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However, the separation and isolation of

bromelain from pineapple are currently in the


development stage with further exploration required
to resolve challenges related to the technology,
purity and economic aspects of large-scale bromelain
production (Arshad et al., 2014).
In general, the bromelain production involves
several
Phase Equilibrium in Aqueous Two-phase
Systems Containing Poly (propylene glycol) and
Sodium Citrate at Different pH
processing steps as illustrated in Figure 1 as
described by Xian Lukee (2015). TheFarshad Rahimpour*, Ali Reza Baharvand steps include:
(i)
AbstractThe phase diagrams and compositions of coexisting These salts, however, lead to high phosphate or sulfate
e phases have been determined for aqueous two-phase systems concentration in the effluent streams and therefore, to
containing poly(propylene glycol) with average molecular weight of environmental concern. One way to reduce the salt discharged
xt 425 and sodium citrate at various pH of 3.93, 4.44, 4.6, 4.97, 5.1, in the wastewater is to recycle the chemicals. In this regard,
r 8.22. The effect of pH on the salting-out effect of poly (propylene Hustedt [8] and Greve and Kula [9] have published useful
glycol) by sodium citrate has been studied. It was found that, an
International papers. Recently, as an alternative approach, Vernau and Kula
a increasing
Science Index, in pH caused the expansion of two-phase region. [10] and Rahimpour et al. [11] have investigated citrates as a
Chemical and
Increasing pH also increases the concentration of PPG in the PPG-
c
Molecular
substitute for inorganic salts and found that sodium and
rich phase, while the salt-rich phase will be somewhat mole diluted. potassium citrates form aqueous two-phase systems with PEG
t
Engineering
which is suitable for biomolecules extraction. Citrate is
Vol:3, No:11,
2009i biodegradable and nontoxic and could be discharged into
KeywordsAqueous two-phase system, Phase equilibrium,
waset.org/Publ biological wastewater treatment plants and therefore the
o Biomolecules
ication/4396 purification citrates can be considered as a substitute for inorganic salts,
n because the citrate forms aqueous two-phase system with PPG
I. INTRODUCTION which are suitable for biomolecules extraction.
, As far as we know, there is no report on the pH effect on
A N aqueous two-phase system (ATPS) is produced when
(i appropriate small amounts of two chemically different
water soluble polymers, or a water soluble polymer and
phase diagram of the PPG425 + sodium citrate + water system
in the literature. This work is devoted to obtaining phase
i) inorganic salts, are added to water, causing the system to equilibrium data for the PPG425 + sodium citrate + water
system at pH of 3.93, 4.44, 4.6, 4.97, 5.1, and 8.22. These results
re separate into two immiscible water-rich phases [1]. The high can be used to develop thermodynamic models of aqueous
water content in aqueous two-phase systems, typically greater
c than 80 wt.%, coupled with low interfacial tension provide a two-phase systems and also to obtaining experimental data for
o benign environment for biomolecules not attained in solvent choosing suitable systems for purification of biomolecules.
extraction. The system offers a technically simple, energy
v efficient, easily scalable and mild separation technique for
er product recovery in biotechnology [2].
y, ofIts major use has been in the concentration and purification
proteins and in the extractive bioconversion of enzymes. II. EXPERIMENTS
(i The separation technique is also becoming important in non-
A. Materials
ii biotechnology areas such as industrial waste remediation. PPG of molecular weight 425, tri-sodium citrate, and citric
Notable examples include utilization of aqueous phase
) systems in removal of color from textile plant wastes [3], acid were obtained from Merck. The polymer and salt were
p metal ions [4] and organic pollutants from the environment used without further purification, and double distilled,
[5], and aromatics from crude oil [4]. Liquidliquid deionized water was used. All other materials used were of
urequilibrium (LLE) data for some aqueous PPG + inorganic salt analytical grade.
itwo-phase systems have been reported in the literature [6-7].
c In these studies, salting out of PPG has been accomplished by
the use of either carbonate or sulfates. B. Preparation of the Aqueous Two-Phase Systems
at Aqueous two-phase systems (ATPS) were prepared by a
ion and (iv) mixture of PPG425 and sodium citrate salt solution at required
pH. The pH of the salt solution was adjusted by mixing
drying and grinding, in order to produce bromelain appropriate ratio of sodium citrate and citric acid.
powder for commercialization purpose. All components were added into a graduated 15 ml test tube
as stock solution at constant pH, resulting in a 10 g system.
Farshad Rahimpour, Assistant Professor, Biotechnology research lab,
Chemical Engineering Department, Faculty of Engineering, Razi University, All experiments were carried out at 25 C. The pH values of
In Kermanshah 67149-67346, Iran (phone: +98-831-4274530; fax: +98-831- the solutions were measured precisely with a pH meter of
th 4274542; e-mail: f_rahimpour@razi.ac.ir). JENWAY 3345 model. In order to speed up phase separation
Ali Reza Baharvand, MSc, Biotechnology research lab, Chemical the resulted solution was mixed by rigorous vortexing the test
e Engineering Dept. Faculty of Engineering, Razi Univ. Kermanshah 67149- tube for 2 min. The tubes were placed in a room temperature
e 67346, Iran (e-mail: Baharvand@razi.ac.ir).
This work was supported in part by the research vice departments of Razi for 4 h and then were centrifuges at 1500 rpm for 10 min; the
xt University.
raction step, pineapple parts are washed,
cut into small pieces and undergo mechanical lysis
by simply applying mechanical shear force to disrupt
the plant cells and separate the enzyme from the
cells by solubilizing the enzyme in water or buffer
(Devakate et al., 2009; Lopes et al., 2009; Hebbar
et International Scholarly and Scientific Research & Innovation 3(11) 2009 616 scholar.waset.org/1999.2/4396 al., 2012). This
mechanical disruption approach
is simple and suitable for pineapple cells due to
their rigid structure (Illanes, 2008). However, the
former method generates heat following processing
that leads to enzyme denaturation, suggesting the
process should be performed at low temperatures
(Ahmed, 2005). Various buffers can be used during
the extraction process with sodium phosphate
buffer reported to yield the highest specic activity
compared to potassium phosphate buffer, sodium
potassium phosphate buffer, potassium-hydroxide
buffer and citratephosphate buffer (Chaurasiya and
Hebbar, 2013).
Once the enzyme has been extracted, it is
recovered in the enzyme recovery step, which implies
its separation from the coarse impurities including
bres, undisrupted cells and cell debris. A solid-
liquid separation process is commonly used where
mashed pineapple slurry from the extraction process
is ltered and centrifuged to recover the enzyme and
eliminate all solid components.
Next, the crude extract is subjected to various
purication techniques to remove contaminants
that can interfere with bromelain intended use,
as well as to increase the specic activity of the
enzyme. Generally, any method suitable for protein
fractionation can be used to purify bromelain.
However, for production purpose, the selected
purication methods are restricted to those amenable
for scale-up at a reasonable cost (Illanes, 2008).
Some precaution steps must be taken throughout
the purication process to minimize bromelain
denaturation, modication and degradation and to
maximise yield (Jervis and Pierpoint, 1989).
After the purication of enzymes to the desired

