A simple approach for the determination and characterization of ternary phase diagrams of aqueous two-phase systems composed of water, polyethylene glycol and sodium carbonate

Abstract

In this work, a simple experimental protocol to determine liquid-liquid phase diagrams of aqueous two-phase systems (ATPS) on a Chemical Engineering course is described. Throughout this laboratory set of experiments, the liquid-liquid ternary phase diagrams, tie-lines, tie-line lengths and critical points of ATPS will be determined. Ternary liquid-liquid phase diagrams composed of water, polyethylene glycol (PEG 200, 400 and 600 g·mol^-1) and sodium carbonate (Na₂CO₃) were obtained by cloud-point titration method at room temperature. The respective tie-lines, tie-line lengths and critical points were also determined. Phase diagrams were represented both as conventional ternary phase diagrams and orthogonal phase diagrams. Through the analysis of the results obtained it was identified a higher ability to form ATPS with the increase of the polymer molecular weight. The interpretation of phase diagrams, particularly the most complex, the orthogonal ones, is not always easy to grasp by students, so this novel 3-hour-class educational approach could be potentially used to teach and help understanding 3-component liquid-liquid equilibrium and the formation of biphasic systems to undergraduate students, without requiring the use of volatile organic solvents.

Introduction

An important chapter of Physical Chemistry courses is based on the study of physical transitions that mixtures undergo upon temperature or composition changes. Phase diagrams are an efficient tool to represent this type of information, enabling to grasp at a glance whether two or more substances are mutually miscible, or how pressure or temperature can be tuned to achieve a particular set of equilibrium conditions. They may be developed using the phase rule, which is, according to Peter Atkins, “the most elegant result of the whole chemical thermodynamics” [1]. However mathematically simple, the Gibbs’ formulation, i.e. the meaning and the definition of phase and component, may not always be straightforward; for instance, when one or more species suffer a chemical reaction, the number of components may be difficult to establish. Also, the interpretation of phase diagrams, particularly the most complex ones, is not always easy to grasp by students. Beyond their conceptual pedagogical value, phase diagrams bear also a practical interest since they are essential in the design of separation processes, which have a wide industrial use. Hence, phase diagrams are an important subject to study and have a decisive role in the academic laboratories and in the industry in the fields of Chemical Engineering, Chemistry and Biotechnology.

Industrial liquid–liquid extractions (LLE) often use volatile organic solvents which may not be suitable to separate some biomolecules or bioproducts and pose hazards to the users and the environment. The design of more eco-friendly and cheaper systems for liquid-liquid extraction/purification of (bio)molecules is imperative from an industrial point of view, and a considerable effort has been made in recent years towards this goal [2]. Aqueous Two-Phase Systems (ATPS) are formed when two hydrophilic solutes are dissolved in water above certain concentrations, spontaneously separating into two liquid phases. These systems were proposed by Albertsson [3] and can be formed by polymer/polymer, polymer/salt or salt/salt combinations, dissolved in aqueous media [2]. ATPS have emerged as prominent purification platforms due to the water-richness of the phases, simplicity of preparation, high resolution capacity and ability to be scaled-up [2]. Among the different polymers, polyethylene glycol (PEG) has been widely used in ATPS formulations combined with salts, since it is nontoxic, inexpensive, and biodegradable [4]. Moreover, tuning of ATPS phase diagrams and characteristics may be achieved by varying the molecular weight of PEG over a considerable range. It is generally observed that the higher the molecular weight of the polymer, the stronger its capability to promote phase separation [3]. On the other hand, and amongst the various salts that can be selected to form ATPS with polymers, sodium carbonate has a high ability to promote phase separation, which means that less amount of salt is needed to create a two-phase system. Furthermore, this salt has a diversity of uses, from food additive and acidity regulator to the manufacture of glass, detergents and paper, as well as in brine treatment, water hardness removal and wastewater pH adjustment. Moreover, sodium carbonate is not toxic to the environment, nor to aquatic organisms [5, 6].

In this experimental work the necessary tools are given for the application of this thematic in the courses of Chemical Engineer. It is performed the determination of ternary phase diagrams for ATPS composed of water, sodium carbonate and PEG with several molecular weights. Several laboratory experiments aiming at teaching liquid-liquid extraction have been reported [7–14], yet few involving phase diagrams of ternary liquid-liquid systems [15–19]. To the best of our knowledge, none proposed the use of ATPS. Hence, this work provides also the opportunity to discuss the environmental advantages associated to the use of ATPS instead of ternary systems involving volatile organic solvents. Furthermore, in the work here proposed, the determination of the phase diagrams is achieved by simple procedures, yet allows students to get acquainted with the mostly used methods for the determination of the solubility (binodal) curves, tie-lines (TLs), tie-line lengths (TLLs), and critical points for these systems.

