Microchemical Journal 147 (2019) 429–436

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Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

Extraction of arsenic(III) in aqueous two-phase systems: A new methodology T

for determination and speciation analysis of inorganic arsenic
Roberta C. Assisa,1, Betânia A. de Araújo Fariaa,1, Claudia L. Caldeirab,
Aparecida Barbosa Magestec, Leandro R. de Lemosd, Guilherme D. Rodriguesa, ,1
⁎

a
Universidade Federal de Minas Gerais, DQ/ICEX, Belo Horizonte 31.270-901, MG, Brazil
b
Universidade Federal de Minas Gerais, DEMET, Belo Horizonte 31.270-901, MG, Brazil
c
Universidade Federal de Ouro Preto, DQUI/ICEB, Ouro Preto 35.450-000, MG, Brazil
d
Universidade Federal dos Vales do Jequitinhonha e Mucuri, DEQUI, Diamantina 39.100-000, MG, Brazil

ARTICLE INFO ABSTRACT

Keywords: This work utilizes the liquid–liquid extraction technique of aqueous two-phase systems (ATPS) formed by a
Inorganic arsenic polymer and electrolyte to develop a novel methodology for the extraction, determination, and speciation
Aqueous two-phase systems analysis of inorganic arsenic. To optimize the methodology, the following parameters were studied: pH of the
Extraction system, nature of the ATPS-forming electrolyte and the polymer, tie-line length (TLL) of the system, type and
Speciation analysis
concentration of extractants, and the mass ratio of the top and bottom phases. The highest extraction (%
E = 98%) was obtained for an ATPS composed of L64 + Na2SO4 + H2O, at pH = 6.00, TLL = 33.55% w/w
using the ammonium pyrrolidine dithiocarbamate (APDC) extractant in a molar ratio (APDC/As(III)) of 960,
and with a mass ratio of 1/4. In these same conditions, As(V) was not extracted satisfactorily (%E = 18%) to
ATPS top phase, with a separation factor equal to 530 after three extractions, showing the great potential of
the system for the speciation analysis of inorganic arsenic. Moreover, the method was validated employing
HG-ICP OES with a coefficient of determination of 0.9957 and limits of detection and quantification equal to
0.20 μg kg−1 and 0.66 μg kg−1, respectively, proving that the method is capable of detecting the low con-
centrations required by legislation. The relative standard deviation (RSD) values varied between 4.88 and
9.45%, and the RSD in inter-day precision was 11.07%, which are in accordance with the limits of the vali-
dation guide used by the National Institute of Metrology, Quality and Technology, COD-CGCRE-008.
Furthermore, relative error rates ranged from −7.44 to 9.90% and recovery percentages were between 92.6
and 110% for the accuracy studies. This method was also applied in a spiked tap water sample, presenting a
recovery value equal to 87.10%.

1. Introduction
Arsenic is a solid metalloid that can be found in the environment in
both organic and inorganic forms, and its exposure is a consequence of
natural phenomena or anthropogenic actions. Therefore, arsenic can be
released into the environment by weathering processes and leaching
processes of volcanic rocks, minerals and geothermal waters, or through
industrial waste, fossil fuel processing and, mining activities (especially
gold exploration) [1]. Its application varies from its use as a pre-
servative in leather and wood (copper and chromium arsenate), as an
additive in metal alloys, in the production of insecticides (lead
arsenate), and more recently, in the treatment of patients with leukemia
(arsenic trioxide) [2,3].
The inorganic species, arsenite (As(III)) and arsenate (As(V)), are
considered more toxic than the organic species, such as mono-
methylarsenate (MMA) and dimethylarsenate (DMA). And among the
inorganic species, arsenite is considered more soluble, mobile and
therefore more toxic than arsenate [4]. Due to its high toxicity, the
ingestion, inhalation, or dermal contact with arsenic can cause skin
lesions, cardiovascular diseases, hematological effects, neurological
disorders, and cancer of the lung, bladder, kidneys and liver [1,5].
Therefore, inorganic arsenic is first on the list of priority pollutants of

Abbreviations: APDC, ammonium pyrrolidine dithiocarbamate; ATPS, aqueous two-phase systems; BP, bottom phase; DDTP, ammonium diethyl dithiophosphate;
TL, tie-line; TLL, tie-line length; TP, top phase
⁎
Corresponding author.
E-mail address: guilhermedr@ufmg.br (G.D. Rodrigues).
1
Laboratório de Soluções Analíticas Verdes.