However, the separation and isolation of


bromelain from pineapple are currently in the
development stage with further exploration required
to resolve challenges related to the technology,
purity and economic aspects of large-scale bromelain
production (Arshad et al., 2014).
In general, the bromelain production involves
several processing steps as illustrated in Figure 1 as
described by Xian Lukee (2015). The steps include:
(i) extraction, (ii) recovery, (iii) purication and (iv)
drying and grinding, in order to produce bromelain
powder for commercialization purpose.
In the extraction step, pineapple parts are washed,
cut into small pieces and undergo mechanical lysis
by simply applying mechanical shear force to disrupt
the plant cells and separate the enzyme from the
cells by solubilizing the enzyme in water or buffer
(Devakate et al., 2009; Lopes et al., 2009; Hebbar
et al., 2012). This mechanical disruption approach
is simple and suitable for pineapple cells due to
their rigid structure (Illanes, 2008). However, the
former method generates heat following processing
that leads to enzyme denaturation, suggesting the
process should be performed at low temperatures
(Ahmed, 2005). Various buffers can be used during
the extraction process with sodium phosphate
buffer reported to yield the highest specic activity
compared to potassium phosphate buffer, sodium
potassium phosphate buffer, potassium-hydroxide
buffer and citratephosphate buffer (Chaurasiya and
Hebbar, 2013).
Once the enzyme has been extracted, it is
recovered in the enzyme recovery step, which implies
its separation from the coarse impurities including
bres, undisrupted cells and cell debris. A solid-
liquid separation process is commonly used where
mashed pineapple slurry from the extraction process
is ltered and centrifuged to recover the enzyme and
eliminate all solid components.
Next, the crude extract is subjected to various
purication techniques to remove contaminants
that can interfere with bromelain intended use,
as well as to increase the specic activity of the
enzyme. Generally, any method suitable for protein
fractionation can be used to purify bromelain.
However, for production purpose, the selected
purication methods are restricted to those amenable
for scale-up at a reasonable cost (Illanes, 2008).
Some precaution steps must be taken throughout
the purication process to minimize bromelain
denaturation, modication and degradation and to
maximise yield (Jervis and Pierpoint, 1989).
After the purication of enzymes to the desiredHowever, the separation and isolation of
bromelain from pineapple are currently in the
development stage with further exploration required
to resolve challenges related to the technology,
purity and economic aspects of large-scale bromelain
production (Arshad et al., 2014).
In general, the bromelain production involves
several processing steps as illustrated in Figure 1 as
described by Xian Lukee (2015). The steps include:
(i) extraction, (ii) recovery, (iii) purication and (iv)
drying and grinding, in order to produce bromelain
powder for commercialization purpose.
In the extraction step, pineapple parts are washed,
cut into small pieces and undergo mechanical lysis
by simply applying mechanical shear force to disrupt
the plant cells and separate the enzyme from the
cells by solubilizing the enzyme in water or buffer
(Devakate et al., 2009; Lopes et al., 2009; Hebbar
et al., 2012). This mechanical disruption approach
is simple and suitable for pineapple cells due to
their rigid structure (Illanes, 2008). However, the
former method generates heat following processing
that leads to enzyme denaturation, suggesting the
process should be performed at low temperatures
(Ahmed, 2005). Various buffers can be used during
the extraction process with sodium phosphate
buffer reported to yield the highest specic activity
compared to potassium phosphate buffer, sodium
potassium phosphate buffer, potassium-hydroxide
buffer and citratephosphate buffer (Chaurasiya and
Hebbar, 2013).
Once the enzyme has been extracted, it is
recovered in the enzyme recovery step, which implies
its separation from the coarse impurities including
bres, undisrupted cells and cell debris. A solid-
liquid separation process is commonly used where
mashed pineapple slurry from the extraction process
is ltered and centrifuged to recover the enzyme and
eliminate all solid components.
Next, the crude extract is subjected to various
purication techniques to remove contaminants
that can interfere with bromelain intended use,
as well as to increase the specic activity of the
enzyme. Generally, any method suitable for protein
fractionation can be used to purify bromelain.
However, for production purpose, the selected
purication methods are restricted to those amenable
for scale-up at a reasonable cost (Illanes, 2008).
Some precaution steps must be taken throughout
the purication process to minimize bromelain
denaturation, modication and degradation and to
maximise yield (Jervis and Pierpoint, 1989).
After the purication of enzymes to the desiredHowever, the separation and isolation of
bromelain from pineapple are currently in the
development stage with further exploration required
to resolve challenges related to the technology,
purity and economic aspects of large-scale bromelain
production (Arshad et al., 2014).
In general, the bromelain production involves
several processing steps as illustrated in Figure 1 as
described by Xian Lukee (2015). The steps include:
(i) extraction, (ii) recovery, (iii) purication and (iv)
drying and grinding, in order to produce bromelain
powder for commercialization purpose.
In the extraction step, pineapple parts are washed,
cut into small pieces and undergo mechanical lysis
by simply applying mechanical shear force to disrupt
the plant cells and separate the enzyme from the
cells by solubilizing the enzyme in water or buffer
(Devakate et al., 2009; Lopes et al., 2009; Hebbar
et al., 2012). This mechanical disruption approach
is simple and suitable for pineapple cells due to
their rigid structure (Illanes, 2008). However, the
former method generates heat following processing
that leads to enzyme denaturation, suggesting the
process should be performed at low temperatures
(Ahmed, 2005). Various buffers can be used during
the extraction process with sodium phosphate
buffer reported to yield the highest specic activity
compared to potassium phosphate buffer, sodium
potassium phosphate buffer, potassium-hydroxide
buffer and citratephosphate buffer (Chaurasiya and
Hebbar, 2013).
Once the enzyme has been extracted, it is
recovered in the enzyme recovery step, which implies
its separation from the coarse impurities including
bres, undisrupted cells and cell debris. A solid-
liquid separation process is commonly used where
mashed pineapple slurry from the extraction process
is ltered and centrifuged to recover the enzyme and
eliminate all solid components.
Next, the crude extract is subjected to various
purication techniques to remove contaminants
that can interfere with bromelain intended use,
as well as to increase the specic activity of the
enzyme. Generally, any method suitable for protein
fractionation can be used to purify bromelain.
However, for production purpose, the selected
purication methods are restricted to those amenable
for scale-up at a reasonable cost (Illanes, 2008).
Some precaution steps must be taken throughout
the purication process to minimize bromelain
denaturation, modication and degradation and to
maximise yield (Jervis and Pierpoint, 1989).
After the purication of enzymes to the desired
World Academy of Science, Engineering and Technology
International Journal of Chemical and Molecular Engineering
Vol:3, No:11, 2009

solution reach to equilibrium and the samples of the top and to the solute molecules. Exclusion of ions from the polymer
bottom phase were carefully withdrawn, with care being taken moleculewater interface itself can occur for a number of
to leave a layer of solution at least 0.2 cm thick above the reasons. Both the polymer and the ion are strongly hydrated in
interface. solution. Because of the hydration sheath, the near-surface
region of the polymer may not be accessible to structure-
making ions. The pH could affect the binodal location, either
C. Measurement of Salt and PPG Concentration by changing the charge of the solute or by altering the ratio of
The analysis methods for salt concentrations were determined the charged species present. Fig. 1 shows that the two-phase
by using atomic absorption spectroscopy (AAS), shimatsu AA- area is expanded with increasing pH. In other words, if we
6300 model. take a sample with a known composition on the binodal curve,
The concentration of PPG was determined by refractive this mixture becomes a two-phase system at increasing pH as
index measurements at 298.15K using an ATAGO- has been observed experimentally. It was found that, at higher
DTM1model. Since the refractive index of phase samples pH, hydrogen bond interactions of PPG are weakened [7].
depends on PEG, and salt concentration, calibration plots of Depression of the cloud point by increasing pH is may be
refractive index versus polymer concentration were prepared related to the salting-out phenomenon resulting from the
for deferent concentrations of salt. weakening of the PPGsolvent interaction.
The tie-line length- TLL, calculated by the following equation:

A.Procedure
International
The binodal curves were determined by a titration method. A
Science Index,
salt solution of known concentration was titrated with the
Chemical and
polymer solution or vice versa, until the solution turned turbid.
Molecular
TLL = (w top
s
w sbot ) +( w
2
top w bot )
pp
2
(1)
The composition of the mixture was determined by mass using
Engineering
Vol:3, No:11,
an analytical balance with a precision of 1107 kg. To
2009 determine the compositions of coexisting phases, feed samples
waset.org/Publ
The tie lines are determined by connecting each
(about 10 gr) were prepared by mixing appropriate amounts of
ication/4396 corresponding set of total, bottom-, and top-phase points. As
polymer, salt and water in the tube. After separation of the two an example, the tie lines for the systems with pH of 3.93 and
transparent phases, the concentrations of the salts and PPG in 5.1 are shown in Fig. 2 and 3. As shown in these figures, the
the top and bottom phases were determined. slope and the length of the equilibrium tie-lines increased with
increasing pH. Also, by increasing pH the volume of salt-rich
phase increases at the expense of the PPG-rich phase, as we
III. RESULTS AND DISCUSSION observed experimentally. This is because the compositions of
the phases in equilibrium change with varying pH. Increasing
Experimental results the pH of the aqueous PPG-salt two-phase system will cause
For the aqueous PPG - sodium citrate two-phase system the an increase of the concentration in the PPG-rich phase and a
binodal data obtained from turbidimetric titrations and the tie decrease of the concentration in the salt-rich phase.
line data for the compositions of conjugate solutions at pH of
3.93, 4.44, 4.6, 4.97, 5.1, and 8.22 are shown in Tables 1 and 2,
respectively. The complete phase diagrams of all studied
systems are shown in Figs. 1. TABLE I
TIE LINE DATA FOR SYSTEMS OF
It can be seen that the binodal curve shifts downward as the
PPG425- CITRATE- WATER AT DIFFERENT pH
pH of the medium becomes lower, indicating that smaller Total
concentration of the phase polymers is required to form Top phaseBottom phase
aqueous two-phase system. This behavior is probably due to compositions
decrease in hydrodynamic volume of the polymer as the pH of pH SaltPPGSaltPPGSaltPPG
[w/w%] [w/w%] [w/w%] [w/w%] [w/w%] [[w/w%]
the solution decreases. It was shown that decreasing the pH 8.0215.982.0836.209.2012.80
leads to reduction in intrinsic viscosity of the polymer 9.8513.751.9539.3410.9611.12
solutions [12]. Since it is well known that hydrodynamic 3.93 11.4212.041.6542.1412.609.02
volume of polymers in solution is directly proportional to their 13.2511.021.5045.0513.849.91
intrinsic viscosity, it can be deduced that decreasing the pH 8.4114.022.0037.219.8610.40
10.0012.001.4040.5711.209.60
causes the polymer chains to assume a more compact 12.5010.001.2443.0313.108.25
4.44
structure. 13.509.801.2046.8714.208.40
The formation of aqueous two-phase systems clearly 8.4114.021.6038.7410.67.35
indicates the mutual exclusion of the ions and the polymer and 10.5712.361.0442.8112.007.50
12.510.001.2045.2114.405.18
their high affinity for the solvent. It is possible that even in 4.60 14.918.720.8551.1816.506.40
homogeneous systems (below the phase boundary) the ions 10.3212.220.84740.8511.207.08
are excluded from the near surface region of the polymer in 11.4711.160.6045.6212.607.10
solution. With increase in the concentration of the polymer or 13.3510.140.4052.0514.206.10
the salt, the extent of exclusion will increase. Ultimately, the 4.97 15.518.910.3055.2317.804.90
10.1210.540.8042.6111.605.01
system could reach a state where, for entropic reasons, phase 11.779.260.5148.7712.865.70
formation would become favorable. In fact, salting-out effects 13.458.260.50453.4115.325.04
are related to a reduction in entropy resulting from the 16.048.030.48856.2518.404.07
5.10 14.139.020.2447.3116.403.05
exclusion of ions from volume elements immediately adjacent
15.328.260.3551.7117.503.35
17.507.140.6056.219.502.45
18.356.510.2661.420.202.34
8.22

International Scholarly and Scientific Research & Innovation 3(11) 2009 617 scholar.waset.org/1999.2/4396
International Science Index, Chemical and Molecular Engineering V0123, No: 1 1, 2009
waset.org/Publication/4396

World Academy of Science, Engineering and Technology


International Journal of Chemical and Molecular Engineering
Vol:3, No: 1 1, 2009

Phase Equilibrium in Aqueous Two-phase


Systems Containing Poly (propylene glycol) and
Sodium Citrate at Different pH

Farshad Rahimpour*, Ali Reza Baharvand

AbstractThe phase diagrams and compositions of coexisting


phases have been determined for aqueous two-phase systems
containing poly(propylene glycol) with average molecular weight of
425 and sodium citrate at various pH of 3.93, 4.44, 4.6, 4.97, 5.1,
8.22. The effect of pH on the salting-out effect of poly (propylene
glycol) by sodium citrate has been studied. It was found that, an
increasing in pH caused the expansion of two-phase region.
Increasing pH also increases the concentration of PPG in the PPG-
rich phase, while the salt-rich phase will be somewhat mole diluted.

KeywordsAqueous two-phase system, Phase equilibrium,


Biomolecules purication

I. INTRODUCTION

N aqueous two-phase system (ATPS) is produced when

appropriate small amounts of two chemically different

water soluble polymers, or a water soluble polymer and


inorganic salts, are added to water, causing the system to
separate into two immiscible water-rich phases [1], The high
water content in aqueous two-phase systems, typically greater
than 80 wt,%, coupled with low interfacial tension provide a
benign environment for biomolecules not attained in solvent
extraction, The system offers a technically simple, energy
efcient, easily scalable and mild separation technique for
product recovery in biotechnology [2],

Its major use has been in the concentration and purication


of proteins and in the extractive bioconversion of enzymes,
The separation technique is also becoming important in non-
biotechnology areas such as industrial waste remediation,
Notable examples include utilization of aqueous phase
systems in removal of color from textile plant wastes [3],
metal ions [4] and organic pollutants from the environment
[5], and aromatics from crude oil [4], Liquidiliquid
equilibrium (LLE) data for some aqueous PPG + inorganic salt
two-phase systems have been reported in the literature [6-7],
In these studies, salting out of PPG has been accomplished by
the use of either carbonate or sulfates,

Farshad Rahimpour, Assistant Professor, Biotechnology research lab,


Chemical Engineering Department, Faculty of Engineering, Razi University,
Kermanshah 67149-67346, Iran (phone: +98-831-4274530; fax: +98-831-
4274542; e-mail: f rahimpour@razi.ac.ir).

Ali Reza Baharvand, MSc, Biotechnology research lab, Chemical


Engineering Dept. Faculty of Engineering, Razi Univ. Kermanshah 67149-
67346, Iran (e-mail: Baharvand@razi.ac.ir).

This work was supported in part by the research vice departments of Razi
University.

International Scholarly and Scientic Research & Innovation 3(11) 2009

These salts, however, lead to high phosphate or sulfate


concentration in the efuent streams and therefore, to
environmental concern, One way to reduce the salt discharged
in the wastewater is to recycle the chemicals, In this regard,
Hustedt [8] and Greve and Kula [9] have published useful
papers, Recently, as an alternative approach, Vemau and Kula
[10] and Rahimpour et a1, [11] have investigated citrates as a
substitute for inorganic salts and found that sodium and
potassium citrates form aqueous two-phase systems with PEG
which is suitable for biomolecules extraction, Citrate is
biodegradable and nontoxic and could be discharged into
biological wastewater treatment plants and therefore the
citrates can be considered as a substitute for inorganic salts,
because the citrate forms aqueous two-phase system with PPG
which are suitable for biomolecules extraction,

As far as we know, there is no report on the pH effect on


phase diagram of the PPG425 + sodium citrate + water system
in the literature, This work is devoted to obtaining phase
equilibrium data for the PPG425 + sodium citrate + water
system at pH of 3.93, 4.44, 4.6, 4.97, 5.1, and 8.22. These results
can be used to develop thermodynamic models of aqueous
two-phase systems and also to obtaining experimental data for
choosing suitable systems for purication of biomolecules,

II. EXPERIMENTS
A. Materials
PPG of molecular weight 425, tri-sodium citrate, and citric
acid were obtained from Merck, The polymer and salt were
used without further purication, and double distilled,
deionized water was used, All other materials used were of
analytical grade,

B. Preparation of the Aqueous Two-Phase Systems

Aqueous two-phase systems (ATPS) were prepared by a


mixture of PPG425 and sodium citrate salt solution at required
pH, The pH of the salt solution was adjusted by mixing
appropriate ratio of sodium citrate and citric acid,

