Environmentally Benign and Recyclable Aqueous Two-Phase System

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Environmentally benign and recyclable aqueous two-phase system composed of distillable CO2-based alkyl carbamate ionic liquid Cher Pin Song, Qian Yi Yap, Mon Yin aigness Chong, Ramanan Ramakrishnan Nagasundara, Vijayaraghavan Ranganathan, Douglas R. Macfarlane, Eng-Seng Chan, and Chien Wei Ooi ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01685 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018

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ACS Sustainable Chemistry & Engineering

Environmentally benign and recyclable aqueous two-phase system composed of distillable CO2-based alkyl carbamate ionic liquid

Cher Pin Song,a Qian Yi Yap,a Mon Yin aigness Chong,a Ramanan Ramakrishnan Nagasundara,a Vijayaraghavan Ranganathan,b Douglas R. MacFarlane,b Eng-Seng Chana and Chien-Wei Ooi*a

a

Chemical Engineering Discipline, School of Engineering, Monash University Malaysia,

Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia. b

School of Chemistry, Faculty of Science, Monash University, Clayton, VIC 3800, Australia.

* Corresponding author: Chien-Wei Ooi (Dr.) Chemical Engineering Discipline, School of Engineering, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia Tel: +60 3 55146201; Fax: +60 3 55146207 E-mail address: [email protected] 1 ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 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 Ionic

liquid-based

aqueous

two-phase

systems

(IL-ATPSs)

have

been

studied

comprehensively as a potential alternative method for protein purification. One of the distinct features of IL-ATPS is that the polarity of IL can be adjusted by different combinations of cation and anion, providing the flexibility in governing the partition of proteins between phases. However, the conventional IL-ATPS usually has poor environmental footprint and low recyclability, thereby hampering the systems for practical use in protein separation. In order to address these shortcomings, here we explored the distillable CO2-based alkyl carbamate

ILs

as

phase-forming

components.

N,N-dimethylammonium

N’,N’-

dimethylcarbamate (DIMCARB) was able to form ATPS with polypropylene glycol (PPG). The liquid-liquid equilibrium data of ATPSs composed of PPG with different molecular mass and DIMCARB were determined. As both phase-forming components are thermo-sensitive, the effect of temperature on the phase diagram of the PPG 400 + DIMCARB system was also evaluated. The partition behaviour of model proteins (i.e., bovine serum albumin and lysozyme) in the PPG + DIMCARB systems were assessed. Both model proteins tend to migrate to the more hydrophilic IL-rich phase, when a higher temperature or a higher molecular mass of PPG was used. The DIMCARB was recovered via rotary evaporation, while the PPG 400 was recovered from a secondary two-phase system thermally induced from the primary ATPS. The recovered components were subsequently used to prepare a new batch of ATPS. The phase composition of the new system as well as the partition coefficients of both model proteins in the recycled system were maintained, thereby proving the feasibility of reusing both phase-forming components. Overall, the recyclable and biodegradable phase-forming components used in the newly developed PPG + DIMCARB system make the ATPS a sustainable and eco-friendly system for protein purification.

Keywords: dialkyl carbamate, poly(propylene glycol), liquid-liquid equilibrium, protein partitioning, recycling, secondary aqueous two-phase system

2 ACS Paragon Plus Environment

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Introduction Aqueous two-phase systems (ATPSs) have been widely regarded as a promising alternative to the chromatographic techniques for the purification of biomolecules.1 The advantages of ATPSs include the simplicity, low cost and the ease of scale up. There are numerous research reports in the literature related to the ATPSs that are conventionally made of dual polymers or a polymer with a salt. These systems are considerably biocompatible because of the high water content in both phases, making them suitable for protein separation.1-3 However, most of the conventional polymer-based ATPSs are highly viscous and normally form opaque aqueous solutions, which could interfere with the quantitative and qualitative analysis of the extracted compounds.4 In the past decade, ionic liquids (ILs) have emerged as an alternative phase-forming component for ATPSs because they are less viscous5 and could induce the phase separation more rapidly than polymer-based phase-forming components.6 Furthermore, ILs are well known for their strong solvation capability as well as their tuneable chemical structures and physical properties, thus providing a wide range of hydrophobicity.5,

7-9

Unlike the

conventional ATPSs that possess a limited range of polarity, the properties of an IL-based ATPS can be easily adjusted to control the partition of protein between aqueous phases, making the system ideal for purifying biomolecules.9-18 The recovery of protein using ILbased ATPS has been reviewed in detail elsewhere.19 However, ILs composed of imidazolium or pyridinium cations are frequently used in the IL-based ATPSs, which raise the concerns of toxicity and biodegradability issues of these systems.20 Although the environmentally benign ILs (e.g., cholinium- and amino acids-based ILs) have emerged as an alternative phase-forming component for ATPS, these ILs are still generally less widely available and are relatively more expensive than the typical phaseforming components (e.g., polymer, alcohol and salt). This is mainly attributed to the complex synthesis and purification processes of these ILs.21-22 Therefore, the recycling of ILs from the IL-ATPSs become critical to the sustainable downstream operation in the large-scale bioprocesses. Considerable effort has been devoted to the development of strategies for recovery of the IL, for example by membrane separations,23 addition of salting-out species,2425

or supercritical CO2 extraction.25 Nevertheless, the recovery process usually involves

volatile organic solvents which themselves have an environmental impact.6 The application of supercritical CO2 in the recovery of hydrophilic ILs is not economically viable as the process is very expensive.25 3 ACS Paragon Plus Environment


