Fluid Phase Equilibria 506 (2020) 112367

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Fluid Phase Equilibria

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / fl u i d

Partitioning of cocaine and its adulterants in aqueous two-phase

systems: An alternative drug identification method
Gabriel M.D. Ferreira a, b, Guilherme M.D. Ferreira a, c, Adriano O. Maldaner d,
Luis H. Mendes da Silva a, Maria C. Hespanhol a, e, *
a
Grupo de Química Verde Coloidal e Macromolecular, Departamento de Química, Centro de Ci^ gicas, Universidade Federal de Viçosa,
encias Exatas e Tecnolo
Viçosa, 36570-900, Minas Gerais, Brazil
b
Departamento de Química, Instituto de Ci^ encias Exatas e Biologicas, Universidade Federal de Ouro Preto, Campus Morro do Cruzeiro, s/n
, Bauxita, Ouro
Preto, 35400-000, Minas Gerais, Brazil
c
Departamento de Química, Universidade Federal de Lavras, Campus Universita rio, Lavras, 37200-000, Minas Gerais, Brazil
d
Instituto Nacional de Criminalística, Minist
erio da Justiça, Brasília, BR-70610-200, Distrito Federal, Brazil
e
Group of Analysis and Education for Sustainability, Departamento de Química, Centro de Ci^ encias Exatas e Tecnologicas, Universidade Federal de Viçosa,
Viçosa, 36570-900, Minas Gerais, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The partitioning behaviors of cocaine freebase (CoF), cocaine hydrochloride (CoH), and a range of
Received 30 July 2019 adulterants (procaine (PRO), phenacetin (PHE), caffeine (CAF), benzocaine (BEN), and lidocaine (LID)) in
Received in revised form aqueous two-phase systems (ATPS) were evaluated, with the aim of developing a selective and efficient
14 October 2019
alternative technique for the identification of cocaine in samples confiscated by the police. The partition
Accepted 14 October 2019
Available online 23 October 2019
coefficients (KS) of all the solutes were obtained in ATPS formed using a macromolecule (PEO1500 or L35
copolymer), a salt (Na2SO4 or Li2SO4), and water. The KS values were consistently higher than 1 and
increased with increase of the tie-line length. For the PEO1500 þ Li2SO4 þ water ATPS at pH 6.0, the KS
Keywords:
Cocaine
values increased in the order: KLID < KPRO < KCAF < KCoF < KCoH < KPHE < KBEN. The greatest effect on the
Adulterants partitioning was shown by the polymer hydrophobicity, followed by the nature of the electrolyte and the
Aqueous two-phase systems pH of the ATPS. The ratio between two KS values for two different ATPS types was used as a strategic
Partition coefficient parameter for distinguishing between the two forms of cocaine, as well as between cocaine and the
Forensic chemistry adulterants. The K12/K6 ratio, where K6 and K12 are the KS values for the PEO1500 þ Li2SO4 þ water ATPS
at pH 6.0 and 12.0, respectively, enabled CoH to be distinguished from all the other solutes studied. In
addition, the effect of the hydrophobicity of the polymer on the KS values was fundamental for dis-
tinguishing CoF from the other solutes.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction Consequently, cocaine has become one of the substances most

Cocaine (methyl(1R, 2R, 3S, 5S)-3-(benzoyloxy)-8-methyl-8-
azabicyclo [1e3] octane-2-carboxylate) (Fig. 1), the main alkaloid
extracted from the leaves of Erythroxylum coca, is a stimulant that
affects the central nervous system and is one of the most widely
abused drugs [1]. Medical problems including brain damage, car-
diovascular toxicity, and death often accompany the use of that
drug [2], so its consumption is prohibited in many countries.
* Corresponding author. Group of Analysis and Education for Sustainability,
Departamento de Química, Centro de Cie ^ncias Exatas e Tecnolo
gicas, Universidade
Federal de Viçosa, Viçosa, 36570-900, Minas Gerais, Brazil.
E-mail address: mariacarmo@ufv.br (M.C. Hespanhol).
frequently apprehended by the police, making the development of
methods for its analysis and identification in seized samples an
extremely important topic of research. Knowledge regarding the
constituents of confiscated samples is very important in forensic
drug chemistry, because such information can provide both stra-
tegic and tactical intelligence data, which can enable determination
of the geographic origin of the drug, among other benefits [3].
Different methods are employed by police laboratories to carry
out analyses of cocaine samples, including chromatographic and
spectrometric techniques [4e6], which are laborious and expen-
sive. Colorimetric spot tests are also utilized, for instance the Scott
test, which has been widely applied for the analysis of confiscated
samples in which the presence of cocaine is suspected [7]. This test