All components were added into a graduated 15 m1 test tube


as stock solution at constant pH, resulting in a 10 g system,
All experiments were carried out at 25 0C, The pH values of
the solutions were measured precisely with a pH meter of
JENWAY 3345 model, In order to speed up phase separation
the resulted solution was mixed by rigorous vortexing the test
tube for 2 min, The tubes were placed in a room temperature
for 4 h and then were centrifuges at 1500 rpm for 10 min; the

616 scholarwasetorg/l9992/4396

International Science Index, Chemical and Molecular Engineering Vol:3, No: 1 1, 2009 waset.org/Publication/4396
World Academy of Science, Engineering and Technology
International Journal of Chemical and Molecular Engineering
Vol:3, No: 1 1, 2009

solution reach to equilibrium and the samples of the top and


bottom phase were carefully withdrawn, with care being taken
to leave a layer of solution at least 0.2 cm thick above the
interface,

C. Measurement of Salt and PPG Concentration

The analysis methods for salt concentrations were determined


by using atomic absorption spectroscopy (AAS), shimatsu AA-
6300 model,

The concentration of PPG was determined by refractive


index measurements at 298,15K using an ATAGO-
DTMlmodel, Since the refractive index of phase samples
depends on PEG, and salt concentration, calibration plots of
refractive index versus polymer concentration were prepared
for deferent concentrations of salt,

A. Procedure

The binodal curves were determined by a titration method, A


salt solution of known concentration was titrated with the
polymer solution or vice versa, until the solution turned turbid,
The composition of the mixture was determined by mass using
an analytical balance with a precision of ilx 107 kg, To
determine the compositions of coexisting phases, feed samples
(about 10 gr) were prepared by mixing appropriate amounts of
polymer, salt and water in the tube, After separation of the two
transparent phases, the concentrations of the salts and PPG in
the top and bottom phases were determined,

III. RESULTS AND DISCUSSION

Experimental results

For the aqueous PPG - sodium citrate two-phase system the


binodal data obtained from turbidimetric titrations and the tie
line data for the compositions of conjugate solutions at pH of
3,93, 4.44, 4.6, 4.97, 5.1, and 8.22 are shown in Tables 1 and 2,
respectively, The complete phase diagrams of all studied
systems are shown in Figs, 1,

It can be seen that the binodal curve shifts downward as the


pH of the medium becomes lower, indicating that smaller
concentration of the phase polymers is required to form
aqueous two-phase system, This behavior is probably due to
decrease in hydrodynamic volume of the polymer as the pH of
the solution decreases, It was shown that decreasing the pH
leads to reduction in intrinsic viscosity of the polymer
solutions [12], Since it is well known that hydrodynamic
volume of polymers in solution is directly proportional to their
intrinsic viscosity, it can be deduced that decreasing the pH
causes the polymer chains to assume a more compact
structure,

The formation of aqueous two-phase systems clearly


indicates the mutual exclusion of the ions and the polymer and
their high afnity for the solvent, It is possible that even in
homogeneous systems (below the phase boundary) the ions
are excluded from the near surface region of the polymer in
solution, With increase in the concentration of the polymer or
the salt, the extent of exclusion will increase, Ultimately, the
system could reach a state where, for entropic reasons, phase
formation would become favorable, In fact, salting-out effects
are related to a reduction in entropy resulting from the
exclusion of ions from volume elements immediately adjacent

International Scholarly and Scientic Research & Innovation 3(11) 2009

to the solute molecules, Exclusion of ions from the polymer


moleculeiwater interface itself can occur for a number of
reasons, Both the polymer and the ion are strongly hydrated in
solution, Because of the hydration sheath, the near-surface
region of the polymer may not be accessible to structure-
making ions, The pH could affect the binodal location, either
by changing the charge of the solute or by altering the ratio of
the charged species present, Fig, 1 shows that the two-phase
area is expanded with increasing pH, In other words, if we
take a sample with a known composition on the binodal curve,
this mixture becomes a two-phase system at increasing pH as
has been observed experimentally, It was found that, at higher
pH, hydrogen bond interactions of PPG are weakened [7],
Depression of the cloud point by increasing pH is may be
related to the salting-out phenomenon resulting from the
weakening of the PPGisolvent interaction,

The tie-line length TLL, calculated by the following equation:

TLL = \/(w:P wf)2 + (wa wff (1)

The tie lines are determined by connecting each


corresponding set of total, bottom-, and top-phase points, As
an example, the tie lines for the systems with pH of 3,93 and
5,1 are shown in Fig, 2 and 3, As shown in these gures, the
slope and the length of the equilibrium tie-lines increased with
increasing pH, Also, by increasing pH the volume of salt-rich
phase increases at the expense of the PPG-rich phase, as we
observed experimentally, This is because the compositions of
the phases in equilibrium change with varying pH, Increasing
the pH of the aqueous PPG-salt two-phase system will cause
an increase of the concentration in the PPG-rich phase and a
decrease of the concentration in the salt-rich phase,

TABLE I
TIE LINE DATA FOR SYSTEMS OF
PPG425- CITRATE- WATER AT DIFFERENT pH
Total
compositions Top phase Bottom phase
H Salt PPG Salt PPG Salt PPG
p [w/w%] [w/w%] [w/w%] [w/w%] [w/w%] [[w/w%]
8.02 15.98 2.08 36.20 9.20 12.80
3.93 9.85 13.75 1.95 39.34 10.96 11.12
11.42 12.04 1.65 42.14 12.60 9.02
13.25 11.02 1.50 45.05 13.84 9.91
8.41 14.02 2.00 37.21 9.86 10.40
4.44 10.00 12.00 1.40 40.57 11.20 9.60
12.50 10.00 1.24 43.03 13.10 8.25
13.50 9.80 1.20 46.87 14.20 8.40
8.41 14.02 1.60 38.74 10.6 7.35
4.60 10.57 12.36 1.04 42.81 12.00 7.50
12.5 10.00 1.20 45.21 14.40 5.18
14.91 8.72 0.85 51.18 16.50 6.40
10.32 12.22 0.847 40.85 11.20 7.08
4.97 11.47 11.16 0.60 45.62 12.60 7.10
13.35 10.14 0.40 52.05 14.20 6.10
15.51 8.91 0.30 55.23 17.80 4.90
10.12 10.54 0.80 42.61 11.60 5.01
5.10 11.77 9.26 0.51 48.77 12.86 5.70
13.45 8.26 0.504 53.41 15.32 5.04
16.04 8.03 0.488 56.25 18.40 4.07
14.13 9.02 0.24 47.31 16.40 3.05
8 22 15.32 8.26 0.35 51.71 17.50 3.35
' 17.50 7.14 0.60 56.2 19.50 2.45
18.35 6.51 0.26 61.4 20.20 2.34

