Food and Bioproducts Processing 1 1 9 ( 2 0 2 0 ) 268–276

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Food and Bioproducts Processing

journal homepage: www.elsevier.com/locate/fbp

Selective and continuous recovery of ascorbic acid

and vanillin from commercial diet pudding waste
using an aqueous two-phase system

Alex Viana Veloso a , Brenda Caroline Silva a , Sthefany Araújo Bomfim a ,

Ranyere Lucena de Souza a,b , Cleide Mara Faria Soares a,b ,
Álvaro Silva Lima a,b,∗
aUniversidade Tiradentes, Av. Murilo Dantas, 300, Farolândia, 49032-490, Aracaju, Sergipe, Brazil
bInstituto de Tecnologia e Pesquisa, Av. Murilo Dantas, 300, Prédio do ITP, Farolândia, 49032-490, Aracaju, Sergipe,
Brazil

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

Article history: In this study, ascorbic acid and vanillin were recovered using semi-continuous and contin-
Received 6 May 2019 uous operation of an aqueous two-phase system (ATPS) formed by ethanol (C2 H5 OH) and
Received in revised form 13 dipotassium hydrogen phosphate (K2 HPO4 ). For operation, a column reactor was designed,
November 2019 allowing temperature control by a coating system and the circulation of up to two phases
Accepted 21 November 2019 simultaneously. The reactor was equipped with a stirring system composed of a shaft and
Available online 27 November 2019 impellers known as vanes. In the experiments, the influence of volumetric flow rate and
agitation speed were analysed to enhance recovery of the biomolecules of interest. The best
Keywords: recovery values were obtained in semi-continuous operation at low rotations (60 rpm) and
Continuous column reactor high volumetric flow rate (6.0 mL min−1 ). These parameters were used to verify partition-
Partitioning ing using a real food matrix (Dr. Oetker Diet Pudding), achieving recovery of up to 94% for
Biocompound vanillin in the top phase and ascorbic acid in the bottom phase.
Aqueous two-phase system © 2019 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Food waste
Selective separation

1. Introduction
An aqueous two-phase system (ATPS) is a strategy of liquid–liquid
extraction (LLE), constituted by two immiscible aqueous phases with
different physical and chemical properties (Garza-Madrid et al., 2010).
ATPSs are usually based on polymers (e.g., dextran, polyethylene gly-
col, polyvinylpyrrolidone) and salts (phosphate, citrate, sulfate), and
recently have been formed by ionic liquids, carbohydrates and organic
solvents (e.g., alcohols, organic acids, acetonitrile and tetrahydrofuran)
combined above a critical concentration (Cardoso et al., 2014a; Souza
et al., 2015a; Grilo et al., 2016).
This strategy is widely used for the recovery, partition, concentra-
tion and purification of several biocompounds, such as antibodies (Rosa
et al., 2012; Campos-Pinto et al., 2017), proteins and enzymes (Souza
et al., 2015b; Song et al., 2018a), toxins (Cavalcanti et al., 2008; Gomez
et al., 2016), antibiotics (Marques et al., 2013; Rabieenezhad and Roosta,
2018), antioxidants (Almeida et al., 2014; Lima et al., 2017) and alkaloids
(Flieger and Czajkowska-Żelazko, 2015; Plácido et al., 2018). The use of
an ATPS presents many advantages, such as low interfacial tension,
biocompatibility, the possibility of process integration, high selectivity
(S), easy implementation and scalability (Raghavarao et al., 2003).
However, its industrial implementation is still limited, due to the
difficulty of separating phases when using viscous compounds (poly-
mers, ionic liquids and some carbohydrates), and the low recyclability
and high cost of the constituents (Soares et al., 2015). In order to
overcome these disadvantages, some authors have proposed phase
recycling (Sankaran et al., 2018; Song et al., 2018b), the use of organic
solvents (Santos et al., 2016; Li et al., 2018) and performing the pro-
cess continuously, improving the purification of labile biocompounds
(Espitia-Saloma et al., 2014; Jungbauer, 2013; Vázquez-Villegas et al.,
2015).