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Protic ILs are a subclass of ILs formed on proton transfer from a Brønsted acid to a Brønsted base.26-27 The protic ILs are an attractive alternative to the traditional ILs in numerous applications. The preparation of protic ILs is generally simpler than their aprotic counterparts, rendering a lower production cost.28 Protic ILs such as pyridinium acetate, 1methylimidazolium acetate and pyrrolidinium acetate were found to be an effective extractant for lignin, and were recyclable through distillation.29 King et al. reported that the 1,1,3,3tetramethylguanidine (TMG) salts of carboxylic acids, including formic, acetic, and propionic acids, could be applied for the cellulose extraction, and these protic ILs could also be recovered via distillation.30 The protic CO2-based alkyl carbamate IL, on the other hand, can be synthesized simply by mixing CO2 with a primary or secondary amine28, 31-33 and have recently gained great interest amongst the scientific community. One of attractive features of these carbamate-based ILs is their distillability at ambient temperatures under reduced pressure, which facilitates easy recovery/recycling of the IL. Reports showed that the simplest

form

of

carbamate-based

IL,

namely

N,N-dimethylammonium

N’N’-

dimethylcarbamate (DIMCARB), was able to extract tannins31 and curcuminoids from plant materials.32 In the present study, the phase-forming abilities of alkyl carbamate ILs such as DIMCARB, N,N-dipropylammonium N’N’-dipropylcarbamate (DPCARB), N,N-diallyl ammonium N’N’-diallylcarbamate (DACARB) and bis(2-ethylhexyl)ammonium bis(2ethylhexyl)carbamate (DBCARB) were investigated. Based on the screening results, ILATPSs formed with DIMCARB and poly(propylene) glycol (PPG) were selected for the studies of liquid-liquid equilibrium (LLE) as well as protein partitioning. The LLE data of PPG 400/700/1000 + DIMCARB + water systems at T = (288, 298 and 308) K and ambient temperature were determined experimentally, and were correlated using the established fitting models. To explore the ATPS as the protein separation tool, the partition behaviour of model proteins (i.e., BSA and lysozyme) in PPG + DIMCARB + water systems was analysed. Moreover, the feasibility of recovering both DIMCARB and PPG for multiple cycles of ATPS operation was explored, with the aim of demonstrating the reuse and sustainability of this system. Experimental Materials


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DIMCARB, dipropylamine, diallylamine, bis(2-ethylhexyl)amine, PPG 400 (Mn ~ 446), PPG 700 (Mn ~ 725), PPG 1000 (Mn ~ 1000), dipotassium phosphate (K2HPO4), ammonium sulfate [(NH4)2SO4], ethanol, 1-propanol, 1-butanol, sucrose, BSA and lysozyme were purchased from Sigma-Aldrich (St. Louis, USA). All these chemicals were used without further purification. The synthesis process of DPCARB, DACARB and DBCARB are described elsewhere.32 The synthesized CO2-based alkyl carbamate ILs were characterised by Fourier Transform-Infrared (FT-IR) and carbon-13 nuclear magnetic resonance (13C NMR) spectroscopies. The spectra of FT-IR and 13C NMR are given in Figs. S1 and S2, respectively (refer to Supporting Information). The chemical structures of synthesized ILs are presented in Table 1.

Table 1 Chemical structures of CO2-based alkyl carbamate ILs and their corresponding dissociated forms after distillation. Carbamate-based IL

Chemical structure

Dissociated form

DIMCARB

DPCARB

DACARB

DBCARB

Methods Investigation of the phase-forming ability The stock solutions of (NH4)2SO4, K2HPO4, and sucrose were prepared at 30 wt% using deionized water. DACARB and DBCARB, which are in solid state at room temperature, 5 ACS Paragon Plus Environment


Page 6 of 30

were dissolved in deionized water to obtain the 20 wt% stock solutions. Next, 0.1 g of the corresponding carbamate-based IL stock solution was transferred to a 2-mL micro-centrifuge tube. The tube was incubated in a thermostatic bath (Alpha RA8, Lauda) at 298 K. Then, the potential phase-forming component (i.e., salt, polymer, alcohol or carbohydrate) was added dropwise to the micro-centrifuge tube followed by the brief vortex mixing. If the mixture turned turbid, the formation of two-phase systems was subsequently validated by subjecting the solution to centrifugation at 6000 rpm for 10 min. For the mixture that showed no sign of cloudiness after the addition of 1.9 g of potential phase-forming component, the miscible liquid system was deemed to be incapable of inducing the two-phase formation.

Construction of binodal curves Turbidimetric titration method was used to construct the binodal curves.34 The known mass fractions of DIMCARB, PPG and deionized water were added to a 15-mL centrifuge tube to form a turbid mixture. A thermostatic bath (Alpha RA8, Lauda) was used to maintain the mixture at a specific temperature. Next, the deionized water was added dropwise to the mixture followed by a thorough mixing using a vortex mixer. The titration continued until the turbid solution was first turned clear. The final mass fractions of DIMCARB and PPG were calculated based on the total amount of deionized water that had been added into the solution. Lastly, the binodal curve was plotted using the calculated binodal points.

