1

V.P and A.B have contributed equally to this work --------------------------------------------------------------------------------------------------------------------

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ABSTRACT

Ionic liquids (ILs) based aqueous biphasic systems (ABS) are envisioned to be promising

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separation and environmentally benign extraction media. In this context, novel ternary phase diagrams were determined for two ILs namely, Benzyltrimethylammonium chloride and

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Benzyltributylammonium chloride in presence of various potassium salts, K3PO4, K2HPO4, K2CO3 and KOH at 298.15 K. The influence of benzyl group substitution on the cation of IL and

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nature of various potassium salts on the phase behavior were analyzed. Experimental binodal data were fitted to Merchuk’s equation and tie line compositions and tie line length were also

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determined. Further, these IL based ABS in presence of various potassium salts have been systematically scrutinized for their efficiency to extract Tryptophan. For the better understanding

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of the role of thermophysical properties on phase behavior and extraction capability, density and viscosity of coexisting phases were measured at various compositions in the temperature range

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from 293.15 to 328.15 K. Enhanced extraction coefficients achieved for the studied combinations of ILs and inorganic salts indicate the possible use of these ABS as efficient extraction systems. Further, the study of thermophysical properties suggested that selected ternary systems present more desirable features in terms of density and viscosity as compared to traditional polymer based ABS. --------------------------------------------------------------------------------------------------------------------Key Words: Liquid-Liquid Extraction; Aqueous Biphasic System; Ionic Liquid; Hofmeister series; Partition Coefficient. 1

ACCEPTED MANUSCRIPT 1. Introduction Liquid-liquid extraction (LLE) systems are most widely studied separation and purification technologies with certain advantages like enhanced selectivity, high yields and also lower associated costs [1-2]. The success of extraction/purification technology depends on factors such as: quick and higher selectivity of the process; lower energy inputs for attaining

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equilibrium; absence of adverse effects on chemical structure, activity and stability of

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biomolecules [3]. Typical LLE systems employ volatile organic solvents since they are immiscible with aqueous phase, the other major component of extraction systems. However,

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these organic compounds exhibit high volatility, substantial levels of toxicity and thus poor biocompatibility leading to denaturation of bioactive compounds like proteins [1].Therefore, a

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need to replace traditional LLE with more biocompatible and eco-friendly systems was considered very essential. In this context, Albertson proposed aqueous based substitutes for

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conventional organic solvents based LLE systems [4].

Aqueous Biphasic Systems (ABS) are envisaged as suitable alternatives for conventional

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LLE systems since they consist of water as major constituent (about 80 %) thus providing mild

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environment for extraction and separation of biomolecules including proteins [5], enzymes [6], antibodies [7] etc. ABS are composed either of polymer-polymer, polymer-salt or salt-salt combinations. Though both solutes are water soluble, phase splitting arises due to

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incompatibility between coexisting systems in certain concentrations. After phase splitting one of

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the phases will be rich in one solute and other phase will be rich in second solute. However, there are some disadvantages associated with conventional ABS like higher viscosities and limited polarity due to constituting polymers [8]. Ionic liquids (ILs) were proposed by many researchers,

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as appropriate choice to resolve complications raised by polymers of conventional ABS. Motivated by application of ILs in formation of ABS as reported for the first time by Rogers and co-workers [9], active research has been done to explore the possibility of two phase formation by ILs in combination with various inorganic salts [10]. In recent years, ILs have been made use as possible alternatives for polymer rich phases [11-12], as well as for salt rich phases [13]. ILs consist exclusively of ions that are poorly coordinated because of their low symmetry and charge delocalization. ILs are composed of either organic or inorganic cations and anions 2

ACCEPTED MANUSCRIPT [14]. Significant properties of ILs that facilitate separation processes include excellent solvation capabilities [15], high chemical stability, negligible vapor pressure [16], non-denaturing environment for biomolecules [17] etc. Main drawback with respect to polymer based ABS is in terms of limited polarity difference of coexisting phases which results in restricted applications [18]. The major advantage of using ILs lies in the possibility of altering their structure to obtain required properties and also to employ them in wide range of applications [19-21]. By

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appropriate choice of cations and anions, ILs can be tuned to modify polarities of coexisting

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phases. In addition, extraction efficiencies of IL based ABS can also be enhanced thus

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facilitating separation of biomolecules.

Various classes of ILs were studied [22] for their ability to form ABS and most studied

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class of ILs include imidazolium [23] and comparatively less studied classes of ILs include pyridinium [24], pyrrolidinium [25], phosphonium [26] and ammonium [11] based ILs. Various

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inorganic salts mostly including potassium and sodium [27] based salts and organic salts [28] have been studied in combination with ILs for the ABS formation. Recently, phase studies have

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also been extended for functionalized ILs like magnetic ionic liquids [29]. However, thorough understanding of role of various phase forming components and molecular level interactions

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governing phase splitting behavior is prerequisite to tailor the corresponding phase behavior

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[22].