∗
Corresponding author at: Universidade Tiradentes, Av. Murilo Dantas, 300, Farolândia, 49032-490, Aracaju, Sergipe, Brazil.
E-mail address: alvaro lima@itp.org.br (Á.S. Lima).
https://doi.org/10.1016/j.fbp.2019.11.011
0960-3085/© 2019 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
 Food and Bioproducts Processing 1 1 9 ( 2 0 2 0 ) 268–276
To date, few studies have focused on the operational characteris-
tics, validation of reactor designs and economic analysis of continuous
processing by ATPS. According to Rito-Palomares and Benavides (2017),
there is still no specialised equipment available; therefore, perfor-
mance of the process is limited to the traditional reactor configurations
used for LLE. One can highlight the spray column (Srinivas et al., 2002),
packed column (Igarashi et al., 2004), conventional or perforated rotat-
ing disc contactor (Kalaivani and Regupathi, 2016; Porto et al., 2004),
pulsed-cap microcolumn (Rabelo and Tambourgi, 2003) and column
with impellers (Biazus et al., 2007).
The continuous and semi-continuous modes of ATPS opera-
tion can be particularly effective and versatile for the recovery of
high value-added compounds from food industry waste. Worldwide,
approximately one-third of global food production is lost or wasted,
representing about 1.3 billion tonnes per year of residues (Gustavsson
et al., 2011). Usually, this food waste originates from agricultural activ-
ities, such as the production of fruits, grains and vegetables, and from
industrial activities, such as the discarding of foods with expired dates.
However, these residues are high in added-value compounds, which
can be extracted and purified. Good examples are ascorbic acid (antiox-
idant) and vanillin (flavour), which were used as target biomolecules in
this work.
Ascorbic acid (C6 H8 O6 ) or vitamin C is an odourless crystalline white
solid, water-soluble and poorly soluble in organic solvents. This bio-
compound is present in several vegetables, mainly in citrus fruits,
and it is used as a dietary supplement (Uddin et al., 2001). More-
over, it is well known as an antioxidant used in the preservation of
industrial food. Vanillin (4-hydroxy-3-methoxybenzaldehyde) is also a
crystalline white solid, soluble in organic solvents (alcohols and chloro-
form), and the most widely used flavouring in the food industry (Jadhav
et al., 2009), but it also displays antioxidant and antimicrobial, anticlas-
togenic, antimutagenic and antitumor properties (Mourtzinos et al.,
2009).
In this context, the present work proposes a design for a jacketed
column reactor, with two feeds and two outputs, operating in contin-
uous or semi-continuous mode. Moreover, the system was agitated by
impellers (four vertical blades) adequately located in the reactor. The
effect of volumetric flow rate and agitation speed on the selective parti-
tion of ascorbic acid and vanillin, compounds that are present in several
types of food waste, was analysed. The results were compared with pre-
vious work led by Lima and co-authors (Reis et al., 2012), who used the
same system, but in batch mode.
2. Material and methods
2.1. Material
The ATPS was prepared using ethanol (CAS 64-17-5; 99 wt%)
supplied by Neon (São Paulo, Brazil) and dipotassium hydro-
gen phosphate (K2 HPO4 ; CAS 7758-11-4; > 98 wt%) acquired
from Dinâmica Química (São Paulo, Brazil). The target
biomolecules used in the partitioning, ascorbic acid (>98 wt%;
CAS 50-81-7) and vanillin (> 99 wt%; CAS 121-33-5), were
purchased from Labsynth (São Paulo, Brazil) and Dinâmica
Química, respectively. The compounds’ characteristics are
shown in Table 1. For the experiments with food waste, Dr.
Oetker Diet Pudding (São Paulo, Brazil) was used. Ultrapure
water that was double-distilled, passed through a reverse
osmosis system and further purified by Milli-Q Plus 185 water
purification apparatus (Millipore, Bedford, MA, USA) was used
in all experiments.
2.2. Reactor design
Fig. 1 depicts the design of the glass column reactor of 550 mL
nominal capacity and 440 mL useful volume, which was used
for partitioning the biomolecules. The temperature was con-
269
trolled by a jacketed system (points 1 and 2). The samples were
collected at points 3 and/or 4, and the feeds were supplied
at points 5 and/or 6. The experiments were conducted using
an agitation system, formed by an axis with three impellers
(four vertical blades) made of 304 stainless steel. The number
of impellers is associated with the volumetric ratio between
the phases (Rv = 3), meaning that the top phase is three times
larger than the bottom phase. Therefore, three impellers were
positioned equidistantly in the top phase and the other in
the bottom phase, taking care that agitation did not disturb
the interface between the phases. Nevertheless, the position
of impellers is adjustable along the axis using fixing screws
(point 7), making reactor design versatile. Two Teflon cen-
tralisers were introduced to avoid imbalance of the shaft and
thereby collision of the impellers with the reactor wall; the gap
between them was 20 mm. The number of centraliser pores
was limited due to space for the production of the holes. The
centralisers were positioned above the impellers so that the
closest to the interface was at least 5 cm away, thereby min-
imising the delayed effect of liquid transfer between phases.
2.3. Selective partitioning of biomolecules
The ATPS composition was based on batch experiments
according to our previous study (Reis et al., 2012), which iden-
tified the best conditions to separately recover vanillin and
ascorbic acid (50 wt% C2 H5 OH + 15 wt% K2 HPO4 + 35 wt% H2 O).
The ATPS was prepared by mixing and homogenising the sys-
tem constituents, and then the column reactor was filled and
left to rest for 6 h to reach thermodynamic equilibrium and
form a distinct interface. The operation was carried out in
semi-continuous and continuous modes, with the reactor vol-
ume constant for both cases (inlet volumetric flow = outlet
volumetric flow). In all experiments, the disperse phase was
pumped into the column reactor with a volume similar to
the phases contained in the reactor. For all cases, the target
biomolecules were dissolved in the phase of lower affinity,
aiming to promote retention of the biomolecule in this phase.
Initially, the partition of biomolecules was examined in the
semi-continuous mode; namely, the dispersed phase had the
same composition as the top or bottom phase containing the
biomolecule, and a stationary phase (inside the reactor). In
Protocol 1, vanillin was dissolved in the bottom phase, which
was pumped into the reactor through point 5 using a pump
with volumetric flow rate until reaching 12 mL min−1 (Milan
model BP662, Colombo-Paraná, Brazil), and harvested at point
3. In Protocol 2, ascorbic acid was dissolved in the top phase,
which was pumped into the reactor through point 6 and col-
lected at point 4. In sequence, the continuous mode was also
investigated. In this case, the top phase (containing ascorbic
acid) and bottom phase (containing vanillin) were simultane-
ously and in counter-current pumped into the reactor through
points 6 and 5, respectively. The phases were harvested at
points 4 and 3, respectively (Protocol 3). Finally, the experi-
ment was also carried out operating in semi-continuous mode
and using a real sample (Dr. Oetker Diet Pudding), which was
dissolved in the bottom phase and pumped in at point 5 and
collected at point 3 (Protocol 4). All protocols are demon-
strated, respectively, in Figs. S1, S2, S3 and S4 of the Supporting
information.
The effects of agitation speed (60, 150, 275 and 360 rpm)
at 3 mL min−1 , and volumetric flow rate (1.5, 3.0, 4.5 and
6.0 mL min−1 ) at 60 rpm were explored. All separation experi-
ments were performed under conditions that did not disturb
270 Food and Bioproducts Processing 1 1 9 ( 2 0 2 0 ) 268–276