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Vol:3, No: 1 1, 2009
6.092 6.092 4.609 8.111 4.111 7.307
6.580 6.580 5.048 8.100 4.440 7.291
7.234 7.234 5.525 7.998 4.896 6.955
7.938 7.938 6.094 7.839 5.348 6.396
60 8.569 8.569 6.416 7.143 5.800 6.053
9.244 9.244 6.773 7.008 6.021 5.952
:0 0} IH 393 'IH 4" 9.610 9.610 7.267 6.999 6.299 5.140
i 11H=4,0 pH=4.0' 10.193 10.193 7.595 6.653 6.667 4.842
_\ .4 I 10.707 10.707 8.098 6.605 6.910 4.574
of 40 . A . 411H=5J 0pH= 8.22 11.348 11.348 7.326 4.562
S 11.619 11.619 7.604 4.497
E . u ' 11.977 11.977
Ir, 30 4 12.449 12.449
M I .
F3. ' 4 '
a: 20 0 4 "2 I. i 3 0
Q1 0 . 4 A I | .I i
1. ..; 4 ~. ~ in...
4 t - 4 I '
'IO....>.I.1.4. HII 4:
0 40
0 2 4 6 8 10 12 14 3
30
(inwrdw 10%) :5 25
Fig. 1 Biondals of aqueous two-phase systems of 3
PPG425- citrate- water at different pH. 3, 20
L- 15
TABLE 11 Q 10
EQUILIBRIUM DATA FOR SYSTEMS OF
PPG425- CITRATE- WATER AT DIFFERENT pH 5
pH=3.93 pH=4.44 pH=4.6 0
100mm 100w; 100ml 100w; 100ml 100w;
1.589 45.415 1.351 44.528 0.940 45.761 0 2 4 6 . 3 010 12 14 1
1.612 39.927 1.898 34.426 1.202 39.657 019 (WW 0)
2.386 34.603 2.571 26.992 1.582 34.366 . . .
2.793 30.447 3.084 22.128 2.332 28.895 Fig. 2 Tie lines of aqueous two-phase systems of
3.368 26.528 3.679 20.548 2.718 25.194 PPG425 Citrate' water at PH 0f3-93-
3.742 24.276 4.357 18.750 3.026 21.443
4.533 21.708 5.033 16.884 3.267 19.433 60
5.122 20.089 5.589 15.484 3.703 18.213
5.492 18.954 6.127 15.064 4.207 16.813 :0
5.969 18.496 6.713 14.043 4.799 15.473
6.518 17.712 7.386 13.654 5.433 14.721
6.956 15.756 7.990 12.089 5.727 13.054 r; 40
7.307 15.066 8.475 11.771 6.101 12.524 ?:
7.809 14.409 9.031 10.782 6.901 12.235 E 10
8.368 13.342 9.740 10.124 7.250 10.994 5: i
8.698 12.725 10.489 9.658 7.738 9.864 '5"
9.211 12.071 10.723 9.154 8.302 9.412 9-4 20
9.728 11.145 11.581 8.527 9.057 9.197
10.274 10.677 11.728 8.0522 9.409 8.311 10
10.715 10.202 12.297 7.815 9.855 8.177 "I I III ..
12.746 7.563 10.522 7.904
11.044 7.446 0
11-726 6376 0 2 4 6 8 10 12 14 16 18 20
12.045 6.522 citrate (w/w%)
452 6312 Fig. 3 Tie lines of aqueous two-phase systems of
12'922 6'215 PPG425- citrate- water at pH of5. 10.
pH=4.97 pH=5.1 pH=8.22
100m 100m; 100ml 100w; 100ml 100w;
0.615 0.615 0.471 49.942 0.326 52.855 As mentioned above, the attraction between PPG and water
"-799 "-799 "-531 44-526 "-3958 45395 will decrease with an increase in pH and therefore by
"'8 "'8 0'555 35243 0'421 39557 increasing pH of the aqueous PPG-salt two-phase system
1.108 1.108 0.896 29.341 0.442 33.025 . . . .
1.616 1.616 1.467 22.687 0.585 24.876 water is driven from the PPG-rich phase to the salt-rich phase,
2.594 2.594 1.697 18.525 0.998 19.142 so the PPG concentration of the PPG-rich phase increases,
2-837 2-837 2-051 163796 1325 16-418 while the salt-rich phase will be somewhat more diluted (1e.
3'1 3'1 2356 15'607 L955 ms the salt concentration will be decreased), and the volume of
3.448 3.448 2.588 14.314 2.298 11.261 . . .
3.792 3.792 2.980 12.276 2.617 10521 the salt-rich phase increases at expense of the PPG-rich phase,
4.422 4.422 3.487 11.028 3.014 10.221 Close examination of the tie-line data given in Table 1
4-938 4-938 3-922 -000 3-282 9-066 indicate that, the PPG has been observed to enrich the upper
5.352 5.352 4.241 9.988 3.731 8.951

International Scholarly and Scientic Research & Innovation 3(11) 2009

61

phase, while the salt has been observed to enrich the lower

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World Academy of Science, Engineering and Technology


International Journal of Chemical and Molecular Engineering

Vol:3,No:1l, 2009

phase for the aqueous PPG-salt systems at pH 3.93, 4,44, 4,6,


4,97, 5,1, and 822,

IV. CONCLUSION
LLE data for the system poly(propylene glycol) + sodium
citrate + water, at different pH have been determined
experimentally, It was found that the two phase area is
expanded with increasing pH, It was also observed that the
slope and length of all equilibrium tie-lines increased with
increasing pH for the investigated system,

REFERENCES

[1] PA. Albertsson, Partitioning of Cell Particles and Macromolecules, 3rd


ed., Wileyilnterscience, New York, 1986.

[2] H.Walter, D.E. Brooks, D. Fisher, Partitioning in Aqueous Two-Phase


Systems, Academic Press, New York, 1985.

[3] JG. Huddleston, HD. Willauer, K.R. Boaz, R.B. Rogers, Separation
and recovery of food coloring dyes using aqueous biphasic extraction
chromatographic resins, J. Chromatogr. B, vol. 711, pp. 2377244,
1998.

[4] R. Hatti-Kaul, Aqueous Two Phase Systems: Methods and Protocols,


Humanae Press, New Jersey, 2000.

[5] RB. Rogers, HD. Willauer, S.T. Grifn, J.G. Huddleston, Partitioning
of small organic molecules in aqueous biphasic systems, J.
Chromatogr. B,, vol. 711, pp. 2557263, 1998.
[6] Zafarani-Moattar, M.T., Salabat, A., Thermodynamics of magnesium
sulfateipolypropylene glycol aqueous two-phase system. Experiment
and correlation, Fluid Phase Equilib., vol. 152, pp. 57-65, 1998.

[7] Zafarani-Moattar, M.T., Sadeghi, R., Phase diagram Data for Several
PPG + Salt Aqueous Biphasic Systems at 25 0C, J. Chem. Eng. Data,
vol. 50, pp. 947-950, 2000.

[8] Hustedt, H. Extractive Enzyme Recovery with Simple Recycling of


Phase Forming Chemicals, Biotechnol. Lett., vol. 8, pp. 791-796,
1986.

[9] Greve, A.; Kula, M. R., Recycling of salt from the primary bottom
phase of protein extraction process, J. Chem. Technol. Biotechnol,
vol. 50, pp. 27-42, 1991.

[10] Vernau, J.; Kula, M. R. Extraction of Proteins from Biological Raw


Material Using Aqueous Poly(ethylene) glycol-Citrate Phase Systems,
Biotechnol. Appl. Biochem., vol. 12, pp. 397-404, 1990.

[11] F. Rahimpour., F. Feyzi., S. Maghsodi and R. H. Kaul, Purication of


plasmid DNA with polymer-Salt aqueous two-phase system:
optimization using response surface methodology, Biotechnol. Bioeng.,
vol. 95, pp 627-637, 2006.

[12] Waziri, S.M., Abu-Sharkh, B.F., Ali, S.A., The effect of pH and salt
concentration on the coexistence curves of aqueous two-phase systems
containing a pH responsive copolymer and polyethylene glycol, Fluid
Phase Equilib., vol. 205 pp. 2757290, 2003.

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Vol:3, No: 1 1, 2009

Phase Equilibrium in Aqueous Two-phase


Systems Containing Poly (propylene glycol) and
Sodium Citrate at Different pH

Farshad Rahimpour*, Ali Reza Baharvand

AbstractThe phase diagrams and compositions of coexisting


phases have been determined for aqueous two-phase systems
containing poly(propylene glycol) with average molecular weight of
425 and sodium citrate at various pH of 3.93, 4.44, 4.6, 4.97, 5.1,
8.22. The effect of pH on the salting-out effect of poly (propylene
glycol) by sodium citrate has been studied. It was found that, an
increasing in pH caused the expansion of two-phase region.
Increasing pH also increases the concentration of PPG in the PPG-
rich phase, while the salt-rich phase will be somewhat mole diluted.