https://doi.org/10.1016/j.fluid.2019.112367
0378-3812/© 2019 Elsevier B.V. All rights reserved.
2 G.M.D. Ferreira et al. / Fluid Phase Equilibria 506 (2020) 112367
Fig. 1. Molecular structure of cocaine.
consists of three simple steps. Firstly, a suspected sample of the
drug is mixed with a cobalt thiocyanate aqueous solution. If a blue
solid is observed, the second step consists of treatment of the
mixture with hydrochloric acid. In the final step, if the blue solid
completely disappears, then chloroform solvent is added to the
mixture and the appearance of a blue color in the organic phase
suggest that cocaine may be present.
In forensic analysis, it is important that the experimental results
should be able to confirm the exact nature of the seized samples,
because these results can condemn or absolve those criminally
involved. Therefore, attention should be given to the development
of tests that can be performed safely, quickly, and, most impor-
tantly, reliably. In this respect, the Scott test, although fast and
simple, has many interfering substances that may compromise a
prompt decision about the composition of a seized sample [8].
Among such factors, there are several adulterants that are added to
cocaine to increase its volume, promote the perceived potency of
the drug, or increase its toxicity [9].
Aqueous two-phase systems (ATPS) represent an interesting
emerging technology for cocaine identification. These systems
allow determination of an intrinsic analytical parameter, the
partition coefficient, which can enable discrimination between
cocaine and the adulterants. ATPS are ternary systems that can be
formed by the mixing, under specific temperature, pressure, and
composition, of aqueous solutions of two distinct macromolecules
[10], an electrolyte and a macromolecule [11], a surfactant and a
macromolecule [12], an electrolyte and a surfactant [13], two sur-
factants [14], two electrolytes [15], or an electrolyte and an alcohol
[16]. ATPS are highly attractive because they are easy to handle,
inexpensive, do not require the use of toxic organic solvents, and
usually have water as the major component in the top and bottom
phases. In last years, these systems have been applied to the par-
titioning, extraction, and purification of solutes including cell or-
ganelles [17], dyes [18,19], proteins [20,21], nanoparticles [22], and
ions [23,24].
ATPS formed by low molar mass poly(ethylene oxide), PEO1500,
or poly(ethylene)11-poly(propylene oxide)16-poly(ethylene ox-
ide)11, L35, polymers and sulfate salts have been successfully used
for preconcentration or extraction of solutes with different hydro-
philic/hydrophobic balances [18,24,30]. L35, a triblock copolymer,
can form micelles that can solubilize and favor the transfer of more
hydrophobic molecules to the macromolecule-rich phase. Because
cocaine and adulterants have different hydrophilic/hydrophobic
balances, the use of ATPS formed by those polymers, combined
with sodium or lithium sulfate, can modulate the partition coeffi-
cient of those species in a very distinct way.
In this work, we propose the use of ATPS as an alternative
technique for distinguishing cocaine from the most common
adulterants found in samples apprehended by the police. To this
end, the partitioning behaviors of cocaine and various of its adul-
terants were studied using ATPS formed by a macromolecule, a salt,
and water. Evaluation was made of the effects on the partition
coefficient of the nature of the components forming the ATPS and
the pH of the system. The results were then used to determine the
differences in the partitioning behaviors of the components.
Cocaine was used in freebase and hydrochloride forms, together
with the adulterants phenacetin (PHE), benzocaine (BEN), caffeine
(CAF), procaine (PRO), and lidocaine (LID).
2. Experimental
2.1. Materials
Poly(ethylene oxide) e average molar mass of 1500 g mol1
(PEO1500)  and Poly(ethylene)11-poly(propylene oxide)16-poly(-
ethylene oxide)11 triblock copolymer  average molar mass of
1900 g mol1 (L35)  were purchased from Synth (Brazil) and
Sigma-Aldrich (USA), respectively. Lithium sulfate dihydrate
(Li2SO4.2H2O), sodium sulfate (Na2SO4), and sodium hydroxide
were purchased from Vetec (Brazil). Analytical grade sulfuric acid
was purchased from Merck (Brazil). Samples of cocaine freebase
(CoF) and cocaine hydrochloride (CoH)) were obtained from the
National Criminalistics Institute (Federal Police, Brazil). The adul-
terants benzocaine, procaine, caffeine, phenacetin, and lidocaine
were obtained from Sigma-Aldrich (USA). All chemicals were used
without further purification. Additional information of each
chemical used in this work are presented in Table S1 and the
chemical structures of the adulterants are presented in Fig. S1
(Supporting Information). All the solutions were prepared using
distilled water.
2.2. Preparation of ATPS
The biphasic systems were prepared by mixing appropriate
quantities of aqueous solutions of the macromolecule (PEO1500 or
L35) and the salt (lithium sulfate or sodium sulfate) in 50 mL
centrifuge tubes, in order to obtain the desired global compositions.
The compositions (five for each system selected) were defined so as
to obtain ATPS with equal amounts of top and bottom phases, based
on equilibrium data available in the literature [25,26]. The global
compositions prepared for each ATPS are provided in Tables S2eS4
(Supporting Information). The centrifuge tubes containing the
systems were stirred until they became turbid and were then
placed in a thermostatic bath (MQBTC 99-20, Microquímica, accu-
racy of ±1 K) for a minimum of 24 h at 298.15 K and 0.09 MPa in
order to obtain two clear phases and a well-defined interface. The
systems were prepared at three pH values, in order to evaluate the
effect of pH on the partitioning of the cocaine and the adulterants.
For this, the pH of the distilled water used to prepare the salt and