The vital application of IL based ABS is in separation and extraction processes involved

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in biotechnological applications [4] since it provides rapid, low cost and gentle means for recovery. Further, application of ABS for separation and extractions of bioactive compounds

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opens up better alternatives for conventional methods involving organic solvents. The separation of bioproducts from reaction media is an important step in biotechnology and amino acids are one such useful bioproducts. In this context it is of practical importance to gain understanding about driving forces that determine partitioning of amino acids [22]. IL based ABS were found to be efficient extraction media not only for amino acids but also for various other bioactive compounds like alkaloids, phenolic compounds, nucleic acids, lipids and also for some pharmaceutical compounds [30].

3

ACCEPTED MANUSCRIPT Further, it is also crucial to study properties like density and viscosity of coexisting phases for application of IL based ABS at large scale. Understanding thermophysical behavior helps in predicting the possibility of scaling up of a process from lab to pilot and industrial scales. Density values are important for equipment design while lower viscosities are preferred in order to facilitate mass transfer and lesser energy consumption [22]. Considering the number of reports related to various classes of ILs and salts that have been explored for ABS formation,

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relatively few reports have been found for understanding thermophysical properties of co-

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existing phases [26, 31-32].

In the present work, we evaluate the phase behavior of two Benzylalkylammonium based ILs namely Benzyltrimethylammonium chloride, [BTMA]Cl and Benzyltributylammonium

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chloride, [BTBA]Cl in presence of potassium based inorganic salts including K3PO4, K2HPO4, K2CO3 and KOH. The influence of benzyl group substitution and alkyl chain length on the ABS

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formation has been analyzed. Further, the influence of various potassium salts on the formation of ABS has been evaluated to gain comprehensive idea about their salting out ability. For each

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selected system, phase diagrams along with corresponding tie lines have been determined at 298.15 K and at atmospheric pressure. Further, these systems have been studied for their

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potential in extraction of an essential amino acid of biological interest i.e., Tryptophan. Thermophysical properties like density and viscosity of coexisting phases of these IL based ABS

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have also been studied to gain deeper understanding of thermophysical behavior.

2.1. Materials

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2. Experimental methods

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Ternary phase diagrams were determined using aqueous solutions of inorganic salts such as potassium phosphate tribasic, K3PO4 (≥ 98 wt %), potassium phosphate dibasic, K2HPO4 (≥ 98 wt %), potassium carbonate, K2CO3 (≥ 99 wt %) and potassium hydroxide KOH (≥ 85 wt %) and aqueous solutions of ILs namely Benzyltrimethylammonium chloride, [BTMA]Cl (≥98 wt% pure) and Benzyltributylammonium chloride, [BTBA]Cl (≥98 wt% pure). K3PO4 and KOH were purchased from Sigma Aldrich, K2HPO4 and K2CO3 from Merck and [BTMA]Cl and [BTBA]Cl were supplied by Sigma Aldrich. All chemicals were dried under vacuum for 24 hours prior to experiments to remove any traces of moisture. Aqueous solutions of Tryptophan were used for 4

ACCEPTED MANUSCRIPT extraction experiments, and Tryptophan was used as supplied by Sigma Aldrich (≥98 wt% pure). Structures of [BTMA]Cl, [BTBA]Cl and Tryptophan are given in Fig. 1 2.2. Experimental Procedure 2.2.1 Phase Diagrams and Tie Lines determination

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Binodal curve data was obtained using well established cloud point titration method [33].

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For determination of phase diagrams, aqueous solutions of inorganic salts were prepared at concentration of 40% by weight and ILs were prepared in variable concentrations ranging from

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50% to 80% by weight. All solutions were prepared on weight basis using analytical balance with a precision of ±0.01 mg (Sartorius CPA225D) using Millipore water. Experiments were

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carried out at 298.15 K and at atmospheric pressure. Constant temperature of 298.15 K was maintained using temperature controller bath (F25-ME, Julabo, Germany) within uncertainty of

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±0.01 K. The aqueous salt solution was added drop-wise to the IL aqueous solution until the appearance of turbid solution indicating the formation of biphasic region was observed. It was

M

followed by drop-wise addition of Millipore quality water until clear solution was observed. All additions were performed under constant stirring and weights of salt solutions and water added

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were noted accurately. Exact compositions of all components of ternary systems were determined by weight quantification within ±10-4 g and all experiments were performed in

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triplicate.