https://doi.org/10.1016/j.microc.2019.03.058
Received 27 July 2018; Received in revised form 7 March 2019; Accepted 18 March 2019
Available online 19 March 2019
0026-265X/ © 2019 Elsevier B.V. All rights reserved.
R.C. Assis, et al.
the United States Environmental Protection Agency (EPA) [6] and is
also classified by the International Agency for Research on Cancer
(IARC) as a human carcinogen [7,8].
Furthermore, the Codex Alimentarius (Food and Agriculture
Organization of the United Nations - FAO) established a maximum limit
of 0.2 mg kg−1 of arsenic in rice [9], the Food and Drug Administration
(FDA) of the United States proposed a limit of 0.1 mg kg−1 of inorganic
arsenic in infant rice cereals [10], and the World Health Organization
(WHO) determined a maximum content of 0.01 mg L−1 of total arsenic
in water for human consumption [11].
Thus, there is an urgent need to develop methods that are low im-
pact and efficient for the extraction, determination, and speciation of
arsenic. Furthermore, the growing environmental concerns about ar-
senic make it essential to develop methodologies that follow the prin-
ciples of Green Chemistry [12].
Several studies have been developed for the detection and de-
termination of arsenic species, including: cloud point extraction in rice
samples for total arsenic speciation and As(V) [13], determination of
total arsenic, total inorganic arsenic and inorganic arsenic species in
rice by electrothermal atomic absorption spectrometry [14], pre-
concentration in solid phase extraction disks and inorganic arsenic
speciation in drinking water by X-ray fluorescence spectrometry [15],
arsenic speciation using LC-ICP-MS (Liquid chromatography-in-
ductively coupled plasma mass spectrometry) and HG-ICP OES (Hy-
dride generation-inductively coupled plasma optical emission spectro-
metry) techniques [16–18]. Moreover, some authors have studied
different types of techniques for removal and determination of arsenic,
such as oxidation, adsorption, electrocoagulation, voltammetry, and
electrodeionization, among others [19–21].
The technique of extraction by aqueous two-phase systems (ATPS)
presents great potential for the extraction of several analytes, and is a
simple, low cost, and more environmentally friendly technique [22]. In
addition, the ATPS has several advantages, including the possibility of
preconcentration, a low interfacial tension, which benefits diffusion
through the interface [23], the possibility of large-scale application
[24], and the ability to extract both charged and neutral species [25].
These are systems primarily composed of water, other compounds that
have low toxicity, and, in some cases, biodegradable compounds [26].
The ATPS can be formed by two heterogeneous phases composed of
aqueous solutions of two incompatible polymers [27], a polymer and an
electrolyte [28], or two types of physically incompatible electrolytes
[26]. For a polymer and a salt type ATPS, the resulting system generally
consists of the polymer-rich top phase (TP) and the electrolyte-rich
bottom phase (BP).
This system has already been used in several analytical meth-
odologies for the extraction and/or determination of several ana-
lytes, such as proteins [28], DNA [29], nanoparticles [30], dyes
[31,32], and metals [33–36]. The ATPS was used in 2013 as an ex-
traction technique for inorganic arsenic speciation, where these
species were converted into arsenomolybdenum blues, and then used
in nitrite and nitrate speciation in aqueous solution, since they ex-
hibited different behaviors in the two-phase system [37]. However,
the highlight of this study was not to optimize the conditions of the
two-phase system to maximize the extraction and speciation of in-
organic arsenic.
The most common extraction and separation techniques are already
widespread and consolidated in practice. However, it has disadvantages
that need to be considered, such as cost and toxicity of the reagents.
Conventional liquid-liquid extraction, for example, uses high toxicity
solvents and flammable substances, in addition to formation of emul-
sions between aqueous and organic phases and large generation of
waste [38]. The ATPS due to the difference between the top and bottom
phases can partition several analytes and therefore also separating them
into phases. Moreover, comparing with classical chromatographic
techniques, the ATPS presents significant advantages, mainly in cost
reduction due to non-use of columns which are expensive, have
430
Microchemical Journal 147 (2019) 429–436
specificity depending on the analytes and/or use a large volume of
organic solvents [39].
The present work thus proposes the development of a more en-
vironmentally friendly method for the extraction and speciation ana-
lysis of the inorganic forms of arsenic in aqueous samples, using the
technique of extraction of the aqueous two-phase systems and the de-
termination through an inductively coupled plasma optical emission
spectrometry (ICP-OES).
2. Experimental
2.1. Equipments
During all experiments, a pH meter (HI 2221, Hanna) was used to
perform pH adjustments of the deionized water used in the preparation
of ATPS stock solutions; an analytical balance (AUY 220, Shimadzu), of
uncertainty ± 0.0001 g; a centrifuge (Excelsa II, FANEN) to accelerate
phases separation; and a thermostatic bath of uncertainty ± 0.5 °C (SL
152, Solab) to establish the thermodynamic equilibrium of ATPS assays.
The analytical signals were obtained through an inductively coupled
plasma optical emission spectrometer (Optima 7300 DV, Perkin Elmer)
and coupled to a hydride generator (HG-ICP-OES) when lower con-
centration levels were required during validation. The parameters used
in both instruments are shown in the Table S1 of the Supporting in-
formation (SI).
2.2. Reagents
Hydrochloric acid (HCl, 37%) and sodium hydroxide (NaOH,
99.0%), used for the pH adjustments of the stock solutions, were pur-
chased from Vetec.
The polymer PEO1500 (composition: HO(C2H4O)nH and molar
mass 1500 g mol−1) obtained from Vetec, and the copolymers L64
(composition: (EO)13(PO)30(EO)13 and molar mass 2900 g mol−1) and
L35 (composition: (EO)11(PO)16(EO)11 and molar mass 1900 g mol−1),
both manufactured by Sigma-Aldrich (being PO blocks of propylene
oxide and EO blocks of ethylene oxide) were used in the composition of
the ATPS studied.
The salts anhydrous sodium sulfate (Na2SO4, 99.0%), sodium tar-
trate (Na2C4H4O6·2H2O, 99.5%), sodium citrate (Na3C6H5O7·2H2O,
99.0%) and lithium sulfate (Li2SO4H2O, 99.0%) were obtained from
Vetec, and the magnesium sulfate (MgSO4·7H2O, 98.0%) manufactured
by Synth. The extractants ammonium pyrrolidine dithiocarbamate
(APDC - C5H12N2S2, 98.0%) and ammonium diethyl dithiophosphate
(DDTP - C4H14NO2PS2, 95.0%) were obtained from Sigma-Aldrich, the
potassium iodide (KI, 99.0%) was obtained by Modern Chemistry, and
the ammonium heptamolybdate ((NH4)6Mo7O24·4H2O, 99.0%) pro-
vided by Merck.
The sodium arsenite salt (NaAsO2, 99.0%) was obtained from
Sigma-Aldrich and sodium arsenate heptahydrate (Na2HAsO4·7H2O,
98.5%) was purchased from Merck. In all experiments was used deio-
nized water (resistivity = 18.2 MΩ·cm at 298.15 K) obtained from a
Simplicity UV ultrapure water system (Millipore).
2.3. ATPS composition
2.3.1. Preparation of stock solutions
Stock solutions of polymer (top phase) and electrolyte (bottom
phase) were prepared in deionized water after pH adjustments to pre-
pare the ATPS assays. The concentrations of these solutions were ob-
tained through the predetermined tie-line length (TLL) of the system
(Eq. (1)), being obtained through the phase diagrams found in the lit-
erature (Table 1).
TLL = [(CP TP CP BP)2 + (CS TP CSBP)2]1/2 (1)
where CPTP and CP BP
are the concentrations of the polymer and CS TP
and
R.C. Assis, et al. Microchemical Journal 147 (2019) 429–436