Table 1 – Name, CAS register number, supplier, and mass fraction purity of chemicals used in this work.a
Compounds IUPAC name Molecular Purification CAS Reg. no Supplier Final purity
formula method (wt%)a

Ethanol Ethanol C2 H6 O None 64-17-5 NEON 99

Dipotassium Potassium K2 HPO4 None 7758-11-4 Dinâmica >98
hydrogen hydrogen
phosphate phosphate
Ascorbic acid (2R)-2-[(1S)-1,2- C6 H8 O6 None 50-81-7 Labsynth >98
dihydroxyethyl]-
3,4-dihydroxy-
2H-furan-5-one
Vanillin 4-Hydroxy-3- C8 H8 O3 None 121-33-5 Dinâmica >99
methoxybenzaldehyde

a
Values reported by supplier.

Fig. 1 – Design of the column reactor and its constituents. (a) Dimensions of the reactor; (b) dimensions of impellers, (c)
dimensions of the centraliser, (d) axis and equidistant impellers, (e) assembly of the reactor and (f) real reactor. All
dimensions are in millimetres. Points 1 and 2 are part of the temperature control system provided by the jacketed reactor.
The samples were collected at points 3 and/or 4, and feeds were supplied at points 5 and/or 6. The position of the impellers
on the axis can be adjusted by the screw positioned at point 7.