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The experimental binodal curve data were fitted to Merchuk’s equation [34] by least squares regression. Though this equation was originally proposed for polymer based ABS, it was

[ ]

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found to be applicable for IL based ABS also [11]. The equation is given below: [( [ ]

)

( [ ] )]

(1)

where [Y] and [X] represent weight fractions of IL and salt respectively. A, B and C are constants obtained by regression of equation 1. In order to obtain tie line (TL) data, ternary mixtures of IL, salt and water with their compositions lying in the biphasic region were selected and prepared gravimetrically. These mixtures were vigorously stirred by maintaining temperature at 298.15 K for about 30 min. 5

ACCEPTED MANUSCRIPT Later, these ternary mixtures were left for equilibrium for minimum of 12 hours in order to ensure complete separation of coexisting phases. After 12 hours of equilibrium, both phases were carefully separated and weighed. Each individual TL was determined gravimetrically by applying lever arm rule to relate weight of top phase and bottom phase to weights of overall

)

( [ ] )]

[ ]

[( [ ]

)

( [ ] )]

[ ]

[ ]

[ ]

[ ]

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[ ]

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[( [ ]

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[ ]

T

system. Following set of equations 2-5 [34] were used in determining tie line data:

[ ]

(2) (3) (4) (5)

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where the subscripts T and B represents top and bottom phases respectively and M represents initial mixture composition. The parameter α denotes the ratio between weight of top phase and

)

(

)

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√(

M

total weight of mixture. Further, equation 6 was used to calculate the tie line length (TLL).

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2. 2.2 Partition studies

(6)

The partitioning of Tryptophan was evaluated in selected ternary phase compositions

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belonging to biphasic region. Tryptophan solution was prepared at concentration of 4 mM. Mixtures of selected compositions were prepared, vigorously stirred and further, they were

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allowed to equilibrate for 12 hours at constant temperature of 298.15 K. Later, two co-existing phases were separated and weighed. Quantification of Tryptophan in each phase was carried out by preparing samples from top and bottom phases by appropriate dilutions. UV-Vis spectrophotometer (Shimadzu UV-1300) was used for measuring absorbance of Tryptophan at 280 nm. Concentrations were calculated using calibration curve established previously. In order to avoid interferences caused by IL and salt, blank samples were also prepared for same ternary weight compositions using pure Millipore water instead of Tryptophan aqueous solutions. Extraction experiments and quantification were performed in triplicate and average

values 6

ACCEPTED MANUSCRIPT were considered. The partition coefficient of Tryptophan,

was determined according to

equation 7: [ [

[

]

]

(7)

]

and [

]

represent concentration of Tryptophan in IL and salt rich phases,

T

respectively.

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Further, percentage extraction efficiencies for Tryptophan have been calculated using

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equation 8 and it can be defined as percentage ratio between amount of Tryptophan in IL rich

( )

(8)

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2.2.3 Thermophysical property measurements

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phase to that present in total mixture.

Ternary mixture composition same as that selected for tie line determination was

M

considered for thermophysical property measurements also. Density and viscosity measurements

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of each phase were performed in the temperature range of 293.15 to 328.15 K with 5 K interval and atmospheric pressure for both IL rich and salt rich phases. Density and viscosity of samples

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were simultaneously measured using Anton-Par, DSA 5000 M Density and sound velocity meter attached with Lovis Micro viscometer. The instrument can work in the temperature range of

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273.15 to 343.15 K and in pressure range of 0 to 3 bar. Measuring range of density is 0 to 3 gcm-3 with uncertainty of 1×10-6 gcm-3 and uncertainty related to viscosity is 0.005 mPas.

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Calibration of instrument was done by freshly degassed Millipore water and dry air at atmospheric pressure.

3. Results and Discussion 3.1. Phase Diagrams and Tie lines For each combination of ABS containing IL, salt and water, phase diagrams were determined at 298.15 K and at atmospheric pressure. The corresponding binodal curves of [BTMA]Cl and [BTBA]Cl with different salts namely, K3PO4, K2HPO4, K2CO3 and KOH are 7

ACCEPTED MANUSCRIPT illustrated in Fig. 2 and Fig. 3 respectively. Binodal curves for all systems have been presented in molality units in order to gain comprehensive idea of different components involved thus avoiding deviations caused by mere differences in molecular weights. However, experimental weight fraction data are presented in Table S1 and S2 of supplementary data. In a typical IL based ABS, two cations and two anions exist and during phase separation, they partition in such a way that electro neutrality is maintained. Rogers [11] and coworkers have experimentally

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proved that cloud point titration results based on salt concentrations rather than ion

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concentrations yields sufficient representation of concentration of ions at any ternary

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composition. Thus salts concentrations instead of ion concentrations have been considered throughout the experiment.