Table 1 DAs (III )

S=
Concentrations in % (w/w) of polymer, salt and water in the top phase (TP) and DAs (V ) (3)
bottom phase (BP), and TLL for the ATPS studied, at temperature equal to
298.15 K.
System TP BP TLL (% w/w)
2.4. Method validation

wpolymer wsalt wwater wpolymer wsalt wwater The proposed method was validated for the following analytical
performance parameters: linearity, limit of detection (LOD), limit of
PEO1500 + Na2SO4 + H2O [42]
quantification (LOQ), and accuracy and precision (repeatability and
1 37.94 1.95 60.11 1.86 19.45 78.69 40.10
intermediate precision) according to the validation guide of the
L64 + Na2SO4 + H2O [40]
National Institute of Metrology, Quality and Technology, COD-CGCRE-
2 41.73 2.23 56.05 0.55 9.60 89.85 41.83
008 [43]. For this, an HG-ICP OES was used to obtain the analytical
L64 + Na2C4H4O6·2H2O + H2O [25]
signal.
3 43.03 4.01 52.96 2.70 12.86 84.44 41.28

L64 + Na3C6H5O7·2H2O + H2O [25]

3. Results and discussion
4 42.99 2.84 54.17 1.37 11.72 86.91 42.56

L35 + Na2SO4 + H2O [41]

