Process Biochemistry 38 (2002) 387
/391

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Mass transfer in a spray column during two-phase extraction of

horseradish peroxidase
N.D. Srinivas 1, A.V. Narayan, K.S.M.S. Raghavarao *
Department of Food Engineering, Central Food Technological Research Institute, Mysore 570 013, India

Received 27 November 2001; received in revised form 25 March 2002; accepted 1 April 2002

Abstract

Multistage contactors are often needed to achieve the desired degree of mass transfer. One such contactor is spray column, which
can be conveniently used to isolate and purify proteins using aqueous two-phase extraction. The effects of orifice size and addition
of NaCl on the overall mass transfer coefficient (KLa ) of horseradish peroxidase (HRP) and fractional dispersed phase hold up (o D)
have been studied in a simple spray column using a polyethylene glycol/potassium phosphate aqueous two-phase system. KLa and
o D increased with an increase in dispersed phase superficial velocity (vs) as well as capillary diameter. The presence of NaCl increased
KLa . The overall mass transfer coefficient for HRP normalized for dispersed phase hold up, was found to be independent of vs in the
range studied. # 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Horseradish peroxidase; Mass transfer coefficient; Hold up; Aqueous two-phase systems; Spray column

1. Introduction and do not require centrifugation to achieve faster phase

separation.
In batch mode, protein extraction using aqueous two- Spray columns, the simplest of column contactors can
phase system (ATPS) is carried out by mixing the be conveniently employed for protein extraction using
protein with the phase system and subsequently, allow- ATPSs. One of the phases is made continuous and the
ing the dispersion to separate. When the dispersion other is dispersed in the form of droplets. Spray columns
separates back into two equilibrated phases, the desired are easy to operate, simple and economical in construc-
protein concentrates into one of the phases facilitating tion. Moreover, continuous phase residence time can be
its isolation. However, the dispersion produced in the easily varied. The major drawback with these columns is
agitated vessel is relatively stable making the rate of back mixing which limits the use of these columns to
phase separation very slow under gravity necessitating systems containing less than five theoretical stages and
the use of a centrifuge. The agitated vessel-centrifuge or fortunately is the case with ATPE [1
/4].
the static mixture-centrifuge assembly provides one Jafarabad et al. [3] have reviewed the literature on
theoretical stage and is expensive on a large-scale. In spray columns. At that time there was practically no
information regarding the mass transfer aspects in spray
many aqueous two-phase extractions (ATPEs) more
columns using ATPS, except that of Sawant et al. [1],
than one theoretical stage is required to achieve the
who have reported the mass transfer coefficient for
desired level of purity. Column extractors can be
bovine serum albumin (BSA) in a 9.7 mm internal
employed as alternative extraction equipment as they
diameter spray column. Authors observed that the
are known to provide more than one theoretical stage
dispersed phase hold up (o D) and mass transfer coeffi-
cient (KLa) increased with an increase in dispersed phase
velocity (vs) and true mass transfer coefficient (KL) was
* Corresponding author. Tel.: /91-821-514874; fax: /91-821- independent of vs. Prafulla et al. [4] investigated the
517233
E-mail address: [email protected] (K.S.M.S. Raghavarao).
effects of various parameters on o D and KLa in a sieve
1
Present address: Mycology Laboratory, David Axelrod Institute, plate column of 60 mm internal diameter using a
Albany, NY, USA. polyethylene glycol (PEG)/salt ATPS with amylogluco-
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 0 9 7 - 3
388 N.D. Srinivas et al. / Process Biochemistry 38 (2002) 387
/391