Construction of TLs A series of PPG + DIMCARB + water systems were prepared at different compositions in 2-mL micro-centrifuge tubes. To ensure the complete phase equilibration and separation, the systems were incubated in a thermostatic bath at a desired temperature for 3 h. Once the two-phase systems were established, the samples from both phases were extracted. The concentration of DIMCARB in phase solutions was measured spectrophotometrically using a UV-Vis spectrophotometer at 350 nm. Then, the samples were subjected to vacuum drying using a freeze dryer (CoolSafe series, ScanVac) at 181 K for 24 h.35 Both DIMCARB and water were vaporized after the drying, leaving the PPG as the residue. Subsequently, the concentration of PPG was determined based on the weight of the sample after the freeze drying.


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Partitioning of model proteins in ATPS An ATPS comprising the equal volumes of top and bottom phases was used in this experiment. The ATPS was prepared by adding 50 wt% of PPG, 8 wt% of DIMCARB, 20 wt% of protein solution, and 22 wt% of deionized water to a 2-mL micro-centrifuge tube to make up a total weight of 2 g. The ATPS was then immersed in a thermostatic bath for 3 h at a desired temperature. After attaining the phase equilibrium in ATPS, the volumes of the top phase and bottom phase were measured. Next, the Bradford assay was used to determine the concentration of protein in each phase.36 In summary, 10 µl of phase solution was mixed with 200 µl of diluted Coomassie Brilliant Blue R-250 dye reagent in a 96-well microplate. Next, the absorbance value of the mixture was determined using a microplate reader (Sunrise, Tecan) at wavelength 595 nm. The distribution of protein in the ATPS was assessed by calculating the partition coefficient (K) of protein as shown in Eq. (1):37

K=

(1)

where and are the concentrations of the protein in the bottom phase and the top phase, respectively.

Recovery of phase-forming components The top and bottom phases were extracted from the ATPS, and were transferred separately to 2-mL micro-centrifuge tubes. The solution of PPG-rich top phase was incubated in a thermostatic bath at 313 K for 3 h to induce the secondary ATPS. The homogenous PPG-rich solution was eventually thermo-separated into two phases, in which the PPG was recovered as the bottom phase of the secondary ATPS. On the other hand, the solution of DIMCARBrich bottom phase was subjected to rotary evaporation. The solution was heated to 318 K, and the vapor produced was transferred to a vapor duct immersed in liquid nitrogen at a temperature of 77 K for condensation. The DIMCARB was recovered as condensate obtained from the evaporation. Both the recovered PPG 400 and DIMCARB were then used to prepare the recycling ATPSs. To maintain the similar composition of ATPS, an appropriate amount of the fresh phase-forming components may be added to the recycling ATPSs for compensating the minor losses of PPG 400 and DIMCARB during their recovery processes. 7 ACS Paragon Plus Environment


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Results and discussion Phase-forming ability of carbamate-based ILs The phase-forming ability of CO2-based alkyl carbamate ILs (i.e., DIMCARB, DPCARB, DACARB and DBCARB) was assessed using nine types of conventional phaseforming components, covering inorganic salts, thermo-responsive polymers, alcohol and carbohydrate. The results of screening test are shown in Table 2. Among the pairs of phaseforming components tested, only the PPG category showed promising potential in the generation of ATPSs with carbamate-based ILs. From the screening tests, both (NH4)2SO4 and K2HPO4 were found to be unable to form ATPSs with the carbamate-based ILs. The salting-out effect is one of the main factors that governs the formation of ATPSs composed of ionic phase-forming components, such as salts and ILs. In a typical IL + salt ATPS, one of the phase-forming components (or kosmotrope) typically has water-structure-making nature, while the counterpart (or chaotrope) has water-structure-breaking ability.38 The formation of such an ATPS arises from the competitive hydration of both phase-forming components; the water-structuring component attracts the surrounding water to form the water-ion complexes, thereby salting out and dehydrating the component with the water-structure-breaking nature. With the increasing interfacial tension between aqueous media, the mixture eventually separates into two distinct aqueous phases and an equilibrium is achieved.39-41 In this study, the inorganic salts and the carbamate-based ILs might possess the similar degree of salting-out strength; thus, no components were excluded or salted-out to form phases. Similarly, the investigated carbamate-based ILs were unable to form ATPSs with the aliphatic alcohols and carbohydrates. These phenomena might be associated with the inadequacy of the salting-out effect exerted by the carbamate-based ILs. In the case of mixtures comprising IL and carbohydrate, the success of ATPS formation relies on the salting-out (or known as “sugaring-out”) of a weakly hydrated IL by the carbohydrate, which is more competitive in the formation of hydration complexes.42 Here, the hydration capacities of the examined carbamate-based ILs might be equivalent to sucrose, thus hindering the formation of ATPS. Out of the four types of carbamate-based ILs, DIMCARB could form ATPSs with all the tested PPGs (i.e., Mn of 446, 725 and 1000). This indicates that the DIMCARB, which has the shortest alkyl side chains on both cation and anion, exhibits a stronger salting-out 8 ACS Paragon Plus Environment

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effect than the other carbamate-based ILs. For carbamate-based ILs with longer alkyl side chains (i.e., DPCARB, DACARB and DBCARB), the salting-out effect exerted by their ionic parts could be weakened by the chaotropic effect contributed by the non-polar parts of their alkyl side chains. The shorter alkyl side chain of DIMCARB also has more hydrophilic nature and a better solubility in water. On the other hand, PPG 400 could induce the formation of ATPS with DIMCARB, but not with DPCARB, DACARB and DBCARB. An increase in the polymer chain length stoichiometrically reduces the number of hydrophilic end groups in polymer,43 giving the PPG a more hydrophobic character. Hence, PPG 400 is less hydrophobic and has a higher affinity for water; this caused the salting-out of PPG 400 to be difficult.44 For this reason, PPG 400 was only able to be salted out by DIMCARB, which was postulated to have the stronger salting-out strength in contrast to DPCARB, DACARB and DBCARB.