5 46.91 0.82 52.27 2.59 15.75 81.66
L64 + Li2SO4·H2O + H2O [40]
6 41.72 3.27 55.00 0.41 10.01 89.57
L64 + MgSO4·7H2O + H2O [40]
7 45.58 2.20 52.22 5.15 7.18 87.66
CSBP are the concentrations of the salt in % (w/w) in the top and bottom
phases, respectively.
2.3.2. Parameters optimized in the extraction
Several parameters that influence the partitioning of the analyte
into the TP of the ATPS were evaluated to obtain the optimal extraction
conditions for As(III) and As(V), such as: the pH of the reaction medium
(2.00, 6.00, 8.00, and 11.00), the nature of ATPS-forming electrolyte
(Na2SO4, Li2SO4·H2O, MgSO4·7H2O, Na2C4H4O6·2H2O, and
Na3C6H5O7·2H2O), the nature of the polymer (PEO1500, L64, and L35),
the tie-line length of the system (TLL = 33.55, 41.83, and 47.82% w/
w), the type and concentration of the extractants (KI, APCD, DDTP, and
ammonium heptamolybdate), and the mass ratio of the top and bottom
phases (mBP/mTP: 1/1, 2/1, 4/1, 6/1, 8/1, and 9/1).
2.3.3. ATPS extraction procedure
The extractant agent and arsenic were added to the TP and BP of the
system, respectively, with the amount of extractant varied and the ar-
senic concentration kept constant at 30 mg kg−1 in each of the opti-
mization experiments. A 15 mL glass tube containing 2.0000 g of each
phase was prepared for each experiment, except in experiments where
the masses of the phases were varied to analyze their influence in the
preconcentration step of the analyte. All experiments were performed
in duplicate, and a blank was prepared for each of the different assays.
After the ATPS preparation, the tubes were manually stirred for
3 min and then centrifuged for 20 min at 560 ×g to accelerate the phase
separation. They were then placed in a thermostatic bath for 30 min at
298.15 K to establish thermodynamic equilibrium.
The TP of each tube was collected and diluted for ICP-OES analysis
(approximately 20 times). An analytical curve in polymer solution was
used to obtain the results. The percentage of arsenic extraction (%E)
was calculated from Eq. (2):
nTP
%E = × 100
nTotal
where nTP is the amount of analyte (mmol) in the top phase and nTotal is
the amount of analyte (mmol) added in the system.
In addition, to evaluate the speciation of the inorganic forms of
arsenic the coefficient of distribution of the species (D = %E /
(100 − %E)) was calculated. This coefficient of distribution was used to
determine the factor of separation of these species through Eq. (3):
The formation of a metallic ion-extractant complex in ATPS for its
46.76
subsequent extraction to TP is governed mainly by the competition
between the electrolyte and the extractant by the interaction with the
41.86
analyte. In the absence of extractant the analyte tends to concentrate
preferentially in the BP, where the interactions between the electrolyte
40.73
and the metallic ion generate a thermodynamically stable complex. As
the amount of extractant increases, the number of its complexes formed
with the metallic ion also increases, and consequently higher %E values
of the analyte for TP are observed [33,34].
3.1. Effect of extracting agent
The first extracting agents studied were KI and APDC, in systems
formed by PEO1500 + Na2SO4 + H2O (TLL = 40.10% w/w), with
pH = 1.00 and 3.00, respectively, and at increasing concentrations
approaching the maximum solubility of these extractants in PEO1500
aqueous solution. According to the results presented in Fig. S1 of the SI,
%E did not change significantly with the increase in the amount of KI
when compared to %E in the absence of it. The maximum extraction
using KI was close to 40%. When the APDC was used in this system, the
highest %E was obtained in the absence of the extractant, yielding a
value close to 20%.
Due to the low %E values, the study of the effect of the extractant
nature was also performed in ATPS formed by the triblock copolymer
(L64). Two types of extracting agents, the APDC and DDTP (Fig. 1A),
were evaluated in systems formed by L64 + Na2SO4 + H2O and at
pH = 6.00, with the purpose of determining the best extraction con-
dition for arsenite (HAsO2) to the TP.
Although both extractants were able to complex As(III) [44,45], the
use of the DDTP extractant (Fig. 1A) under these conditions did not
significantly extract As(III) to the TP (%E < 35%). The maximum %E,
around 98%, was obtained using the APDC extractant with an APDC/As
(III) molar ratio of 960. Fig. 1A shows that in the absence of APDC, the
arsenic remains mostly in the BP, with a maximum %E that is close to
30%.
This fact occurs due to the interactions of arsenic in solution with
the ions present in the BP. When this interaction is stronger than the
arsenic-extracting interaction, the arsenic remains in the BP and
therefore is not extracted satisfactorily to the TP. However, the %E
values of arsenic obtained in this work, in the absence of extractant, are
high when compared to other studies involving the extraction of me-
tallic ions using ATPS [22,34,46]. This high extraction in the absence of
(2) APDC can be justified due to the arsenic species at this pH being in
neutral form (HAsO2). Consequently, the interaction of the analyte with
the TP of the ATPS is intensified, thus increasing the %E value of ar-
senic.
Fig. 1B shows the behavior of arsenic extraction with the extractant
ammonium heptamolybdate, which is an extractant that complexes
only the As(V). This study was performed in ATPS composed of
L64 + Na2SO4 + H2O (TLL = 41.83% w/w), at its maximum solubility

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R.C. Assis, et al. Microchemical Journal 147 (2019) 429–436

110 (A) DDTP 100 As (III)

APDC (B)
100 90 As (V)
90 80
80 70
70 60
%E

%E
60 50
50 40
40 30
30 20
20 10
0 200 400 600 800 1000 1.0 1.5 2.0 2.5 3.0 3.5
(molar ratio (extractant)/(As(III)) pH

Fig. 1. Effect of the concentration of APDC and DDTP (A) and ammonium heptamolybdate (B) on the extraction of As(III) and As(V) in ATPS formed by
L64 + Na2SO4 + H2O (TLL = 41.83% w/w).

