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j.foodchem.2017.10.110

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Accepted Manuscript
Improving the extraction of L-phenylalanine by the use of ionic liquids as adjuvants in aqueous biphasic systems
Hongpeng Yang, Li Chen, Cunshan Zhou, Xiaojie Yu, Abu ElGasim A. Yagoub,
Haile Ma
PII:
DOI:
Reference:
S0308-8146(17)31748-X
https://doi.org/10.1016/j.foodchem.2017.10.110
FOCH 21933
To appear in:
Food Chemistry
Received Date:
Revised Date:
Accepted Date:
20 April 2017
19 October 2017
20 October 2017
Please cite this article as: Yang, H., Chen, L., Zhou, C., Yu, X., Yagoub, A.E.A., Ma, H., Improving the extraction
of L-phenylalanine by the use of ionic liquids as adjuvants in aqueous biphasic systems, Food Chemistry (2017),
doi: https://doi.org/10.1016/j.foodchem.2017.10.110
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Improving the extraction of L-phenylalanine by the use of ionic liquids as
adjuvants in aqueous biphasic systems
Running title:
:Extraction of L-Phe with ILs-ATPS
Hongpeng Yang1, Li Chen2, Cunshan Zhou1,*, Xiaojie Yu1 , Abu ElGasim A. Yagoub3, Haile Ma1,*
1
School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China
2
Jiangsu Marine Resources Development Research Institute, Lianyungang, 222005, China.
3
Faculty of Agriculture, University of Zalingei, P.O. Box: 06, Zalingei, Sudan
∗
Corresponding authors.
E-mail: cunshanzhou@163.com (Cunshan Zhou), Tel. & Fax: +86-511-88780201.
E-mail: mhl@ujs.edu.cn (Haile Ma), Tel. & Fax: +86-511-88790958
ABSTRACT
Polyethylene glycol (PEG) is widely used in the polymer-salt systems. However, the low polarity
of the PEG-rich phase limits the application of aqueous biphasic systems (ABS). To overcome this
disadvantage, a small quantity of ionic liquid (IL) was used as an adjuvant in ABS to enlarge the
polarity range. Therefore, an innovative study involving addition of 4 wt% imidazolium-based ILs
to the PEG 600/ NaH2PO4 ABS, aiming at controlling the phase behavior and extraction ability,
was carried out. The phase diagrams, the tie-lines and the partitioning behavior of L-phenylalanine
and ILs were studied in these systems. The results reveal that L-phenylalanine preferentially
partitions for the PEG-rich phase. The addition of 4 wt% IL to ABS controls the partitioning
behavior of L-phenylalanine, which depends on the type of IL employed. Moreover, it is verified
that increasing temperature lead to a decrease in the partition coefficient of L-phenylalanine.
Keywords: Aqueous biphasic systems; Ionic liquids; Polyethylene glycol; L-phenylalanine
1
1.
Introduction
Aqueous biphasic systems (ABS) for extraction of biomolecules have been widely studied and
used as an alternative to traditional liquid-liquid extraction techniques over the past few decades
(Albertsson, 1958). The separation of bio-products from bio-reaction media is an important step in
biotechnology. Extraction can be applied to the first-stage separation or purification processes.
Conventional ABS typically consists of two immiscible aqueous-rich phases based on
polymer/polymer, polymer/salt or salt/salt combinations dissolved in aqueous media that might be
used in liquid–liquid extraction processes (Azevedo et al., 2009; Almeida et al., 2014). ABS forms
two aqueous phases that coexist in equilibrium due to the dissolution, above a critical
concentration of the phase-forming components, of pairs of solutes in water (Freire et al., 2012a).
However, both phases are mostly composed of water, offering a bio-compatible medium for
biologically active molecules, and hence recognized as a promising technique to separate and
purify biomolecules (Ferreira, Faustino, Mondal, Coutinho, & Freire, 2016). ABS is characterized
by low interfacial tension, rapid phase separation, low viscosity, good biocompatibility and low
cost; therefore it is widely used for extraction of the biological molecules (de Souza et al., 2010;
Barbosa et al., 2011). Factors, such as the characteristics of target material, system characteristics
and the economic costs, have to be taken into account when deciding to choose ABS. The
polymer/polymer aqueous two-phase system is mainly studied extensively earlier. This system has
limited industrial application due to its relatively large viscosity and to the higher operating cost.