the interface formed between the phases at 298.15 ± 0.1 K and KVAN
S= (3)
0.10 ± 0.01 MPa. KAA
During operation, samples of the bottom phase were har-
vested (at pre-set times) in order to measure the concentration mT
RT = × 100 (4)
of biomolecules; and with the phase volume, the bottom mass mT + mB
was determined. The mass of the biomolecule in the top phase
was determined through mass balance (Eq. (1)), and divided by mB
RB = × 100 (5)
the volume of the top phase to determine the concentration mT + mB
in this phase.
where m corresponds to the masses of the biomolecules.
The mass transfer coefficient (KD a) (min−1 ) was evaluated
min = mT + mB + mout (1) according to the material balance proposed by Pawar et al.
(1997) and shown briefly in Eq. (6):
where m corresponds to the masses of the biomolecules, and
FD
 C − KC 
the subscripts in, T, B and out mean input, top, bottom and Di S
KD a = × ln (6)
exit, respectively. V CDf − KCS
For semi-continuous mode (Protocol 2), the samples were
returned to the reactor after measurement using a syringe where FD is the volumetric flow rate (mL min−1 ); V is the vol-
(point 6), aiming to maintain the volume of the ATPS. ume of ATPS (mL); CDi is the biomolecule concentration in
The partition coefficient (K) was determined by the ratio inject solution, CDf is final biomolecule concentration in the
between the concentration of biomolecules (C) in the top dispersed phase (mg mL−1 ); K is the partition coefficient of the
and bottom phases (Eq. (2)); the selectivity (S) of the system biomolecule (dimensionless); and CS is the concentration of
was measured by the ratio between the partition coefficients biomolecule in the stationary phase in steady state (mg mL−1 ).
of vanillin (KVAN ) and ascorbic acid (KAA ) (Eq. (3)), and the In order to understand the effect of the dispersed phase
recovery of biomolecules in the top and bottom phases was flow rate in the kind of flow regime, Reynolds number (Re) was
determined using Eqs. (4) and (5), respectively, as described by calculated as the ratio between the inertial forces and viscous
Albertsson (1970). force, as described in Eq. (7):