(~94,000 mg kg−1), and at different acid pH values (1.50, 2.00, 2.50,
and 3.00), since the formation of the complex with As(V) has been
shown to occur within this range of values [47].
Fig. 1B shows that the ammonium heptamolybdate, besides not
extracting As(III), also did not satisfactorily extract As(V), with its
maximum %E close to 50% at pH = 1.5.
These results obtained with the extractant DDTP and heptamo-
lybdate of ammonium highlight that the ATPS is a more complex ex-
traction method than the solvent extraction technique due to the
greater number of variables (salt, polymer, and extractant, among
others), and that more studies are required to better understand the
different behaviors and interactions exhibited by some analytes in the
ATPS. As the APDC generated higher values of %E it was the extractant
chosen for the next experiments with As(III) in solution.
3.2. Influence of the ATPS-forming electrolyte and the copolymer
Systems formed by L64 + H2O and at pH = 6.00, with the electro-
lytes Na2SO4, Li2SO4·H2O, and MgSO4·7H2O, and TLL = 41.83, 41.86
and 42.05% w/w, respectively, were studied to analyze the influence of
cations on As(III) extraction (Fig. 2A).
It was observed that As(III) exhibited a similar extraction behavior
in the presence of Na2SO4 and Li2SO4·H2O, with maximum %E values
close to 100%, whereas the ATPS assays composed of MgSO4·7H2O
presented lower values of %E (%E = 76.36%).
The lower %E values of the analyte are likely due to the +2 charge
of the magnesium ion, which may have intensified its interaction with
arsenic (HAsO2) in the BP, decreasing the partition of the analyte. This
interaction between ions and HAsO2 in BP occurs by ion-dipole inter-
action.
Three different electrolytes were used under the same conditions to
analyze the influence of the anions on the extraction of arsenic
(Fig. 2B): Na2SO4, Na3C6H5O7·2H2O, and Na2C4H4O6·2H2O.
The key observation from Fig. 2B is that the anion of the electrolyte
does not exert a significant influence on the %E of As(III), since the
resultant extractions involving the three electrolytes exhibited a similar
behavior along the variation of APDC concentration.
These observed similarities are likely due to the arsenic species
being in neutral form (HAsO2) at this pH value. Considering this simi-
larity of the behavior of arsenic extraction in the presence of the dif-
ferent electrolytes studied, the Na2SO4 salt was chosen to compose the
ATPS due to its greater practicality in the preparation of the assays
containing the L64 copolymer, especially the lower viscosity of the
system that is obtained with this salt. Furthermore, the cost of this salt
is lower than the others used in this study, which make it a more
practical choice for extraction applications.
The nature of the ATPS-forming macromolecule was studied with
two triblock copolymers, L64 and L35, in the ATPS composed of
Na2SO4 + H2O (TLL = 41.83 and 46.76% w/w, respectively) at
pH = 6.00 (Fig. 2C).
The highest As(III) extraction occurs in the presence of the L64
copolymer (99.35%). L64 is a triblock copolymer that possesses a
higher percentage of propylene oxides ((EO)13(PO)30(EO)13) than co-
polymer L35 ((EO)11(PO)16(EO)11). The L64 copolymer has 60%

100 100 (B) 100

(C)
(A)
90 90 90

80
80 80
70
70 70
%E

%E

60
%E

60 60
50
50 50
40
Li2SO4 . H20 Na3C6H5O7
40 40 30
MgSO4 . 7H20 NaC4H4O6·4H 2O L35
30 30 Na2SO4 20
Na2SO4 L64
20 20 10
0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200

(molar ratio (APDC)/(As(III)) (molar ratio (APDC)/(As(III)) (molar ratio (APDC)/(As(III))

Fig. 2. Effect of the cation (A) and anion (B) nature of the ATPS-forming electrolyte, and effect of the nature of the ATPS-forming copolymer (C) on As(III) extraction
in the presence of the APDC extractant and at pH = 6.00.

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R.C. Assis, et al. Microchemical Journal 147 (2019) 429–436

110
100
100
90
90
80
80
70
70
%E

60 60

%E
50 50

40 40
pH = 2.00
pH = 6.00 30 TLL = 33.55% w/w
30 pH = 8.00
pH = 11.00 20 TLL = 41.83% w/w
20 TLL = 47.82% w/w
0 400 800 1200 1600 2000 2400 10
0 200 400 600 800 1000 1200
(molar ratio (APDC)/(As(III)))
(molar ratio (APDC)/(As(III))
Fig. 3. Influence of the pH of the reaction medium on the extraction of As(III) in
Fig. 4. Effect of TLL = 33.55; 41.83; and 47.82% w/w in the extraction of As
the ATPS composed of L64 + Na2SO4 + H2O (TLL = 41.83% w/w), in the
presence of the APDC extractant. (III) in ATPS composed of L64 + Na2SO4 + H2O, at pH = 6.00, in the presence
of the APDC extractant.