ABS is typically formed by polymer/inorganic salt has some advantages over conventional
polymer/polymer ABS (Lin, Wu, Mei, Zhu, &. Yao, 2003). Polyethylene glycol (PEG) is
commonly used as one of the phase-forming polymers in ABS; it is characterized by high
biodegradability, low toxicity, low volatility, low melting temperature, large water miscibility and
low cost (Pereira, Lima, & Freire, 2010). Polymer-salt ABS provides advantages over systems
formed by polymer–polymer combinations, such as a low interfacial tension, low viscosity, good
biocompatibility, fast and high phase separation rates and low cost, which makes them practical
for downstream processing (Pereira, Lima, & Freire, 2010). Despite all these advantages, the
narrow tailoring nature of PEG, which can be achieved only by changes in the molecular weight or
by the polymer structural modification, limits its applicability through the complete extraction of
several biomolecules to the polymer-rich phase (Pereira, Lima, & Freire, 2010). To overcome this
2
limitation, recent works have introduced ionic liquids (ILs) to tune the physicochemical properties
of the PEG-rich phase, either by using them as adjuvants or as an agent to improve functionality of
PEG, aiming at getting high extraction yields (Wu et al., 2008; Azevedo et al., 2009; Jiang, Xia, Yu,
Guo, & Liu, 2009).
Ionic liquids (IL) are salts composed of asymmetric organic cation and organic or inorganic
anions and liquefy below a temperature of 373.15 K (Wilkes, 2002). ILs have remarkable
characteristics, such as a negligible vapor pressure, non-flammability under ambient conditions,
which contribute to their “green solvents” characterization, good solubility, high ionic
conductivity, high thermal stability, large liquid temperature range and good chemical stability
(Zakrzewska, Bogel-Łukasik, & Bogel-Łukasik, 2010; Freire et al., 2012b; Passos, Ferreira,
Cláudio, Coutinho, & Freire, 2012). Moreover, there are large variations in the cation and anion
chemical structures which further allow the fine-tuning of their physicochemical properties (Freire
et al., 2012b). One of the main advantages of ABS composed of ionic liquid relays on the
possibility of controlling its phase polarity by an appropriate choice of the ions that combined to
form a given ionic fluid (Pereira, Lima, & Freire, 2010). Due to these advantages, some
researchers have studied the application of the IL as based salt or as an adjuvant in ABS in the
extraction of variety of bio-molecules (Dreyer, Salim, & Kragl, 2009; Pei, Wang, Wu, Xuan, &
Luet, 2009; Pereira, Lima, & Freire, 2010; de Souza, R.A. Limaa, J.A.P. Coutinho, C.M.F. Soares,
Á.S. Lima, 2015; Ferreira, Faustino, Mondal, Coutinho, & Freire, 2016).
Amino acids are a class of useful bio-products for various applications. In this context, the
partition behavior of amino acids in ABS is both of the academic and practical importance; since
amino acid residues determine the surface properties of proteins, further understanding of the
driving forces for the partitioning of proteins in given ABS can be obtained from the study of
single amino acids (Freire et al., 2012b). Furthermore, amino acids are very important
bio-products, so their recovery via ABS extraction from bio-reaction media may represent realistic
alternatives to more traditional methods (Freire et al., 2012b). Aqueous biphasic systems of IL-salt
and polymer-salt with or without IL have been successfully applied to the extraction of amino
acids (Shang, Li, Jia, & Li, 2004; Salabat, Abnosi, & Motahari, 2008; Ventura et al., 2009; Neves,
Ventura, Freire, Marrucho & Coutinho, 2009;
Louros et al., 2010; Salabat, Sadeghi, Moghadam,
& Jamehbozorg, 2011; Zafarani-Moattar, & Hamzehzadeh, 2011; Freire, Louros, Rebelo, &
3
Coutinho, 2011).
The amino acid, L-phenylalanine, is one of the essential amino acids, and used as a
nutritional enhancer, amino acid infusion, and complex amino acid preparations. In addition, this
amino acid is used as a raw material of a variety of anti-cancer drugs and sweet taste agents. In the
present paper, L-phenylalanine is engaged as a model biomolecule to study the extraction ability
of the aqueous biphasic systems. However, these novel systems can be further studied to expand
their selective extraction ability and the range of their application. In this context, the partitioning
behavior of phenylalanine, has been studied in aqueous biphasic systems of (PEG 600 + sodium
phosphate salt (NaH2PO4·2H2O) + H2O, using imidazolium-based ILs as adjuvants at 4 wt% mass
concentration ([C2mim]Cl, [C4mim]Cl, [C6mim]Cl, [C8 mim]Cl, [C10mim]Cl, [C4mim][CF3SO3],
[C4mim]OAC ). Phase diagrams and tie-lines have been studied for better understanding the
characteristic of the ABS. For better and accurate understanding the partition ability of these
systems, the pH of the phase was tested and the partition coefficient of L-phenylalanine and ILs
was studied. Moreover, the influence of the temperature on the extraction of L-phenylalanine was
also investigated.
