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Bovine Serum Albumin Partitioning in Aqueous Two-Phase Systems: Effect of

Variables and Optimization

Article in BioProcessing Journal · April 2013

DOI: 10.12665/J121.Regupathi

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 TRENDS & DEVELOPMENTS IN BIOPROCESS TECHNOLOGY

Bovine Serum Albumin Partitioning

in Aqueous Two-Phase Systems:
Effect of Variables and Optimization
By Nangarthody Sindhu, Sivakumar Kalaivani, and Iyyaswami Regupathi

Introduction
Abstract The challenges which the biotechnology industry faces today are ever

T
he objective of this study more demanding. In the case of therapeutic protein production for human
was to ­o ptimize process use, extremely high purity standards of 99.9% or higher are required.
conditions for the effective Proteins must be separated from a very large number of production con-
partitioning of bovine serum taminants such as host cell proteins, nucleic acids, and polysaccharides
albumin (BSA) using response surface present in the cell culture or cell lysate. Separation techniques used for
methodology (RSM). ­Initially, four downstream purification should be relatively simple, provide high reso-
different salts (tripotassium c­ itrate, lution, speed, ease of scale-up, and if needed, operated continuously. In
tripotassium phosphate, sodium this regard, aqueous two-phase systems (ATPS) are gaining in importance
carbonate, and sodium sulphate) were and researchers are focusing in this area to develop more environmentally
tested for the ability to partition BSA. friendly and economical systems to optimize product recovery.
Among the salts chosen, tripotassium ATPS are formed when two mutually incompatible polymers, or a polymer
citrate was observed to yield a high and salt, are mixed above a critical concentration.[1] Mixtures of proteins
partition coefficient. added to this ATPS tend to partition unequally between the phases, thus
The effect of phase forming com- allowing for the extraction of a particular protein. Several researchers were
ponents: concentration, PEG molecu- employed to study the purification characteristics of proteins using bovine
lar weight, and pH were studied for a serum albumin as a model protein through ATPS. Alves et al.[2] used poly-
PEG/tripotassium citrate system and ethylene glycol (PEG)/maltodextrin (MD) ATPS for the partitioning of BSA
the information obtained was utilized where BSA prefers the MD-rich phase over the PEG-rich phase. However, they
to fix the ranges in RSM studies. Four did not assess the pH and salt concentration effect on protein partitioning.
different independent variables (PEG Conversely, the partitioning characteristics of the ATPS were dependant
2000, tripotassium citrate, NaCl con- on many operating variables like pH, salt/PEG concentration, addition of
centrations, and pH) were considered neutral salt (sodium chloride [NaCl]) and their concentration, tie line length
for RSM studies and the responses (TLL), temperature, etc. The effects of these parameters on the partitioning
generated were partition coefficient coefficients of a variety of proteins for different ATPS have been reporteded
(k) and percentage yield. A statistical in literature in recent years.
model was developed and the values Haghtalab et al.[3] investigated the partitioning of lysozyme, BSA, and
obtained were 99% within the confi- α-amylase in ATPS of PEG/dipotassium hydrogen phosphate (K 2HPO4)/water
dence level. Optimal conditions of the (H2O) and PEG/sodium sulphate (Na2SO4)/H2O at 298.15 K. They found that
system were found as: 0.25 M sodium pH and salt concentration also affected protein partitioning. Capezio et al.[4]
chloride, 32% PEG 2000 (w/w), 16% analysed the influence of PEG molecular mass, pH, and NaCl concentration
tripotassium citrate (w/w), pH 6.0, a on the partitioning of bovine whey proteins (BSA, α-lactalbumin [α-LA] and
partition coefficient of 6.03, a recovery β-lactoglobulin [β-LG]) and α-1 antitrypsin [α1AT] using PEG (1000, 1500,
of 91.76%, and a controlled operating 3350)/potassium phosphate (K 3PO 4). BSA and α-LA concentrated in the
temperature of 303.15 K. PEG-rich phase, while β-LG and α1AT showed affinity for the phosphate-rich
phase. They also found that an increase in the medium’s pH induced the