The composition of the system in the saturation curve is experimentally determined by weighing the components corresponding to the two-phase formation that is visually detected. A curve is then fitted to those experimental solubility points that can be used to extrapolate data beyond the experimental range. The students need to perform a non-linear regression analysis and to solve a series of equations required to determine the tie-lines based on material balances. Depending on the goals of the course, and on the purpose of the lab experiments, the software used by the students to build the phase diagrams may demand none or little programming, or they may be asked to write a program to carry the correlation of the binodal curve and the determination of the tie-lines. So, this set of experiments is very versatile and adequate to be carried out by 3^rd or 4^th year students of Chemical Engineering, Chemistry and Biotechnology degrees.

The binodal curve for the ATPS formed by water, Na₂CO₃ and PEG 600 g·mol^-1 obtained experimentally may be represented in a triangular ternary phase diagram, in weight fraction or weight fraction percentages (Figure 1.a). However, to define a ternary system only the amount of two components is needed, the third being dependent; since one of the components is water, the composition of the system may also be represented on an orthogonal diagram (Figure 1.b) highlighting the concentration of the other two components. In fact, the use of triangular coordinates is not straightforward, and “it may be difficult to estimate the composition represented by a specific point, simply by taking a quick glance at the diagram” [20]. Moreover, the orthogonal representation is the most used approach by researchers in the field of ATPS extractions [2], thus being also important for students to become acquainted with it. It must be remarked that for high concentration values, the solubility limit of one of the solutes may eventually be reached; in this case, other lines corresponding to the precipitation of that solute should be taken into account, but those three-phase regions of the diagram are outside of the scope of this work.

Figure 1.

Phase diagrams for the ternary system composed of PEG 600 + Na₂CO₃ + H₂O at 293 K: (a) ternary representation and (b) orthogonal representation. CP represents the critical point of the diagram, M is a random mixture point in the biphasic region of the system, and X_T and X_B represent the compositions of top and bottom phase of the system, respectively.

In the phase diagram represented in Figure 1, the biphasic region is localized in the region where the PEG and salt contents are higher, i.e. below the solubility curve in Figure 1a, and above the solubility curve in Figure 1b. For a given total mixture composition in the biphasic region (M), the mixture suffers phase separation and forms two coexisting phases, each being represented by the points X_T and X_B, which are the end-points (nodes) of a specific tie-line (TL). In the studied ATPS, the less dense phase is the PEG-rich one. The composition difference between these two phases is expressed by the tie-line length (TLL). This numerical indicator is an important parameter in separation procedures since it correlates trends in the partitioning of solutes between the two phases. Mixtures with total compositions along a specific tie-line have different mass or volume ratios from those of the two coexisting phases; yet, the composition of each phase remains the same. The critical point of the ternary system is point C, where the compositions of the two coexisting phases become equal, and the biphasic system ceases to exist.

Laboratory Description

This laboratory work is divided in two parts: part 1 - determination of binodal curves of ternary systems constituted by water, PEG (200, 400 and 600 g·mol^-1) and sodium carbonate (Na₂CO₃); and part 2 – determination of tie-lines and critical points of each ATPS. The students work in groups of two, and a different molecular weight PEG (200, 400 and 600 g·mol^-1) can be assigned to each group. Two 3-hour periods (classes) are needed. The first period/class dedicates 30 minutes to discuss/explain the details of the experiment; then, approximately 60 min are required to collect experimental data for the first part; a computational period for each group to compute and represent the phase diagram follows; finally, the results obtained for all systems are analyzed and compared. The second period/class comprises 60-70 minutes for the preparation of the several mixtures (at least 5) by each group and the rest of the time is devoted to computational work: determination of mixture compositions, TLs, TLLs and critical points.

Determination of binodal curves of ternary systems constituted by PEG + Na₂CO₃ + H₂O

The ternary phase diagrams are determined at ambient temperature and pressure by cloud point titration. Stock solutions of sodium carbonate (100 w_salt = 21,21 wt%), slightly below the salt solubility saturation in water, should have been previously prepared and used for the determination of the phase diagrams. Repetitive drop-wise addition of the salt solution to the PEG (100 w_PEG = 80,80 wt%) solution is carried out until the detection of a cloudy solution, followed by the drop-wise addition of distilled water until the detection of a monophasic region (clear and limpid solution), as illustrated in Figure 2.