Table 2 Screening of carbamate-based ILs and other components for the formation of ATPSs. Carbamate-based ILs Phase-forming species (NH4)2SO4 K2HPO4 PPG 400 PPG 700 PPG 1000 Ethanol 1-propanol 1-butanol Sucrose

DIMCARB

DPCARB

DACARB

DBCARB

× ×

× × ×

× × ×

× × ×

× × × ×

× × × ×

× × × ×

× × × ×

Note: , two-phase formation observed; ×, no two-phase formation.

Binodal data of PPG + DIMCARB + water system and correlations The binodal data of the PPG 400/700/1000 + DIMCARB + water systems determined at 298 K are shown in Fig. 1 and Table S1 (refer to Supporting Information). As the molecular weight of PPG increased, the binodal curve of the systems shifted closer to the origin (see Fig. 1). This indicated that the PPG + DIMCARB + water systems could be induced by a higher-molecular-weight PPG at a lower concentration. The PPG with a higher molecular weight exhibited a greater hydrophobicity and thus a lower affinity for water.44 In other words, the solubility and the hydrogen-bonding donor sites per molecule of the polymer 9 ACS Paragon Plus Environment


decreased.45 As a result, the PPG with a higher molecular weight was favourably salted out by DIMCARB, and was more readily separated from aqueous media to form ATPS.

50

40

30

100 1

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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20

10

0 0

2

4

6

100 2

8

10

12

14

Fig. 1 Binodal curves for PPG 400/700/1000 (1) + DIMCARB (2) + water (3) systems at 298 K.  PPG 400; PPG 700; PPG 1000

To investigate the effect of temperature on the phase diagram, the LLE data of PPG 400 + DIMCARB + water systems were determined at two additional temperature points, i.e.,

T = (288 and 308) K. The binodal curves for PPG 400 + DIMCARB + water systems at different temperatures are presented in Fig. 2 and Table S2 (refer to Supporting Information). In general, an increase in temperature led to an expansion of the two-phase region, as indicated by the position of the binodal curve moving closer to the origin at higher temperature. The observed trend corroborated the results observed from other aqueous polymer + IL systems reported by Song et al.,46 Zafarani-Moattar et al.,47 Li et al.,48 Liu et al.49 and Pereira et al.45 Contrastingly, the STL decreased when the temperature increased. The PPG exhibits lower critical solution temperature (LCST)-type phase behaviour in polymer solutions, a phenomenon in which the polymer is more hydrophobic at a higher temperature.50-51 As the temperature increased, the interaction between PPG and solvent decreased and the solubility of PPG in water medium was reduced. As a result, the hydrogen bonding between the hydrophobic moieties along the PPG and the caged water molecules 10 ACS Paragon Plus Environment

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around the hydrophobic group of PPG become less favourable. Subsequently, the liquidliquid demixing occurred as the water molecules were drawn from the PPG-rich phase to the DIMCARB-rich phase. In short, an increase in temperature facilitates an easier formation of the PPG 400 + DIMCARB + water systems.

60

50

40

100 1

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


30

20

10

0 0

2

4

6

8

10

100 2

12

14

16

Fig. 2 Binodal curves for PPG 400 (1) + DIMCARB (2) + water (3) systems at T = 288 K (); 298 K (); 308 K ()

A four-fitted-parameter nonlinear expression that has been widely adopted to correlate the experimental binodal data of IL-based ATPSs9, 47, 52-53 was used here:

= exp + . + +

(2)

where and are the mass fractions of the PPG and IL, respectively, while a, b, c and d are the fitting parameters. To correlate the experimental binodal data at different temperatures with Eq. (2), the linear form of each fitting parameter in Eq. (2) was incorporated as a function of temperature and was expressed in the forms of Eq. (3):54

= exp + | − | + + | − | . + + | − | + + | − |

(3)



Page 12 of 30

where and are the absolute temperature and the reference temperature (i.e., room temperature = 298 K), respectively. The fitting parameters in Eqs. (2) and (3) was estimated by the least-square regression analysis of the experimental binodal data. The values of fitting parameters of Eqs. (2) and (3), along with the square of correlation coefficients (R2) and the standard deviations (sd), are shown in Table 3. In general, the R2 values obtained from the correlation with Eqs. (2) and (3) are close to unity, showing the satisfactory fitting of the experimental data using these equations. The comparison between the experimental and the calculated binodal data for PPG + DIMCARB + water systems can be found in Figs. S3 and S4 of Supporting Information.

Table 3 Values of parameters of Eq. (2) for PPG 400/700/1000 + DIMCARB + water systems at T = 298 K and Eq. (3) for PPG 400 + DIMCARB + water systems at T = (288 and 308) K.