Spring 2013 BioProcessing Journal • 34 • www.bioprocessingjournal.com

 partition coefficient of all these proteins while the increase Materials
in PEG molecular mass induced the negative behaviour. and Methods
Boaglio et al.[5] studied the influence of PEG concentration,
TLL, molecular mass, and pH on the partitioning of bovine Materials
whey proteins (BSA, α-LA, and β-LG) and α1AT for PEG (1000,
Bovine Albumin Fraction V was purchased from
1450, and 3350)/sodium citrate systems and found that
HiMedia Laboratories. Polyethylene glycol of different
the partition coefficient decreased with increasing TLL for
average molecular weights of 2000, 3000, 4000, and
all these proteins. They demonstrated that the change in
6000 were p ­ urchased from Merck Specialities Pvt. Ltd.
TLL affects the free volume available for a different solute
Sodium ­sulphate, sodium carbonate, dipotassium hydro-
to accommodate in a given phase. Perumalsamy et al.[6]
gen ­phosphate, tripotassium citrate, and sodium chloride
investigated the effect of phase-forming components, pH,
were also procured from Merck Specialties Pvt. Ltd. All salts
and neutral salt additions on BSA partitioning in PEG 2000/
were of analytical grade with a purity of more than 99 %,
sodium citrate-based ATPS at 298.15, 308.15, and 318.15 K.
and used without further purification. Bradford Reagent
They found that the partition coefficient of BSA decreased
from Sigma-Aldrich was used for protein analysis. Double
with an increase in PEG 2000 concentration and tempera-
distilled water (ddH2O) was used for all purposes.
ture. Further partition behaviour of BSA was investigated
by Salabat and Batcha[7] in a PEG 4000, 6000, 8000/sodium Binodal Curve
citrate ATPS. The effect of different polymers (polypropyl-
ene glycol [PPG 425] and PEG 6000), and salts (magnesium The cloud point method was used to determine the
sulphate [MgSO 4], ammonium sulphate [(NH4)2SO 4], and phase equilibrium concentrations for establishing binodal
Na2SO4) on partition behaviour of different proteins (BSA, curves.[10] The experiment was carried out in a jacketed
β-LG, and zein) as model proteins have been studied in glass vessel. The temperature of the working vessel, a
detail by Salabat et al.[8] The recovery results shows a trend ­r efrigerating bath (JEIO Tech, model RW-0525G), was
in the ­salting-out power of these salts (Na2SO4, MgSO4, and ­maintained at 303.14 ± 0.1 K by circulating water through
(NH4)2SO4). the external jacket. Aqueous stock solutions of 50% PEG
Literature has shown that the process of partitioning (w/w) and 30% salt (w/w) were prepared and used for the
in ATPS is influenced by many factors. Varied results for experiments. To ensure uniform concentration of the ATPS
ap ­ articular protein in different ATPS is to be expected constituents in the jacketed vessel, constant stirring was
because of the interactions that exist between the applied using a magnetic stirrer. A salt solution of known
­factors inherent in the system itself. Such factors respon- concentration was titrated against the polymer solution
sible for the interactions include: (1) the choice of system (or vice versa) until the clear solution turned turbid and
­components (polymers and salts); (2) polymer molecular then water was added until turbidity was eliminated. The
weights and concentrations; (3) the types and concentra- procedure was repeated to get the second binodal point.
tions of phase-forming salts, ionic strength, concentration An analytical balance with a precision of ± 0.1 mg (Ohaus-
of ­neutral salt; (4) phase volume ratios, temperature, and Essae-Teraoka, model AR2140) was used to determine the
pH; and (5) those of the target protein, such as surface composition of the mixture.
hydrophobicity, charge, structure, specific bonding sites,
and molecular weight.[9, 10] Thus, the partitioning of a Tie Line and Partition Coefficient
­p articular protein is highly dynamic with respect to an For the determination of tie lines and the partition
ATPS. Although the ­available literature provides some coefficient, 10 g of systems (total weight) were prepared
­information, no ­comprehensive theory currently exists to by mixing appropriate amounts of PEG, salt, protein, and
guide the design of systems for the separation of specific water in graduated centrifuge tubes. The BSA concen­tration
proteins and particle mixtures. Therefore, applying the was maintained at 0.5 mg/mL as a feed for all the experi-
ATPS technique requires extensive experimentation to mentations. The samples were thoroughly combined with
design an adequate phase system for optimal partitioning a vortex mixer at lower rpm for 10 min. The solution was
of a particular protein. allowed to settle for 24 h in a constant temperature bath.
This study attempts to investigate the influence and Top and bottom phase aliquots were taken using a pipette.
­o ptimization of such process variables like the type While withdrawing the sample from bottom phase, a small
and ­c oncentration of salt, PEG molecular weight and positive pressure was maintained to avoid contamination
­concentration, pH, and how the addition of neutral salt at from top phase.[11,12] Thus, the top and bottom phases were
303.15 K influence the partitioning behaviour of the well- separated, diluted, and analyzed for PEG, salt, and protein
established model ­protein, BSA, in ATPS. concentrations.

Spring 2013 BioProcessing Journal • 35 • www.bioprocessingjournal.com

PEG Concentration Estimation interference of PEG and salt in the sample, extensive calibra-
Determining PEG concentration was done through ­a tion was done by varying the concentrations of PEG, salt,
­measuring refractive index using a digital refractometer and protein, and the absorbance was measured using the
(Atago Co. Ltd, model RX-5000α) with a precision of Bradford Assay at 595 nm in a Spectro UV-Vis double beam
± 0.00004 (range 1.3–1.7 ηD). A correlation was devel- spectrophotometer (Labomed, Inc., model UVD-3500). The
oped to measure the refractive index at various known following correlation was determined through regression
compo­sitions of PEG and salt. For dilute aqueous solutions analysis:
containing polymer and salt, the relation between the
Absorbance = a0wBSA + a1w pb1 + a 2w sb2 (2)
refractive index (ηD) and the mass fractions of polymer
(wp) and salt (ws) is given by: where a 0, a1, and a2 are the fitting parameters for different
systems and are tabulated in Table 2. This is applicable in
ηD = a0 + a1wP + a 2wS (1)
the concentration range of PEG < 10% and salt < 5%. For the
where a 0, a1, and a 2 are the fitting parameters and are determination of protein concentration, samples withdrawn
tabulated in Table 1 for different ATPS used in the from each phase were diluted and the absorbance was
present study. For PEG analysis, Equation 1 was used measured in the spectrophotometer at 595 nm using a
successfully in literature.[13-15] The correlation is valid in the Bradford Assay.
concentration range of PEG < 10% and salt < 5%. Further,
the salt concentration was quantified by the estimation Partition Coefficient (k)
of sodium and potassium concentration in the solution Partitioning between the two phases is a complex
through a Flamephotometer (Elico Ltd., model CL 378, phenomenon, guided mainly by the interaction of the
range 1–100 ppm). partitioned substance and the phase components through
hydrogen bonds (e.g., van der Waals, hydrophobic, and
Protein Concentration electrostatic interactions, and steric and conformational
The determination of protein in both phases was done effects, etc.). The partition coefficient is defined as the ratio
through the Bradford Protein Assay methodology. To avoid of protein concentration in the top phase to that in the

Table 1. Coefficients of Equation 1 for different systems.