sidase (AMG) as solute. They observed that both KLa 3. Materials and methods
and o D increased with an increase in vs, number and size
of orifice and decreased with an increase in phase Polyethylene glycol (MW 6000), K2HPO4, KH2PO4,
composition. Authors calculated the contributions to NaCl, guaiacol, and H2O2 were all procured from
mass transfer during the formation, rise and coalescence Ranbaxy Chemical (Punjab, India). HRP was purchased
of droplets. The present study is aimed at investigating from Sigma Chemicals (USA).
the extraction capacity of a simple spray column for a
new solute, horseradish peroxidase (HRP) using a PEG/ 3.1. Phase system preparation and partition coefficient
potassium phosphate ATPS. Horseradish root tubers measurements
are commonly employed as a source for production of
peroxidase, which has many commercial applications. Predetermined, weighed quantities of polyethylene
The effects of dispersed phase superficial velocity, orifice glycol and potassium phosphates (K2HPO4:KH2PO4 /
size and NaCl concentration on KLa and oD were 1.82:1, to obtain the required pH of 7.0) were added to a
studied. The effect of NaCl was studied for the first known quantity of distilled water, so as to make the
time considering the fact that addition of NaCl has a total weight of the system 100% on w/w basis. After
significant effect on partition coefficient of enzymes in dissolving the components, the system was mixed
ATPS [5]. thoroughly for 1 h and allowed to separate in a
separating funnel overnight. The equilibrated and sepa-
rated phases were then collected and used as stock
solutions for further experiments. In this way 3000 g of
systems were prepared at a phase composition of PEG
2. Theoretical aspects
15% and potassium phosphate 11%. Similarly, to study
the effect of NaCl, 3000 g of phase system comprising of
In equipment such as spray columns, where the
PEG 15%, potassium phosphate 11% and NaCl 2% was
interfacial area is not known, the following equation
prepared, individual phases were separated and used as
can be used to calculate overall protein transfer coeffi-
stock for further experiments. The partition coefficient
cients from experimental data [1]:
of HRP was calculated as the ratio of the equilibrium
dN KL a(CT CT ) dv (1) concentration of the enzyme in the top phase to that in
the bottom phase [6].
where C T is equilibrium concentration of protein/
enzyme in PEG phase; KLa , the overall mass transfer 3.2. Mass transfer experiments
coefficient for a protein/enzyme.
Integration of the above equation with suitable A schematic diagram of the experimental set up for
assumptions and rearrangement results in, mass transfer is shown in Fig. 1. The internal diameter
  of the column was 18 mm and the length 400 mm. HRP
L (CTi  mCTs ) transfer experiments from dispersed phase to continuous
KL a ln (2)
VD (CTo  mCTs ) phase was carried out with salt-rich (heavy) phase as
continuous phase and PEG-rich (light) phase as dis-
where L is the flow rate of the dispersed phase, VD is the persed phase, which formed droplets in the continuous
dispersion volume (calculated from HD value), CTi and phase. These droplets coalesced at the top of the
CTo are the inlet and outlet concentration of HRP in the continuous phase and the coalesced layer was collected
PEG-rich dispersed phase, CTs is the concentration of through the overflow outlets. In all the experiments the
HRP in the salt-rich continuous phase after extraction solute HRP was dissolved in PEG-rich phase at a
and m is the partition coefficient of HRP. predetermined concentration (7.5 U/ml) and the stock
The fractional dispersed phase hold up (o D) was solution was stored in a glass tank. The extent of
calculated by using the equation, deactivation of the enzyme was negligible during the
time of experimentation (
/3 h). The flow of the PEG-
HD  H rich phase was monitored using a peristaltic pump
oD  (3)
HD (Pharmacia, Sweden). The PEG phase was removed
from the top through the overflow line and the flow rate
where H is the initial height of continuous phase before was calculated by measuring the amount of PEG phase
the start of the sparging of the dispersed phase and HD collected for a known period of time. In all experiments
is the height of the continuous phase when dispersed 110 ml of the continuous phase was added into the
phase is sparged into the continuous phase or in other column and the flow of the dispersed phase continued
words the height of the dispersion (continuous phase for a period which was always 10 times greater than the
containing dispersed phase). residence time of PEG droplets. This time was provided
 N.D. Srinivas et al. / Process Biochemistry 38 (2002) 387
/391 389

mPa s and densities (top and bottom phase) 1135.2 and

1190.7 kg/m3, respectively.