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Reports based on formation of ABS by various classes of ILs suggested that ILs consisting of tetra alkyl ammonium and phosphonium cations were competent in formation of

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ABS in presence of inorganic salts by easily salting out from aqueous solutions [11, 26]. Further the alkyl chain substituents of ILs were found to have significant influence on their phase

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behavior [22] and thus their systematic study is crucial for deeper understanding of phase formation. However, such methodical analysis is available only for limited classes of

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imidazolium and pyridinium based ILs [22]. Studies related to the effect of substituent of ammonium based ILs are few and there is still scope for developing clear understanding in this

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perspective. In case of biocompatible choline based ILs, the effect of hydrophilic hydroxyl groups on phase splitting behavior was reported [35-36] and suggested that such groups increase

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hydrophilicity of ILs thus reducing their relative phase splitting ability.

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In this context, present study is focused on analyzing the effect of benzyl group substituted on ammonium cation on formation of ABS. Phase behavior for selected benzyl group containing compounds, [BTMA]Cl and [BTBA]Cl in presence of potassium based salts can be analysed from binodal curves as given in Fig 2 and Fig 3 respectively. Binodal curves of [BTMA]Cl and [BTBA]Cl with K3PO4 are further compared in Fig 4 with already reported binodal curves for alkyl ammonium, alkyl phosphonium, choline, imidazolium and pyridinium based ILs [11, 35, 37] along with their benzyl substituted counterparts. It can be clearly observed from Fig 4 that, introduction of benzyl group in side chain of cations led to drastic change in 8

ACCEPTED MANUSCRIPT phase behavior of ILs i.e. the benzyl group has led to better phase separation and reason could be attributed to the presence of aromatic ring and corresponding increase in carbons that leads to weaker affinity for water [35]. As it can be observed from Fig 4, binodal curves of Benzyl choline chloride, [Bz Ch]Cl, 1-Benzyl 3-methyl imidazolium chloride [C7H7mim]Cl and Benzyltrimethylammonium chloride, [BTMA]Cl lie more towards origin as compared to their unsubstituted counterparts. Thus, appending the cation with benzyl group in ammonium based

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ILs has indeed promoted phase segregation leading to larger biphasic region as observed in case

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of imidazolium and pyridinium based ILs also [38]. The effect of benzyl group substitution was

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seen to be of greater extent in case of hydrophilic choline based ILs as compared to imidazolium based ILs which is depicted as larger gap between positions of binodal curves of [Ch]Cl and

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[BzCh]Cl

Further, with increase in alkyl chain length of cation, the hydrophobic nature of ILs

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increases and such compounds show lesser affinity for water. On this basis, it can be concluded that [BTBA]Cl which consists of longer butyl chains is more hydrophobic and shows higher

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ability in phase formation. This behavior can be confirmed from Fig. 4 by observing the position of binodal curve of [BTMA]Cl being away from origin in contrast to [BTBA]Cl whose binodal

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curve lies closer to origin. Similar behavior was observed for other salts also and it is represented

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in Fig S1.

In the present work, the influence of potassium salts on phase formation ability of

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[BTMA]Cl and [BTBA]Cl has also been analyzed. As can be seen from Fig. 2 and Fig. 3, for same IL, binodal curve of systems with K3PO4 salt is closest to origin and thus has largest

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biphasic region indicating that lower quantities of salt is sufficient to form ABS. For various salts with same cation but different anions, the phase splitting ability was found to follow the order: K3PO4> K2HPO4> K2CO3> KOH. This shows that salting-out ability of the anions follows the ordering PO4

3-

>HPO4 2- > CO3

2-

> OH-. The observed order is in accordance with well-known

Hofmeister series [39] and can be directly correlated with Gibbs free energy of Hydration (∆Ghyd) values for corresponding anions : −2835 kJmol-1, −1125 kJmol-1, −1300 kJmol-1 , −430 kJmol-1 for PO43-, HPO42- , CO32- and OH- respectively [11, 20]. These ions are known as kosmotropic ions as described in Hofmeister series and exhibit stronger interactions with water 9

ACCEPTED MANUSCRIPT molecules and readily form ABS by salting out ILs that are usually chaotropic in nature [40]. Further, the order observed implies that anions with higher valencies hydrate more water molecules thus decreasing the amount of water available to hydrate ILs [22] and therefore are better salting-out agents. The kosmotropicity of studied ions can also be explained based on ionic viscosity B

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coefficient values. Ionic viscosity B coefficient values helps in understanding the hydration of

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ions as well as their effects on structure of water present near solute components [41]. Bcoefficient values for ions involved in this study are: 0.495, 0.382, 0.294 and 0.122 dm3mol-1

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respectively for PO43- , HPO42- , CO32- and OH- [42]. Ions with higher positive B values are considered as kosmotropes because strongly hydrated ions exhibit a larger change in viscosity

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with concentration whereas negative values suggest that ions are chaotropic in nature [43].