Systems a0 a1 a2 R2
PEG 4000/Sodium Sulphate 1.3325 0.1509 0.1578 0.9968
PEG 4000/Sodium Carbonate 1.3325 0.1409 0.1827 0.9992
PEG 4000/Dipotassium Hydrogen Phosphate 1.3325 0.1106 0.1392 0.9944
PEG 2000/Sodium Citrate/Water 1.3325 0.1314 0.2430 0.9949
PEG 3000/Potassium Citrate/Water 1.3325 0.1416 0.2290 0.9901
PEG 4000/Potassium Citrate/Water 1.3325 0.1079 0.1596 0.9955
PEG 6000/Potassium Citrate/Water 1.3325 0.1383 0.1227 0.9970
R : regression coefficient
2

Table 2. Coefficients of Equation 2 for different systems.

Systems a0 a1 a2 b1 b2 AARD %
PEG 4000/Sodium Sulphate 1965.903 0.5116 -0.1599 0.9968 0.4611 1.9286
PEG 4000/Sodium Carbonate 500.3408 0.0484 -0.0499 0.9992 2.0199 1.0072
PEG 4000/Dipotassium Hydrogen Phosphate 1296.198 3.4429 23.227 0.9944 2.7122 2.5921
PEG 2000/Sodium Citrate/Water 1056.873 0.6702 0.1511 0.9949 0.5873 4.0862
PEG 3000/Potassium Citrate/Water 425.0001 0.1048 0.0303 0.9901 0.1239 1.6408
PEG 4000/Potassium Citrate/Water 1036.741 0.2164 0.2736 0.9955 0.9497 1.5434
PEG 6000/Potassium Citrate/Water 215.2484 0.1096 0.4005 0.9970 0.9664 1.9361
AARD %: average arithmetic relative deviation (AARD) = (Σ| (Exptl – Cal)/ (Exptl)|)/ (N) x 100.

Spring 2013 BioProcessing Journal • 36 • www.bioprocessingjournal.com

bottom phase, as shown in this equation:[16] with a full quadratic polynomial model is a powerful combi-
k = Cptop / C pbot (3) nation that efficiently provides an adequate representation
of most continuous responses without expending many
where k is the partition coefficient, CpT and CpB are protein resources. In this present study, design of experiments
concentrations (mg/mL) in top and bottom phases, (DoE) was performed based on the central composite
respectively. rotatable design with four independent variables: (1) PEG %
(w/w); (2) tripotassium citrate % (w/w); (3) pH; and (4) NaCl
Volume Ratio (M) at a temperature of 303.15 K. The ranges of variables
Volume ratio (VR) is the ratio of top phase volume (V T in in the form of coded and noncoded values are shown in
mL) to bottom phase volume (V B in mL) that is measured Table 3. For each of the four factors, high (coded value: +2)
in all the experiments as follows: and low (coded value: −2) set points were determined at an
axial distance (= [2k]1/4) dependent on the number of factors
VR = V T/ V B (4)
(k). The experimental design consisted of 31 experiments,
The volume ratio of a phase system can be modified by including 16 factorial points, eight axial points, and seven
shifting system initial feed compositions along a particular replications at the center points to evaluate the pure error.
tie line to get the larger top phase volume or larger bottom The experiments at the design points were performed, and
phase volume. responses such as partition coefficient and % recovery were
obtained for the selected system. Further, to obtain the
Protein Recovery optimum values of the independent variables for response,
The percentage recovery of protein in the top phase is a full quadratic equation (or the diminished form of this
calculated as follows: equation) represented (as follows) was developed by fitting
the experimental values:
% Recovery =  Cptop V top (5)
                         Cp0 V 0           k k
y = β 0 + ∑ βi xi + ∑ βi xi2 + ∑ ∑ βj xi xj (6)
i=1 i=1 i<j
where, Cp0and Cptop are the concentration of protein (mg/mL)
in feed mixture and the top phase, respectively. V 0 and V T where y is the response, xi and xj are noncoded independent
are volume (mL) of feed sample and top phase, respectively. variables, k is the number of independent variables, and β 0,
βi, βii and βij are intercept, linear, quadratic, and interaction
Partitioning Optimization coefficients, respectively. Response surface analysis, used to
The common method of experimental design and opti- determine the conditions that maximize the % yield of the
mization, where operating conditions are optimized by protein, experimental data fitting of quadratic polynomial
changing one variable at a time, gives no guarantee for the equation through least square technique, analysis of
optimal parameter determination. [17, 18] RSM consists of a variance (ANOVA) and the lack-of-fit test for the final
group of mathematical and statistical techniques for seeking predictive equation were done using statistical software
the optimum conditions in a system which had multi-variable (Minitab, Inc., version 16).
and complex interactions existing between the parameters.
It can be used to define the relationships between the
Results and Discussion
response variables (% recovery, partition coefficient) and the
independent variables (PEG % [w/w], tripotassium citrate % The Effects of Variables on BSA Partitioning
[w/w], NaCl concentration, and the pH) and also generates a In the present study, partitioning of BSA was carried out
mathematical model that describes the process. in a series of PEG/salt/water-based ATPS. There are many
A central composite rotatable design (CCRD) coupled factors influencing the partitioning in ATPS. Differences in

Table 3. Independent variables and levels used for experimental design.