macromolecule solutions was previously adjusted to 1.00 or 12.00
by the addition of sodium hydroxide or sulfuric acid aqueous so-
lution. Otherwise, distilled water at pH 6.00 was used to prepare
the ATPS.
2.3. Partitioning behaviors of adulterants and cocaine in the ATPS
After the ATPS had reached thermodynamic equilibrium, the top
and bottom phases were collected and were used to prepare new
ATPS with compositions of the top and bottom phases equal to
those of the systems prepared in section 2.2, but with smaller
masses of the phases. For this, 3.0000 ± 0.0001 g portions of each
phase were added in 10 mL glass tubes containing
0.1000 ± 0.0001 g of a cocaine or adulterant solution prepared us-
ing aqueous solutions of PEO1500 or L35 at the same mass per-
centage as the ATPS top phase. The concentrations of the solutes in
these solutions were 30.0 ± 0.3, 2.0 ± 0.2, 5.0 ± 0.5, 10.0 ± 0.1,
20.0 ± 0.2, 35.0 ± 0.4, and 100 ± 1 g kg1 for CoH, CoF, PHE, BEN,
CAF, PRO, and LID, respectively. The concentrations of the solutes in
the solutions were selected so as to allow their spectrophotometric
 G.M.D. Ferreira et al. / Fluid Phase Equilibria 506 (2020) 112367
determination in the top and bottom phases. The ATPS were stirred
and placed in a thermostatic bath at 298.15 K and 0.09 MPa for a
minimum of 24 h, in order to obtain two clear phases and a well-
defined interface.
2.4. Determination of the partition coefficients of the solutes
After the ATPS containing the solutes (cocaine or adulterants)
had attained thermodynamic equilibrium, portions of the top and
bottom phases were collected and were appropriately diluted for
determination of the absorbances at the maximum absorption
wavelengths of the solutes (245 nm for phenacetin, 282 nm for
benzocaine, 272 nm for caffeine, 291 nm for procaine, 262 nm for
lidocaine, 232 nm for cocaine freebase, and 233 nm for cocaine
hydrochloride). The absorbances were measured using a Shimadzu
UV-2550 digital double beam spectrometer. The diluted phases of
the systems prepared without solute addition were utilized as
blanks. Since the molar absorptivities of the solutes were the same
in the top and bottom phases, the partition coefficients of the
cocaine and adulterants (KS) could be calculated by the following
equation:
AbsT ,fT
KS ¼
AbsB ,fB
where AbsB and AbsT are the absorbances of the bottom and top
phases, respectively, and fB and fT are the dilution factors of the
respective phases. All the experiments were performed in duplicate
and the coefficients of variation for KS were smaller than 5.0%.
The partitioning behaviors of the solutes were studied as a
function of the tie-line length (TLL), calculated using the following
equation:
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 2  2
TLL ¼ C TM  C BM þ C TS  C BS
Where CM and CS are the macromolecule and salt concentrations (%
w/w), with the superscripts B and T indicating the bottom and top
phases, respectively. The TLL is an important parameter that ex-
presses how different are the intensive thermodynamic properties
of the two ATPS phases, at constant temperature and pressure. The
TLL values for the ATPS studied are presented in Tables S2eS4
(Supporting Information).
Fig. 2. Partitioning behavior of the solutes in the ATPS formed using PEO1500 þ Li2SO4 þ water, at pH 6.0, 298.15 K, and 0.09 MPa: (a) ( ) LID, ( ) PRO, ( ) CAF, ( ) PHE, and ( )
BEN; (b) ( ) CoF and ( ) CoH. The reported pH was that of the distilled water used to prepare the ATPS.
3
3. Results and discussion
3.1. Partitioning behaviors of the cocaine and the adulterants in the
ATPS
The partitioning of an analyte between the ATPS phases is the
result of a complicated and delicate balance of interactions be-
tween the partitioned analyte and the ATPS components present
in the top and bottom phases [27,28]. Therefore, the KS value
for an analyte in an ATPS can supply information about the in-
teractions that determine its distribution between the two phases
coexisting at equilibrium. Fig. 2 shows the KS values for all the
solutes studied, according to the TLL, in the ATPS formed using
PEO1500 þ Li2SO4 þ water, at pH 6.0, 298.15 K, and 0.09 MPa. The
experimental data are presented in Tables S5 and S6 (Supporting
Information).
For the entire TLL range studied, the KS values were higher than
1, indicating that the cocaine and the adulterants were preferen-
tially transferred to the top phase of the ATPS, which was enriched
in macromolecules. This behavior was observed for all the condi-
tions tested. Moreover, the partition coefficients of all the investi-
gated solutes increased with increase of the TLL, showing that the
distribution of the solutes between the two phases became more
(1)
unequal as the difference in composition between the two phases
increased. The KS values for the solutes increased in the order
KLID < KPRO < KCAF < KCoF < KCoH < KPHE < KBEN, showing dependence
on the pH, the hydrophobicity of the polymer, and the nature of the
electrolyte. Interestingly, the partition coefficients for phenacetin
(66.8 < KPHE < 1000) and benzocaine (101 < KBEN < 2450) were
much higher than the partition coefficients for the other solutes
(values varying between 4.07 and 19.0).
3.2. Driving forces determining partitioning of the solutes
(2)
Evaluation of the driving forces responsible for the preferential
partitioning of an analyte to a specific phase of a biphasic system is
important, because it enables identification of the parameters that
can modulate the partition coefficients, such as the pH, the polymer
hydrophobicity, and/or the nature of the electrolyte. Therefore, a
model based on Flory-Huggins theory was used to understand the
partitioning behaviors of cocaine and the adulterants in the ATPS.
This model was suggested by Johansson et al. [29] and consists of
4 G.M.D. Ferreira et al. / Fluid Phase Equilibria 506 (2020) 112367