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However, recent studies have emphasized that molar entropy of hydration plays an important role in determining phase formation capacity and showed that salts with highest values of molar entropy of hydration showed strongest salting-out effect. Molar entropy of hydration,

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∆Shyd values for PO4 3- , HPO4 2- , CO3 2- and OH- ions are as follows: −421, −272, −245 and −161

splitting behavior of salts.

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JK-1mol-1 respectively [44]. Thus ∆Shyd values provide additional support to the observed phase

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Experimental binodal curve data obtained by cloud point titration method were fitted using equation 1. Regression parameters of equation 1 i.e., A, B and C for all studied ABS

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systems are given in Table 1. Obtained regression coefficient values suggest that experimental data were well fitted to equation 1. Tie lines for each system were determined using equations 2-

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5. Ternary mixture compositions selected for tie line determination, with corresponding tie line compositions in top and bottom phases along with tie line length are given in Table 2. Two different TLs obtained for [BTBA]Cl and K3PO4 system at different concentrations denoted as TL5 and TL6 in Table 2 are represented in Fig. 5. Tie line length denotes the difference between IL and inorganic salt concentrations in top and bottom phases, therefore higher TLL indicates the higher concentration of IL in the top phase and consequently higher salt concentration in bottom phases. In presently studied ABS systems, top phase corresponds to IL rich phase and bottom phase is mainly composed of inorganic salts similar to typical ABS systems [22, 24]. 10

ACCEPTED MANUSCRIPT 3.2. Partitioning of Tryptophan The partition coefficients of Tryptophan,

in studied ABS depend on various

interactions that exist in system including dispersive forces, hydrogen bonding, electrostatic interactions, hydrophobic interactions and π-π interactions [4, 37] among which hydrophobic interactions play dominant role [45]. Higher values of partition coefficients indicate the higher

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tendency of selected biomolecule to migrate to the aqueous IL-rich phase.

Ternary system compositions selected for extraction experiments in present work are: 25

% water. Partition coefficients of Tryptophan, (

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wt % [BTMA]Cl + 22 wt % salt + 53 wt % water and 22 wt % [BTBA]Cl + 13 wt % salt + 65 wt ) for these selected ternary systems are

is given in Fig. 6 and 7 respectively. Further

values along with extraction efficiencies

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calculated using equation 8 are given in Table S3.

values for [BTMA]Cl and [BTBA]Cl

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calculated using equation 7. Graphical presentation of

values obtained for studied ABS are significantly higher than those obtained by

values obtained for water immiscible imidazolium ILs like [C6mim][BF4] and

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to

≈ 1-7 [46]. Further, they are also higher compared

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conventional polymer-salt based ABS :

[C8mim][BF4] [47] as well as water miscible imidazolium ILs [37] with variable anions [48].

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The presence of benzene ring in ILs lead to π-π stacking between aromatic rings of IL and Tryptophan and in addition, strong N-H---π interactions also exist where pyrrole group in

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Tryptophan acts as NH donor and aromatic benzyl group as acceptor [49] and as a consequence, significantly higher partition coefficient values were obtained. The enhancement of

values

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due to the presence of benzyl group was observed in case of imidazolium based ILs also [37]. Furthermore,

values obtained are also appreciable as compared to hydrophobic

imidazolium [49] as well as phosphonium based ILs [26] thus indicating that the nature of anions and chemical structures of cations have significant influence on partition coefficient values. Various studies have reported the analysis of extraction capabilities of different classes of ILs based ABS for Tryptophan [22]. However, systematic evaluation of effect of salts on extraction capabilities for Tryptophan is also important though less explored. In the present 11

ACCEPTED MANUSCRIPT work, we have attempted to analyze and gain some insights into effect of salts on

values. In

case of both [BTM]Cl and [BTBA]Cl containing systems, highest partition coefficients were obtained for K3PO4 and the order of effect of salts observed was as follows: K3PO4 > K2HPO4> K2CO3> KOH. This indicates that influence of salts on partitioning of Tryptophan is based on their salting out ability trend. values has also been

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The influence of salts other than usual inorganic salts on the

values (ranging from 3 to 67) with

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combination with ILs resulted in appreciably higher

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explored. In a study based on biodegradable citrate salt as phase forming component in extraction efficiencies well above 70% [50]. The results indicated that apart from π-π

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interactions, partition coefficients depends on various other factors like hydrogen bonding interactions, hydrophobic interactions as well as balance between amount of charged species and compositions of coexisting phases. However, ILs in combination with carbohydrates resulted in values with extraction efficiencies of 50% for all studied carbohydrates

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much lower to

[51]. This suggests that interactions between carbohydrates and Tryptophan were insignificant in

M

this case.

