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Odd-even effect in the formation and extraction performance of ionic-liquid-based aqueous biphasic systems Diana Cléssia Belchior, Mafalda R. Almeida, Tânia E. Sintra, Sonia P.M. Ventura, Iola F Duarte, and Mara G. Freire Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.9b00663 • Publication Date (Web): 18 Apr 2019 Downloaded from http://pubs.acs.org on April 21, 2019

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Industrial & Engineering Chemistry Research

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Odd-even effect in the formation and extraction

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performance of ionic-liquid-based aqueous

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biphasic systems

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Diana C. V. Belchior a, Mafalda R. Almeida a, Tânia E. Sintra a, Sónia P. M. Ventura a, Iola F.

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Duarte a, and Mara G. Freire a*

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a CICECO

- Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810193 Aveiro, Portugal

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* Corresponding author. Tel: +351-234-370200; Fax: +351-234-370084; E-mail address:

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[email protected]

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1 ACS Paragon Plus Environment

Industrial & Engineering Chemistry Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Aqueous biphasic systems constituted by ionic liquids (IL-based ABS) are a target of investigation

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in the separation of high-value biomolecules. However, the identification of the molecular-level

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mechanisms ruling two-phase formation and extraction performance of these systems is crucial to

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successfully design effective separation processes. In this work, IL-based ABS formed by K2HPO4

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and cholinium carboxylate ILs ([Ch][CnCO2] with n = 1 to 7, comprising anions with odd and even

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alkyl chain length) were investigated. The corresponding ternary phase diagrams, including

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binodal curves, tie-lengths, tie-line lengths and critical points, as well as the Setschenow salting-

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out coefficients (ks), which quantitatively describe the two-phase formation ability, were

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determined at 298 K. The extraction capability of these systems was then evaluated for four amino

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acids (L-tryptophan, L-phenylalanine, L-tyrosine, L-3,4-dihydroxyphenylalanine/L-dopa). It was

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found that ILs composed of anions with even alkyl chains display slightly higher ks values,

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meaning that these ILs are more easily salted-out or more easily phase separate to form ABS,

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whereas ABS formed by ILs with anions comprising odd alkyl chains lead to slightly higher

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partition coefficients of amino acids. Beyond the neat ILs odd-even effect resulting from their

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nanostructuration, being this a well-known phenomenon occurring in a series of their

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thermophysical properties, it is here shown the existence of an odd-even effect displayed by the

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IL anion aliphatic moiety in aqueous solution, visible both in the two-phase formation ability and

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extraction performance of ABS. These findings contribute to elucidate the molecular-level

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mechanisms governing ABS formation and partitioning of biomolecules, ultimately contributing

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to the design of proficient separation platforms.

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Keywords: Aqueous two-phase systems, cholinium carboxylate ionic liquids, salting-out

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coefficient, extraction, partition coefficient, odd-even effect. 2 ACS Paragon Plus Environment

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1. Introduction

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Separation and purification steps within biotechnological processes are still major

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challenges due to difficulties in purifying and recovering the target compounds from the original

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complex media in which they are produced.1 Furthermore, these should be cost-effective and

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should be able to keep the products biological activity. Research on alternative separation

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techniques has been carried out with Aqueous Biphasic Systems (ABS), which correspond to

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liquid-liquid extraction systems formed by water and two solutes (e.g. a polymer and a salt, two

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polymers, two salts, etc.) that undergo phase separation above defined concentrations.2-5 In ABS,

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both phases are majorly constituted by water, meaning that such media may be compatible with

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biologically active molecules.2

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In addition to more conventional ABS formed by polymers, in the last decade, a substantial

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interest has been placed in ionic-liquid-based ABS, initially demonstrated to be formed by adding

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ionic liquids to inorganic salts aqueous solutions.6 Ionic liquids (ILs) are poorly coordinated

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organic salts, and hence may be liquid at or close to room temperature. Besides other relevant

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properties, the major advantage associated to IL-based ABS relies on these compounds designer

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ability, resulting in a virtually endless opportunity of tuning the ILs chemical structures for specific

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applications.7 As a result, IL-based ABS have been successfully applied in the separation of a wide

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number of biomolecules, such as amino acids, proteins, antioxidants, among others.8 Still, most of

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the ILs studied up to date are imidazolium-based, most of the times combined with [BF4]-, which

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may raise some toxicity and biodegradability concerns.8 The ample flexibility of ILs should

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however permit the preparation of new compounds with an acceptable environmental footprint and

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biocompatibility to be used in the creation of novel ABS. Cholinium-based ILs are a promising

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family of ILs, which can be prepared from natural sources, and may present high biodegradability 3 ACS Paragon Plus Environment


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and, in some cases, marginal (eco)toxicity.9-11 In particular, cholinium chloride is an essential

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nutrient with an important role, namely as precursor in the synthesis of vitamins (e.g., thiamine)

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and enzymes that participate in the carbohydrates metabolism.12-13 Taking into account their

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advantageous features, several works have been published reporting the use of cholinium-based

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ILs to form ABS with salts. Xie et al.14 investigated novel ABS formed by four cholinium-based

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ILs comprising carboxylate anions and K3PO4 to extract tryptophan, phenylalanine, and caffeine.