CT ×v×d
K= (2) Re = (7)
CB 
 Food and Bioproducts Processing 1 1 9 ( 2 0 2 0 ) 268–276
where ␳ and ␮ are phase density (kg m−3 ) and viscosity (Pa s),
respectively; v is the dispersed phase velocity (m s−1 ); and d is
the diameter of the reactor (m).
Density and dynamic viscosity for each phase were deter-
mined at different temperatures (293.15–323.15 K), with an
uncertainty of ±0.02 K, using an automated Anton Paar
SVM 3000 rotational Stabinger viscosimeter–densimeter (Graz,
Austria). The density assays have an absolute uncertainty of
0.5 × 10−3 g cm−3 , and the relative uncertainty of the dynamic
viscosity measurements is 0.35%. The equipment was cali-
brated using a standard solution.
2.4. Determination of biomolecule concentration
Vanillin and ascorbic acid concentrations in the bottom phase
were evaluated by the same protocol as in our previous work
(Reis et al., 2012). The concentrations of ascorbic acid were
quantified by UV spectroscopy using a UV-Vis Hack DR 5000
spectrophotometer (Loveland, Colorado, USA) at a wavelength
of 280 nm. Vanillin concentration was measured by UV spec-
troscopy at two different wavelengths (280 and 347 nm), in
order to eliminate the influence of the pH of the phases and
consequent change to the surface charge of vanillin (Reis
et al., 2012). Standard curves were properly established for
each wavelength considered, and the mass balance of vanillin
was calculated and confirmed in each experiment. As blank
solution, either water (calibration curve) or the corresponding
phase under analysis (partitioning process) was used.
In experiments using a real sample (Dr. Oetker Diet
Pudding), the ascorbic acid was quantified using Tillman’s
method (Lutz, 2008) that is based on the oxidation of 2,6-
dichlorophenolindophenol sodic (DCPIP), in order to eliminate
the interference of other compounds presents in the feed-
stock. For vanillin, determination using the two wavelengths
minimises the interference.
3. Results and discussion
Previously, our research group reported selective parti-
tion of ascorbic acid and vanillin using a batch ATPS,
based on different alcohols and inorganic salts (Reis et al.,
2012). The optimised operational conditions were 50 wt%
C2 H5 OH + 15 wt% K2 HPO4 + 35 wt% H2 O, at 298.15 ± 0.1 K and
0.1 MPa, which provided recovery of 99.93 ± 0.01% vanillin in
the top phase (R(VAN)T ) and 90 ± 1% ascorbic acid in the bottom
phase (R(AA)B ). When these conditions were applied to a real
sample (diet pudding), the results were R(VAN)T = 99.62 ± 0.05%
and R(AA)B = 38 ± 3% (Reis et al., 2012). The current study
adopted the same conditions for selective recovery of ascor-
bic acid and vanillin in evaluating the column reactor design
operated in semi-continuous and continuous modes.
Initially, the viscosity and density of each phase were mea-
sured at several temperatures (Fig. 2, and Tables S1 and S2 of
the Supporting information). Determination of the Reynolds
number is a very important tool to investigate the kind of
flow regime in a reactor (Quental et al., 2015) at several tem-
peratures, even though our experiments were carried out at
298.15 ± 0.1 K and 0.10 ± 0.01 MPa. Moreover, we provide a com-
parison of the density and viscosity of phases with other
systems.
The resultant linear and exponential trends for den-
sity and viscosity, respectively, are typical for ATPS (Oliveira
et al., 2019). For the studied system, the density ranges were
271
Table 2 – Reynolds number of each phase of the studied
aqueous two-phase system composed by 50 wt%
ethanol + 15 wt% K2 HPO4 + 35 wt% H2 O under all
operation conditions at 298.15 and 0.1 MPa.a
FD (mL min−1 ) ReT ReB
1.5 0.23 0.10
3.0 0.47 0.17
4.5 0.70 0.26
6.0 0.94 0.34
a
Expanded uncertainties for 0.95 level of confidence are U(T) = 1 K
and U(p) = 10 kPa. T and B correspond to top and bottom phase.
0.839–0.866 (ethanol-rich phase) and 1.506–1.524 g m−3 (salt-
rich phase). For the solvent-rich phase, the density obtained
is similar to those found in other systems based on organic
solvents, such as 0.873–0.905 g m−3 for tetrahydrofuran (Sousa
et al., 2017) and 0.806–0.837 g m−3 for acetonitrile (Oliveira
et al., 2019), whereas higher densities have been observed
in polymers, such as polyethylene glycol-1000 (1.089–1.091 g
m−3 ; Snyder et al., 1992). The viscosity of the ethanol-rich
phase (1.033–2.286 mPa s) was greater than that of acetonitrile
(0.312–0.505 mPa s; Oliveira et al., 2019) and a tetrahydrofuran-
rich phase (0.420–0.651 mPa s; Sousa et al., 2017) while
the salt-rich phase (4.902–11.060 mPa s) was less viscous
than those using polyvinylpyrrolidone (10.556–30.056 mPa s;
Oliveira et al., 2019) and glucose (6.900–24.551 mPa s; Sousa
et al., 2017).
The Reynolds number calculated from these results was
very low for all process conditions (Table 2), due to the low
volumetric flow rate and consequently the dispersed phase
velocity. Therefore, the kind of flow regime was classified as
laminar (Re «« 2000) as described by Bird et al. (2004). Thus, it
can be inferred that laminar flow, associated with low agita-
tion speeds and phase characteristics (density and viscosity)
did not disturb the interface between the continuous phases
in the reactor.
3.1. Recovery of standard biomolecules
Our experiments were carried out using Protocols 1 (vanillin
−0.05 mg mL−1 dissolved in the bottom phase) and 2 (ascorbic
acid −0.02 mg mL−1 dissolved in the top phase). The agitation
speed ranged between 60 and 360 rpm at a volumetric flow rate
of 3 mL min−1 , at 298.15 ± 0.1 K and 0.10 ± 0.01 MPa (Fig. 3, and
Tables S3 and S4 of the Supporting information). These initial
biomolecule concentrations were established by the solubility
of each one in the respective phases. The parameter evaluated
was the recovery in the phase opposite its entry (top phase
vanillin and bottom phase ascorbic acid).
Vanillin moved from the bottom or salt-rich phase (feed) to
the top or ethanol-rich phase because of its hydrophobic char-
acter (logarithm of octanol–water coefficient: log Kow = 1.19;
Noubigh et al., 2010). The top continuous phase was able
to retain fully the vanillin (R(VAN)T = 100%) for up to 20 min
(150 rpm). On increasing the agitation speed, the time required
for total recovery of vanillin decreased, reaching 6 min at
360 rpm. After this time, part of the vanillin migrated to the
top phase due to continuous feeding. We considered that the
steady state of the process was reached at 37 min, in which the
recovery of vanillin in the top phase decreased slightly from
98.4 ± 0.1% (60 rpm) to 97.446 ± 0.003% (360 rpm).
In contrast to vanillin, ascorbic acid is hydrophilic (log
Kow = −1.85; Takács-Novák and Avdeef, 1996) and migrated
272 Food and Bioproducts Processing 1 1 9 ( 2 0 2 0 ) 268–276

Fig. 2 – Experimental (a) density and (b) viscosity for aqueous two-phase system based on 50 wt% ethanol + 15 wt%
K2 HPO4 + 35 wt% H2 O at different temperatures (283.15–338.15 K) and 0.10 ± 0.01 MPa. bottom phase, top phase.