Levels
Factors Variables
-2 -1 0 +1 +2
PEG % (w/w) 1965.903 24 28 32 36 40
Tripotassium Citrate % (w/w) 500.3408 12 14 16 18 20
pH 1296.198 4.0 5.0 6.0 7.0 8.0
Salt Molality 1056.873 0.05 0.15 0.25 0.35 0.45

Spring 2013 BioProcessing Journal • 37 • www.bioprocessingjournal.com

partitioning result from the interaction of fac- interaction between the protein and PEG molecules, thus improving
tors inherent in the system itself, such as choice extraction. As the PEG 2000/tripotassium citrate system demonstrated
of system components (polymer/salt), type and a better partitioning behaviour of BSA, this was chosen as the system
concentration of phase forming salts, polymer for further investigation.
molecular weight and concentration, pH, con-
centration of neutral salt, etc. The effects are PEG Molecular Weight in Tripotassium Citrate System
analyzed in detail at a temperature of 303.15 K. For this study, partitioning of BSA was conducted in the PEG/
Initially, the following ATP combinations with tripotassium citrate system for different molecular weights of PEG
salts like sodium sulphate, sodium carbonate, (2000, 3000, 4000, and 6000) and the variation of partition coefficient
dipotassium hydrogen phosphate, and tripo- is shown in Figure 2. It was observed that as the molecular weight of
tassium citrate, and polymers PEG 2000, 3000, the PEG increased, the partition coefficient decreased, which may be
4000, and 6000 were considered for partitioning
of BSA at 303.15 K.

Salts
When the anion and cation of a salt have
different relative affinities for different phase-
forming polymers, and consequently for each
phase, the requirement of electro-neutrality in
each phase results in a Donnan-type electro-
static potential difference between the phases.
This potential difference appears to have large
effects on partitioning of charged solutes (such
as proteins). Therefore, the choice of salt is an
easy way to influence the target biomolecule
partitioning. The most hydrophobic anions
or cations will drive the partitioning of their
counter-ions to the most hydrophobic phase.
Co-ions, which are less hydrophobic, will parti-
tion to the hydrophilic phase. FIGURE 1. Variation of partition coefficient k against PEG concentration for
different salts (8 % salt concentration) at a temperature of 303.15 K:  K 2HPO4;
The variation of partition coefficient k  K 3C6H5O7;  Na2CO3; 0 Na2SO 4.
against PEG 2000 concentration for different
salts (sodium sulphate, sodium carbonate,
dipotassium hydrogen phosphate, and tripo-
tassium citrate) with a salt concentration of
8% (w/w) at 303.15 K is shown in Figure 1. BSA,
being hydrophilic in nature, was observed to
be attracted to the bottom phase, in the case
of sodium carbonate and sodium sulphate, due
to the lessened hydrophobicity of the salt-rich
phase. Since the phosphate salt phase is more
hydrophobic than the polymer phase, water
molecules were expelled to the top phase,
resulting in increased partitioning of BSA to
that phase. Citrate also has similar properties
to phosphate in the PEG/phosphate system.[19]
The PEG/citrate system extracted the highest
amount of BSA to the top phase suggesting
that the extraction of BSA in ATPS was influ-
enced by hydrophobicity. Increasing the dif- FIGURE 2. Partition coefficient k against PEG concentration for different
ference in hydrophobicity between the phases molecular weights of PEG with an 8 % C6H5K 3O7 concentration at 303.15 K:
would increase the strength of hydrophobic  PEG 2000; 0 PEG 3000; * PEG 4000;  PEG 6000.

Spring 2013 BioProcessing Journal • 38 • www.bioprocessingjournal.com

due to the excluded volume effects. Johansson[20] observed that in gen- weight of PEG. Per Huddleston et al.,[24] because
eral, the protein partition coefficient could be increased by reducing the hydrophobic character of PEG increases with
the molecular weight of the polymer in the top phase. A decrease in the chain length, the BSA is also liable to transfer
the partition coefficient while increasing the PEG molecular mass was to the top PEG-rich phase with the decrease
found for other proteins (reported earlier).[5, 7, 21] This behaviour was in of PEG molecular weight, substantiating the
agreement with an exclusion effect owing to the diminution of the free hydrophilic character of the BSA. The maximum
volume available in the top phase. Published literature has shown that partition was obtained using PEG 2000 suggest-
the exclusion volume effect[22, 23] can lead to the protein’s movement ing that the lower PEG molecular weight was
from the top phase to the bottom phase and then lessened as the favorable for the partitioning of BSA (Figure 2).
molecular weight of PEG is lowered. BSA showed a high affinity for the
top phase in PEG 2000/tripotassium citrate due to the lower molecular PEG 2000 Concentration
The concentration of the phase-forming
polymer would greatly inf luence the
partitioning behaviour of target products.
Generally, the partition coefficient tends
to become exceedingly higher or lower,
beyond the critical polymer concentration.
The concentration of PEG 2000 was varied for
different tripotassium citrate concentrations
and the partition coefficient was analyzed and
plotted in Figure 3. It was found that the partition
coefficient would increase at first, reach a peak
value, and then decrease with the increased
concentration of phase-forming polymer. The
same trend was also reported by Wu et al.[25]
As salt becomes more hydrophobic than
PEG 2000 (at lesser PEG 2000 concentrations),
when the PEG 2000 concentration is increased,
salt will expel the water molecules to the top
FIGURE 3. The effect of PEG 2000 concentration on the partitioning of BSA in phase, providing more water volume for the
PEG 2000/C6H5K 3O7 at 303.15 K: 0 8 % salt;  12% salt;  16 % salt; × 20 % salt.
protein, thus increasing the k value. Beyond a
particular composition, the protein transfer to
the salt-rich phase may be due to a decrease
in the free volume available in the top phase
as a consequence of the increased PEG 2000
concentration.[5]