KeywordsAqueous two-phase system, Phase equilibrium,


Biomolecules purication
I. INTRODUCTION

N aqueous two-phase system (ATPS) is produced when

appropriate small amounts of two chemically different

water soluble polymers, or a water soluble polymer and


inorganic salts, are added to water, causing the system to
separate into two immiscible water-rich phases [1], The high
water content in aqueous two-phase systems, typically greater
than 80 wt,%, coupled with low interfacial tension provide a
benign environment for biomolecules not attained in solvent
extraction, The system offers a technically simple, energy
efcient, easily scalable and mild separation technique for
product recovery in biotechnology [2],

Its major use has been in the concentration and purication


of proteins and in the extractive bioconversion of enzymes,
The separation technique is also becoming important in non-
biotechnology areas such as industrial waste remediation,
Notable examples include utilization of aqueous phase
systems in removal of color from textile plant wastes [3],
metal ions [4] and organic pollutants from the environment
[5], and aromatics from crude oil [4], Liquidiliquid
equilibrium (LLE) data for some aqueous PPG + inorganic salt
two-phase systems have been reported in the literature [6-7],
In these studies, salting out of PPG has been accomplished by
the use of either carbonate or sulfates,

Farshad Rahimpour, Assistant Professor, Biotechnology research lab,


Chemical Engineering Department, Faculty of Engineering, Razi University,
Kermanshah 67149-67346, Iran (phone: +98-831-4274530; fax: +98-831-
4274542; e-mail: f rahimpour@razi.ac.ir).

Ali Reza Baharvand, MSc, Biotechnology research lab, Chemical


Engineering Dept. Faculty of Engineering, Razi Univ. Kermanshah 67149-
67346, Iran (e-mail: Baharvand@razi.ac.ir).

This work was supported in part by the research vice departments of Razi
University.

International Scholarly and Scientic Research & Innovation 3(11) 2009

These salts, however, lead to high phosphate or sulfate


concentration in the efuent streams and therefore, to
environmental concern, One way to reduce the salt discharged
in the wastewater is to recycle the chemicals, In this regard,
Hustedt [8] and Greve and Kula [9] have published useful
papers, Recently, as an alternative approach, Vemau and Kula
[10] and Rahimpour et a1, [11] have investigated citrates as a
substitute for inorganic salts and found that sodium and
potassium citrates form aqueous two-phase systems with PEG
which is suitable for biomolecules extraction, Citrate is
biodegradable and nontoxic and could be discharged into
biological wastewater treatment plants and therefore the
citrates can be considered as a substitute for inorganic salts,
because the citrate forms aqueous two-phase system with PPG
which are suitable for biomolecules extraction,

As far as we know, there is no report on the pH effect on


phase diagram of the PPG425 + sodium citrate + water system
in the literature, This work is devoted to obtaining phase
equilibrium data for the PPG425 + sodium citrate + water
system at pH of 3.93, 4.44, 4.6, 4.97, 5.1, and 8.22. These results
can be used to develop thermodynamic models of aqueous
two-phase systems and also to obtaining experimental data for
choosing suitable systems for purication of biomolecules,

II. EXPERIMENTS
A. Materials
PPG of molecular weight 425, tri-sodium citrate, and citric
acid were obtained from Merck, The polymer and salt were
used without further purication, and double distilled,
deionized water was used, All other materials used were of
analytical grade,

B. Preparation of the Aqueous Two-Phase Systems

Aqueous two-phase systems (ATPS) were prepared by a


mixture of PPG425 and sodium citrate salt solution at required
pH, The pH of the salt solution was adjusted by mixing
appropriate ratio of sodium citrate and citric acid,

All components were added into a graduated 15 m1 test tube


as stock solution at constant pH, resulting in a 10 g system,
All experiments were carried out at 25 0C, The pH values of
the solutions were measured precisely with a pH meter of
JENWAY 3345 model, In order to speed up phase separation
the resulted solution was mixed by rigorous vortexing the test
tube for 2 min, The tubes were placed in a room temperature
for 4 h and then were centrifuges at 1500 rpm for 10 min; the

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solution reach to equilibrium and the samples of the top and


bottom phase were carefully withdrawn, with care being taken
to leave a layer of solution at least 0.2 cm thick above the
interface,

C. Measurement of Salt and PPG Concentration

The analysis methods for salt concentrations were determined


by using atomic absorption spectroscopy (AAS), shimatsu AA-
6300 model,

The concentration of PPG was determined by refractive


index measurements at 298,15K using an ATAGO-
DTMlmodel, Since the refractive index of phase samples
depends on PEG, and salt concentration, calibration plots of
refractive index versus polymer concentration were prepared
for deferent concentrations of salt,

A. Procedure

The binodal curves were determined by a titration method, A


salt solution of known concentration was titrated with the
polymer solution or vice versa, until the solution turned turbid,
The composition of the mixture was determined by mass using
an analytical balance with a precision of ilx 107 kg, To
determine the compositions of coexisting phases, feed samples
(about 10 gr) were prepared by mixing appropriate amounts of
polymer, salt and water in the tube, After separation of the two
transparent phases, the concentrations of the salts and PPG in
the top and bottom phases were determined,

III. RESULTS AND DISCUSSION

Experimental results

For the aqueous PPG - sodium citrate two-phase system the


binodal data obtained from turbidimetric titrations and the tie
line data for the compositions of conjugate solutions at pH of
3,93, 4.44, 4.6, 4.97, 5.1, and 8.22 are shown in Tables 1 and 2,
respectively, The complete phase diagrams of all studied
systems are shown in Figs, 1,

It can be seen that the binodal curve shifts downward as the


pH of the medium becomes lower, indicating that smaller
concentration of the phase polymers is required to form
aqueous two-phase system, This behavior is probably due to
decrease in hydrodynamic volume of the polymer as the pH of
the solution decreases, It was shown that decreasing the pH
leads to reduction in intrinsic viscosity of the polymer
solutions [12], Since it is well known that hydrodynamic
volume of polymers in solution is directly proportional to their
intrinsic viscosity, it can be deduced that decreasing the pH
causes the polymer chains to assume a more compact
structure,

The formation of aqueous two-phase systems clearly


indicates the mutual exclusion of the ions and the polymer and
their high afnity for the solvent, It is possible that even in
homogeneous systems (below the phase boundary) the ions
are excluded from the near surface region of the polymer in
solution, With increase in the concentration of the polymer or
the salt, the extent of exclusion will increase, Ultimately, the
system could reach a state where, for entropic reasons, phase
formation would become favorable, In fact, salting-out effects
are related to a reduction in entropy resulting from the
exclusion of ions from volume elements immediately adjacent

International Scholarly and Scientic Research & Innovation 3(11) 2009

to the solute molecules, Exclusion of ions from the polymer


moleculeiwater interface itself can occur for a number of
reasons, Both the polymer and the ion are strongly hydrated in
solution, Because of the hydration sheath, the near-surface
region of the polymer may not be accessible to structure-
making ions, The pH could affect the binodal location, either
by changing the charge of the solute or by altering the ratio of
the charged species present, Fig, 1 shows that the two-phase
area is expanded with increasing pH, In other words, if we
take a sample with a known composition on the binodal curve,
this mixture becomes a two-phase system at increasing pH as
has been observed experimentally, It was found that, at higher
pH, hydrogen bond interactions of PPG are weakened [7],
Depression of the cloud point by increasing pH is may be
related to the salting-out phenomenon resulting from the
weakening of the PPGisolvent interaction,

The tie-line length TLL, calculated by the following equation:

TLL = \/(w:P wf)2 + (wa wff (1)

The tie lines are determined by connecting each


corresponding set of total, bottom-, and top-phase points, As
an example, the tie lines for the systems with pH of 3,93 and
5,1 are shown in Fig, 2 and 3, As shown in these gures, the
slope and the length of the equilibrium tie-lines increased with
increasing pH, Also, by increasing pH the volume of salt-rich
phase increases at the expense of the PPG-rich phase, as we
observed experimentally, This is because the compositions of
the phases in equilibrium change with varying pH, Increasing
the pH of the aqueous PPG-salt two-phase system will cause
an increase of the concentration in the PPG-rich phase and a
decrease of the concentration in the salt-rich phase,