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In present study, systems consisting of K3PO4 salt showed highest efficiency in extraction and this appreciable efficacy can be related to its higher salting-out ability. The phase-forming

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salt strongly influences the hydrophobic character of the IL rich phase as well as partitioning of amino acids through its salting–out effects. Thus presence of benzyl group in cation of ILs in

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combination with inorganic salt with higher salting-out ability facilitated in the enhancement of

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partition coefficient values. 3.3. Density and viscosity

Important thermophysical properties like density and viscosity were measured for selected ternary compositions as a function of temperature in the range of (293.15-328.15) K and at atmospheric pressure. Compositions of IL and salt selected for this study are specified in Table 2. For systems composed of [BTMA]Cl, weight fraction compositions selected for measuring density and viscosity was 22 wt % of salt+ 25 wt % of [BTMA]Cl and 53 wt % water and it is indicated as TL1 in Table 2. Few more compositions were also selected as follows: 19 wt% of 12

ACCEPTED MANUSCRIPT K3PO4+ 22 wt % of [BTMA]Cl (denoted as TL2); 21 wt% of K2HPO4+ 21wt % of [BTMA]Cl (denoted as TL3) and 25 wt% of KOH+ 25 wt % of [BTMA]Cl (denoted as TL4). Similarly for systems composed of [BTBA]Cl, weight fraction compositions selected for measuring density and viscosity was 13 wt % of salt+ 22 wt % of [BTBA]Cl and 65 wt % water and it is indicated as TL5 in Table 2. One more compositions was also selected for comparison

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and is as follows: 12 wt % of K3PO4 + 22 wt % of [BTBA]Cl (denoted as TL6)

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The plot of variation of density data for systems containing [BTMA]Cl for selected

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ternary compositions (represented as TL1) are presented in Fig. 8 and Fig. 9 represents variation in density for systems with [BTBA]Cl (at composition represented as TL5). Corresponding

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experimental density data are presented in Tables S4 and S5. In general, densities of all systems were observed to decrease with increase in temperature. Same behavior was observed for both

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top and bottom phases of all ABS systems studied. In addition, density of bottom phases or salt rich phases was observed to be higher in comparison with top or IL rich phases. The reason

M

could be attributed to dominant presence of inorganic salts of higher densities in bottom phases. Further, to understand the comparison between both studied ILs, the plot demonstrating variation

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in density for systems containing both [BTMA]Cl and [BTBA]Cl is given in Fig. S3. For systems denoted as TL1, density values observed for top phases were quite similar,

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whereas density values of bottom or salt rich phases varied over comparatively broader range. For eg, in [BTMA]Cl systems, at 298.15 K, densities range between 1.0704 gcm-3 (K3PO4) and

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1.1043 gcm-3 (KOH) for top phase. Whereas in case of bottom phases values range between 1.4185 gcm-3 and 1.2681 gcm-3. Similar trend was observed for [BTBA]Cl containing systems

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also. This variation can clearly be observed from Fig. 8 and Fig. 9. The observed difference in densities is due to the presence of higher concentration of salts in bottom phases but in less concentration in top phases. Variation in density data with change in tie line length or in other terms, with change in water composition has also been analyzed. It was observed that for IL rich phases, increase in TLL lead to decrease in density values, whereas opposite trend was observed in bottom phases i.e. increase in TLL lead to increase in density values. This can be further observed clearly from 13

ACCEPTED MANUSCRIPT Fig. 10 A and 10 B in which densities of [BTMA]Cl +K3PO4 (TL1 and TL2) and [BTBA]Cl +K3PO4 (TL5 and TL6) systems were represented at variable TLL. Similar behavior was also observed in case of ABS formed by imidazolium based ILs with sodium and potassium acetate salts [31]. In comparison with conventional polymer-salt ABS, for systems studied in this work, the difference between densities of coexisting phases was higher thus facilitating easier and

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faster separation of top and bottom phases [52].

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The other important property that has been measured for selected ternary systems is

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viscosity. Viscosity of a system mainly depends on various inter and intra molecular interactions and most dominating type of interactions involved in present aqueous systems includes hydrogen bonding and columbic interactions. As expected, viscosity values of studied systems decreased

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with increase in temperature over observed temperature range of 293.15 to 328.15 K.