Tripotassium Citrate Concentration

The effect of tripotassium citrate con­
cen­­tra­t ion on the partitioning of BSA was
investigated and is shown in Figure 4. It
was observed that the partition coefficient
first increased and then decreased as the
salt concentration was raised. The available
free volume in bottom phase decreases and
biomolecular partition occurs more fully in the
top or interphase (salting-out). After the con-
centration of BSA in the top phase increases
to a certain point, the propulsion among the
FIGURE 4. The effect of C6H5K 3O7 concentration on the partitioning of BSA
biomolecules and salt ionic attraction results
in PEG 2000/ C6H5K 3O7 at 303.15 K:  30 % PEG; 0 32% PEG;  34 % PEG; in a salting-in effect.[26] More BSA molecules
* 36% PEG. go to the lower phase at this point, leading

Spring 2013 BioProcessing Journal • 39 • www.bioprocessingjournal.com

to a decrease in the partition coefficient. In a aqueous two-phase methodology application for biological separa-
secondary event, a high concentration of salt tions. Generally, the neutral salts have the capacity to modify the water
in the ATPS may damage the target molecule, structure. The cation always decreases the partition coefficient of BSA
resulting in lowered activity of the recovered in ATPS.[29] This behaviour can be explained on the basis that the ions
molecules. of the salt have different affinities for the two phases, giving rise to an
electrostatic potential difference between phases.[1, 30] The variation of
pH partition coefficient of BSA upon addition of NaCl is shown in Figure 6.
The variation of partition coefficient with pH The partition coefficient of the BSA decreases with increasing NaCl
for constant PEG 2000 and tripotassium citrate concentrations in the small salt concentrations (up to 0.15 M NaCl) as
concentration is shown in Figure 5. The change has been observed with α-LA and β-LG in literature.[31] In this range of
in partition coefficient can be explained by NaCl concentration, the interaction of the protein molecules with the
considering the protein surface charge com- ions and the PEG molecules in the polymer-rich phase is increased. For
pared to its isoelectric point (pI). The protein NaCl concentrations above 0.15 M, higher partition coefficients of BSA
carries net negative charge when the pH of
the system is higher than the pI of the protein.
It was observed that the increase and decrease
of the partition coefficient of BSA was directly
correlated with the pH of the system while
in the range of 4.0 to 7.0. As pH increased, so
did the partition coefficient, as was the case
upon decrease. The reduction in the partition
coefficient beyond pH 7.0 may be due to the
electrostatic force overcoming the hydrophobic
force, as explained in the following equation
by Albertsson:
1n K = 1n K0 + (FZ∆φ/RT )           (7)

According to Formula 7, protein partition

is driven by two effects: the electrostatic
component (FZ∆φ /RT) determined by the net
electrical protein charge, and a hydrophobic
FIGURE 5. The effect of pH on the partitioning of BSA in 34 % PEG 2000/34 %
component (K0) which has a maximal effect
tripotassium citrate at 303.15 K.
when the medium pH is near the isoelectric
point (for BSA, the pI is 4.7). When pH is
increased from 4.0 to 7.0, the hydrophobic
component is greater than the electrostatic
forces. Therefore, the PEG tends to interact
with the protein to create the increase of the
partition coefficient.[27]
When pH increases beyond 7.0, the net
charge of protein (Z ) becomes negative and the
electrostatic potential difference (∆φ ) between
the phases acquires positive value due to the
accumulation of citrate anions in the bottom
phase. Hence, the electrostatic term (FZ∆ φ /
RT) will be less than zero. As a result, the net
negative protein charge increases with > 7.0 pH,
decreasing the k value.[28]

Adding Neutral Salt

Salts have been added to systems to increase FIGURE 6. The effect of NaCl concentration on the partitioning of BSA in 34 %
the selectivity of protein partitioning in the PEG 2000/34 % tripotassium citrate, pH 7.0, 303.15 K.

Spring 2013 BioProcessing Journal • 40 • www.bioprocessingjournal.com

 to form the two phases is more, or solubility is
increased with the addition of BSA.