Figure 2.

Illustration of the cloud point titration method.

To complete the binodal curve, an indirect method is attempted: the repetitive drop-wise addition of the PEG solution to the salt solution until the detection of a cloudy solution, followed by the drop-wise addition of distilled water until the detection of a monophasic region (clear and limpid solution). All these additions are carried out under continuous stirring. The ternary system compositions are determined by weight quantification of all components added, for which the samples should be always weighted after identifying each cloudy and limpid solution. After the experimental procedure of this first experiment, the students should correlate the binodal curves obtained with an equation proposed by Merchuk et al. [21], that has been almost universally used by researchers in the field [2].

$$100w_{PEG}=A\mathit{\text{exp}}\left[\left(B\times 100{w_{salt}}^{0.5}\right)-\left(C\times 100{w_{salt}}^3\right)\right]$$ (1)

where 100 w_PEG and 100 w_salt are, respectively, the PEG and salt weight percentages and A, B and C are parameters obtained by the regression. This adjustment can be performed using any commercial software provided with non-linear regression and fitting tools; in this work, SigmaPlot software was used to visualize and plot the curve that best describes the shape and behavior of the data, through the “Curve Fitting and Regression” tool.

Determination of tie-lines, tie-line lengths and critical points of each phase diagram

The tie-lines (TLs) are determined by a gravimetric method originally described by Merchuk and collaborators [21]. The ternary mixtures at the biphasic region described in Table 1 are gravimetrically prepared in tubes, by weighing the appropriate amounts of PEG, salt and water, vigorously stirred, and allowed to reach the equilibrium by the separation of the two phases through centrifugation for 30 minutes at 3500 rpm, at ambient temperature.

Table 1. Mixture points for each system constituted by PEG + Na₂CO₃ + H₂O used for the determination of TLs.

Molecular weight PEG / g∙mol-1	Mixture Points
	100 wPEG	100 wsalt
200	30	8.5
		9
		10
		11
		12
400	25	8
		9
		10
		11
		12
		13
		14
600	15	8
		10
		12
		16
		18

After the thermodynamic equilibrium is accomplished and the two-phase formation is visually detected (Figure 3), each top and bottom phase is quantitatively transferred to a vial using a syringe or a Pasteur pipette and its weight is determined.

Figure 3.

Visual appearance of the two phases formed after the thermodynamic equilibrium is accomplished: top (PEG-rich) and bottom (salt-rich) phases.

Finally, to determine each individual TL, the composition of both phases is needed, and this can be carried out using different methodologies. In this work, the composition of each phase is computed through the application of mass balances to compute the values of four unknowns $\left(100w_{PEG}^{PEG},100w_{salt}^{PEG},100w_{PEG}^{salt},100w_{salt}^{salt}\right),$ by solving the following four equations system:

$$100w_{PEG}^{PEG}=A\mathit{\text{exp}}\left[\left(B\times 100{w_{salt}^{PEG}}^{0.5}\right)-\left(C\times 100{w_{salt}^{PEG}}^3\right)\right]$$ (2)

$$100w_{PEG}^{salt}=A\mathit{\text{exp}}\left[\left(B\times 100{w_{salt}^{salt}}^{0.5}\right)-\left(C\times 100{w_{salt}^{salt}}^3\right)\right]$$ (3)

$$100w_{PEG}^{PEG}=\frac{100w_{PEG}^M}{\alpha }-\frac{1-\alpha }{\alpha }\times 100w_{PEG}^{salt}$$ (4)

$$100w_{salt}^{PEG}=\frac{100w_{salt}^M}{\alpha }-\frac{1-\alpha }{\alpha }\times 100w_{salt}^{salt}$$ (5)

The superscripts PEG and salt designate the PEG-rich (top) and salt-rich (bottom) aqueous phases, respectively, and M represents the initial mixture composition. The parameter α is the ratio between the top weight and the total weight of the mixture (experimentally determined). By solving the above system the concentration values of PEG and of salt in the top and the bottom phases are obtained. This system of four equations can be solved using any commercial software package that allows the creation and optimization of algorithms to solve this type of system (e.g. Matlab, Datafitting, NCSS). Our students used Matlab software, since this is a software tool they are familiar with.