2. Experimental section
2.1. Materials
The present study was carried out using polyethylene glycol polymer (average molecular
weight of 600 g· mol-1), abbreviated as PEG 600. The polymer was supplied by Aladdin Industrial
Corporation and was used as received. The salt used in the formation of the phase diagrams was
sodium dihydrogen phosphate dehydrate (NaH2PO4·2H2O). The salt was purchased from
Sinopharm Chemical Reagent Co., Ltd (Shanghai, China), with purities higher than 99 wt%. The
ionic liquids used in this work to study the formation of ABS were the following:
1-ethyl-3-methylimidazolium chloride,
[C2mim]Cl;
1-butyl-3-methylimidazolium chloride,
[C4mim]Cl; 1-hexyl-3-methyl- imidazolium chloride, [C6mim]Cl; 1-octyl-3-methylimidazolium
chloride,
[C8 mim]Cl;
1-butyl-3-methylimidazolium
1-decyl-3-methylimdazolium
acetate,
[C4 mim]OAC;
chloride,
[C10mim]Cl;
1-butyl-3-methylimidazolium
trifluoromethanesulfonate, [C4 mim][CF3 SO3]. All ionic liquids were supplied by Chenjie
Chemical Co., Ltd. (Shanghai, China) with purities of above 0.99 mass fraction and were used
without further purification. The L-phenylalanine (C9H11NO2 ) was purchased from Sinopharm
4
Chemical Reagent Co., Ltd (Shanghai, China). Double distilled water was used for the preparation
of solutions.
2.2. Methods
2.2.1. Determination of the phase diagrams and tie-lines
The phase diagrams were determined by the cloud point titration method at atmospheric
pressure and at a temperature of 298.15 K, which maintained constant by using a water thermostat
(KW-1000DC, Jintan Zhongda Instrument Factory, China). Aqueous solutions composed of 40
wt% NaH2PO4·2H2O + 4 wt% of each IL, aqueous solutions of PEG 600 at 60 wt% + 4 wt% of
the same ILs, and aqueous solutions of 4 wt% of each IL were prepared and used for
determination of the binodal curves. The binodal curve of the control system, without IL, is also
prepared for the comparison. It should be mentioned that the concentration of IL was kept constant
in all systems. The experimental method was previously validated in the literature (Jiang, Xia, Yu,
Guo, & Liu, 2009; Ventura et al., 2009; Neves, Ventura, Freire, Marrucho & Coutinho, 2009). For
determination of the phase diagrams of the aqueous solutions in IL, each IL was added to the
solution and kept at a constant concentration (at 4 wt%) during the experiment. The aqueous
inorganic salt solution (containing 4 wt% IL) was added drop-wise to the PEG 600 aqueous
solution (containing 4 wt% IL) until the detection of a cloudy solution (biphasic region), followed
by the drop-wise addition of IL solution until the formation of a clear solution (monophasic
region). The weights of the added components were recorded. The drop-wise addition was carried
out under constant shaking. The composition of each quaternary system was determined by weight
quantification of all components within ± 10-4 g (using an analytical balance, PRACTUM124-1CN
from Sartorius Scientific Instruments Co., Beijing, China). The phase diagram experiments were
repeated three times and the average weights were obtained. The curves are reported in molality
units for better understanding of the impact of the imidazolium-based ILs in formation of the ABS.
The binodal data of these systems were correlated using the Merchuk equation (Eq. (1)) (Merchuk,
Andrews, & Asenjo, 1998).
Y=Aexp(BX0.5-CX3)
(1)
Where Y and X are the PEG and salt weight percentages, respectively, and A, B, and C are the
fitting parameters obtained by regression of the data.
The tie-lines (TLs) of each phase diagram were determined by a gravimetric method
5
described by Merchuk, Andrews, and Asenjo, (1998). Hereby, a mixture of the biphasic region
was prepared, vigorously stirred and allowed to reach equilibrium by the separation of both phases
for 24 h at 298.15 K. After the equilibration, the bottom and the top phases were separated and
weighed. The determination of the TL was solved by the following system of four equations (Eqs
(2) to (5)) and four unknown values of YT, YB, XT, XB.