Tie Line
At equilibrium, the composition of phases
along the TLL are especially important in the
contest of the partitioning study. To obtain the
equilibrium phase composition at a known feed
composition of PEG and salt, the Othmer-Tobias
(Equation 8) and Bancroft (Equation 9) relation-
ships were used and the equilibrium studies were
made for the current ATPS (with the presence of
BSA). The two-phase systems were formed at
several compositions of tripotassium citrate and
PEG 2000 by mixing the individual stock solu-
tions and allowing them to reach equilibrium. A
number of tie lines were created, and the feed
FIGURE 7. Phase diagram of  PEG 2000/tripotassium citrate ATPS without compositions, along with equilibrium composi-
BSA at 303.15 K: , PEG 2000/tripotassium citrate ATPS with BSA at 303.15 K;
tion of the individual phases (Figure 7) with TLL
 tie line; ---- tie line predicted by Equations 8 and 9.
are reported in Table 4. The reliability of tie line
compositions was ascertained by Equations 8
have been observed at different pH values where the BSA partition and 9).[14, 15] The regression coefficient found for
is determined by its hydrophobic nature. It appears likely that the the present system is 0.988 for Equation 8 and
partition coefficient is totally independent of protein surface charge 0.9702 for Equation 9.
in combination with hydrophobic characteristics. It was confirmed that
bot n
 1 – wp  = K  1 – ws
top
the PEG, being more hydrophobic in nature, tends to strongly interact 
    (8)
with the non-polar regions of BSA. Thus, the partition coefficient of  wptop   wsbot 
r
BSA increases beyond 0.15 M NaCl concentrations.[6]  ww  = K1  ww
bot top
 (9)
 wsbot   wptop 
Phase Diagram and Equilibrium of PEG 2000/Tripotassium
Citrate System with BSA at 303.15 K
Should we show your phase diagram?

Binodal Table 4. Tie line data for PEG 2000/tripotassium

In order to further understand the applica- citrate system at 303.15 K with TLL.
bility of ATPS for any application, the phase Feed Top Phase Bottom Phase
diagram of the system is essential to explaining Composition Composition Composition
TLL
the equilibrium characteristics of the system wp % ws % wp % ws % wp % ws %
components (PEG and salt). The phase dia- (w/w) (w/w) (w/w) (w/w) (w/w) (w/w)
gram also explains the critical concentrations 0.3000 0.0800 0.3150 0.0742 0.1851 0.1288 0.1409
of PEG and salt required to form the ATPS. In 0.3400 0.0800 0.3873 0.0551 0.0856 0.1881 0.3296
the literature, the binodal curve for PEG 2000/ 0.3600 0.0800 0.4419 0.0456 0.0608 0.2109 0.4153
tripotassium citrate system without BSA at
0.2400 0.1200 0.3722 0.0601 0.0825 0.1856 0.3157
303.15 K was reported by Yan-Min, et al.[20] In
0.3000 0.1200 0.4904 0.0315 0.0502 0.2262 0.4814
the present study, the binodal curve was pre-
pared through the cloud point method with 0.3400 0.1200 0.5509 0.0232 0.0499 0.2457 0.5482

0.05 mg/mL of BSA. The experimental binodal 0.2400 0.1600 0.5500 0.0228 0.0495 0.2440 0.5472
data and literature binodal data[20] were plotted 0.3000 0.1600 0.6093 0.0174 0.0427 0.2709 0.6207
and compared in Figure 7. With the figure, it 0.3400 0.1600 0.6845 0.0116 0.0384 0.2862 0.7021
was found that the binodal curve was shifting 0.2600 0.2000 0.6964 0.0124 0.0370 0.2949 0.7174
towards the two-phase region when protein
0.3000 0.2000 0.7280 0.0104 0.0385 0.3103 0.7519
was incorporated. The results suggested that
0.3400 0.2000 0.7786 0.0066 0.0330 0.3302 0.8128
the critical concentration of PEG/salt required

Spring 2013 BioProcessing Journal • 41 • www.bioprocessingjournal.com

Where wptop represents the weight fraction of partition coefficient k and % recovery, and the independent variables,
PEG 2000 in top phase, wsbot represents the namely the PEG % (w/w), tripotassium citrate % (w/w), pH, and NaCl
weight fraction of salt in bottom phase, wwbot and concentrations, the linear model, the linear + squares model, and
wwtop are weight fractions of water in bottom and the quadratic model have been tested. Results determined that the
top phase respectively, and K, n, K1, and r are quadratic model represented the data with a > 99 % confidence level
the fit parameters, found to be 0.1084, 1.7526, for both the responses with the regression coefficient of 98.7 % and
3.3287, and 0.4909 respectively. 97.4% for Equations 11 and 12, respectively. The predicted quadratic
models in terms of noncoded (actual) factors are:
Tie Line Length
k = – 245.342 + 6.929 A + 9.764 B + 17.137 C +
The % TLL provides the phase characteristics
78.677 D – 0.091 A2 – 0.254 B2 – 0.931 C 2 –
including the physical properties and chemical (11)
85.464 D 2 – 0.011 AB – 0.123 AC – 0.479 AD –
compositions at its equilibrium. Further, the 0.134 BC – 1.489 BD + 0.260 CD
volume ratio of the phases can be modified by
altering the feed composition along a particular
tie line. The TLL are estimated using the follow-
ing relationship:
TLL (%) = [(w p(T) – w p(B))2 + (ws(T) – ws(B))2] 1/2   (10)
From Figure 8, it is evident that the partition
coefficient increases with an increase in TLL
value for a particular salt concentration at
constant temperature and molecular weight
of PEG. However, for a specific TLL value, the
lower salt concentration gave better partition
at the same temperature and molecular weight.
For the large value of TLL, the equilibrium
values of the salt and PEG concentration in both
the phases were high (top most region of the
binodal curve) where the partition behaviour
was purely derived from the net force due
to electrostatic force offered by the salt and FIGURE 8. Effect of tie line length on partition coefficient in PEG 2000/tri-
hydrophobicity offered by the PEG. However, potassium citrate at 303.15 K:  8% salt; 0 12 % salt;  16% salt;  20% salt.
handling of these phases further for protein
recovery might become difficult.
Table 6. ANOVA for the quadratic models predicted
Response Surface Methodolgy for the partition coefficient k and % recovery.
Optimization Source DF SS MS F Value Probability > F
The central composite rotatable design was
Partition Coeffient