Once a TL is determined, the calculation of the tie-line length (TLL) is a straightforward procedure, using the equation shown below:

$$TLL=\sqrt{{\left(100w_{salt}^{PEG}-100w_{salt}^{salt}\right)}^2+{\left(100w_{PEG}^{PEG}-100w_{PEG}^{salt}\right)}^2}$$ (6)

As mentioned before, several tie-lines are obtained for each system (we obtained at least five, Table 1). The critical points of each ternary system are then estimated by extrapolation from the TLs compositions applying the following linear equation:

$$100w_{PEG}=f+g\times w_{salt}$$ (7)

where f and g are fitting parameters that may be obtained using simple software of calculus and statistics (e.g. Microsoft Excel, SigmaPlot, MatLab).

To provide a thorough understanding of the phase diagram, each group should determine several TLs, the respective TLLs, and the critical point for the selected system, using the experimentally determined binodal data.

Hazards

Both PEG 200, 400, 600 and sodium carbonate are non-toxic and only slightly hazardous compounds in case of skin contact (irritant, permeator), eye contact (irritant), or inhalation. However, since students always wear laboratory coats, gloves and goggles, risks are minimal.

Data Analysis

The systems investigated in this work are formed by water, Na₂CO₃ and PEG with three different molecular weights: 200, 400 and 600 g·mol^-1. The binodal curves for all studied systems are presented in the conventional triangular form (Figure 4) and orthogonal based form (Figure 5). The experimental weight fraction data of each phase diagram are given in the Supporting Information (Tables SI.1 – SI.4).

Figure 4.

Ternary phase diagram for the systems composed of PEG + Na₂CO₃ + H₂O at 293 (± 1) K: (●) PEG 200; (▾) PEG 400; (◾) PEG 600; (a) whole ternary phase diagram and (b) zoom of the diagram by changing the axis grade.

Figure 5.

Orthogonal phase diagram for the systems composed of PEG + Na₂CO₃ + H₂O at 293 (± 1) K: (●) PEG 200; (▾) PEG 400; (◾) PEG 600.

When the orthogonal representation is considered (Figure 5), it may be appreciated that the closer the solubility (binodal) curve is to the axes, the larger is the region above the curve and thus the higher is the ability of PEG to undergo liquid-liquid demixing in presence of Na₂CO₃ in aqueous media. As expected, the influence of the length of the polymer’s chains on the phase diagram is notorious. For lower molecular weight PEG, the phase separation occurs at higher concentrations of both the polymer and the salt. In general, the ability of PEG to form ATPS in presence of the inorganic salt decreases in the following order: PEG 600 > PEG 400 > PEG 200. This behavior is a consequence of the higher hydrophobicity displayed by PEGs of higher molecular weight because they present a lower affinity for water, being thus more easily salted-out by sodium carbonate. Similar trends have been observed in other ATPS composed of polymer/salt described in the literature [22, 23]. Salting-out (or salting-in) effects are most significant in solution chemistry and have paramount importance in ATPS, so this is a subject that students should be familiarized with.

For the studied systems, the experimental binodal data were further correlated by the empirical relationship described by Equation 1. The regression parameters were estimated by the least-squares regression method, and their values and the corresponding standard deviations are provided in Table 2. In general, good correlation coefficients were obtained for all systems, indicating that these fittings can be used to predict data in a given region of the phase diagram where no experimental results are available.

Table 2. Correlation parameters used to describe the experimental binodal data by Equation 1.

Ternary system PEG + Na2CO3 + H2O
PEG	A ± σ	B ± σ	C ± σ (x 10-5)	R2
PEG 200	100.4 ± 1.4	-0.4279 ± 0.007	8.54 ± 0.69	0.99580
PEG 400	96.1 ± 1.1	-0.4555 ± 0.007	28.16 ± 1.35	0.99586
PEG 600	81.8 ± 0.6	-0.5334 ± 0.004	41.47 ± 1.05	0.99883

The experimental TLs, along with their respective length (TLLs), are reported in Table 3. The tie-line length (TLL) is a numerical indicator of the difference between the compositions of the two phases and is generally used to correlate trends in the partition of solutes between both phases. The mixtures with total compositions along a specific tie-line have different mass or volume ratios, but the composition of each of the two phases in equilibrium is the same. The parameters f and g of Equation 7 were determined for each ternary system and are presented in Table 4, as well as the critical point (100 w_PEG and 100 w_salt), resulting from the interception of the linear equation (a result of the extrapolation from the TLs composition) with the binodal curve. All the TLs and critical points obtained for each system are depicted in Figures 6 – 8.