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The authors reported novel ABS liquid-liquid phase diagrams and showed their enhanced

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extraction performance, as well as the presence of liquid crystal structures in ABS formed by ILs

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with long alkyl chain anions. Shahriari et al.15 determined the phase diagrams of novel ABS formed

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by a series of cholinium-based ILs with different anions and K3PO4, having evaluated their

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performance to extract antibiotics. This type of ABS has also been used to extract antibiotics from

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real fermentation broths,16 and it was latter demonstrated that they represent a more economical

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alternative to recover tetracycline from the fermentation broth than more traditional ABS formed

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by polymers.17

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To design effective ABS for separation processes, it is relevant to determine and

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characterize their phase diagrams. The respective ternary phase diagrams provide valuable data on

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the phase-forming components compositions required to form two-phase systems, and on the

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composition of each phase for a given overall mixture. Accordingly, a significant number of works

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have focused on the determination of novel IL-based ABS ternary phase diagrams.8 Furthermore,

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it is well known that ILs comprising long alkyl side chains tend to self-aggregate in aqueous media,

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a trend that was further demonstrated to occur in ABS18 and that may lead to unfavorable extraction

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results.19 In a previous work, the impact of the IL cation alkyl side chain length in two-phase

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separation and creation of ABS has been comprehensively investigated by employing the 1-alkyl4 ACS Paragon Plus Environment

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3-methylimidazolium chloride series, with both odd and even alkyl side chains.20 An odd-even

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effect was identified on the Setschenow salting-out coefficient (ks), which quantitatively addresses

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the two-phase formation capacity, as a function of the number of methylene groups at the longest

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IL cation alkyl side chain.20 ILs comprising cations with even alkyl side chains have higher molar

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volumes and are thus more easily salted-out, meaning that more easily undergo phase separation

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in aqueous media. Although this trend was detected for the IL cation, the effect of the IL anion on

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ABS formation is scarcely studied. Some reports exist on the effect of the IL anion alkyl chain

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length on the formation of ABS,14,21–24 but most of these studies employed ILs with chemically

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different anions or only with even alkyl chains at the anion. In general, it is understood that ILs

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with longer aliphatic moieties at the anion are more easily salted-out and need less inorganic salt

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to undergo phase separation. However, no studies on the self-aggregation impact of long alkyl

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chain anion ILs and on the possibility of an odd-even effect have been performed. Furthermore, to

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the best of our knowledge, the impact of the odd-even effect in the partitioning of target molecules

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in ABS was never investigated. It should be remarked that although the odd-even effect is scarcely

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studied in aqueous solutions, the ILs odd-even effect was already reported in a series of neat ILs

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properties, such as molar volumes, viscosities and entropy and enthalpy of vaporization.25-28 Santos

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and co-workers26-28, by studying several series of imidazolium-based ILs with cations of different

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alkyl side chain length, demonstrated that the odd-even effect is a result of the ILs nanosegregation

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and consequent impact in these fluids cohesive energy.

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Aiming at better understanding the influence of the IL anion alkyl chain length on the

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creation of ABS, in this work, seven cholinium-based ILs with carboxylate anions, from acetate to

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octanoate and comprising both even and odd alkyl side chains ([Ch][CnCO2], n = 1 to 7), were

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synthesized and used in ABS formation with dipotassium hydrogen phosphate (K2HPO4). The 5 ACS Paragon Plus Environment


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corresponding ternary phase diagrams were determined at (298 ± 1) K, including tie-lines and

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critical points, and further evaluated in terms of their extraction capability for amino acids, namely

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L-tryptophan, L-phenylalanine, L-tyrosine and L-3,4-dihydroxyphenylalanine/L-dopa. The

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Setschenow coefficient of each ABS was determined, and the odd-even effect was addressed in

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both ABS formation and amino acids partitioning between the coexisting phases.