propylene oxide and a molar mass of 2900 g mol−1, while L35 has 50%
3.4. Influence of tie-line length (TLL)
propylene oxide and a molar mass of 1900 g mol−1, which creates a
greater hydrophobic character in the L64 molecule than in L35, pro-
The tie-line length was varied between 33.55, 41.83, and 47.82%
viding the formation of an ATPS with a more hydrophobic TP.
w/w in the ATPS composed of L64 + Na2SO4 + H2O at pH = 6.00
Furthermore, an ATPS whose solubilization of these complexes of less
(Fig. 4).
polarity within the copolymer micelles is favored, which justifies the
It is noticed from Fig. 4 that the maximum extraction percentage of
behavior observed in Fig. 2C.
As(III) obtained was 93.89% for TLL = 47.82% w/w, which was lower
than the maximum extractions obtained for TLL = 33.55% w/w (%
E = 98.33%) and TLL = 41.83% w/w (%E = 99.35%).
3.3. Effect of pH
The differences between the compositions of the top and bottom
phases of the system (i.e., thermodynamic intensive properties) in-
After selecting APDC as extracting agent and ATPS-forming elec-
crease for TLL = 47.82% w/w, such that the amount of polymer in-
trolyte and the copolymer for the extraction of As(III), different pH
creases in TP and the amount salt increases in the BP [48]. Therefore,
values (2.00, 6.00, 8.00, and 11.00) of the deionized water were ad-
there is a competition between the electrolyte and the extractant for the
justed (Fig. 3) used in the preparation of the polymer and salt solutions
interaction with the arsenic, thus reducing the %E of arsenic to the TP.
in the ATPS formed by L64 + Na2SO4 + H2O (TLL = 41.83% w/w).
It is worth mentioning that the copolymer in the TP of the ATPS also
Fig. 3 illustrates that the arsenite extraction is influenced by the pH
plays an important role in the extraction of the analyte (as shown in
of the system, with the maximum extraction percentage attained for
Section 3.3), but under these conditions, the partition of the arsenic-
pH = 6.00. Here 99.35% extraction was obtained using a smaller pro-
APDC complex is not significantly modulated by the L64 concentration.
portion of extractant when compared with the other studied values.
The maximum percentage of arsenic extraction obtained in the
This behavior may be associated to the dependence of the molecular
study of TLL influence (%E = 99.35%) was reached in the second TL
structures of the extractant, the electrolyte, and the arsenic species as a
(TLL = 41.83% w/w). However, in the first TL (TLL = 33.55% w/w), in
function of pH. The molecular structures at each pH value (2.00, 6.00,
the same proportion of APDC, the percentage of arsenic extraction was
8.00, and 11.00) are shown in Table S2 of the SI.
98.33%, which was not significantly different from the observed max-
At pH = 2.00, there is a balance of the ionized species of electrolyte
imum. The first tie-line length (TLL = 33.55% w/w) was chosen for the
(HSO4−/SO42−) in the BP, and in the TP the extractant is in the neutral
next studies since the overall compositions of the system are smaller for
form, which does not favor the extraction of the analyte (HAsO2) to TP.
this TLL, thus reducing the reagent costs.
At pH = 11.00, the change that occurs is in the molecular structure of
arsenic (AsO2−) and of the APDC. In anionic form, the interaction of
3.5. Speciation analysis of inorganic arsenic
this analyte with the extractant decreases, requiring a greater amount
of APDC to obtain the maximum extraction, as can be seen in Fig. 3. At
It is possible to evaluate the differences in the partitioning behavior
pH = 8.00, the molecules have the same structure as at pH = 6.00, but
of the inorganic arsenic by varying the ATPS parameters. The speciation
there was a lower formation of arsenic-extracting complexes and,
capacity of As(III) and As(V) was studied at concentrations of
therefore, lower values of %E were obtained at this pH.
30 mg kg−1 and under the conditions optimized for As(III) extraction
At pH = 6.00, the extraction of the analyte into the TP of the system
(Fig. 5A). These ATPS conditions are: L64 + Na2SO4 + H2O, with
was the most favored. In these conditions, the interaction of arsenic in
TLL = 33.55% w/w, at pH = 6.00, and in the presence of the APDC
solution (HAsO2) with the extractant molecule (total load equal to −1)
extractant at 298.15 K.
was more intense even the electrolyte molecule was predominantly in
Fig. 5A shows that the highest extraction of As(V) occurred in the
the form of SO42−. The number of complexes formed between ex-
absence of APDC (%E = 21.62%), confirming that there is no complex
tractant and analyte was higher, and consequently, the maximum %E of
formation of the APDC extractant in the presence of the arsenate ion
As(III) was obtained under these conditions. It was thus decided to use
(HAsO42−), in agreement with previous studies [44]. Therefore, these
the stock solutions at pH = 6.00 for the following studies due to the
results demonstrate that As(V) cannot be extracted to the TP under the
maximum %E%E obtained at this pH value.
current conditions satisfactorily.

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R.C. Assis, et al. Microchemical Journal 147 (2019) 429–436

110 600

(A) As (III) (B)

100
As (V) 500
90

80

S As(III)/As(V)
400
70

60 300
%E

50
200
40

30
100
20

10 0
0 200 400 600 800 1000 1 2 3

(molar ratio (APDC)/(As)) Extractions

Fig. 5. Single extraction of As(III) and As(V) (A) and successive extractions (B) in the ATPS composed of L64 + Na2SO4 + H2O (TLL = 33.55% w/w) at pH = 6.00
and in the presence of the APDC extractant.