TABLE I
TIE LINE DATA FOR SYSTEMS OF
PPG425- CITRATE- WATER AT DIFFERENT pH
Total

compositions Top phase Bottom phase


H Salt PPG Salt PPG Salt PPG
p [w/w%] [w/w%] [w/w%] [w/w%] [w/w%] [[w/w%]
8.02 15.98 2.08 36.20 9.20 12.80
3.93 9.85 13.75 1.95 39.34 10.96 11.12
11.42 12.04 1.65 42.14 12.60 9.02
13.25 11.02 1.50 45.05 13.84 9.91
8.41 14.02 2.00 37.21 9.86 10.40
4.44 10.00 12.00 1.40 40.57 11.20 9.60
12.50 10.00 1.24 43.03 13.10 8.25
13.50 9.80 1.20 46.87 14.20 8.40
8.41 14.02 1.60 38.74 10.6 7.35
4.60 10.57 12.36 1.04 42.81 12.00 7.50
12.5 10.00 1.20 45.21 14.40 5.18
14.91 8.72 0.85 51.18 16.50 6.40
10.32 12.22 0.847 40.85 11.20 7.08
4.97 11.47 11.16 0.60 45.62 12.60 7.10
13.35 10.14 0.40 52.05 14.20 6.10
15.51 8.91 0.30 55.23 17.80 4.90
10.12 10.54 0.80 42.61 11.60 5.01
5.10 11.77 9.26 0.51 48.77 12.86 5.70
13.45 8.26 0.504 53.41 15.32 5.04
16.04 8.03 0.488 56.25 18.40 4.07
14.13 9.02 0.24 47.31 16.40 3.05
8 22 15.32 8.26 0.35 51.71 17.50 3.35
' 17.50 7.14 0.60 56.2 19.50 2.45
18.35 6.51 0.26 61.4 20.20 2.34

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Vol:3, No: 1 1, 2009

6.092 6.092 4.609 8.111 4.111 7.307


6.580 6.580 5.048 8.100 4.440 7.291
7.234 7.234 5.525 7.998 4.896 6.955
7.938 7.938 6.094 7.839 5.348 6.396
60 8.569 8.569 6.416 7.143 5.800 6.053
9.244 9.244 6.773 7.008 6.021 5.952
:0 0} IH 393 'IH 4" 9.610 9.610 7.267 6.999 6.299 5.140
i 11H=4,0 pH=4.0' 10.193 10.193 7.595 6.653 6.667 4.842
_\ .4 I 10.707 10.707 8.098 6.605 6.910 4.574
of 40 . A . 411H=5J 0pH= 8.22 11.348 11.348 7.326 4.562
S 11.619 11.619 7.604 4.497
E . u ' 11.977 11.977
Ir, 30 4 12.449 12.449
M I .
F3. ' 4 '
a: 20 0 4 "2 I. i 3 0
Q1 0 . 4 A I | .I i
1. ..; 4 ~. ~ in...
4 t - 4 I '
'IO....>.I.1.4. HII 4:
0 40
0 2 4 6 8 10 12 14 3
30
(inwrdw 10%) :5 25
Fig. 1 Biondals of aqueous two-phase systems of 3
PPG425- citrate- water at different pH. 3, 20
L- 15
TABLE 11 Q 10
EQUILIBRIUM DATA FOR SYSTEMS OF
PPG425- CITRATE- WATER AT DIFFERENT pH 5
pH=3.93 pH=4.44 pH=4.6 0
100mm 100w; 100ml 100w; 100ml 100w;
1.589 45.415 1.351 44.528 0.940 45.761 0 2 4 6 . 3 010 12 14 1
1.612 39.927 1.898 34.426 1.202 39.657 019 (WW 0)
2.386 34.603 2.571 26.992 1.582 34.366 . . .
2.793 30.447 3.084 22.128 2.332 28.895 Fig. 2 Tie lines of aqueous two-phase systems of
3.368 26.528 3.679 20.548 2.718 25.194 PPG425 Citrate' water at PH 0f3-93-
3.742 24.276 4.357 18.750 3.026 21.443
4.533 21.708 5.033 16.884 3.267 19.433 60
5.122 20.089 5.589 15.484 3.703 18.213
5.492 18.954 6.127 15.064 4.207 16.813 :0
5.969 18.496 6.713 14.043 4.799 15.473
6.518 17.712 7.386 13.654 5.433 14.721
6.956 15.756 7.990 12.089 5.727 13.054 r; 40
7.307 15.066 8.475 11.771 6.101 12.524 ?:
7.809 14.409 9.031 10.782 6.901 12.235 E 10
8.368 13.342 9.740 10.124 7.250 10.994 5: i
8.698 12.725 10.489 9.658 7.738 9.864 '5"
9.211 12.071 10.723 9.154 8.302 9.412 9-4 20
9.728 11.145 11.581 8.527 9.057 9.197
10.274 10.677 11.728 8.0522 9.409 8.311 10
10.715 10.202 12.297 7.815 9.855 8.177 "I I III ..
12.746 7.563 10.522 7.904
11.044 7.446 0
11-726 6376 0 2 4 6 8 10 12 14 16 18 20
12.045 6.522 citrate (w/w%)
452 6312 Fig. 3 Tie lines of aqueous two-phase systems of
12'922 6'215 PPG425- citrate- water at pH of5. 10.
pH=4.97 pH=5.1 pH=8.22
100m 100m; 100ml 100w; 100ml 100w;
0.615 0.615 0.471 49.942 0.326 52.855 As mentioned above, the attraction between PPG and water
"-799 "-799 "-531 44-526 "-3958 45395 will decrease with an increase in pH and therefore by
"'8 "'8 0'555 35243 0'421 39557 increasing pH of the aqueous PPG-salt two-phase system
1.108 1.108 0.896 29.341 0.442 33.025 . . . .
1.616 1.616 1.467 22.687 0.585 24.876 water is driven from the PPG-rich phase to the salt-rich phase,
2.594 2.594 1.697 18.525 0.998 19.142 so the PPG concentration of the PPG-rich phase increases,
2-837 2-837 2-051 163796 1325 16-418 while the salt-rich phase will be somewhat more diluted (1e.
3'1 3'1 2356 15'607 L955 ms the salt concentration will be decreased), and the volume of
3.448 3.448 2.588 14.314 2.298 11.261 . . .
3.792 3.792 2.980 12.276 2.617 10521 the salt-rich phase increases at expense of the PPG-rich phase,
4.422 4.422 3.487 11.028 3.014 10.221 Close examination of the tie-line data given in Table 1
4-938 4-938 3-922 -000 3-282 9-066 indicate that, the PPG has been observed to enrich the upper
5.352 5.352 4.241 9.988 3.731 8.951

International Scholarly and Scientic Research & Innovation 3(11) 2009

61

phase, while the salt has been observed to enrich the lower

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World Academy of Science, Engineering and Technology
International Journal of Chemical and Molecular Engineering

Vol:3,No:1l, 2009

phase for the aqueous PPG-salt systems at pH 3.93, 4,44, 4,6,


4,97, 5,1, and 822,

IV. CONCLUSION
LLE data for the system poly(propylene glycol) + sodium
citrate + water, at different pH have been determined
experimentally, It was found that the two phase area is
expanded with increasing pH, It was also observed that the
slope and length of all equilibrium tie-lines increased with
increasing pH for the investigated system,

REFERENCES

[1] PA. Albertsson, Partitioning of Cell Particles and Macromolecules, 3rd


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Systems, Academic Press, New York, 1985.

[3] JG. Huddleston, HD. Willauer, K.R. Boaz, R.B. Rogers, Separation
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[4] R. Hatti-Kaul, Aqueous Two Phase Systems: Methods and Protocols,


Humanae Press, New Jersey, 2000.

