3. Contents
TITLE: Aqueous biphasic systems composed of ionic liquids as a sample purification stepin the detection and quantification of phenolic contaminants in biological tissues
MEMBERS:

Helena Passos, University of Aveiro, CICECO, Chemistry Department
Ana Catarina Sousa, University of Évora, Department of Biology and Comprehensive

Health Research Centre (CHRC)
Mara G. Freire, University of Aveiro, CICECO, Chemistry DepartmentJoão A.P. Coutinho, University of Aveiro, CICECO, Chemistry Department
Nguyen Minh Tue, Center for Marine Environmental Studies (CMES), Ehime University
Tatsuya Kunisue, Center for Marine Environmental Studies (CMES), Ehime University
AIM: This project aims to use liquid-liquid extraction based on more efficient,environmental benign and economically viable systems, such as aqueous biphasicsystems (ABS) composed of ionic liquids (ILs) or solvent extraction with hydrophobic
eutectic solvents, as extraction and purification techniques in the detection of phenoliccontaminants in biological tissues.
PROCEDURE: In line with previous results, the ability of hydrophobic eutectic solvents(HES) to extract and purify bisphenol A (BPA) in chicken blood samples was evaluated.
BPA was used as a model phenolic contaminant, since its identification andquantification can be easily attained by using UV-Vis spectroscopy allowing a fastercharacterization of the systems studied here. HES of trioctylphosphine oxide(TOPO):decanoic acid (1:1 mol:mol) and thymol:menthol (1:1), mixed with citrate buffer
aqueous solutions at different volume ratios (1:2, 1:1, 2:1) were evaluated. All systems
were spiked with BPA at known concentrations. Chicken blood was commerciallyacquired at a local supermarket.
RESULTS: The results obtained last year demonstrated the remarkable ability of HES toinduce the precipitation of most blood matrix during solvent extraction step. Theseresults agreed with previous data reported in the literature.[1] Thus, in the current year,
a more detailed studied was carried-out by using these systems. HES composed oftrioctylphosphine oxide (TOPO):decanoic acid (1:1 mol:mol) and thymol:menthol (1:1),
mixed with citrate buffer aqueous solutions (pH 7) at different volume ratios (1:2, 1:1,2:1) were evaluated (cf. Figure 1). In a first set of experiments, BPA extraction efficiency
percentage (EE %) was determined in these systems in the absence of chicken blood. The

EE % was calculated by Eq. 1 and the obtained results are presented in Figure 2.

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𝐸𝐸 % =$%&$%&𝑚!"#!%''%(𝑚!"# + 𝑚!"#(Eq. 1)
With 𝑚!"# representing the weight of BPA determined on the HES phase (Top) andaqueous phase (Bottom).

+TOPO+

Decanoic acid
ThymolMenthol

Figure 1. Chemical structure of HES components.

100%

EE %

Thymol:Menthol

TOPO:DA

90%
80%
70%
60%50%
1: 2

1: 1

2: 1

HES:Aq. Phase vol. ratio
Figure 2. Extraction efficiency percentage (EE %) of BPA for HES phase by using

different HES (thymol:menthol and TOPO:decanoic acid(DA)) and organic:aqueous
phase volumetric ratios.
Results presented in Figure 2 demonstrated that both HES used in this work have goodability to extract BPA. The best results were attained with TOPO:Decanoic acid mixture,probably due to its more hydrophobic character which is favorable to the extraction ofBPA. Furthermore the higher is the HES:Aqueous phase volumetric ratio, the highest is
the extraction of BPA to the HES phase. Thus the best result was attained by using
TOPO:Decanoic acid HES at HES:Aqueous phase 2:1 volumetric ratio: EE % = 92 % ±
1 %. Despite the remarkable results obtained, it is important to refer that HES
composition was not optimized in this step, which may have a positive impact on BPA
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extraction efficiency. This should be evaluated as future work.
Since previous results were obtained in the absence of chicken blood a simple test was
carried-out to verify how the addition of chicken blood to the extraction system can affect
the quantification of BPA on the aqueous phase through UV-Vis spectroscopy. Figure 3
discloses the UV-Vis spectrum of the blank aqueous phase, i.e. without BPA addition, inthe presence and the absence of chicken blood to infer on blood matrix impact in thequantification technique. Since BPA is quantified at the wavelength of 277 nm, as it ispossible to infer from Figure 3, chicken blood matrix will have an influence on this regionof the spectra. Thus, it is important to take into account this influence and carry-out theprevious experiments in the presence of chicken blood to see if BPA EE % is also affected.

Abs (u.a.)This will be evaluated as future work.

0.6277 nm

0.40.20.0250300350400450500

550600

Wavelength (nm)Figure 3. Blank aqueous phase UV-Vis spectrum in the presence (orange) or absence(blue) of blood chicken. Macroscopic aspect of chicken blood matrix precipitation bysolvent extraction with HES.
PERSPECTIVES IN FUTURE: The results reported here demonstrate the potential of solvent

extraction based on HES to be used in the pre-treatment of blood samples for a moreaccurate detection of phenolic contaminants. To the good development of thismethodology, the future work should focus on the following aspects: (i) evaluation of HES
composition effect on BPA extraction efficiency; (ii) evaluation of blood matrix impact onBPA extraction efficient; (iii) use of the optimized system for the extraction of more
relevant phenolic contaminants, such as triclosan and 1-OH-pyrene; (iv) evaluation ofHES phase composition analysis by GC-MS: direct analysis vs back-extraction.

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REFERENCES:[1] Schaeffer N, Kholany M, Veloso TLM, Pereira JL, Ventura SPM, Nicaud JM, Coutinho
JAP (2019) Temperature-responsive extraction of violacein using a tuneable anionicsurfactant-based system. Chemical Communications 55: 8643–8646.

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