YT=Aexp[(BXT0.5 )-(CXT3)]
(2)
YB =Aexp[(BXB0.5)-(CXB3)]
(3)
YT=YM/α-[(1-α)/α]YB
(4)
XT=XM/α-[(1-α)/α]XB
(5)
Where Y and X are respectively, the PEG and inorganic salt weight percentages, the subscript
letters T, B and M represent the top, the bottom and the mixture phase, respectively. The parameter
α is the ratio between the top and the total mass of the mixture. The tie-line length (TLL) was
determined according to Eq (6),
TLL = ( X T − X B )2 + (YT − YB )2
(6)
2.2.2. Partitioning of L-phenylalanine and ILs in PEG 600-phosphate salt ABS
The partitioning behavior of the L-phenylalanine and ILs in the quaternary system was
studied at a given point in the biphasic region. An aqueous solution of L-phenylalanine at a
concentration of 5 mg mL-1 was used in the water content composition. To achieve a complete
partitioning of L-phenylalanine and ILs between the two phases, the quaternary aqueous mixture
was stirred for 5 min, left to equilibrate for 24 h at a predetermined temperature (278.15K,
288.15K, 298.15K, 308.15K, and 318.15K) in a thermostat-controlled water bath (KW-1000DC,
Jintan Zhongda Instrument Factory, China). Thereafter, the phases were separated carefully and
the concentrations of L-phenylalanine and IL were determined in each phase. The contents of
L-phenylalanine and IL in the top and the bottom phase were assayed by using a
UV-spectrophotometer (T6 series, Persee Analytics, Beijing, China), at 257 nm and 211 nm,
respectively. The concentrations of the partitioned substances were calculated from standard
calibration curves. For accurate quantification of L-phenylalanine, possible interferences of the
PEG 600, ILs and NaH2PO4·2H2O, especially of the aromatic ILs (maximum absorption at 211
nm), were monitored. Accordingly, no PEG or IL interference in the absorbance at 257 nm was
6
detected, at the dilutions used. Three partitioning experiments were done and the average amounts
of L-phenylalanine and IL in each phase were obtained.
Based on the previous partitioning data, the partition coefficients of IL, KIL, and
L-phenylalanine, KPhe, (the ratio of the amount of IL or L-phenylalanine in the PEG 600-rich top
phase and that in the NaH2PO4·2H2O-rich bottom phase) were calculated using Eqs. (7) and (8),
respectively.
K IL = [ IL]PEG600 [ IL]Salt
(7)
K Phe = [ Phe]PEG600 [ Phe ]Salt
(8)
where [IL]PEG600 and [Phe]PEG600 are the concentrations of IL and L-phenylalanine in the PEG
600-rich phase, respectively, and [IL]Salt and [Phe]Salt are the concentrations of IL and
L-phenylalanine in the phosphate-rich phase, respectively.
2.2.3. Determination of the pH
Aqueous biphasic systems, containing 40 wt% PEG 600, 12 wt% NaH2PO4 ·2H2 O, 44 wt%
water and 4 wt% of each ionic liquid, were employed for pH measurements. Hereby, ABS
mixtures were prepared gravimetrically (within ± 10-4 g), vigorously stirred, left to separate into
two phases for 24 h, at 298.15 (± 1 K). The top and the bottom phase were separated and then the
pH of the top phase (PEG-rich phase) was measured at 298.15 K using a pH Meter (Hanna
Instruments, Model 2011, Beijing, China).
2.2.4. Examining the extraction of L-phenylalanine in ABS at different temperatures
To assess the effect of temperature on the extraction of L-phenylalanine, KPhe was determined
at different values of temperature (278.15, 288.15, 298.15, 308.15 and 318.15 K). Extraction
measurements were done in aqueous biphasic systems composed of 40 wt% PEG 600 + 12 wt%
NaH2PO4· 2H2O + + 44 wt% water + 4 wt% IL (no IL, [C4 mim]Cl, [C8 mim]Cl, [C4 mim]OAC and
[C4mim][CF3SO3]. The extraction experiments were repeated three times and the average of KPhe
was obtained.
The thermodynamic parameters of the partitioning of L-phenylalanine in ABS were also
determined at 298.15 K by using Eqs. (9) to (11):
∆PheG0 m = -RTln(KPhe)
(9)
∆PheG0 m =∆pheH0 m -T∆Phe S0 m
(10)
7
ln( K P h e ) =-[ ∆ p h e H 0 m /R]/T+[ ∆ P h e S 0 m] /R
(11)
where ∆PheG0 m is the standard molar Gibbs free energy, ∆pheH0m is the standard molar enthalpy,
∆PheS0m is the standard molar entropy of transfer, KPhe is the partition coefficient of
L-phenylalanine between the PEG-rich phase and the salt-rich phase, R is the universal gas
constant, and T is the temperature.