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The variation in viscosity of systems containing [BTMA]Cl with various salts is presented in Fig. 11 and the representative plot of [BTBA]Cl is given in Fig. 12. In general, for

M

ABS systems studied in present work, viscosities of IL rich phases were found to be higher in comparison with those of bottom salt rich phases. For IL rich top phases, viscosity values were

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found to decrease with decrease in the IL content and corresponding increase in water content. For ABS system of [BTMA]Cl and K3PO4 at 298.15 K, viscosity values of top phase follow the

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order: 6.287 mPas (56.67 wt % IL + 42.61 wt % water)> 4.512 mPas (44.06 wt % IL + 53.04 wt % water). Similar behavior was observed for [BTBA]Cl and K3PO4 at 298.15 K, 7.999 mPas

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(38.96 wt % IL + 57.3 wt % water)> 6.716 mPas (35.22 wt % IL + 60.05 wt % water). Similarly, viscosity values decreased with increase in water content and corresponding decrease

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in salt content. The trend in viscosity values can also be observed from Tables S6 and S7. Viscosity values for IL rich phases were observed to be much lower (2-15 mPas), when compared to typical polymer based ABS (around 50 mPas) [8] and were in the same range of imidazolium [31] and phosphonium [26] IL based systems. The lower viscosity values are significant since they simplify the mass transfer as well as phase separation processes. 4. Conclusions

14

ACCEPTED MANUSCRIPT Ionic Liquids are currently considered as potential phase forming promoters of ABS that can be used in liquid-liquid separation processes. With an intention to develop highly potential separation systems, we report here new set of ternary phase diagrams composed of [BTMA]Cl and [BTBA]Cl in combination with inorganic salts like K3PO4, K2HPO4, K2CO3 and KOH. Ternary phase diagrams for all systems and corresponding tie line compositions along with tie line lengths have been studied and reported in present work. The effect of salts on phase

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behavior of [BTMA]Cl and [BTBA]Cl have been analyzed and their phase splitting ability have

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been found to follow the order: K3PO4> K2HPO4> K2CO3> KOH. The experimental order

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determined in present work was observed to follow well known Hofmeister series. Further, these ternary systems were scrutinized for their efficiency to extract Tryptophan. Systems in

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combination with salt of highest salting-out ability i.e., K3PO4 was observed to show the highest extraction efficiency among studied ABS systems. In addition, densities and viscosities of

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coexisting phases were also determined. For all studied systems, densities of bottom phases were found to be higher when compared to that of top phases due to the higher concentration of salts

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present in bottom phases. Viscosities of coexisting phases of all studied ternary systems were observed to be much lower as compared to viscosities exhibited by polymer based ABS thus

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raising their industrial importance. Experimentally obtained partition coefficient values and values of densities and viscosities suggested that ABS systems based on [BTMA]Cl and

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[BTBA]Cl and potassium based inorganic salts are potential candidates for extraction of bioactive compounds like Tryptophan.

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Acknowledgments

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Authors thank IIT Madras for the financial support through Institute Research and Development Award (IRDA): CHY/15-16/833/RFIR/RAME.

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ACCEPTED MANUSCRIPT List of symbols

wt %

weight fraction in percentage

T

Temperature

∆Shyd

Entropy of Hydration

ρ

Density

η

Viscosity

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Gibbs free energy of Hydration

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M

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∆Ghyd

T

Partition coefficients of Tryptophan

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ACCEPTED MANUSCRIPT Figure Captions:

Figure 1: Structures of (A) Benzyltrimethyl ammonium chloride (B) Benzyltributylammonium chloride and (C) Tryptophan. Figure 2: Phase diagram for systems composed of [BTMA]Cl and K3PO4, K2HPO4, K2CO3

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T

and KOH at 298.15 K and atmospheric pressure.

KOH at 298.15 K and atmospheric pressure.

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Figure 3: Phase diagram for systems composed of [BTBA]Cl and K3PO4, K2HPO4, K2CO3 and

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Figure 4: Phase diagram for systems composed of [N4444]Cl (ref. 11), [P4444]Cl (ref. 11), [Ch]Cl (ref. 35), [BzCh]Cl (ref. 35), [C1mim]Cl (ref. 37), [C4mim]Cl (ref. 37),

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[C7H7mim]Cl (ref. 37), [C4Py]Cl (ref. 37), [BTMA]Cl and [BTBA]Cl with K3PO4 at 298.15 K and atmospheric pressure.

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Figure 5: Phase diagram representing tie-line data for systems composed of [BTBA]Cl and

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and K3PO4. Solid line and dotted line represents ternary composition denoted as TL5 and TL6 respectively.