two analytical equations that express the KS value in terms of the enriched in component i with which the analyte interacts more
enthalpic and entropic contributions. According to the Johansson favorably (wiS is more negative). The double summation
model, the entropic contribution to the KS value in a biphasic sys- Pm1 Pm T T
i¼1ðisSÞ j¼iþ1ðisSÞ ð4i 4j Þwij expressed in the second
tem can be predicted using the following equation:
termi ¼ 1isPm-1j ¼ iþ1jsPmFiTFjTwij, is the total enthalpy for
  formation of the biphasic system top phase (in the absence of the
MS nT nB
lnKS ¼ (3) partitioned analyte) divided by the number of lattice sites in that
r VT VB phase. The insertion of an analyte into a phase requires breaking of
the interactions between the original components of the phase, in
where MS is the molar mass of the partitioned analyte (LID, CAF,
order to create a cavity into which the analyte fits, which is
BEN, PRO, PHE, CoH, or CoF); nB and nT are the total number of
dependent on the volume of the analyte. As shown by da Silva et al.
molecules in the bottom and top phases, respectively, which when
[30], the phase energy alone does not contribute significantly to the
divided by the phase volumes, VB and VT, generates the numerical
analyte partitioning behavior. Consequently, the partitioning be-
densities of the phases; and r is the number of lattice sites per unit
haviors of the solutes studied in this work could be attributed to the
volume of the ATPS. Equation (3) shows that in the absence of
favorable enthalpic interactions among them and the macromole-
enthalpic contributions, the analyte will be transferred to the phase
cules, which were concentrated in the top phases of the ATPS.
that has the highest number of molecules per unit volume (the
Since increase of the TLL was associated with increased amounts
phase with the highest number density).
of the major components of the phases, with KS increasing for all
In the ATPS studied in this work, the number density of the salt-
the solutes in all the systems, the preferential partitioning of the
rich phase was higher than that of the macromolecule-rich phase,
solutes to the top phase could be attributed to the favoring of in-
mainly due to the greater water content in the bottom phase [18].
teractions between the solutes and the macromolecules, relative to
Hence, in accordance with Equation (3), an entropic force should
the solute-salt interactions.
drive the analyte into the bottom phase, in order to increase the
entropy of the system. However, this was not shown by the
experimental data obtained for the partitioning of cocaine and the 3.3. Influence of polymer hydrophobicity on the partitioning
adulterants in the ATPS studied here, where all the solutes were behavior
preferentially transferred to the macromolecule-rich phase (KS > 1).
Since the transfer of an analyte from the bottom phase to the top Fig. 3 shows the influence of the polymer hydrophobicity on the
phase decreases the entropy of the system, entropic increase was partitioning behaviors of CoF and CoH in the systems formed using
not the driving force governing the partitioning of the solutes in the a macromolecule (PEO1500 or L35) þ lithium sulfate þ water, at pH
ATPS. 6.0, 298.15 K, and 0.09 MPa.
Together with the entropic contribution, there is an enthalpic It should be noted that the KCoH values for the ATPS containing
driving force that determines the unequal distribution of an analyte L35 were smaller than the KCoH values for the ATPS containing
in an ATPS. This driving force is the result of the complicated bal- PEO1500. This general behavior was observed for all the solutes,
ance of the intermolecular interactions between the molecules of with the exception of CoF, which presented a higher partition co-
the partitioned analyte and the ATPS components (salt, macro- efficient for the ATPS formed by PEO1500 than for the ATPS formed
molecule, and water), and of the interactions among each compo- by L35. This was an important finding, because it allowed CoF to be
nent of the system. Therefore, analyte transfer to the distinguished from the other solutes in ATPS with varying hydro-
macromolecule-rich phase is driven by an enthalpic contribution, phobicity. The L35-rich phase was more hydrophobic than the PEO-
which in the Johansson model is expressed by the following rich phase, due to the presence of propylene oxide segments (PO) in
equation: the L35 macromolecules. Under specific conditions of pressure,
temperature, and composition, these macromolecules can form
2
X
m   X1
m micelles by self-assembly [31], generating aggregates that are
MS 4
lnKS ¼ 4Ti 4Bi wiS formed of a hydrophilic corona region consisting of water and
RT
i¼1ðisSÞ i¼1ðisSÞ
3
X
m  
 4Ti 4Tj 4Bi 4Bj wij 5 (4)
j¼iþ1ðisSÞ