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2. Materials and methods

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2.1. Materials

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ABS were prepared using aqueous solutions of potassium phosphate, K2HPO4 (>98% of

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purity), purchased from Sigma–Aldrich, and aqueous solutions of each IL. The investigated

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cholinium-based ILs comprise anions derived from carboxylic acids, corresponding to cholinium

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acetate ([Ch][C1CO2], > 98 wt % pure) from Iolitec, and cholinium propanoate ([Ch][C2CO2]),

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cholinium butanoate ([Ch][C3CO2]), cholinium pentanoate ([Ch][C4CO2]), cholinium hexanoate

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([Ch][C5CO2]), cholinium heptanoate ([Ch][C6CO2]), and cholinium octanoate ([Ch][C7CO2]) that

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were synthetized in our laboratory following protocols previously described.29 All ILs synthesized

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showed high purity (>97 wt%) (1H and 13C NMR results are given in the Supporting Information).

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The required precursors, namely cholinium hydroxide solution (46 wt%, Sigma-Aldrich),

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propanoic, butanoic, pentanoic, hexanoic and heptanoic acids (99 %, Acros Organics), and

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octanoic acid (98 %, Sigma-Aldrich), were commercially acquired. Briefly, the corresponding

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acids were added drop wise into an aqueous solution of [Ch][OH] (1.1 equivalents of acid) at 298

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K, being the resultant mixture stirred at room temperature and under inert atmosphere for at least

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12 h to produce the cholinium-based ILs. The mixtures were then dried for 4 h under vacuum. The


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where A, B, and C are constants obtained by the regression, and [IL] and [Salt] correspond to the

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IL and salt weight fraction percentages, respectively.

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Tie-lines (TLs) associated to each binodal curve, i.e the composition of each phase for a

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target mixture composition, were determined by a gravimetric method.30. Several ternary mixtures

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constituted by IL + K2HPO4 + water at the biphasic region were prepared by weight and vigorously

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mixed. Each mixture was centrifuged for 30 min at 298 K aiming for the separation of the two

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phases. Both phases were carefully separated and weighed. Each TL was determined by the lever-

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arm rule30 according to the following equations (2-5):

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[IL]T = exp [( × [Salt]0.5 T )

( × [Salt]3T)]

(2)

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[IL]B = exp [( × [Salt]0.5 B )

( × [Salt]3B)]

(3)

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[IL]T =

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[Salt]T =

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where the subscripts M, T and B represent the initial mixture, the top and bottom phases,

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respectively. K corresponds to the ratio between the weight of the top phase and the total mass of

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the system. In all systems, the top phase is enriched in IL (IL-rich phase), whereas the salt is

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majorly enriched in the bottom phase.

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[IL]M

1

[Salt] M

(4)

× [IL]B 1

(5)

× [Salt]B

Each tie-line length (TLL) was determined by the following equation: TLL = ([Salt]T

[Salt]B)2 + ([IL]T

[IL]B)2

(6)


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The critical point of each system, i.e. when the composition of the two aqueous phases is

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identical, was determined through the application of the Sherwood method using the TLs slopes,31

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according to equation (7),

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(7)

!" = # $%&' + (

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where f and g are obtained from the fitting, and [IL] and [salt] are the weight fraction percentages

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of IL and Salt.

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The magnitude of salting-out may be quantified by correlating solubility values with the

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empirical equation of Setschenow.32 In this work the modified version of the original Setschenow

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equation proposed by Hey et al.33, previously used to characterize polymer-based ABS34 and of

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IL-salt-based ABS34-35, was applied:

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&)[IL]B = *!"([IL]B

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where T and B designate the top (IL-rich) and bottom (salt-rich) phases, respectively, and [IL] and

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[Salt] correspond to the molality of IL and salt. kIL and ks parameters are related with the IL activity

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coefficient to its concentration and the salting-out coefficient, respectively.

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[IL]T

([IL]T) + *$([Salt]B [Salt]T)

(8)

2.3. Cation-anion interaction energies

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The cation-anion total interaction energies of each IL, EInt, were determined using the

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COSMO-RS thermodynamic model. This model combines quantum chemistry, based on the

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dielectric continuum model known as COSMO (COnductor-like Screening MOdel for Real

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Solvents), with statistical thermodynamic calculations. The process employed in this work to

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determine the interaction energies was described by Kurnia et al.36 The COSMO quantum 9 ACS Paragon Plus Environment


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chemical calculations were performed with the TURBOMOLE 6.1 program package at the density

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functional theory (DFT) level. The BP functional B88-P86 with a triple-z valence polarized basis

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set (TZVP) and the resolution of identity standard (RI) approximation were applied.37 The

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COSMOthermX program and the parameter file BP_TZVP_C20_0111 (COSMOlogic GmbH &

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Co KG, Leverkusen, Germany) were used.38

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2.4. Extraction of amino acids

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ABS containing amino acids aqueous solutions (5 g.L-1 for L-tryptophan, 15 g.L-1 for L-