Fig. 3 – Effect of agitation speed ( - 60, - 150, - 275, and - 360 rpm) on the recovery of (a) vanillin (top phase; R(VAN)T )
and (b) ascorbic acid (bottom phase; R(AA)B ) from an aqueous two-phase system composed by 50 wt% ethanol + 15 wt%
K2 HPO4 + 35 wt% H2 O at 298.15 ± 1 K and 0.10 ± 0.01 MPa. Dispersed phase flow rate of 3 mL min−1 of vanillin at
0.05 mg mL−1 (dissolved in the bottom phase) or 0.02 mg mL−1 ascorbic acid (dissolved in the top phase).

from the top (feed) to bottom phase. Under the conditions

Table 3 – Effect of agitation speed on the mass transfer
of Protocol 2, the R(AA)B in the steady state condition also parameters for biomolecules in the aqueous two-phase
decreased on increasing the agitation speed, from 92.2 ± 0.5% system composed by 50 wt% ethanol + 15 wt%
(60 rpm) to 40 ± 3 (275 rpm); however, the time required for the K2 HPO4 + 35 wt% H2 O at 298.15 and 0.10 after the steady
bottom continuous phase to retain the total amount of ascor- state.a
bic acid remained constant with the agitation speed. For this Agitation speed (rpm) K KD a (min−1 )
case, the steady state was observed at 110 min. Pourebrahimi
Partition of vanillin (Protocol 1)
et al. (2015) studied the vanillin partition in aqueous two-
60 20.0 ± 0.4 4.50 × 10−2
phase system, and observed that vanillin migrated to top 150 18.9 ± 0.4 4.40 × 10−2
phase (choline chloride rich-phase) with KVAN = 12.56 ± 0.47 275 12.46 ± 0.08 5.30 × 10−2
and RVAN = 94.02 ± 0.20 % using choline chloride (20 wt%) and 360 12.72 ± 0.02 6.67 × 10−2
K3 PO4 (38 wt%) at 298 K and ambient temperature. By replac- Partition of ascorbic acid (Protocol 2)
ing choline chloride with imidazolium-based ionic liquids, 60 0.028 ± 0.002 3.14 × 10−2
150 0.085 ± 0.007 3.37 × 10−2
the best partitioning for the IL-rich top phase occurred using
275 0.50 ± 0.06 4.31 × 10−2
25 wt% of [C7 H7 mm]Cl + 15 wt% of K3 PO4 (KVAN = 98.69) at 298 K
360 0.174 ± 0.007 3.71 × 10−2
(Claudio et al., 2010). Our previous articles using acetonitrile
a
report recovery of vanillin and partition coefficients ranging Expanded uncertainties for 0.95 level of confidence are U(T) = 1 K
from 79.8 % and 2.24 (ATPS based on 49 wt% acetonitrile and and U(p) = 10 kPa.
9 wt% poly(vinyl alcohol) – Cardoso et al., 2014b) to 95 % and
11.3 (ATPS based on 30 wt% acetonitrile and 18 wt% dextran
100 – Cardoso et al., 2015), respectively.
and presented the highest recovery of biomolecules in the
The effect of agitation speed on recovery of vanillin in the
steady state. For this reason, the effect of the volumetric phase
top continuous phase and ascorbic acid in the bottom contin-
flow rate (1.5, 3.0 and 4.5 and 6.0 mL min−1 ) on R(VAN)T and
uous phase agrees with the KD a variation under steady state
R(AA)B was studied (Fig. 4, and Tables S5 and S6 of the Sup-
conditions (Table 3). According to Sarubbo et al. (2003) and
porting information).
Biazus et al. (2007), the KD a increases with agitation speed,
For these conditions, the increase of volumetric flow rate
which provides a smaller drop size and larger superficial area
and consequently the disperse phase velocity increased the
for mass transfer (Porto et al., 2004). In this sense, the migra-
mass transference (Table 4), which according to Cavalcanti
tion of vanillin to the bottom phase and ascorbic acid to the
et al. (2008) is a consequence of smaller drop size at higher
top phase is facilitated, and consequently provides a decrease
velocity. Similar to the previous results, the increase in KD a
of recovery in the top and bottom phases, respectively.
was inversely proportional to the total time required to
In the further experiments, an agitation speed of 60 rpm
retain the biomolecule in the corresponding phase (top phase
was chosen because the continuous phase retained the total
vanillin and bottom phase ascorbic acid), as well as the time
amount of biomolecules for longer (R(VAN)T = R(AA)B = 100%),
required for the system to operate in steady state conditions.
 Food and Bioproducts Processing 1 1 9 ( 2 0 2 0 ) 268–276
Fig. 4 – Effect of volumetric flow rate ( 1.5, 3.0, 4.5 and 6.0 mL min−1 ) of vanillin at 0.05 mg mL−1 (dissolved in the
−1
bottom phase) or 0.02 mg mL ascorbic acid (dissolved in the top phase) on the recovery of (a) vanillin (top phase; R(VAN)T )
and (b) ascorbic acid (bottom phase; R(AA)B ) from an aqueous two-phase system composed by 50 wt% ethanol + 15 wt%
K2 HPO4 + 35 wt% H2 O at 298.15 ± 1 K and 0.10 ± 0.01 MPa. Agitation speed of 60 rpm.
Table 4 – Effect of dispersed phase flow rate on the mass
transfer parameters of biomolecules in the aqueous
two-phase system composed by 50 wt% ethanol + 15 wt%