1200 extraction (SPE) cartridges for the speciation of four arsenic species
(DMA, MMA, As(V) and As(III)), and the determination was performed
using FI-HGAA (Flow Injection Hydride Generation Atomic Absorption
1000 Spectrometry). The authors used a silica-based SAX cartridge to sepa-
rate As(V) and MMA of As(III), and elution was performed with hy-
800 drochloric acid. Under these conditions, As(III) and As(V) were retained
in the cartridge around 22 ± 1% and 100 ± 5%, respectively.
Therefore, from the selective retention using specific cartridges the
Signal

600
400
200
0 2 4 6
Mass ratio mBP/mTP
Fig. 6. Influence of mass ratio of BP and TP (mBP/mTP = 1/1, 2/1, 4/1, 6/1, 8/
1, and 9/1) in the extraction of As(III) in ATPS composed of
L64 + Na2SO4 + H2O (TLL = 33.55% w/w), at pH = 6.00 and in the presence
of APDC extractant.
At the molar ratio of APDC/As(III) equal to 960, where the max-
imum As(III) extraction occurs (%E = 98.33%), the As(V) extraction
percentage was close to 18%. Therefore, it is perceived that there was a
significant separation of As(III) and As(V) using the ATPS under these
conditions. Arsenite was almost fully partitioned to the TP, while the
arsenate preferentially remained in the BP.
Despite the considerable separation of arsenic species, which con-
firms the potential of the ATPS for inorganic arsenic speciation analysis,
it is possible to further increase the separation factor (S) of these species
through successive system extractions (Fig. 5B).
Two more extractions were performed to further decrease the per-
centage of As(V) in the TP, similar to the extraction procedure de-
scribed in Section 2.3. In the successive system extractions, the TP of
the first extraction stage was used successively in the next extraction,
obtaining a final separation factor (S) value of 530, as show in Fig. 5B.
This separation factor is a value considered high when compared with
other literature results [35].
The results obtained using ATPS as a liquid-liquid extraction tech-
nique for speciation analysis of inorganic arsenic are comparable to the
results of other studies. Yalcin and Chris Le [49] used solid phase
authors were able to separate the inorganic species of arsenic using SPE
[49].
Wangkarn and Pergantis [50] also studied arsenic speciation. The
authors used a narrow-bore column HPLC (high performance liquid
chromatography) coupled on-line with ICP-MS for the speciation of
arsenic compounds. Some parameters, such as pH and flow rate, were
As(III)
optimized to increase the separation and the resolution of the peaks.
8 10 The arsenic species (As(III), As(V), DMAA and MMAA) were separated
on a C18 column with tetrabutylammonium hydroxide as mobile phase.
Although the high sensitivity obtained and the high speed of separation,
the used technique (HPLC-ICP-MS) is significantly more expensive than
the present work [50].
Thus, ATPS approach to inorganic arsenic speciation analysis pro-
vided competitive results comparable to more robust, expensive and
less green techniques reported in the literature.
3.6. Effect of phases mass ratio
The influence of the phase masses on the values of the analytical
signals was studied to determine As at lower concentrations. Here the
BP mass was increased relative to the TP mass to obtain a higher pre-
concentration of the analyte. The following mass ratios between the
phases were evaluated: mBP/mTP = 1/1, 2/1, 4/1, 6/1, 8/1, and 9/1 for
the extraction of As(III) under the already optimized conditions of the
system (Fig. 6).
Fig. 6 shows that there was a progressive increase of the analytical
signal of As(III) to a mass ratio of BP/TP of 4/1, where mBP/mTP = 4/1
caused the greatest increase in the analytical signal when compared to
other mass ratios of the phases. The analyte concentration did not in-
crease for mBP/mTP > 6/1, suggesting a possible saturation of the TP
by the arsenic-APDC complex as the BP mass continued to increase
relative to the TP mass.
The validation studies were then conducted with mBP/mTP = 4/1
(mBP = 12.000 g and mTP = 3.000 g), chosen as a compromise between
arsenic preconcentration and its efficiency of extraction, to validate the
methodology with maximum sensitivity in As(III) determinations.

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R.C. Assis, et al. Microchemical Journal 147 (2019) 429–436