where 4j and 4i are the volume fractions of the j and i components

of the ATPS; T is the absolute temperature; R is the universal gas
constant; and wij is the effective pair-wise interchange energy. The
subscript S is used to refer to the partitioned analyte. The effective
pair-wise interchange energy is defined by Equation (5):


1
wij ¼ z εij  εii þ εjj (5)
2

where εij is the potential energy for the formation of one i-j pair and
z is the number of nearest neighbors.
In accordance with Equation (4), two terms determine the
enthalpic contribution to the analyte partitioning. The first of
P
them, m i¼1ðisSÞ ð4i  4i ÞwiS i ¼ 1isPmFiT-FiBwiP, expresses the
T B
Fig. 3. Partitioning behaviors of CoH ( , ) and CoF ( , ) in the systems formed using
energy difference between all interactions that the analyte creates L35 (unfilled symbols) or PEO1500 (filled symbols) þ Li2SO4 þ water, at pH 6.0,
with the components forming the ATPS, in both the top and bottom 298.15 K, and 0.09 MPa. The reported pH was that of the distilled water used to prepare
phases. From this term, the analyte will be transferred to the phase the ATPS.
 G.M.D. Ferreira et al. / Fluid Phase Equilibria 506 (2020) 112367 5

ethylene oxide segments (EO) and a hydrophobic core of PO units.
The lower values of the KS for LID, CAF, BEN, PRO, PHE, and CoH
in the ATPS formed using L35 indicated that it was more favorable
for these solutes to interact with the EO blocks of the macromol-
ecule than to solubilize in the hydrophobic cores of the micelles. In
contrast, the CoF molecules moved toward the inner micelles, due
to the higher hydrophobicity of CoF, compared to the other solutes.
Hence, the transfer of CoF molecules to the top phase was more
favorable in the ATPS formed using the copolymer than in the ATPS
formed using PEO1500.
3.4. Influence of pH on partitioning of the solutes
The charges of the analytes studied in this work are
dependent on the pH of the medium, so alteration of the system
pH can affect the partitioning behavior. Fig. 4 shows plots of KS
against TLL for CoH, BEN, PRO, and CoF in ATPS formed using
PEO1500 þ Li2SO4 þ water, at different pH values, at 298.15 K and
0.09 MPa.
Alteration of the pH of the system affected the partitioning
behavior of the solutes in different ways. For CoH (Fig. 4a), the
partition coefficients did not change when the pH was increased
from 1.0 to 6.0. However, the partition coefficients increased up to
eight-fold when the pH was changed to pH 12.0. The same behavior
was observed for LID, although the influence of pH on KS was lower
(Fig. S2, Supporting Information). For PRO (Fig. 4b), the KS values
increased in the order KPRO (pH 1.0) < KPRO (pH 6.0) < KPRO (pH 12.0).
When BEN was used as the solute (Fig. 4c), an influence of pH was
only notable for higher TLL values. Under such conditions, the KS
values increased in the order KBEN (pH 1.0) < KBEN (pH 6.0) z KBEN
(pH 12.0), which was the same as the behavior observed for PHE
(Fig. S3, Supporting Information). For CoF (Fig. 4d), the order was
KCoF (pH 6.0) < KCoF (pH 1.0) < KCoF (pH 12.0). Finally, the variation of
pH did not affect the partitioning behavior of CAF (Fig. S4, Sup-
porting Information).
The model proposed by da Silva and Loh [32] can help in un-
derstanding these results. It was demonstrated that when poly(-
ethylene oxide) and sulfate salts are mixed to form a biphasic
system, an interaction occurs between the macromolecules and the
cations, associated with increased entropy of the system caused by
release of some of the water molecules solvating both the ions and
macromolecules. After splitting of the phases, the macromolecule-
rich phase contains PEO macromolecules with bound cations,
forming positively charged macromolecules called pseudopolyca-
tions, which can interact with charged species by means of elec-
trostatic interactions (analogously to some of the solutes studied
here). The magnitude of these interactions is dependent on the net
charge of the solutes and can consequently be modulated by vari-
ation of the pH.
The pKa values of the adulterants evaluated are 0.6 and 14.0
(CAF), 2.5 (BEN), 2.2 (PHE), 9.1 (PRO), and 7.9 (LID). The pKa of CoH is
8.6. When the pH values of the phases are below the pKa values, the