3. Results and discussion
3.1. Analysis of the phase diagrams and tie-lines
In the present study, the influence of the alkyl chain length and anion of the
imidazolium-based IL on the phase-forming ability of ABS were investigated. The phase diagrams
for the quaternary systems composed of PEG 600 + NaH2 PO4 + H2O + 4 wt% IL, at 298.15 K and
atmospheric pressure, are shown in Fig. 1. To study the effect of the IL cation alkyl chain length,
phase diagrams of quaternary systems comprising different ILs, viz, [C2mim]Cl, [C4 mim]Cl,
[C6mim]Cl, [C8mim]Cl, [C10mim]Cl (see chemical structures in Supporting information Fig. 1)
were used as adjuvants in formation of the ABS. It can be seen that an increase in the cation alkyl
chain length from [C4 mim]Cl to [C8 mim]Cl increases the liquid-liquid demixing ability of ABS,
but this phenomenon is not particularly obvious for [C2mim]Cl and [C10mim]Cl (Fig. 1). This
trend of improvement in the phase-forming ability in ABS is probably attributed to the increase in
the hydrophobicity of IL with the increase in the alkyl chain length, leading to a lower affinity for
water (Huddleston et al., 2001; Freire et al., 2007; Ventura et al., 2009; Neves, Ventura, Freire,
Marrucho & Coutinho, 2009). Similar enhancement in phase separation is observed in PEG 600 Na2 SO4 ABS containing ILs with the cation chain length larger than [C4mim]Cl, compared with
ABS with no IL (Pereira, Lima, Freire, and Coutinho, 2010). On the other hand, the effect of the
IL anion on the phase separation is displayed in Fig. 1. [C4mim][CF3SO3] and [C4mim]OAC
increase the phase immiscibility, compared to the ABS with no IL. However, [C4mim]OAC shows
better phase-forming ability at lower concentrations of the salt and [C4mim][CF3SO3] at higher
concentrations of the salt. It is reported that the phase-forming ability of ABS decreases with the
increase in the IL anion affinity for water, coinciding with the trend of the anion hydrogen bond
acidity (Ventura et al., 2009), and thus confirming the importance of the hydrophobicity of the IL
anion as a driving force of the phase separation.
The fitted parameters, the experimental tie-lines (TLs) measured for each system together
8
with the respective tie-line lengths (TLLs) are presented in Table 1 and Supporting information
Table 1.
3.2. Analysis of the partitioning behavior of ILs in the ABS
For the ABS containing ILs at fixed concentration, the partition coefficients of ILs were
determined to understand their impact on the phase-forming ability and extractability of the ABS.
The mass fraction composition of each component and the partition coefficient of each IL, KIL, at
298.15 K are presented in Table 2. Except for [C2mim]Cl and [C10mim]Cl, all ILs have KIL higher
than 1, suggesting the advantageous migration of IL to the PEG600-rich phase. As seen in Table 2,
the partition coefficient of [C10mim]Cl is the lowest (KIL = 0.567) and therefore KIL becomes
higher as the imidazole alkyl chain length increases, reaching its highest value in [C8mim]Cl (KIL
= 13.587). Pereira, Lima, Freire, and Coutinho (2010) have ascribed the efficiency of partitioning
of IL between the two phases to the IL salting-in/-out capability. Consequently, [C2 mim]Cl and
[C8mim]Cl are respectively considered the strongest salting-out and salting-in inducing ILs,
therefore preferentially extracted in the NaH2PO4 -rich phase and PEG600-rich phase, respectively.
However, the partition coefficient of [C10mim]Cl is abnormal and less than [C8 mim]Cl, which
might be related to the nature of the ionic liquid. On the other hand, the KIL of [C4 mim]OAC is
higher (5.302) than that of [C4 mim]Cl (2.516) while the KIL of [C4mim][CF3SO3 ] is lower (2.258).
These variations in the KIL values indicate that the anion of OAC- is characterized by lower
hydrogen bond basicity value than Cl- (Crowhurst, Mawdsley, Perez-Arlandis, Salter, & Welton,
2003). The lower hydrogen bond basicity results in an increase in the hydrophobicity of the ionic
liquid and hence is more easily excluded to a second liquid phase by the salting-out effect of the
organic salt (Cláudio, Ferreira, Shahriari, Freire, & Coutinho, 2011; Almeida et al., 2014).
Therefore, the affinity of ions for water defines the migration of ionic liquids to a particular phase,
resulting in changes in the physical and chemical properties of the phase (Freire et al., 2010), and
thus regulating the extractability of ABS. Although the ionic liquid partition coefficient has a
relatively large change in different systems, but the systems’ biphasic region does not change too
much, which is consistent with the conclusions obtained on the phase diagram. In conclusion, it
could be noticed that the IL cation shows prominent effect on the behavior of PEG-phosphate salt
phase diagrams when compared with the IL anion influence. Similar results are observed during
extraction of L-phenylalanine in PEG 400 + Na2SO4 + IL ABS (Pereira, Lima, Freire, & Coutinho,
9
2010).