K2 HPO4 + 35 wt% H2 O at 298.15 and 0.10 after the steady
state.a
FD (mL min−1 ) K KD a (min−1 )
1.5 13.8 ± 0.2 2.09 × 10−2
3.0 20.0 ± 0.4 4.50 × 10−2
4.5 22.2 ± 0.3 5.57 × 10−2
6.0b – –
1.5 0.078 ± 0.001 1.68 × 10−2
3.0 0.028 ± 0.002 3.14 × 10−2
4.5 0.103 ± 0.002 4.62 × 10−2
6.0 0.023 ± 0.005 6.31 × 10−2
a
Expanded uncertainties for 0.95 level of confidence are U(T) = 1 K
and U(p) = 10 kPa.
b
Completed migration to the top phase; thus, K (partition coeffi-
cient) and KD a (mass transfer coefficient) could not be calculated.
It should be highlighted that for Protocol 1 there was no
transfer of vanillin to the bottom phase (C(VAN)B =0 mg L−1 ), and
therefore it was not possible to calculate the KD a. Based on the
results presented, the operating conditions in the next exper-
iments to carry out a continuous process were an agitation
speed of 60 rpm and volumetric flow rate of 6 mL min−1 .
3.2. Partition of standard biomolecules in continuous
mode
In the continuous mode (Protocol 3), we applied the best
parameters of agitation speed (60 rpm) and volumetric
flow rate (6.0 mL min−1 ) determined in the previous exper-
iments while maintaining the concentrations of vanillin at
0.05 mg mL−1 (dissolved in the bottom phase) and ascor-
bic acid at 0.02 mg mL−1 (dissolved in the top phase) at
298.15 ± 0.1 K and 0.10 ± 0.01 MPa. Fig. 5 (Table S7 of the
Supporting information) depicts recovery and K for both
biomolecules and the S of the system.
Recovery of vanillin in the top phase (R(VAN)T ) reached
100%, while the recovery of ascorbic acid (R(AA)B ) diverged
from that expected, by remaining largely in the top phase
(R(AA)B = 14.39 ± 0.01). The top phase containing ascorbic acid
was disperse with the droplets rising and coalescing in the
top continuous phase. The viscosity of the bottom contin-
uous phase (9.450 mPa s) was higher than that of the top
disperse phase (1.995 mPa s), resulting in large and non-
spherical shape, and consequently provided the drag effect
according Rosa et al. (2012). Moreover, the simultaneous
273
counter-current flow contributed to this phenomenon, which
was not observed in semi-continuous mode. Therefore, the
ascorbic acid migrated to the top phase (R(AA)T = 85.61 ± 0.01%).
Recovery of ascorbic acid in the bottom phase reduced
from 8.8 ± 0.2% to 1.98 ± 0.02% throughout the operation.
For vanillin, an increased KVAN was verified, reaching
220.75 ± 0.03%. In this way, an increase in the S between
the phases to a maximal 93.5 ± 0.6 (KAA = 2.36 ± 0.02 and
KVAN = 220.75 ± 0.03) at the end of the process could be
obtained. It should be noted that Reis et al. (2012)
obtained the best partition coefficient values (KAA = 0.034
and KVAN = 430) in a batch system, which corresponded to
S = 12,647.
The continuous mode was ineffective for selective separa-
tion of the biomolecules, because it did not allow high recovery
of ascorbic acid in the continuous bottom phase when com-
pared to the semi-continuous mode (94 ± 1%), although the
recovery of vanillin in the top continuous phase was main-
tained at 100%. For this reason, the following experiments with
food waste were performed in semi-continuous mode using a
volumetric flow rate of 6.0 mL min−1 and agitation speed of
60 rpm at 298.15 ± 0.1 K and 0.1 ± 001 MPa.
3.3. Partition of biomolecules from food waste
Finally, to prove the ability of the continuous ATPS formed
by ethanol and K2 HPO4 to simultaneously separate vanillin
and ascorbic acid from a food waste source, Protocol 4 was
used, which involved the dilution of Dr. Oetker Diet Pud-
ding at an initial average concentration of 0.015 mg mL−1 .
The choice of this food matrix was based on the pres-
ence of both biomolecules in significant (non-residual)
quantities, providing the necessary conditions for its quan-
tification. Again, the best recovery parameters were used
(volumetric flow rate of 6 mL min−1 and agitation speed of
60 rpm).
According to Fig. 6 (Table S8 of the Supporting informa-
tion), the system provided recovery of 95.9 ± 0.7% for vanillin
in the top continuous phase and 100% for ascorbic acid in the
bottom continuous phase at steady state, despite the pres-
ence of several biomolecule in the diet pudding composition
(lipids, sugars, vanillin as aroma, ascorbic acid as preserva-
tive, among others). In the steady state, the KAA decreased
from 1.00 ± 0.06 to 0.009 ± 0.001, and KVAN also decreased,
from 30.244 ± 0.001 to 7 ± 1; however the selectivity reached
1361 ± 207. As mentioned above, Reis et al. (2012) obtained
the best partition coefficient values (KAA ∼= 2.82 and KVAN ∼
= 63;
S = 22.34) in a batch system with a food sample. Therefore, the
274 Food and Bioproducts Processing 1 1 9 ( 2 0 2 0 ) 268–276