Fig. 4. Influence of pH on the partitioning of (a) CoH, (b) PRO, (c) BEN, and (d) CoF in ATPS formed using PEO1500 þ Li2SO4 þ water, at 298.15 K and 0.09 MPa: ( ) pH 1.0, ( ) pH 6.0,
and ( ) pH 12.0. The values of reported pH were the values of pH of the distilled water used to prepare the ATPS.
6 G.M.D. Ferreira et al. / Fluid Phase Equilibria 506 (2020) 112367

partitioned solutes have higher degrees of protonation, acquiring
positive net charge. Therefore, alteration of the pH of the phases
can lead to a change in the solute charge, consequently altering the
magnitude of interaction between the components of the ATPS and
solute.
For BEN, PRO, PHE, CoF, CoH, and LID, the KS values were highest
at pH 12.0, since these solutes are totally deprotonated at this pH.
Hence, the solute charge became negative or zero, favoring the
solute-pseudopolycation interaction. Consequently, the summation
Pm T B
i¼1ðisSÞ ð4i 4i ÞwiS in Equation (4) was more negative (or less
positive) and contributed to an increase in the KS values.
Variation of pH within the range considered did not significantly
affect the KS of CAF, because the net charge of the caffeine molecule
remained almost unaffected. Consequently, there was little effect
on the magnitudes of the water-CAF, macromolecule-CAF, and salt-
CAF interactions.
Here, it is important to recognize that the systems prepared
using water with pH adjusted to 1.0 or 12.0 can have their phase
compositions changed in comparison to those reported in
Tables S1eS3, although this change should be low. This is clear to
see through our results of ratio between partition coefficients
(Fig. 8 in section 3.6), in which, for some of the solutes, there was
almost no change in the partition coefficient when the pH was
changed (ratio between partition coefficients close to 1). This can
be observed, e.g., for the solutes LID, CoH, and PHE which did not
have its charge changed within the pH range evaluated. Otherwise,
if the phase compositions were changed in a large extension, the K
values for these solutes would have change a lot.
values were observed for the ATPS formed using sodium cations.
The other solutes studied (CAF, PHE, and LID) showed results
similar to those observed for PRO, especially at higher TLL values
(Fig. S5, Supporting Information).
These results could also be interpreted based on the model
developed by da Silva and Loh [32], who showed that lithium
cations (with higher charge density than sodium cations) inter-
acted more favorably, compared to sodium cations, with the EO
segments of the macromolecules of L35 and PEO1500, generating
higher positive charge density on the macromolecule chain. For this
reason, if the partitioned solute has a positive charge, the repulsive
electrostatic interaction energy between the pseudopolycation and
the solute is higher in the presence of lithium cations than in the
presence of sodium cations, consequently leading to smaller KS
values in ATPS formed using Li2SO4 electrolyte.
In the ATPS formed using L35 þ Li2SO4 þ water or
L35 þ Na2SO4 þ water, the phase pH was lower than 7.0. Within this
range of pH, LID and PRO are positively charged, so the partition
coefficients were higher for these solutes in the ATPS formed using
Na2SO4 electrolyte, compared to the ATPS formed using Li2SO4, in