288 298 308

-0.607 3.154 0.000

1.466 0.066

2.023 1.466 0.048

-1.020 0.383

-2.423 -0.651 -0.013

0.422 -0.134

1.497 0.015 0.388

-0.153 -0.036

0.996 0.999 0.999

sd 0.559 0.669 0.485

PPG 700 + DIMCARB + water

298

18.36

-

-24.23

-

10.05

-

-0.531

-

0.995

0.286


298

6.435

-

-5.336

-

1.348

-

0.010

-

0.997

0.119

System

T (K)


a

sd = ∑'() ! − "#$ /& number of binodal data.

.

R2

, where represents the concentration of PPG 400 (wt%) and n is the

TL data of PPG + DIMCARB + water system and correlations A TL indicates the compositions of the phase-forming components present in top and bottom phases. The TL length (TLL) and the slope of the TL (STL) were calculated using Eqs. (4) and (5), respectively:

.

TLL = ,- . − / 0 + -. − / 0 1

(4)


a

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STL =

. − / . − /

(5)

where is the mass fraction of phase component; the subscripts ‘1’ and ‘2’ refer to PPG and DIMCARB, respectively; the superscripts ‘t’ and ‘b’ refer to top and bottom phases of the system, respectively. The plait point is a critical point located on the binodal curve at where the TTL has decreased to zero, indicating that the composition and volume of two phases become theoretically identical.55 The estimated plait points for PPG + DIMCARB + water systems were determined based on the intercept point between the extrapolated auxiliary straight line [i.e., obtained from the TL compositions in the phase diagram using Eq. (6)]56 and the binodal curve [i.e., derived from Eq. (3)].

= 3 + 4

(6)

where and are mass fractions for PPG and DIMCARB, respectively. The 3 and 4 are the fitting parameters. The experimental TL data, together with the calculated TLL, STL and plait points for PPG 400/700/1000 + DIMCARB + water systems at T = 298 K are reported in Table 4. Also, the phase diagrams showing the experimental TL data and plait points are given in Figs. S3 and S4 of Supporting Information. An increase in the TLL caused an increase in the concentrations of PPG 400 and DIMCARB in the top and bottom phases, respectively. Conversely, the STL decreased when the TLL increased. To examine the effect of temperature on TL of the systems, the TL data for PPG 400 + DIMCARB + water systems at

T = (288 and 308) K were generated and presented in Table 4. When the temperature increased, the TLL increased correspondingly. This trend corresponded well to the position of binodal curve under the influence of temperature. As mentioned in the previous section, a higher temperature rendered the shift of binodal curve towards the origin and expanded the two-phase region. Concomitantly, the composition nodes of top and bottom phases in the phase diagram were pushed closer to the axes, thus lengthening the TL of the system.



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Table 4 Experimental TL data in mass fraction, TLL, STL and plait point for PPG 400/700/1000 (1) + DIMCARB (2) + water (3) systems at T = (288a,b, 298a and 308a,b) K and pressure (p) = 101 kPaa. System

T (K)


288

Total composition

Top phase

Bottom phase

100 TLL

STL

100

100

100

100

100

100

50.0 45.0 40.0 35.0 30.0

8.00 12.0 16.0 20.0 24.0

71.3 80.7 86.4 90.4 93.1

4.12 3.84 3.62 3.51 3.46

13.2 11.6 10.1 8.37 7.13

15.0 19.2 23.5 27.7 31.5

59.1 70.8 78.8 85.5 90.5

-5.37 -4.49 -3.84 -3.38 -3.06

55.0 50.0 45.0 40.0 35.0

4.00 8.00 12.0 16.0 20.0

63.8 79.8 85.5 90.1 93.5

2.61 2.28 2.10 1.96 1.86

17.9 11.3 8.99 7.67 4.55

9.21 15.4 20.3 24.9 29.4

46.4 69.8 78.7 85.6 93.1

-6.95 -5.21 -4.20 -3.60 -3.23

55.0 50.0 45.0 40.0 35.0

4.00 8.00 12.0 16.0 20.0

70.6 85.0 89.5 93.4 96.0

1.58 1.25 1.17 1.13 1.11

10.3 8.57 6.94 4.02 2.45

10.6 16.0 21.1 26.1 30.6

61.0 77.8 84.9 92.8 98.1

-6.72 -5.19 -4.14 -3.58 -3.17

7.00 8.00 9.00 10.0 11.0

4.00 5.00 6.00 7.00 8.00

92.7 94.2 95.0 95.6 95.9

0.59 0.58 0.57 0.56 0.55

2.70 1.40 0.90 0.63 0.52

4.14 5.35 6.44 7.77 8.93

90.0 92.9 94.3 95.2 95.8

-25.3 -19.5 -16.0 -13.2 -11.4

6.00 7.00 8.00 9.00 10.0

3.00 4.00 5.00 6.00 7.00

95.0 95.6 96.2 97.1 97.6

0.41 0.40 0.39 0.38 0.37

2.14 0.87 0.59 0.57 0.40

3.11 4.31 5.33 6.44 7.82

92.9 94.8 95.8 96.7 97.4

-34.3 -24.2 -19.4 -15.9 -13.0

(60.0, 4.51)

298

(58.0, 2.88)

308


298


298

Plait point (100 , 100 )

(64.0, 1.89)

(91.0, 0.60)

(94.0, 0.40)

a

Standard uncertainty of temperature, u(T) = 1 K and pressure u(p) = 0.5 kPa. Expanded uncertainty: for PPG 400 + DIMCARB + water system, Uc are Uc(PPG 400) = Uc(DIMCARB) = 0.0027 (95% level of confidence); for PPG 700 + DIMCARB + water system, Uc(PPG 700) = Uc(DIMCARB = 0.0018 (95% level of confidence); for PPG 1000 + DIMCARB + water system, Uc(PPG 1000) = Uc(DIMCARB) = 0.0025 (95% level of confidence)

b

The effect of temperature on TL data was evaluated only for PPG 400 + DIMCARB + water system.