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[6] Zafarani-Moattar, M.T., Salabat, A., Thermodynamics of magnesium


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[8] Hustedt, H. Extractive Enzyme Recovery with Simple Recycling of


Phase Forming Chemicals, Biotechnol. Lett., vol. 8, pp. 791-796,
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[9] Greve, A.; Kula, M. R., Recycling of salt from the primary bottom
phase of protein extraction process, J. Chem. Technol. Biotechnol,
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[10] Vernau, J.; Kula, M. R. Extraction of Proteins from Biological Raw


Material Using Aqueous Poly(ethylene) glycol-Citrate Phase Systems,
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[11] F. Rahimpour., F. Feyzi., S. Maghsodi and R. H. Kaul, Purication of


plasmid DNA with polymer-Salt aqueous two-phase system:
optimization using response surface methodology, Biotechnol. Bioeng.,
vol. 95, pp 627-637, 2006.
World Academy of Science, Engineering and Technology
International Journal of Chemical and Molecular Engineering
Vol:3, No:11, 2009 [12] Waziri, S.M., Abu-Sharkh,
B.F.,
phase for the aqueous PPG-salt systems at pH 3.93, 4.44, 4.6, 6.092 6.092 4.609 8.111 4.111 7.307 concentra
4.97, 5.1, and 8.22. 6.580
7.234
6.580
7.234
5.048
5.525
8.100
7.998
4.440
4.896
7.291
6.955
tion on
the coexistence curves of aqueous two-phase systems 7.938 7.938 6.094 7.839 5.348 6.396
8.569 8.569 6.416 7.143 5.800 6.053
containing a pHIV. CONCLUSION responsive 9.244 9.244 6.773 7.008 6.021 5.952
9.610 9.610 7.267 6.999 6.299 5.140
LLE data for the system poly(propylene glycol) + sodium 10.193 10.193 7.595 6.653 6.667 4.842
copolyme
citrate + water, at different pH have been determined 10.707 10.707 8.098 6.605 6.910 4.574 r and
experimentally. It was found that the two phase area is 11.348 11.348 7.326 4.562
expanded with increasing pH. It was also observed that the 11.619 11.619 7.604 4.497
slope and length of all equilibrium tie-lines increased with 11.977
12.449
11.977
12.449 polyethylene glycol, Fluid
increasing pH for the investigated system.
Phase Equilib., vol. 205 pp. 2757290,
2003.
International REFERENCES Scholarly and Scientic Research & Innovation 3(11) 2009
[1] P.A. Albertsson, Partitioning of Cell Particles and Macromolecules, 3rd
ed., WileyInterscience, New York, 1986.
619
[2] H.Walter, D.E. Brooks, D. Fisher, Partitioning in Aqueous Two-Phase scholarwasetorg/l 9992/4396
Systems, Academic Press, New York, 1985.
[3] J.G. Huddleston, H.D. Willauer, K.R. Boaz, R.B. Rogers, Separation
and recovery of food coloring dyes using aqueous biphasic extraction
International
chromatographic resins, J. Chromatogr. B, vol. 711, pp. 237244,
Science Index,
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Chemical and
[4] R. Hatti-Kaul, Aqueous Two Phase Systems: Methods and Protocols,
Molecular Humanae Press, New Jersey, 2000.
Engineering Fig.H.D.
[5] R.B. Rogers, 1 Biondals
Willauer,ofS.T.
aqueous
Griffin,two-phase systems
J.G. Huddleston, of
Partitioning
Vol:3, No:11, PPG425-
of small organic citrate-
molecules waterbiphasic
in aqueous at different pH.J.
systems,
2009 Chromatogr. B,, vol. 711, pp. 255263, 1998.
waset.org/Publ
[6] Zafarani-Moattar, M.T.,IISalabat, A., Thermodynamics of magnesium
TABLE
ication/4396sulfatepolypropylene
EQUILIBRIUM glycol
DATAaqueous two-phaseOF
FOR SYSTEMS system. Experiment
and correlation, Fluid Phase
PPG425- CITRATE- Equilib.,
WATER ATvol. 152, pp. 57-65,
DIFFERENT pH 1998.
[7] Zafarani-Moattar, M.T., Sadeghi, R., Phase diagram Data for Several
pH=3.93pH=4.44pH=4.6
PPG
100 + Salt
1100 Aqueous
2100 1100Biphasic
21001100Systems
2 at 25 C, J. Chem. Eng. Data,
vol. 50, pp. 947-950, 2000.
1.58945.4151.35144.5280.94045.761
[8] Hustedt, H. Extractive Enzyme Recovery with Simple Recycling of
1.61239.9271.89834.4261.20239.657
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2.38634.6032.57126.9921.58234.366
1986.
2.79330.4473.08422.1282.33228.895
[9] Greve, A.; Kula, M. R., Recycling of salt from the primary bottom
3.36826.5283.67920.5482.71825.194 Fig. 2 Tie lines of aqueous two-phase systems of
phase of protein extraction process, J. Chem. Technol. Biotechnol.,
3.74224.2764.35718.7503.02621.443 PPG425- citrate- water at pH of 3.93.
vol. 50, pp. 27-42, 1991.
4.53321.7085.03316.8843.26719.433
[10] Vernau, J.; Kula, M. R. Extraction of Proteins from Biological Raw
5.12220.0895.58915.4843.70318.213
Material Using Aqueous Poly(ethylene) glycol-Citrate Phase Systems,
5.49218.9546.12715.0644.20716.813
Biotechnol. Appl. Biochem., vol. 12, pp. 397-404, 1990.
5.96918.4966.71314.0434.79915.473
[11] F. Rahimpour., F. Feyzi., S. Maghsodi and R. H. Kaul, Purification of
6.51817.7127.38613.6545.43314.721
plasmid DNA with polymer-Salt aqueous two-phase system:
6.95615.7567.99012.0895.72713.054
optimization using response surface methodology, Biotechnol. Bioeng.,
7.30715.0668.47511.7716.10112.524
vol. 95, pp 627-637, 2006.
7.80914.4099.03110.7826.90112.235
[12] Waziri, S.M., Abu-Sharkh, B.F., Ali, S.A., The effect of pH and salt
8.36813.3429.74010.1247.25010.994
concentration on the coexistence curves of aqueous two-phase systems
8.69812.72510.4899.6587.7389.864
containing a pH responsive copolymer and polyethylene glycol, Fluid
9.21112.07110.7239.1548.3029.412
Phase Equilib., vol. 205 pp. 275290, 2003.
9.72811.14511.5818.5279.0579.197
10.27410.67711.7288.05229.4098.311
10.71510.20212.2977.8159.8558.177
12.7467.56310.5227.904
11.0447.446
11.7266.876
12.0456.522
12.4526.312
12.9226.215
pH=4.97pH=5.1pH=8.22
100110021001100210011002
0.6150.6150.47149.9420.32652.855 Fig. 3 Tie lines of aqueous two-phase systems of
0.7990.7990.53144.5260.395845.895 PPG425- citrate- water at pH of 5.10.
0.8060.8060.55535.2430.42139.557
1.1081.1080.89629.3410.44233.025
1.6161.6161.46722.6870.58524.876 As mentioned above, the attraction between PPG and water
2.5942.5941.69718.5250.99819.142 will decrease with an increase in pH and therefore by
2.8372.8372.05116.7961.32516.418 increasing pH of the aqueous PPG-salt two-phase system
3.1043.1042.35615.6071.95514.025
3.4483.4482.58814.3142.29811.261 water is driven from the PPG-rich phase to the salt-rich phase,
3.7923.7922.98012.2762.61710.521 so the PPG concentration of the PPG-rich phase increases,
4.4224.4223.48711.0283.01410.221 while the salt-rich phase will be somewhat more diluted (i.e.
4.9384.9383.92211.0003.2829.066 the salt concentration will be decreased), and the volume of
5.3525.3524.2419.9883.7318.951
the salt-rich phase increases at expense of the PPG-rich phase.
Close examination of the tie-line data given in Table 1
indicate that, the PPG has been observed to enrich the upper
phase, while the salt has been observed to enrich the lower

International Scholarly and Scientific Research & Innovation 3(11) 2009 618
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