3.5. Influence of electrolyte on partitioning of the solutes in the

ATPS formed using L35

The influence of the cation on the partitioning behavior was

investigated using the solutes in ATPS formed using
L35 þ Li2SO4 þ water or L35 þ Na2SO4 þ water, at pH 6.0, 298.15 K,
and 0.09 MPa. Fig. 5 shows the KS versus TLL curves for some of the
systems studied.
The nature of the electrolyte had different effects on the parti-
tion coefficients, depending on the analyte partitioned. For CoH, the
KS values were not affected when the lithium cation was replaced Fig. 6. KS,PEO1500-Li/KS,L35-Li ratios versus TLL0 for ( ) LID, ( ) PRO, ( ) PHE, ( ) CAF, ( )
with sodium cation. In contrast, the KS values for BEN and CoF were BEN, ( ) CoF, and ( ) CoH, where KS,PEO1500-Li and KS,L35-Li are the partition coefficients
for the PEO1500 þ Li2SO4 þ water and L35 þ Li2SO4 þ water ATPS, at pH 6.0,
higher for the ATPS formed using lithium cations, compared to the respectively. TLL0 is the average value of two similar TLL values for the two ATPS for
ATPS formed using sodium cations. The opposite was observed for which the corresponding KS values were determined. The reported pH was that of the
the partitioning of PRO, for which higher partition coefficient distilled water used to prepare the ATPS.