Table 2 Recovery assays were analyzed in triplicate at the same con-

Analytical parameters of the validation step of the proposed centrations of arsenic from the precision study to evaluate the accuracy
method for extraction of As(III) analyzed by HG-ICP OES. of the extraction method. This method is also considered accurate,
Parameters Values because it generated relative errors equal to −7.44% (5.00 μg kg−1),
6.58% (25.0 μg kg−1), 9.90% (40.0 μg kg−1), and 7.73%
Linearity 0.66–80 μg kg−1 −1
(70.0 μg kg ), as well as recovery percentages between 92.6 and
R2a 0.9957
110%.
LODb 0.20 μg kg−1
LOQc 0.66 μg kg−1 The sample used for the application of the method was a public
RSDd (%) (n = 6) 8.31 (5 μg kg−1) water supply sample collected at the tap in the Department of
5.85 (25 μg kg−1) Chemistry of the Federal University of Minas Gerais, in the city of Belo
9.45 (40 μg kg−1)
Horizonte - MG. The pH was adjusted to 6.00, according to the opti-
4.88 (70 μg kg−1)
RSDd (%) inter-days 11.1 (25 μg kg−1)
mization study, and was centrifuged at 560 ×g for 20 min, so that any
Relative errors (%) −7.44 (5 μg kg−1) undissolved particle was separated from the sample. For the recovery
6.58 (25 μg kg−1) studies, the sample was spiked with arsenic concentration equal to
9.90 (40 μg kg−1) 80 μg kg−1 in the BP of the system and diluted 25-times to obtain the
7.73 (70 μg kg−1)
analytical signal from the recovery assays. This study presented a re-
Recovery (%) 92.6 (5 μg kg−1)
107 (25 μg kg−1) covery value equal to 87.10%, which is in accordance with previous
110 (40 μg kg−1) studies reported in the literature that validated methods of inorganic
108 (70 μg kg−1) arsenic speciation in different samples, which presented recovery rates
a
that varied between 88 and 105% [52,53].
R2: coefficient of determination.
b
LOD: limit of detection.
c 4. Conclusions
LOQ: limit of quantification.
d
RSD: relative standard deviation.
A liquid–liquid extraction method employing ATPS for the extrac-
3.7. Method validation tion and speciation analysis of inorganic arsenic was developed in this
work. The primary objective of extraction inorganic arsenic was
This validation was necessary to determine if the methodology achieved with an %E close to 98% for As(III). Due to the different be-
presented here was able to detect the low limits for arsenic in en- havior of As(V) in the system, which remained mostly in the BP (%
vironmental samples, such as 0.01 mg L−1 in drinking water [11]. Due E = 18%), the ATPS presented a great potential for inorganic arsenic
to the need for a more sensitive technique, an HG-ICP OES was used to speciation analysis.
obtain the analytical signal of the assays with standard solutions, blanks The best value of the %E for As(III) was obtained in the ATPS
and spiked sample. composed of L64 + Na2SO4 + H2O, using the solvent at pH = 6.00,
employing APDC as the extractant at a concentration of
63,150 mg kg−1, and with the compositions of the phases in the first tie-
3.7.1. Analytical parameters line length. This proposed extraction method has been validated fol-
Employing the previously established optimal conditions, the main lowing the main analytical parameters. The method is considered linear
analytical parameters of the proposed extraction method were de- with a coefficient of determination equal to 0.9957, it is precise
termined, and the results are presented in Table 2. (RSD < 15%), it is accurate (relative errors < 10%) and it yields sa-
To evaluate the linearity of the method the ATPS assays were pre- tisfactory recovery (92.6–110%). Furthermore, the LOQ of 0.66 μg kg−1
pared in triplicate, with the As(III) concentration varying from 0 to is in accordance with current legislation, such as the maximum allow-
80 μg kg−1 in the BP of the system, and the concentration of the ex- able arsenic in water for human consumption (10 μg L−1).
tractant APDC kept constant in the TP (63,150 mg kg−1). The coeffi- This method also presented an adequate recovery value, 87.10%,
cient of determination (R2 = 0.9957), together with the outliers tests within the acceptance criteria according to the validation guide used
(Jackknife test) and homoscedasticity (Brown–Forsythe test), showed (National Institute of Metrology, Quality and Technology, COD-CGCRE-
that the relation between analytical signal and concentration of the 008), when applied in a spiked tap water sample.
method studied follows a strong linear trend [51]. From the analysis of Thus, the aqueous two-phase system method is an environmentally
variance (ANOVA), the final regression equation obtained for this friendly and efficient alternative to liquid–liquid extraction, when
analytical curve was y = 7.94845x − 18.34793, where y is the in- conducted following the methodology outlined here, for the extraction
tensity of the emission line of arsenic and x is the concentration of and speciation analysis of inorganic arsenic.
arsenic in μg kg−1.
Seven blanks were prepared for the calculation of LOD Acknowledgements
(LOD = 3 × (s/S)) and LOQ (LOQ = 10 × (s/S)), where s is the stan-
dard deviation of the blanks and S is angular coefficient of the analy- The authors acknowledge the Fundação de Amparo à Pesquisa do
tical curve, yielding LOD = 0.20 μg kg−1 and LOQ = 0.66 μg kg−1 from Estado de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento
the analytical curve. This LOQ value highlights the sensitivity of this de Pessoal de Nível Superior (CAPES) and Conselho Nacional de
method, smaller than the WHO maximum permitted level of Desenvolvimento Científico e Tecnológico (CNPq) for financial support
0.01 mg L−1 of total arsenic in water for human consumption [11]. in this project, and to the Laboratório de Análises Químicas LAQ/
In the precision (repeatability) study, the relative standard devia- DEMET/UFMG - INCT Acqua (Instituto Nacional de Ciência e
tion (RSD) was determined for four different concentration levels of As Tecnologia em Recursos Minerais, Água e Biodiversidade) and
(III) in six replicates each. Values of 8.31, 5.85, 9.45, and 4.88% were Laboratório de Biohidrometalurgia e Tratamento de Efluentes – UFOP
found for As(III) concentrations of: 5.00, 25.0, 40.0, and 70.0 μg kg−1, for conducting the analysis on the equipment.
respectively. These values are within the acceptable limits
(RSD < 15%), according to the validation guide used [43]. Further- Appendix A. Supplementary data
more, an RSD of 11.1% was determined for the inter-day precision
(nn = 15) of a 25.0 μg kg−1 As(III) concentration, which is also an ac- Supplementary data to this article can be found online at https://
ceptable limit [43]. doi.org/10.1016/j.microc.2019.03.058.

435
R.C. Assis, et al.
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