4.1. Effect of superficial dispersed phase velocity

The effect of superficial dispersed phase velocity on

KLa and oD was studied at various nozzle diameters in a
given phase system. In all the cases the values of KLa
and o D increased with an increase in vs as shown in Fig. 2
and Fig. 3, respectively. It was shown that the drop size
decreases with an increase in vs [7]. Visual observations
also indicated a decrease in drop size as well as an
increase in drop frequency (number of drops/min) with
an increase in vs. This decrease in drop size decreases the
drop rise velocity and increases the drop residence time
in the column and hence o D as well as KLa increases. In
addition, the increase in drop frequency and a decrease
Fig. 1. Schematic diagram of the experimental set-up for HRP transfer
in drop size, increase the interfacial area available for
studies in a simple spray column using a PEG/potassium phosphate
ATPS. protein transfer, resulting in an increase in KLa . Similar
results were reported in previous works [1
/4].
to ensure a steady state with respect to dispersion
characteristics.
The dispersed phase flow rate was varied over the
range 2.98 /109 to 9.73 /109 m3/s. The fractional
dispersed phase hold up (o D) was calculated using Eq.
(3). To determine the protein/enzyme transfer coeffi-
cients, all parameters in Eq. (2) need to be measured.
The column was filled with the salt phase. The PEG
phase containing HRP was pumped through the sparger
at a known flow rate. Samples of the PEG phase were
collected from the overflow line and analysed for
enzyme activity. Similarly, samples of salt phase were
collected before and after the experiment. The enzyme
concentration was taken to be average of the initial and
final value. As the enzyme concentrations were very low
and the amount of enzyme extracted was also low, a
quasi steady state prevailed during the experiment.
The densities of the phases were measured with a Fig. 2. Effects of dispersed phase superficial velocity and orifice
diameter on the mass transfer coefficient of HRP in a PEG/potassium
density bottle and the viscosities by an Oswald-U tube
phosphate ATPS. Orifices diameter (mm): j, 0.50; ', 0.92; m, 1.32.
viscometer of 10 ml capacity. Every precaution was
taken to maintain the cleanliness of the internal parts of
the column as well as other apparatus, because inter-
facial tension is very sensitive to the presence of small
amounts of impurities.
All experiments were carried out in triplicates and
average values represented. All experiments were per-
formed at 279/0.5 8C (ambient temperature).

4. Results and discussion

Fig. 3. Effects of dispersed phase superficial velocity and orifice
diameter on the fractional dispersed phase hold up in a PEG/
The physical properties of 15/11 phase composition potassium phosphate ATPS. Orifices diameter (mm): j, 0.50; ',
are viscosities (top and bottom phase) 24.47 and 1.41 0.92; m, 1.32.
390 N.D. Srinivas et al. / Process Biochemistry 38 (2002) 387
/391