Table 3. Experimental data of TLs and TLLs of ATPS constituted by PEG + Na₂CO₃ + H₂O.

Weight fraction composition (100 w)
PEG	$100w_{PEG}^{PEG}$	$100w_{salt}^{PEG}$	$100w_{PEG}^M$	$100w_{salt}^M$	$100w_{PEG}^{salt}$	$100w_{salt}^{salt}$	TLL
PEG 200	45.49	3.39	29.91	12.15	1.57	28.07	50.38
	34.36	6.06	30.02	8.49	5.03	22.45	33.59
	36.97	5.31	30.14	9.14	3.89	23.87	37.93
	39.55	4.65	29.92	10.04	3.08	25.05	41.79
	43.12	3.86	30.03	11.08	2.34	26.36	46.58
PEG 400	29.58	6.02	24.29	7.96	8.43	13.81	22.54
	37.72	4.05	25.20	9.09	2.52	18.20	37.94
	41.70	3.28	25.01	10.04	1.58	19.53	43.28
	43.91	2.91	24.98	10.98	0.78	21.29	46.89
	48.11	2.29	25.12	12.00	0.46	22.43	51.72
	49.96	2.05	24.92	13.21	0.18	24.24	54.50
	52.55	1.75	25.00	14.07	0.11	25.19	57.44
PEG 600	22.23	5.39	15.33	8.18	7.13	11.50	16.29
	33.55	2.74	15.25	10.20	2.09	15.56	33.97
	39.85	1.81	15.08	12.30	0.62	18.42	42.60
	48.70	0.94	15.32	15.93	0.05	22.78	53.33
	53.08	0.66	14.87	17.82	0.01	24.49	58.17

Table 4. Fitting parameters (f and g) in the determination of the critical point (PEG and salt compositions) by Equation 7.

PEG	Fitting parameters		Critical point
	f	g	100wPEG	100wsalt
PEG 200	2.06	-11.89	13.76	16.43
PEG 400	2.02	1.59	9.04	19.85
PEG 600	2.32	-3.50	7.92	14.85

Figure 6.

Phase diagram for the ternary system composed of PEG 200 + Na₂CO₃ + H₂O at 293 (± 1) K, with the binodal curve data adjusted through Equation 1, TLs data and critical point determination.

Figure 8.

Phase diagram for the ternary system composed of PEG 600 + Na₂CO₃ + H₂O at 293 (± 1) K, with the binodal curve data adjusted through Equation 1, TLs data and critical point determination.

Conclusions

This laboratory experiment was composed by two main tasks: the determination of binodal (solubility) curves of ternary systems constituted by water, PEG (200, 400, 600 and 1000 g·mol^-1) and sodium carbonate (Na₂CO₃), and the determination of tie-lines, tie-line lengths and critical points for each polymer-salt ATPS. The first task was entirely experimental, and the second required the combination of experimental data with calculations. The experimental procedure used for determining the binodal curves was based on the cloud point titration method, in which the endpoint is the appearance (or disappearance) of turbidity when the system changes from one phase to two phases (or vice versa). The determination of the tie-lines by the combination of experimental and calculated data allows a substantial reduction in laboratory time, which is a most important issue not only for this lab experiment but in general, when designing new industrial separation processes. Also, the cloud point titrations may often present ill-defined endpoints, a drawback that may be overcome by the correlation of the experimental data.

If lectured in laboratory, this experimental exercise will allow students to become acquainted with the principles behind the basic thermodynamics of ternary liquid-liquid systems and/or ATPS and with the necessary tools for the characterization of such systems, in particular by determining the binodal curve, tie-lines, tie-line lengths and the critical point - fundamental aspects for the characterization of any ternary liquid-liquid system. In addition, they will have the opportunity of to infer about the influence of using different polymers as phase-forming components in two main areas: the ability to form ATPS and how they influence the parameters that characterize each ATPS.

When applied this experimental laboratory exercise, lab techniques along with computational calculations will be practiced by students. The relatively intense use of computational resources, both for calculations and parameters estimation, is a distinct feature from previously proposed experiments but most important in today’s laboratories, both in academic and industrial environments. The students use commercial spreadsheets that have advanced numerical tools packages that allow sophisticated calculations in multivariate statistics, as well as linear and nonlinear optimizations, that are essential tools in laboratory techniques and scientific research. Non-linear curve fitting is a most used tool in Chemistry, from deconvolutions of overlapping bands in vibrational or electronic spectra to the analysis of chemical kinetics, and thus it is important that students are acquainted with these procedures.