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The consistency of the determined TL compositions was ascertained by correlating the experimental TL data using the Othmer–Tobias [Eq. (7)] and Bancroft [Eq. (8)] equations.57

1 − . 1 − / = 6 7 8 . / 9/ 9. = 6 7 . 8 /

'

(7)

:

(8)

where is the mass fraction of phase component; the subscripts ‘1’, ‘2’ and ‘3’ refer to PPG, DIMCARB and water, respectively; the superscripts ‘t’ and ‘b’ refer to top and bottom

phases of the system, respectively; 6 , & , 6 and ; are the fitting parameters. The fitting

parameters of Eqs. (7) and (8), along with the respective R2 and sd values, are given in Table 5. The values of R2 indicated that the plot of log [(1− . )/ . ] against log [(1−/ )/ / ] from

Eq. (7) and the plot of log (9/ // ) against log (9. / . ) from Eq. (8) are linear, which means that the experimental results obtained are relatively consistence. Moreover, on the basis of sd values, the TL data were well-fitted using the Other-Tobias equation than the Bancroft equation.

Table 5 Values of fitting parameters in Eqs. (7) and (8) for PPG 400/700/1000 (1) + DIMCARB (2) + water (3) systems at T = (288, 298 and 308) K.

288 298 308

Othmer-Tobias equation R2 sd a & 6 PPG 400 + DIMCARB + water system 0.020 1.743 0.998 0.002 7.120 0.021 1.450 0.992 0.007 10.76 0.012 1.682 0.991 0.007 11.34

298

0.007

0.755

298

0.004

T (K)

a

6

= ∑'() !

sd of TL data.

−

Bancroft equation R2 ;

sd a

0.407 0.547 0.492

0.985 0.985 0.980

0.058 0.063 0.082

PPG 700 + DIMCARB + water system 0.989 0.002 461.5 1.146

0.988

0.019

PPG 1000 + DIMCARB + water system 0.784 0.955 0.003 726.6 1.081

0.955

0.044

. "#$ /& ,

where represents the concentration of PPG 400 (wt%) and n is the number



Page 16 of 30

Effect of PPG molecular mass on the partitioning of model protein The effect of PPG molecular mass on the partition behaviour of BSA and lysozyme in 50 wt% PPG + 8 wt% DIMCARB systems were investigated. The selected compositions of ATPS, which yields a top-to-bottom volume ratio of 1:1, excludes the effects of phase volume on the partitioning of proteins. The K values of BSA and lysozyme are shown in Fig. 3. Two general trends were observed on the partition behaviour of both model proteins: (i) K values increased as the temperature is increased, (ii) K values increased when the molecular weight of PPG is increased. It can be concluded that the increase in hydrophobicity of PPG favoured the partition of protein to the top PPG-rich phase. Overall, the K values of lysozyme were lower than that of BSA; this could be explained in terms of the protein’s surface hydrophobicity. As determined using the ammonium sulphate precipitation method,58 lysozyme is relatively more hydrophobic than BSA. Accordingly, the hydrophobic interaction between BSA and the PPG-rich top phase was more pronounced, resulting in a lower K values (note: a lower K value denotes a greater partition of protein to the top phase). Moreover, the excluded volume effect could also be an important driving force for the distribution of protein in the systems.59 An increase in polymer chain length enhances the excluded volume effect, which reduces the volume available for accommodating the proteins in polymer-rich phase. Among the studied ATPSs, the systems made of PPG 1000 has the highest K values. This result we believe to be related to the limited free volume in the (PPG 1000)-rich phase as an effect of excluded volume; therefore, more protein molecules move to the bottom phase.


Page 17 of 30 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


Fig. 3 K values of model proteins in 50 wt% PPG 400 (blue) / PPG 700 (orange) / PPG 1000 (grey) + 8 wt% DIMCARB + water systems at T = (288-308) K. (a) BSA and (b) lysozyme.

and used in the calculation of K values for BSA and lysozyme are expressed as a mean of triplicate readings with estimated error of less than 5%.