3.3. Extraction of L-phenylalanine in the ABS
The partition coefficients of L-phenylalanine (KPhe) and mass fraction compositions of the
ABS are shown in Table 2. While the effects of the IL cation and anion on KPhe and the pH of the
top phase of the different ABS are depicted in Fig. 2. The results from Table 2 show that the
studied ABS with or without IL have KPhe values higher than one, suggesting the advantageous
partitioning of L-phenylalanine for the PEG600-rich phase. So far, there is a noticeable decrease
in the KPhe in ABS using [C2mim]Cl as adjuvant compared with the system with no IL. Moreover,
addition of imidazolium-based IL with butyl- to octyl-chain length ([C4mim]Cl to [C8mim]Cl) to
ABS improves gradually KPhe over that of no IL (Fig. 2). This tendency towards improvement
coincides with the partitioning behavior of the IL (KIL), resulting from the IL salting-out/salting-in
inducing ability that discussed previously in section 3.2. In addition, the hydrophobic nature of
L-phenylalanine arising from the aromatic ring may facilitate its migration to the hydrophobic
polymer-rich phase (see the chemical structure shown in the Supporting information, Fig.1.). As
formerly observed for KIL, the KPhe of the ABS containing [C10mim]Cl is smaller than that of the
system with [C8mim]Cl, which is consistent with the anomalous partitioning behavior of
[C10mim]Cl. This abnormal phenomenon may be related to the nature of [C10mim]Cl and the
system.
Moreover, the ABS containing [C4 mim]OAC displays higher KPhe value (4.458) compared
with the system containing [C4mim]Cl, on the contrary the system containing [C4 mim][CF3SO 3]
shows lower KPhe value (2.813) (Table 2). The impact of the IL anion on KPhe follows the trend of
the anion hydrophobicity, emanating from the differences in hydrogen-bond basicity as discussed
before. The above findings suggest the importance of the excluded IL in the PEG 600-rich phase
(Table 1) in enhancing the partitioning of L-phenylalanine, which is imputed to the IL aptitude for
changing the type of solute-solvent interactions (Tomé et al., 2010) and hence permitting to shape
the extraction ability of the PEG 600-rich phase. However, the IL cation seems to have the
greatest impact on regulating the extraction of L-phenylalanine to PEG-rich phase than the IL
anion, which agrees with previous studies on the partitioning behavior of IL-based ABS (Ventura
et al., 2009; Neves, Ventura, Freire, Marrucho & Coutinho, 2009). In general, the partition
coefficients of biomolecules in ABS depend on several factors such as (i) the interactions between
10
the hydrophobic characteristics of ABS and the hydrophobicity of the biomolecule, (ii) the
molecular size, conformation, solubility, electrostatic forces and the affinity of the biomolecule for
the two phases, (iii) the composition of the two phases and the nature of the biomolecules and (iv)
the interactive role of the electrical potential for the two phases and the charge of the biomolecule
on the separation process (Spelzini, Picó, & Farruggia, 2006; Asenjo, & Andrews, 2012; Pereira et
al., 2015; Cláudio et al., 2015). Most of the studied aqueous biphasic systems show pH of 4.55,
but the ABS containing [C4 mim]OAC shows a pH of 5.76 (Fig. 2).
3.4. The effect of temperature on the extraction of L-phenylalanine in ABS
The results of the effect of different temperatures (278.15 - 318.15 K) on the partition
coefficient of L-phenylalanine, KPhe, in aqueous biphasic systems composed of 40 wt% PEG 600
+ 12 wt% NaH2PO4·2H2O + 4 wt% IL (those containing no IL, [C4mim]Cl, [C8 mim]Cl,
[C4mim]OAC and [C4 mim][CF3SO3 ]), and the mass fraction compositions for each system are
displayed in Fig. 3 and Supporting information, Table 2. It can be seen that at all levels of
investigated temperature the KPhe values of the studied ABS are larger than 1.0, illustrating the
impact of the temperature on the extraction of L-phenylalanine. Except for ABS containing
[C8mim]Cl, all other ABS with or without IL show the maximum KPhe values at 278.15 K.
However, the increase in temperature higher than 278.15 K leads to decrease in the KPhe values of
all ABS. Similar effect of temperature on the extraction of methionine in polymer-phosphate salt
ABS (Salabat, Sadeghi, Moghadam, & Jamehbozorg, 2011) and proteins in IL-based ABS has
been reported (Pei, Wang, Wu, Xuan, & Luet, 2009). It is worth noticing that ABS with
[C4mim]OAC displays the highest KPhe value at 278.15 K compared with the other systems.