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phenylalanine, 2.5 g.L-1 for L-tyrosine and 1.5 g.L-1 for L-dopa, defined according to the amino

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acids solubility in water), were prepared and used to study the amino acids partitioning between

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the ABS coexisting phases. The compositions of the ternary systems applied in the partitioning

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experiments were defined according to the determined phase diagrams and TLs. To circumvent

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inconsistencies in results, which could result from the different phase compositions and, with the

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goal of addressing the odd-even effect in the amino acids partitioning, all extraction studies were

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carried out at a constant TLL (ca. 58 ± 2). Each mixture was vigorously stirred to allow

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equilibration and then centrifuged for 30 min at 298 K to reach complete separation between the

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coexisting phases. Both phases were separated, and the amount of amino acid in each phase was

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quantified by UV-spectroscopy, using a BioTeck Synergy HT microplate reader using calibration

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curves previously determined (at a wavelength of 280 nm for L-tryptophan, L-tyrosine and L-

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dopa, and at 260 nm for L-phenylalanine). At least three ABS with each amino acids were

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prepared, and at least three samples of each phase were quantified.

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The ABS capability to extract different amino acids to the IL-rich phase was evaluated by

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their partition coefficient (KAA). This parameter corresponds to the ratio of the concentration of


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each amino acid in the phase enriched in IL to that in the salt-rich phase, according to equation

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(9).

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+AA =

!"

(9)

,%&'

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where [AA]IL and [AA]Salt correspond to the concentration of each amino acid (AA) in the phases

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enriched in IL and salt, respectively.

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The pH of the IL- and K2HPO4-rich aqueous phases was determined at 298 K using a

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Mettler Toledo S47 SevenMulti™ dual meter pH/conductivity equipment (data shown in the

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Supporting Information).

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3. Results and Discussion

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3.1. Phase diagrams and critical points

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New ternary phase diagrams of ABS composed of water, K2HPO4 and cholinium-based

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ILs with carboxylate anions of different alkyl chain length were determined in this work at (298 ±

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1) K. Their orthogonal representations, both in percentage weight fraction and in molality units,

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are shown in Figure 2. The weight fraction detailed results are given in the Supporting Information

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(Table S1-S7). Experimental binodal data were fitted by Equation (1), whose fitting is given in

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Figure 2. The fitted constants A, B and C were estimated by the least squares regression, being teir

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values given in the Supporting Information (Table S8). At least 4 TLs and respective length (TLL)

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were determined for each system by Equations (2) to (6), with the detailed results shown in the

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Supporting Information (Table S9). In addition, the critical point of each system was determined,

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being provided in Figure 2 (cf. the Supporting Information for detailed data - Table S10).



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Since all ILs are composed of a common cholinium cation, the results depicted in Figure 2

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mirror the effect of the IL anion on the formation of ABS with K2HPO4. The two-phase region is

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located above the solubility curve, and the larger this region is, the more prone the IL is to be

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salted-out by K2HPO4 and to form ABS. It is well documented39 that small IL anions derived from

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organic acids are excellent hydrogen bond acceptors and exhibit high capability to establish

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hydrogen bonds with water. Furthermore, the hydroxyl group at the cholinium cation can establish

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strong hydrogen bonds or dipole-dipole interactions with water molecules.40,41 Therefore,

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cholinium carboxylate ILs display a high affinity for water and only moderate to strong salting-

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out salts able to create ABS. Accordingly, it was previously shown that these ILs form ABS with

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K3PO4,14 and in this work we show that they form ABS with K2HPO4. However, the studied

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cholinium carboxylate ILs do not form ABS with weaker salting-out salts, such as KH2PO4 or

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K2HPO4/KH2PO4 at pH 7 (as experimentally attempted by us in this work - data not shown).

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The investigated ILs capacity to form ABS at ~ 22 wt% of K2HPO4 follows the order:

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[Ch][C1CO2] S [Ch][C7CO2] < [Ch][C6CO2] < [Ch][C2CO2] < [Ch][C5CO2] < [Ch][C3CO2] S

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[Ch][C4CO2]. When weight fraction is used [Figure 2(a)], a straight relationship of the ILs’

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aptitude to be salted-out cannot be straightforwardly assessed. When using molality units, the

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differences arising from different molecular weights of ILs are circumvented, consenting a more

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accurate analysis of the effect of the several phase-forming components on endorsing liquidWliquid

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demixing. The binodal curves represented in molality units, shown in Figure 2(b), prove that the

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ILs performance to create ABS, e.g. at 2.0 mol·kgW1 of K2HPO4, follows the rank: [Ch][C1CO2]

Odd-even effect in the formation and extraction ... - ACS Publications

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