4.2. Effect of orifice diameter 4.4. Effect of NaCl

Effect of orifice diameter on KLa and o D was studied It is evident from the literature [5] that addition of
at 3 different orifice diameters viz: (i) 0.50 mm; (ii) 0.92 NaCl to the system has a profound effect on the
mm; and (iii) 1.33 mm and the results were depicted in partition coefficient of enzymes, and it is worthwhile
Fig. 2 and Fig. 3, respectively. It can be seen that the to study its effect on mass transfer. Experiments on
values of KLa and o D increased with an increase in HRP partition coefficient studies at various NaCl
orifice diameter. This is explained based on the visual concentrations indicated that lowest partition coefficient
observations, which indicated that at a given dispersed could be obtained at 2% NaCl (Fig. 5). The addition of
phase velocity, drop size decreased and drop frequency NaCl changed the interaction of peroxidases with the
increased with an increase in orifice diameter. This system, resulting in significant decrease in partition
decrease in drop size and an increase in drop frequency coefficient [5]. Hence, this concentration was selected
are eventually responsible for an increase in the values to study its effect on mass transfer. It was observed that
of KLa and o D as explained earlier. KLa increased in the presence of 2% NaCl (Fig. 6).
However, o D remained practically the same (data not
shown). Although, the exact explanation is not known
4.3. True mass transfer coefficient at this stage, it is thought that attractive electrostatic
interactions between NaCl and HRP may be responsible
As the behaviour of KLa and o D were similar with for this increased in KLa . However, detailed investiga-
respect to vs (Fig. 2 and Fig. 3), a plot of KLa/o D (HRP tions on the effect of NaCl on interfacial tension of the
transfer per unit hold up) versus vs was plotted as shown system and on the internal circulation of the drops have
in Fig. 4. The ratio was independent of vs indicating that to be performed to provide a proper explanation for the
the true mass transfer coefficient was independent of observed enhancement of KLa in presence of NaCl.
superficial velocity. This result is consistent with that of
the earlier one [1] and is important in designing scale-up
aspects of spray column extraction.
With an increase in superficial velocity (or flow rate)
of the dispersed phase KLa product increases. The
increase could be either due to increase in KL or increase
in a . The increase in KL will be due to the factors, such
as dispersed phase superficial velocity, diffusion coeffi-
cient etc. The increase in a will be due to increase in o D.
In order to determine which is contributing to the
increase in KLa , KLa /o D was plotted against vs and
found that o D is contributing and not KL. This is
consistent with literature. However the situation is
different when NaCl is incorporated. Same analysis
(KLa /o D vs. vs) indicated a constant increase (unlike
Fig. 5. Effect of NaCl concentration on partition coefficient of HRP in
constant in the previous case) as discussed in Section a PEG/potassium phosphate ATPS.
4.4.

Fig. 4. Effects of dispersed phase superficial velocity on KLa /oD in a Fig. 6. Effect of NaCl (2% w/w) on mass transfer coefficient of HRP in
PEG/potassium phosphate ATPS. Orifices diameter (mm): j, 0.50; ', a spray column (orifice diameter: 0.92 mm) using a PEG/potassium
0.92; m, 1.32. phosphate ATPS: ', 0% NaCl, ^, 2% NaCl.
 N.D. Srinivas et al. / Process Biochemistry 38 (2002) 387
/391 391

5. Conclusions NDS acknowledges CSIR, Govt. of India for providing

Senior Research Fellowship.
This study deals with the extraction of HRP in a
simple spray column of 18 mm internal diameter using a
PEG/salt ATPS. The main advantage of the spray References
column extraction method is that the phases separate
easily and quickly by gravity without the need of a [1] Sawant SB, Sikdar SK, Joshi JB. Hydrodynamics and mass
centrifuge. In addition, the process is simple and easy to transfer characteristics of spray column for two-phase aqueous
operate in continuous mode. KLa and o D were found to extraction. Biotechnol Bioeng 1990;36:109
/15.
[2] Joshi JB, Sawant SB, Raghavarao KSMS, Patil TA, Rostami KM,
increase with an increase in vs as well as orifice size. The Sikdar SK. Continuous countercurrent two-phase extraction.
true mass transfer coefficient, represented by (KLa/o D) Bioseparation 1990;1:311
/24.
was independent of vs. KLa increase in the presence of [3] Jafarabad KR, Kale DD, Joshi JB. Effect of viscosity and drag
NaCl in the system. Detailed investigations on the effect reducing agent on mass transfer coefficient in liquid-liquid spray
of NaCl on interfacial tension and internal circulation of column. Solvent Extraction Ion Exchange 1990;8:669
/94.
[4] Prafulla CMB, Pandit AB, Sawant SB, Joshi JB. Enzyme mass
the drops are required to provide an extensive explana- transfer coefficient in a sieve plate extraction column. The Chem
tion for this observed increase in KLa . Eng J 1994;55:B1
/B17.
[5] Srinivas ND, Aqueous two-phase extraction for downstream
processing of enzymes. Ph.D. Thesis, University of Mysore,
Mysore 2000.
Acknowledgements
[6] Albertson PA. Partition of Cells, Particles and Macromolecules,
2nd Ed.. New York: Wiley, 1986.
Authors wish to thank Dr V. Prakash, Director, [7] Kumar A, Hartland S. Prediction of drop size produced by a
CFTRI, Mysore for his support and interest in ATPS. multiorifice distributor. Trans Instn Chem Engrs 1982;60:35
/9.