This experiment may be part of Chemical Engineering course (e.g. 3^rd year) and similarly oriented courses where separation processes are important. It was designed to be taught in two practical laboratory classes and also to include more modern computational methods in the elaboration of the phase diagrams. Students were able to translate the theory on how to determine phase diagrams and characterize them to the practice, and also on the real application of calculus software to solve chemical engineering challenges.

This lab work was thought as a way to update the curriculum so as to reflect environmental issues for Biotechnology, Chemical Engineering, Chemistry and Biochemistry students at an intermediate level. The ATPS presented herein will familiarize students with a viable and more eco-friendly alternative to conventional liquid-liquid extractions and separation processes in which the use of organic volatile solvents is avoided, and using techniques that from an administrative point of view offer low operational costs.

Supplementary Material

All the experimental weight fraction data of each phase diagram presented in the Results Section are given in Supporting Information.

Figure 7.

Phase diagram for the ternary system composed of PEG 400 + Na₂CO₃ + H₂O at 293 (± 1) K, with the binodal curve data adjusted through Equation 1, TLs data and critical point determination.

Biographies

Biographical sketches

Emanuel V. Capela: Emanuel V. Capela graduated in Biochemistry in 2014, by University of Aveiro, Portugal, where he also obtained his MSc in Biochemistry – Biomolecular Methods, in 2016. He worked as Research Assistant at Institute for Bioengeneering and Biosciences (iBB), Instituto Superior Técnico, University of Lisbon, Portugal, and currently he is a PhD student in Chemical Engineering – Bioengineering at University of Aveiro, Portugal, working on the development of new platforms for the extraction and purification of biopharmaceuticals and other value-added compounds using ionic liquids.

João H. P. M. Santos: João H. P. M. Santos graduated in Biotechnology at the University of Aveiro, Portugal, and concluded his MSc in Industrial and Environmental Biotechnology at the same university. He conducted a 7-month period of exchange, enabling him to develop part of his MSc thesis at the Laboratory of Pharmaceutical Biotechnology, in the Faculty of Pharmaceutical Sciences of the São Paulo University (USP), Brazil. Currently, he is a Joint PhD student in Chemical Engineering at Aveiro University (UA), working in collaboration with São Paulo University. His research interests are focused on PEGylation strategies for the development of analytical and therapeutic proteins.

Isabel Boal-Palheiros: Isabel Boal-Palheiros received her PhD degree in 1997 in Inorganic Chemistry from the University of Aveiro, Portugal. Since then she is a lecturer at Department of Chemistry at the same University, teaching mainly Physical Chemistry subjects. Her current research interests are in the field of properties and applications of ionic liquids.

João A. P. Coutinho: João A. P. Coutinho graduated in Chemical Engineering at University of Porto, Portugal, and further studied Thermodynamics and Petroleum Technology at Technical University of Denmark where he got his PhD in Chemical Engineering in 1995, and was then a researcher at IFP, France. He is nowadays Full Professor at the Chemistry Department of University of Aveiro, Portugal, and he is active on the development of more sustainable purification processes to be applied at the biorefinery.

Sónia P. M. Ventura: Sónia P.M. Ventura received her PhD degree in 2011 in Chemical Engineering from the University of Aveiro, Portugal. From 2012 to 2014 she was a post-doctoral researcher at University of Aveiro. From 2014 to 2015 she was an Assistant professor in the same institution and after one year she resumed her post-doctoral research until 2016. Since November of 2016, she is an Assistant Researcher at CICECO, at the Chemistry Department of University of Aveiro. She is nowadays actively working under the Blue Biorefinery concept, in the development of efficient bioprocessing tools and processes to recover bioactive compounds from marine biomass using alternative solvents.

Mara G. Freire: Mara G. Freire received her PhD degree in 2007 in Chemical Engineering from the University of Aveiro, Portugal. From 2008 to 2013 she was a post-doctoral researcher at Instituto de Tecnologia Química e Biológica, ITQB2, the New University of Lisbon, Portugal. Since the beginning of 2014 she is a Coordinator Researcher at CICECO, at the Chemistry Department of University of Aveiro, and the principal investigator of a European Research Council (ERC) Starting Grant regarding the development of cost-effective purification platforms for biopharmaceuticals using ionic liquids.