Page 18 of 30

Effect of temperature on partition of model proteins The partition of protein between phases could be influenced by the temperature because both PPG and DIMCARB used in this study are temperature sensitive. It is also evident that the LLE of the PPG + DIMCARB + water systems was affected by temperature, and therefore the partition behaviour of protein in the ATPS can be expected to be temperature dependent. BSA and lysozyme, which differ in hydrophobicity, were chosen as the model proteins in this study. The partition behaviour of model proteins in the ATPS composed of 50 wt% PPG 400 and 8 wt% DIMCARB was investigated at T = (288, 293, 298, 303 and 308) K. As shown in Fig. 3, an increase in the temperature led to a greater partition of BSA and lysozyme to the DIMCARB-rich bottom phase, which was indicated by the increasing values of the K of BSA and lysozyme. The effect of temperature on partition behaviour of the proteins can be due to many factors, including the steric exclusion effect and the variations in polymer structure as well as phase composition. When the polymer becomes more hydrophobic with increasing temperature,51 the strong polymer-polymer interactions exert a volume exclusion effect that reduces the interaction between polymer and protein.60 On a side note, the decrease in K value of lysozyme with increasing temperature was more prominent than that of BSA, showing that the partition of lysozyme was more prone to the influence of temperature. This may be attributed to the fact that the total surface hydrophobicity of lysozyme is greater than that of BSA. The lysozyme could still preferentially interact with the increasingly hydrophobic PPG-rich phase when the temperature increased.

Recovery of PPG 400 and DIMCARB The recovery and reuse of phase-forming components from ATPS could potentially minimize the environmental and economic impact of the ATPS-based separation systems. Moreover, the removal of phase components from the phase containing the target proteins would further increase the purity of the final product. Here, we demonstrate the recycling schemes for PPG 400 and DIMCARB using thermo-separation and distillation, respectively. PPG 400 is a type of thermo-responsive polymer which allows the polymer solution to be thermally separated into two clear phases.61 At temperatures higher than the LCST of 18 ACS Paragon Plus Environment

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PPG 400, the phase separation can be induced to yield a polymer-rich phase that coexists with water-rich phase. The strategy for recovering a LCST-type polymer via thermoseparation has been studied and adopted46,62 in the sustainable ATPS-based bio-separation area. Among the studied polymers, PPG 400 was chosen as the subject of this study because it has a relatively higher LCST (313 K) than PPG 700 and PPG 1000 (304 K and 300 K, respectively). Although a lower temperature for thermo-separation of PPG would be attractive from the perspective of economics, the LCSTs of PPG 700 and PPG 1000 are close to room temperature; this necessitates the maintenance of their LLE by cooling the systems during the processing of ATPS. This requirement reduces the attractiveness of PPG 700 and PPG 1000 for practical use in ATPS. In this study, the (PPG 400)-rich top phase originated from the ATPS composed of PPG 400 and DIMCARB (herein referred to as the primary ATPS) was thermo-separated into the secondary ATPSs at T = 313 K. The compositions of the PPG 400 and DIMCARB in the secondary ATPS are tabulated in Table 6. Five sets of primary ATPSs with different total compositions were prepared initially. The (PPG 400)-rich bottom phases from the corresponding systems were subjected to thermo-separation. It was found that the PPG 400 together with the minimum traces of water and DIMCARB were predominantly concentrated into the top phase of the thermo-separated secondary ATPS. This demonstrated the feasibility of recovering PPG 400 from the primary ATPS for the next preparation of a new ATPS. Additionally, the secondary ATPS induced from the primary ATPS could be used to further partition the proteins between the secondary phases formed.

Table 6 Compositions of secondary ATPSs [PPG 400 (1) + DIMCARB (2) + water (3)] formed from the top phase of the primary ATPSs at 313 Ka and p = 101 kPaa. Primary systemb Top phase

100 63.8 79.8 85.5 90.1 93.5

100 2.61 2.28 2.10 1.96 1.86

Secondary system Top phase

100 86.2 84.7 82.1 80.9 77.8

100 1.73 1.92 2.11 2.34 2.59

Bottom phase

100 10.7 12.3 14.1 15.9 17.7

100 1.42 1.30 1.22 1.14 1.05

a

Standard uncertainty of temperature, u(T) = 1 K and pressure u(p) = 0.5 kPa. Expanded uncertainty: for PPG 400 + DIMCARB + water system, Uc are Uc(PPG 400) = Uc(DIMCARB) = 0.0027 (95% level of confidence) b

Binodal data for PPG 400 + DIMCARB + water at 298 K (adopted from Table 4)



Page 20 of 30

The IL-rich bottom phase of the primary ATPS was subjected to distillation for the recovery of DIMCARB. The DIMCARB-rich phase was held at 318 K in a water bath to dissociate DIMCARB into the respective gaseous amine and CO2. When the gasses passed through a vapor duct immersed in liquid nitrogen, they reformed into DIMCARB and were collected. Table 7 shows the composition and the percentage of recovery of the DIMCARB condensate. A satisfactory recovery of ~91% DIMCARB (95 wt% concentration) was attained from the DIMCARB-rich bottom phase of a primary ATPS.