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Figure 6: Bar diagram representing partition coefficients of Tryptophan,

between

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[BTMA]Cl rich and salt rich phases. Figure 7: Bar diagram representing partition coefficients of Tryptophan,

between

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[BTBA]Cl rich and salt rich phases. Figure 8: Experimental density as a function of temperature for systems containing [BTMA]Cl and K3PO4 (■), K2HPO4 (▲), K2CO3 (◄) and KOH(♦). Open symbols represent top phase and closed symbols represent bottom phases.

20

ACCEPTED MANUSCRIPT Figure 9: Experimental density as a function of temperature for systems containing [BTBA]Cl and K3PO4 (●), K2HPO4 (★), K2CO3 (►) and KOH(▼). Open symbols represent top phase and closed symbols represent bottom phases. Figure 10: Experimental density as a function of temperature for ternary systems represented as TL1

and TL5 (closed symbols); TL2 and TL6 (open symbols) for [BTMA]Cl+

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K3PO4 (■) and [BTBA]Cl +K3PO4 (●). A- top phase ; B- bottom phase.

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Figure 11: Experimental viscosity as a function of temperature for systems containing [BTMA]Cl with K3PO4 (■), K2HPO4 (▲), K2CO3 (◄) and KOH(♦). Open symbols

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represent top phase and closed symbols represent bottom phases. Figure 12: Experimental viscosity as a function of temperature for systems containing

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[BTBA]Cl with K3PO4 (●), K2HPO4 (★), K2CO3 (►) and KOH(▼).Open symbols

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represent top phase and closed symbols represent bottom phase.

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ACCEPTED MANUSCRIPT Table 1: Fitting Parameters obtained from Equation 1 along with standard deviations and regression coefficients.

A±σ

B±σ

105×(C ± σ)

R2

K3PO4

72.7 ± 0.4

-0.294 ± 0.002

5.33 ± 0.05

0.9999

K2HPO4

81.9 ± 0.8

-0.314 ± 0.004

3.22 ± 0.09

0.9993

K2CO3

89.2 ± 0.8

-0.304 ± 0.003

4.12 ± 0.06

0.9998

KOH

104.7 ± 2.7

-0.261 ± 0.008

3.98 ± 0.08

0.9996

K3PO4

84.8 ± 0.6

-0.399 ± 0.004

10.00 ± 0.30

0.9995

K2HPO4

85.1 ± 0.4

-0.399 ± 0.003

8.25 ± 0.16

0.9997

K2CO3

93.6 ± 0.8

-0.426 ± 0.005

10.00 ± 0.45

0.9991

KOH

108.5± 1.0

-0.407 ± 0.004

20.00 ± 0.28

0.9998

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M

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[BTBA]Cl

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[BTMA]Cl

T

Salt

IL

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ACCEPTED MANUSCRIPT Table 2: Weight fraction compositions of salt [X] and IL [Y] in mixture (M), top (T) and bottom (B) phases along with tieline length (TLL) and IL

Salt

XT

YT

XB

YB

TLL

ID

0.69

67.08 TL1

19.04 22.00 2.90 44.06 33.95

1.63

52.58 TL2

K2HPO4 22.00 24.99 2.19 51.42 39.56

1.55

62.31 TL1

21.07 21.07 4.96 40.53 36.29

2.66

49.16 TL3

K2CO3

22.09 24.99 2.58 54.75 37.42

1.60

63.55 TL1

KOH

22.61 24.83 6.42 53.52 33.85

4.90

55.83 TL1

24.98 24.98 5.03 58.04 38.85

2.00

65.45 TL4

13.06 21.99 3.74 38.96 23.14

3.60

40.34 TL5

11.99 22.00 4.73 35.22 21.24

5.17

34.29 TL6

K2HPO4 13.05 21.98 4.85 34.97 24.86

3.29

37.47 TL5

K2CO3

13.16 21.93 3.75 40.80 21.84

4.51 40.55 TL5

KOH

13.06 21.99 3.99 47.54 19.36

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K3PO4

T

21.79 24.76 0.72 56.67 37.68

[BTMA]Cl

4.24

45.95 TL5

AC

CE

PT

ED

[BTBA]Cl

YM

M

K3PO4

XM

values for studied ternary systems.

23

(B)

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Fig. 1

(C)

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(A)

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Fig. 3

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Fig. 4

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Fig. 5

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Fig. 6

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Fig. 7

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Fig. 8

31

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Fig. 9

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(B)

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(A)

Fig. 10 (A) and 10 (B) 33

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Graphical abstract

36

ACCEPTED MANUSCRIPT Research Highlights:

Analyzed the phase behavior for ternary systems: water+ionic liquid+potassium salt.



Phase splitting capability explored in terms of benzyl group substitution in IL.



Evaluated the effect of potassium salts in terms of Hofmeister series.



Enhanced partition coefficients have been observed for Tryptophan.

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

37