Regression 14 115.465 8.2475 343.62 0.0001

used to design the experiments by consider- Linear 4 64.847 16.2170 675.45 0.0005
ing four independent variables: (1) PEG 2000 %
Square 4 105.659 26.4140 1100.55 0.0002
(w/w); (2) tripotassium citrate % (w/w); (3) pH;
Interaction 6 7.188 1.1980 49.91 0.0008
and (4) NaCl (M) at a temperature of 303.15 K
Lack of Fit 10 0.382 0.0382 31.98 0.0004
(Table 3). The range of variables was chosen
according to the respective individual param- Pure Error 6 0.002 0.0004 — —
eter studies in the previous sections. In total, 31 Source DF SS MS F Value Probability > F
experiments were employed. The experiments Regression 14 13640.00 974.29 197.89 0.0001
were carried out in triplicate and the average
% Recovery

Linear 4 8815.64 2203.91 447.64 0.0000

values of responses (partitioning coefficient k Square 4 4908.14 1227.04 249.23 0.0001
and % recovery) are reported in Table 5 (follow-
Interaction 6 6493.60 1082.26 219.82 0.0005
ing page). A regression analysis was carried out
Lack of Fit 10 78.37 7.84 106.75 0.0003
to fit the experimental data in the mathematical
model. In order to develop a functional rela- Pure Error 6 0.40 0.007 — —
tionship between the responses, namely the SS: sum of squares; DF: degree of freedom; MS: mean square

Spring 2013 BioProcessing Journal • 42 • www.bioprocessingjournal.com

 % recovery = – 2539.68 + 59.65 A + 103.54 B + adequately explained by the model equation. Generally, p
81.85 C + 2222.81 D – 0.68 A2 – values lower than 0.001 indicate that the model is consid-
1.78 B 2 – 3.87 C 2 – 536.68 D 2 – (12) ered to be statistically significant at the 99 % confidence
0.29 AB – 1.20 AC – 7.78 AD –
level. Both the predictive models were statistically vali-
4.00 BC – 59.63 BD – 126.99 CD
dated since the F value of each model was higher than
The ANOVA in Table 6 (previous page) shows that for the F value of the corresponding lack-of-fit. The partition
both, models explained the process with significant values partition coefficient and % recovery predicted through
of p at 99% confidence level. From this, Table 6 proves evi- the model is also reported in Table 5. Further, the F and p
dence that the F values of linear and squared regression values of the individual terms (Equations 11 and 12) reveal
were much higher than the lack-of-fit. These large values that all the individual variables had significant influence
imply, that the partition coefficient and % recovery can be in the partition coefficient and % recovery. However, the

Table 5. Experimental central composite rotatable design (CCRD) runs using four independent variables and corresponding results.