Accordingly, the variations in KPhe, at the different levels of temperature, are resulting from the
influence of the IL cation and anion. Since electrostatic contributions are rather independent of
temperature, the deviations in KPhe as a function of temperature could be arisen from dispersive
forces and H-bonding interactions taking place in those systems (Pereira, Lima, Freire, &
Coutinho, 2010).
Furthermore, the thermodynamic functions of standard molar enthalpy (∆pheH0m), molar
entropy (∆PheS0 m) and molar Gibbs free energy (∆PheG0 m) of the extraction of L-phenylalanine at
298.15K was obtained from the plots of ln(KPhe) versus T-1 (KPhe > maximum values, Pereira,
Lima, Freire, & Coutinho, 2010) and the results are shown in Table 3. The linearity of the plots of
11
ln(KPhe) versus T-1 reveals the temperature independence of the molar enthalpy of migration of
L-phenylalanine. As seen from Table 3 the absolute values of ∆PheG0 m of the ABS containing ILs
(except [C4mim][CF3SO3 ]) are higher than that of the system with no IL. The negative values of
∆PheG0 m for all ABS demonstrate the spontaneous extraction of L-phenylalanine to the phases with
a preference to the PEG-rich phase. Moreover, the findings of this study illustrate that the
spontaneous selective migration of L-phenylalanine to the two phases is an exothermic process as
proved from the negative values of ∆pheH0 m of the ABS (with ILs or no IL). It seems that the
increase in the IL alkyl chain length has an impact on the absolute value of ∆pheH0 m, which
increases from 12.2424 for the ABS containing [C4mim]Cl to 20.0309 for the system containing
[C8mim]Cl. This increase agrees with a similar increase in the partition coefficient of
L-phenylalanine (from 2.950 to 5.403). Moreover, the effect of the IL anion on the standard molar
enthalpy is observed in the ABS containing [C4mim]OAC, which shows an absolute value of
33.3233 versus 12.2424 for the system containing [C4mim]Cl. Accordingly, the standard molar
enthalpy of ABS looks to widely rely on both the IL cation and anion. The changes in the molar
entropy of the studied ABS are similar to the changes in the molar enthalpy in regard to the effects
of IL cations and anions. It is therefore the L-phenylalanine molar entropy of transfer of
[C4mim]OAC is the highest, followed by [C8mim]Cl, then followed by [C4 mim]Cl and finally by
[C4mim][CF3SO3].
As a consequence of the lower absolute values of T×∆PheS0 m compared to ∆pheH0 m, the molar
enthalpy could be the main thermodynamic factor that controls the extraction of L-phenylalanine.
Thus, the thermodynamic study of the extraction of L-phenylalanine ascertains its exclusion from
the salt-rich phase to the PEG-rich phase. The results obtained in this work illustrate that the
characteristics of the polymer-rich phase of the ABS can be manipulated by using a small amount
of an adequate IL as adjuvant. The characteristics of the IL cation and anion can be used to change
the hydrophobic characteristics of the PEG-rich phase, aiming at expanding the scope of
application of ABS and at improving the extraction rate of the target substance.
4. Conclusions
The results here reported indicate that the use of imidazolium-based IL as an adjuvant in the
PEG600/NaH2PO4 ABS can provide an enhanced extraction efficiency of L-phenylalanine. This
can be maximized by a correct selection of the chemical structure of the IL employed and the
12
extraction temperature, which can enlarge the scale of application of the ABS. Thus a new route
for less ‘‘conventional’’ ABS can be opened.
Acknowledgments
The authors are grateful for the support provided by the National Natural Science Foundation of
China (21676125), the National Key Research and Development Program of China
(2016YFD0400705-04, 2017YFD0400903-01), the Special Fund of Jiangsu Province for the
Transformation of Scientific and Technological Achievements (BA2016169), the Policy Guidance
Program (Research Cooperation) of Jiangsu (BY2016072-03) and the Social Development
Program (General Project) of Jiangsu (No. BE2016779).
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17
Figure captions
Fig. 1. Phase diagrams for the imidazolium-based quaternary systems composed PEG 600 +
NaH2PO4 + H2O + 4 wt% IL, at 298.15K.
Fig. 2. Partition coefficients of L-phenylalanine (KPhe) and of each IL(KIL) and the pH of the top
phase for the quaternary systems composed of 40 wt% PEG 600 + 12 wt% NaH2 PO4·2H2O + 44
wt% H2O+ 4 wt% IL, at 298.15K.
Fig. 3. Partition coefficient of L-phenylalanine (KPhe) at different temperatures.