Fig. 5 – Recovery index (IR) of ascorbic acid in the bottom phase ( ) and vanillin in the top phase ( ), (a) partition coefficient
(K) of ascorbic acid ( ) and vanillin ( ), and selectivity (S) ( ) (b) using an aqueous two-phase system (Protocol 3) composed by
50 wt% ethanol + 15 wt% K2 HPO4 + 35 wt% H2 O at 60 rpm, 6.0 mL min−1 , 298.15 ± 1 K and 0.1 MPa.

Fig. 6 – Recovery of ascorbic acid in the bottom phase ( ) and vanillin in the top phase ( ) from Dr. Oetker Diet Pudding (a);
and partition coefficient (K) of ascorbic acid ( ) and vanillin ( ), and selectivity (S) ( ) (b) from aqueous two-phase system
(Protocol 4) composed by 50 wt% ethanol + 15 wt% K2 HPO4 + 35 wt% H2 O at 60 rpm, 6.0 mL min−1 , 298.15 ± 1 K and 0.1 MPa.

semi-continuous mode to operate the system agrees with the Tiradentes University for the scholarship given to S.A. Bomfim,
batch mode values. and the financial support provided to A.V. Veloso.

4. Conclusion Declarations of interest

It has been shown that agitation speed and volumetric flow
rate influence mass transference, which can be identified as
the driving force to allow the selective partition of vanillin
and ascorbic acid in a continuous ATPS. The semi-continuous
mode is more effective for selective recovery than the con-
tinuous mode under the operating conditions used because
the bottom continuous phase did not retain the ascorbic acid.
Additionally, in the semi-continuous mode, the reactor design
was also favourable for the separation of the biomolecules
under study, similarly to the batch conditions of previous
studies. Finally, vanillin can be selectively separated from
ascorbic acid in a diet pudding (S = 1361 ± 207) using an ATPS
based on 50 wt% ethanol + 15 wt% K2 HPO4 + 35 wt% H2 O at
298.15 ± 1 K and 0.10 ± 0.01 MPa carried out in a column reac-
tor with three impellers (four vertical blades) at 60 rpm and
6.0 mL min−1 .
Funding
This work was supported by the Fundação de Amparo
à Pesquisa e Inovação Tecnológica do Estado de Sergipe
(FAPITEC/SE) and the Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES); (Grant number 88887-
15791/2017-00), and the Conselho Nacional de Desenvolvi-
mento Científico e Tecnológico (CNPq) their financial support
and the scholarship awarded to B.C. Silva and Á.S. Lima
(Grant number 304672/2015-7). Moreover, we are thankful to
None.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at doi:https://doi.org/10.1016/j.fbp.
2019.11.011.
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