Feed Top Phase Bottom Phase Recovery

Salt Phase
pH TLL Volume kexp kpredicted
PEG 2000 C6H5K3O7 Molality wp % ws % wp % ws % exp predicted
% (w/w) % (w/w) (w/w) (w/w) (w/w) (w/w) Ratio (VR)
% %
32 16 6.0 0.25 0.5715 0.0302 0.0365 0.3082 0.6029 1.8333 6.0190 6.0253 91.90 91.76
36 14 7.0 0.35 0.5803 0.0286 0.0304 0.3099 0.6177 2.4400 1.4080 1.4744 78.63 78.31
28 14 7.0 0.35 0.4991 0.0336 0.0334 0.2639 0.5196 1.7742 2.1309 2.3290 70.29 71.53
24 16 6.0 0.25 0.4791 0.0360 0.0360 0.2672 0.4998 1.3889 0.1628 -0.1650 33.51 30.15
36 14 7.0 0.15 0.5747 0.0290 0.0445 0.3028 0.5968 2.5417 1.6009 1.5403 89.63 88.16
36 18 5.0 0.15 0.6330 0.0236 0.0190 0.3712 0.7056 2.4000 3.6821 3.5730 83.83 83.19
32 16 6.0 0.25 0.5716 0.0302 0.0375 0.3074 0.6017 1.8333 6.0203 6.0253 91.92 91.76
32 16 6.0 0.25 0.5720 0.0298 0.0373 0.3078 0.6026 1.8333 6.0246 6.0253 91.97 91.76
32 12 6.0 0.25 0.4911 0.0340 0.0324 0.2614 0.5120 2.7500 1.6153 1.5229 70.63 69.61
32 16 6.0 0.25 0.5727 0.0294 0.0374 0.3074 0.6032 1.8333 6.0190 6.0253 91.90 91.76
32 20 6.0 0.25 0.6286 0.0232 0.0232 0.3625 0.6940 1.6129 2.4300 2.4047 59.70 56.98
36 18 5 0.35 0.6521 0.0211 0.0294 0.3703 0.7140 2.2800 2.2198 2.2118 76.20 76.43
28 14 5 0.35 0.5023 0.0340 0.0322 0.2643 0.5235 2.6957 0.8015 0.8868 68.36 69.15
28 18 7 0.15 0.5431 0.0323 0.0324 0.3256 0.5889 1.2857 2.1708 2.3026 75.69 75.08
36 14 5 0.35 0.6047 0.0253 0.0343 0.2887 0.6284 3.2500 2.0474 2.0046 93.99 95.20
28 14 5 0.15 0.4854 0.0331 0.0457 0.2631 0.4962 2.4000 0.1901 0.2901 16.33 15.77
32 16 6 0.05 0.5633 0.0294 0.0299 0.3086 0.6020 2.1482 2.8400 2.9369 74.22 72.38
40 16 6 0.25 0.6661 0.0203 0.0258 0.3616 0.7256 3.1000 0.3032 0.5133 66.55 66.17
28 18 5 0.35 0.5497 0.0286 0.0366 0.3264 0.5933 1.3611 1.2961 1.4457 57.55 59.63
28 14 7 0.15 0.4851 0.0344 0.0382 0.2701 0.5053 1.8667 1.5914 1.6281 66.04 68.94
32 16 8 0.25 0.5692 0.0282 0.0307 0.3032 0.6047 1.8333 2.3153 2.1671 79.74 78.41
28 18 7 0.35 0.5609 0.0286 0.0247 0.3152 0.6080 1.4286 1.7028 1.8123 28.83 29.97
36 14 5 0.15 0.5911 0.0257 0.0282 0.2970 0.6249 2.6250 2.2554 2.1747 52.27 54.25
32 16 6 0.25 0.5722 0.0298 0.0363 0.3082 0.6038 1.8333 6.0104 6.0253 91.75 91.76
32 16 6 0.25 0.5715 0.0302 0.0371 0.3078 0.6021 1.8333 6.0169 6.0253 91.69 91.76
32 16 6 0.45 0.5764 0.0286 0.0314 0.3099 0.6133 1.8966 2.4913 2.2766 70.12 68.21
36 18 7 0.15 0.6233 0.0240 0.0207 0.3732 0.6965 2.2400 1.9198 1.8632 82.72 85.06
28 18 5 0.15 0.5459 0.0290 0.0278 0.3289 0.5986 2.5417 2.0778 2.0401 50.49 53.95
32 16 4 0.25 0.5693 0.0286 0.0398 0.3066 0.5980 2.4000 2.4044 2.4349 76.58 74.16
32 16 6 0.25 0.5722 0.0298 0.0384 0.3070 0.6015 1.8333 6.0668 6.0253 91.22 91.76
36 18 7 0.35 0.6531 0.0207 0.0283 0.3712 0.7164 1.9286 0.6172 0.6061 26.34 27.51

Spring 2013 BioProcessing Journal • 43 • www.bioprocessingjournal.com

interaction between the PEG and salt concentrations and on the model equations. The plots (Figures 9A and 9E) are
pH and NaCl molality terms had minimal influence on the represented as a function of two factors at a time, holding
responses. other factors at a fixed level of zero. All of the response plots
The partition coefficient of the present ATPS over differ- revealed that at low and high variables levels, the partition
ent combinations of independent variables were visualized coefficient was minimal. However, it was noted that the
through contour plots (Figures 9 and 10), obtained based higher values of the partitioning coefficient represented

A B C

D E F
FIGURE 9. Contour plot for the partition coefficient of BSA in the top phase as a function of: (A) tripotassium citrate and PEG con-
centration; (B) pH and PEG concentration; (C) NaCl and PEG concentration; (D) pH and tripotassium citrate concentration; (E) NaCl
and tripotassium citrate concentration; and (F) NaCl concentration and pH (0 level values of the other variables were considered as
a constant for all figures (i.e., 32 % PEG 2000/16 % tripotassium citrate/0.25 M NaCl, pH 6).

A B C
FIGURE 10. Contour plot for the % recovery of BSA in the top phase as a function of: (A) __% (w/w) PEG/ 16 % (w/w) NaCl molality, pH
6; (b) __% (w/w) PEG 2000/16 % (w/w) tripotassium citrate salt/0.25 M NaCl, pH _; and (c) __% (w/w) PEG 2000/__% (w/w) tripotassium
citrate salt/0.25 M NaCl, pH 6.

Spring 2013 BioProcessing Journal • 44 • www.bioprocessingjournal.com

the middle regions of the contours. This phe- Acknowledgement
nomenon proves that there was an existence of The authors acknowledge the grant (Scheme number: BT/PR11935/
optimization for the independent variables in PID/06/456/2009 and August 16, 2010) from the Department of Biotechnology
order to maximize the partitioning and % recov- (DBT), Government of India, for this research work.
ery. Also, the contours confirmed the existence
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The optimal system consisted of 32% PEG 2000
(w/w), 16% tripotassium citrate (w/w), and 0.25 M About the Authors
NaCl, pH 6.0, and 303.15 K. Maximum partition of Nangarthody Sindhu*, M Tech; Sivakumar Kalaivani*, M Tech;
6.03 and a recovery of 91.76 % in the top phase and Iyyaswami Regupathi*†, PhD, Assistant Professor
were obtained under these conditions. Study * Department of Chemical Engineering
National Institute of Technology Karnataka
results show that ATPS can also be used for the
Surathkal, Mangalore 575 025, India
recovery and concentration of other proteins by
Corresponding Author: Email: [email protected]
†
optimizing process parameters. http://nitk.ac.in/departments/chemical-engineering
Phone: 08242474000 | Fax: 0824-2474057

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