18
0.8
no IL
[C2mim]Cl
0.7
[C4mim]Cl
[C6mim]Cl
0.6
PEG600/(mol/kg)
[C8mim]Cl
0.5
[C10mim]Cl
[C4mim][CF3SO3]
0.4
[C4mim]OAC
0.3
0.2
0.1
0.0
0.0
0.5
1.0
1.5
2.0
2.5
NaH2PO4/(mol/kg)
Fig. 1.
KPhe
14
KIL
10
pH
12
8
10
6
pH
K
8
6
4
4
2
2
0
C8
IL
19
CF3
OAC
S
3
Fig. 2.
C10
0
C
A
]O
im
m
4
]
[C
O3
C6
CF
][
im
m
[C 4
C4
l
]C
im
m
10
[C
C2
l
]C
im
m
8
[C
l
]C
im
m
6
[C
l
]C
im
m
4
[C
l
]C
im
m
2
[C
o
N
NO IL
No IL
12
[C4mim]Cl
[C8mim]Cl
10
[C4mim][CF3SO3]
[C4mim]OAC
KPhe
8
6
4
2
0
278.15K
288.15K
298.15K
Fig. 3.
20
308.15K
318.15K
Table 1 Initial mass fraction compositions for the TLLs determination and compositions of the
respective top (T) and bottom (B) phases at 298.15K
Quaternary
system
Mass fraction composition (wt%)
PEG600(YM)
NaH2 PO4·2H 2O(XM)
No IL
39.95
12.01
48.858
[C2 mim]Cl
40.02
11.98
[C4 mim]Cl
39.99
[C6 mim]Cl
YT
XT
YB
XB
TLL
2.9979
0.4448
51.9742
68.8659
48.4993
3.6742
1.1291
50.0816
66.3142
11.99
47.7949
3.6340
0.7775
53.9822
68.8881
40.05
12.03
47.9033
3.3572
0.5943
55.6145
70.4909
[C8 mim]Cl
40.02
12.01
48.8584
2.6779
0.2957
53.9517
70.6210
[C10mim]Cl
39.98
12.01
51.2948
1.9027
0.7799
47.1079
67.7284
[C4 mim][CF3 SO3]
40.02
11.98
49.4038
2.6284
0.0152
51.8401
69.7211
[C4 mim]OAC
40.04
11.99
49.0595
1.5250
0.3053
58.0906
74.6769
Table 2 Partition coefficients of each IL (KIL), the pH of the top phase and L-phenylalanine (KPhe)
and mass fraction compositions of the systems at 298.15 K.
Quaternary
Mass Fraction Composition/(wt%)
System
PEG600(YM)
NaH2 PO 4·2H2O(X M)
IL
KPhe
KIL
pH
No IL
39.95
12.01
—
2.718±0.045
—
4.54±0.05
[C 2mim]Cl
40.02
11.98
4.01
1.836±0.088
0.683±0.098
4.53±0.06
[C 4mim]Cl
39.99
11.99
4.02
2.950±0.061
2.516±0.106
4.55±0.04
[C 6mim]Cl
40.05
12.03
3.99
5.076±0.083
12.005±0.24
4.56±0.04
[C 8mim]Cl
40.02
12.01
4.00
5.403±0.109
13.587±0.25
4.53±0.04
[C 10mim]Cl
39.98
12.01
4.01
1.457±0.064
0.567±0.087
4.56±0.07
[C 4mim][CF3 SO3]
40.02
11.98
3.99
2.813±0.100
2.258±0.125
4.65±0.07
[C 4mim]OAC
40.04
11.99
4.00
4.458±0.098
5.302±0.213
5.77±0.07
Table 3 Standard molar thermodynamic functions of transfers of L-phenylalanine at 298.15K.
Quaternary system
∆pheH0m
∆PheS0m
(kJmol-1)
(Jmol-1K-1)
T×∆PheS0 m
∆PheG0 m
(kJmol-1)
(kJmol-1)
ln(KPhe)
No IL
-6.2900
-12.2989
-3.6669
-2.6231
1.0582
[C4mim]Cl
-12.2424
-32.0571
-9.5578
-2.6845
1.0830
[C8mim]Cl
-20.0309
-53.6112
-15.984
-4.0468
1.6325
[C4mim][CF3SO3]
-11.2946
-30.6429
-9.1362
-2.1584
0.8707
[C4mim]OAC
-33.3233
-99.2442
-29.590
-3.7337
1.5062
21
Highlights
ILs as additives to extract L-phenylalanine based on PEG/Salt ABS.
The IL’s structure and temperature play important role on extraction process.
ILs as adjuvants open a new route for conventional ABS.
22
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