Fig. 5. KS versus TLL curves for (a) CoH ( , ) and CoF ( , ), and (b) PRO ( , ) and BEN ( , ), in systems formed using L35 þ Li2SO4 þ water (filled symbols) or L35 þ Na2SO4 þ water
(unfilled symbols), at pH 6.0, 298.15 K, and 0.09 MPa. The reported pH was that of the distilled water used to prepare the ATPS.
 G.M.D. Ferreira et al. / Fluid Phase Equilibria 506 (2020) 112367
Fig. 7. KS,L35-Na/KS,L35-Li ratios versus TLL0 for ( ) LID, ( ) PRO, ( ) PHE, ( ) CAF, ( ) BEN,
( ) CoF, and ( ) CoH, where KS,L35-Na and KS,L35-Li are the partition coefficients for the
L35 þ Na2SO4 þ water and L35 þ Li2SO4 þ water ATPS, respectively, at pH 6.0. TLL0 is
the average value of two similar TLL values for the two ATPS for which the corre-
sponding KS values were determined. The reported pH was that of the distilled water
used to prepare the ATPS.
accordance with the above description. However, within the same
pH range, BEN, CAF, and PHE are neutral and could therefore
interact more intensely with the pseudopolycations formed using
lithium cation for each ion-dipole interaction, compared to the
pseudopolycations formed using sodium cation, resulting in higher
partition coefficients for these solutes in the ATPS formed using
Li2SO4.
3.6. Identification parameters for cocaine
The different ways in which the partitioning behaviors of the
solutes were affected by alteration of the ATPS features (hydro-
phobicity of the polymer, nature of the salt, TLL, and pH) enabled
cocaine to be distinguished from the adulterants. In order to
simplify this distinction, a specific parameter was defined that re-
flected the effects that the changes in the features of the ATPS had
on the KS values. This parameter was the ratio between the parti-
tion coefficients for a solute in two different ATPS, given by KS,1/KS,2,
where KS,1 and KS,2 are the partition coefficients of the solute in
Fig. 8. Partition coefficient ratios plotted against TLL for ( ) LID, ( ) PRO, ( ) PHE, ( ) CAF, ( ) BEN, ( ) CoF, and ( ) CoH: (a) K12/K1 and (b) K12/K6, where K1, K6, and K12 are the
partition coefficients for the PEO1500 þ Li2SO4 þ water ATPS at pH 1.0, 6.0, and 12.0, respectively. The values of reported pH were the values of pH of the distilled water used to
prepare the ATPS.
7
ATPS 1 and 2, respectively. When the ATPS contained the same
components, this ratio was calculated using values of partition
coefficients obtained at the same TLL values. When the ATPS were
formed using different components, the ratio was calculated using
KS values obtained at TLL values that were as close as possible.
Fig. 6 shows plots of the KS,PEO1500-Li/KS,L35-Li ratios against TLL0 ,
where KS,L35-Li is the KS value obtained for the system formed using
L35 þ Li2SO4 þ water and KS,PEO1500-Li is the KS value for the system
formed using PEO1500 þ Li2SO4 þ water (both at pH 6.0). TLL0 is the
average value of two similar TLL values in the two ATPS for which
the corresponding KS values were determined. The TLL values
chosen to compare the KS values, together with the corresponding
TLL’ values, are presented in Tables S7 and S8 (Supporting
Information).
When the partitioned solute was CoF, the KS,PEO1500-Li/KS,L35-Li
ratio was consistently smaller than 1, while it was always higher
than 1 for all the other solutes. This reflected the fact that only CoF
presented a partition coefficient that was smaller for the system
formed using PEO1500, compared to the system formed using L35,
which was opposite to the trend observed for the other solutes. This
was an important finding, because it allowed CoF to be distin-
guished from CoH and the adulterants.
Fig. 7 shows the KS,L35-Na/KS,L35-Li ratios plotted against TLL’,
where KS,L35-Na and KS,L35-Li are the KS values for the
L35 þ Na2SO4 þ water and L35 þ Li2SO4 þ water systems,
respectively.
The KS values for cocaine and the adulterants were affected
differently by alteration of the cation used to form the ATPS. As
shown in Fig. 7, this could be used to distinguish both forms of
cocaine from the adulterants, particularly at higher TLL’ values,
where the KS,L35-Na/KS,L35-Li ratios were lower than unity for both
CoH and CoF, but higher than unity for the adulterants.
Fig. 8 shows the K12/K6 and K12/K1 ratios plotted against TLL,
where K1, K6, and K12 are the partition coefficients for the
PEO1500 þ Li2SO4 þ water ATPS at pH 1.0, 6.0, and 12.0,
respectively.
As shown in Fig. 8a, the parameter K12/K1 enabled CoH
(5.86 < K12/K1 < 7.97) to be reliably distinguished from BEN, PHE,
CAF, CoF, and LID (K12/K1  2.89). The best distinction between PRO
and CoH was observed for TLL of 41.71% (w/w). Although this was a
satisfactory result, the K12/K6 parameter (Fig. 8b) provided better
performance, enabling safe distinction between CoH (6 < K12/
K1 < 9.5) and the other solutes (K12/K1  2) for the entire TLL range
8 G.M.D. Ferreira et al. / Fluid Phase Equilibria 506 (2020) 112367
Fig. 9. Ratios that provided the best distinction of the cocaine forms and the adul-
terants, considering the solutes cocaine hydrochloride (CoH), cocaine freebase (CoF),
phenacetin (PHE), benzocaine (BEN), caffeine (CAF), procaine (PRO), and lidocaine
(LID).
evaluated.
Interestingly, the ratio between two partition coefficients ob-
tained under two different conditions was not a more specific
parameter than the partition coefficient. However, it could be
concluded that this ratio was an adequate parameter that could be
used to distinguish cocaine, in its different forms (CoF or CoH), from
the adulterants studied. Fig. 9 shows a scheme summarizing the
best use of the parameters for distinguishing the various solutes.
4. Conclusions
The partitioning behaviors of cocaine and some of its adulter-
ants were evaluated using aqueous two-phase systems formed
using a macromolecule, a salt, and water. All the solutes were
predominantly transferred to the top phase of the ATPS, due to
specific enthalpic interactions between the solutes and the mac-
romolecules mainly present in this phase. The partition coefficients
of the species studied were significantly affected by the natures of
the macromolecule and the salt forming the ATPS, as well as by the
pH of the system.
Based on the different partitioning behaviors of the solutes
studied, the findings demonstrated the potential of ATPS as an
alternative method for distinguishing between cocaine and some of
its adulterants. Modulation of the characteristics of these systems
(considering pH, TLL, nature of the electrolyte, and hydrophobicity
of the polymer) provided a strategic parameter that could be
employed for such distinction, namely the ratio between two
partition coefficient values obtained for two different types of
ATPS. Cocaine in its freebase form was distinguished from all the
other solutes, as a result of its different relative partitioning
behavior in systems formed using L35 þ Li2SO4 þ water and
PEO1500 þ Li2SO4 þ water. Cocaine in its hydrochloride form could
be distinguished from the other solutes by employing ATPS
composed of PEO1500 þ Li2SO4 þ water at different pH values.
These results open up a new field of applications for ATPS in
forensic chemistry, where the technique can offer both economic
benefits and practical viability.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
The authors thank Coordenaçaeo de Aperfeiçoamento de Pessoal
de Nível Superior (CAPES/Pro -Forenses), Instituto Nacional de
^ncias e Tecnologias Analíticas Avançadas (INCTAA), Conselho
Cie
Nacional de Desenvolvimento Cientıfico e Tecnolo  gico (CNPq), and
Fundaçaeo de Amparo  a Pesquisa do Estado de Minas Gerais
(FAPEMIG) for their financial support of this research. G.M.D.F. and
G.M.D.F. thank CAPES for the research studentships. M.C.H. and
L.H.M.S. acknowledge CNPq for their research fellowships. The
authors acknowledge Instituto Nacional de Criminalística (Polícia
Federal) for the samples provided and for monitoring the drug
experiments.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.fluid.2019.112367.
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