Table 7 Concentrations of DIMCARB in the condensate distilled from the bottom phase of primary ATPSs composed of PPG 400 and DIMCARB at 77 Ka and p = 101 kPaa. Bottom phase of primary ATPS Total mass (g) 6.00 6.00 6.00 6.00 6.00

Concentration of DIMCARB (wt%) 9.21 15.4 20.3 24.9 29.4

Mass of DIMCARB (g) 0.55 0.92 1.22 1.49 1.76

Condensate Total mass (g) 0.53 0.88 1.16 1.43 1.67

Concentration of DIMCARB (wt%) 94.6 94.9 95.3 95.7 95.8

Mass of DIMCARB (g) 0.50 0.84 1.11 1.37 1.60

Percentage recovery (%) 91.2 90.8 91.1 91.4 90.6

a

Standard uncertainty of temperature, u(T) = 1 K and pressure u(p) = 0.5 kPa. Expanded uncertainty: for PPG 400 + DIMCARB + water system, Uc(DIMCARB) = 0.0036 (95% level of confidence)

Fig. 4 shows the schematic diagram of the two successive recycling processes. The PPG 400 and DIMCARB recovered from the first ATPS were used to prepare the subsequent ATPSs (denoted as Recycling 1 and Recycling 2). As given in Fig. 5, the FT-IR spectroscopic analyses indicated the presence of symmetric carbamate (approximately 1408 cm-1) and carbamate C−O stretching (approximately 1621 cm-1) peaks in the pure DIMCARB and the DIMCARB fraction recovered from the bottom phases of previous ATPSs. In addition, the 13C NMR conducted on both the pure and recycled DIMCARB (see Fig. S5 of Supporting Informaiton) show the carbamate signals at around 162 ppm. These analyses confirmed that the DIMCARB was successfully reformed and recovered via evaporation. The separation efficiency of this new ATPS was tested by analysing the partition behaviour of model proteins (i.e., BSA and lysozyme) in the system. Table 8 shows the K values for BSA and lysozyme in the recycling ATPSs along with their phase volumes. Based on the K values, there was no significant difference in the separation efficiencies of proteins from all the tested systems. Similarly, the phase volumes of all the ATPSs did not deviate greatly, hinting that 20 ACS Paragon Plus Environment

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the similar compositions across the ATPSs were well maintained. Therefore, the recycling of phase-forming components for the successive ATPSs used in protein separation was proved successful.

Fig. 4 Schematic diagram of the recycling of the phase-forming components used in PPG 400 + DIMCARB + water systems.



(a) Pure

~1621 cm-1

~1408 cm-1

(b) Recycling 1 % Transmittance

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

Page 22 of 30

(c) Recycling 2

3700

3100

2500

1900

1300

700

Wavelength (cm-1)

Fig. 5 FT-IR spectra for (a) pure DIMCARB, (b) DIMCARB obtained from Recycling 1 and (c) DIMCARB obtained from Recycling 2.

Table 8 K values of BSA and lysozyme in the recycling ATPSs composed of 50 wt% PPG and 8 wt% DIMCARB at T = 298 K. Cycle First ATPS Recycling 1 Recycling 2

a

K

Volume (ml) Top phase 1.01 1.07 1.04

Bottom phase 0.99 0.93 0.96

a

BSA 9.35 9.27 9.23

Lysozymea 6.01 5.92 5.84

and used in the calculation of K values for BSA and lysozyme are expressed as a mean of triplicate readings with estimated error of less than 5%.


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Conclusion The formation of ATPS with CO2-based alkyl carbamate ILs and other conventional phase-forming components was studied. The ATPSs composed of PPG and DIMCARB were further explored as the environmentally benign and recyclable systems for protein separation. The effects of PPG molecular mass and temperature on the LLE of PPG + DIMCARB + water systems were evaluated, and the experimental results were well correlated with the four-fitted-parameter nonlinear expression. Moreover, the partition behaviour of the model proteins in the system was also found to be influenced by the type of PPG employed and the temperature. The excluded volume effect became more significant when the molecular mass of PPG was increased, as evidenced by the affinities of both proteins for the DIMCARB-rich bottom phase. Also, the hydrophobic interaction between the protein and PPG 400 became less prominent at higher temperature. As a result, the model protein gradually shifted from the PPG-rich top phase to the DIMCARB-rich bottom phase. The recovery of PPG 400 from the primary top phase was proven to be feasible. Moreover, DIMCARB was also successfully recovered from the primary bottom phase. The newly formed ATPS using the recycled components have the nearly identical compositions of top and bottom phase as the primary ATPS, even after two successive recycling. More importantly, the partition behaviour of both model proteins in the newly formed ATPSs remained almost unchanged, affirming that the separation performance of the system is not compromised. Overall, this environmentallyfriendly PPG + DIMCARB ATPS has shown a great potential to serve as a sustainable protein separation technique for the bioprocess industries.

Associated Content Supporting Information Binodal data for PPG 400/700/1000 + DIMCARB + water at T = 298 K, binodal data for PPG 400 + DIMCARB + water at T = (288 and 308) K, phase diagrams for PPG 400/700/1000 + DIMCARB + water at T = 298 K, and phase diagrams for PPG 400 + DIMCARB + water at T = (288 and 308) K. This material is available free of charge via the Internet at http://pubs.acs.org. Note The authors declare no competing financial interest. 23 ACS Paragon Plus Environment


Page 24 of 30

Acknowledgements This work was supported by the Fundamental Research Grant Scheme (FRGS) (Ref. no. FRGS/1/2015/SG05/MUSM/02/4) which was awarded by the Ministry of Higher Education (MOHE) Malaysia. Cher Pin Song acknowledges the MyBrain 15 Scholarship granted by the Ministry of Higher Education, Malaysia.

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For Table of Contents Use Only

The recyclable ATPS-forming components, namely PPG 400 and DIMCARB, can be recovered via thermo-separation and distillation, respectively.


Environmentally Benign and Recyclable Aqueous Two-Phase System

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