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# 2006 Institution of Chemical Engineers
www.icheme.org/fbp Trans IChemE, Part C, March 2006
doi: 10.1205/fbp.05159 Food and Bioproducts Processing, 84(C1): 51 – 58


Process Development Group, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, Eindhoven, The Netherlands
Department of Biotechnology, Delft University of Technology, Delft, The Netherlands

rotein partitioning based on micellar-induced excluded-volume interactions is studied
for different aqueous nonionic micellar systems. In particular, protein partitioning in
the traditional aqueous micellar two-phase system is compared with a system where
a gel phase is combined with a micellar phase and with a membrane system with micelles
at the feed side of the membrane. A two-phase system is created using cylindrically-
shaped n-decyl tetra(ethylene oxide) (C10E4) micelles that are too large to diffuse into the
gel-bead or across the membrane. Results are reported for the partition coefficient of
single-component protein systems with myoglobin, ovalbumin, and BSA, and for binary
protein mixtures. The individual protein partition coefficients in the mixtures follow the
same protein concentration dependence as the partition coefficient obtained for the single-
component protein solutions.

Keywords: nonionic surfactant; excluded-volume interactions; protein; partitioning;

membrane; sephacryl gel.

INTRODUCTION or solid agent is the most wide spread (Lightfoot and Moscar-
iello, 2004). This includes size exclusion chromatography
Separation and purification of biomolecules makes up the (Barth et al., 1998; Wu, 1999), hollow fibres (Gabelman
major part of the production costs for biological products and Hwang, 1999) and membranes (Pujar and Zydney,
(Goetheer et al., 1999; Cunha and Aires-Barros, 2002). 1998; Persson et al., 2003), integrative membrane chromato-
Considering the range of biomolecules to be separated, graphy (Thömmes and Kula, 1995; Ghosh, 2002), adsorption
there is a clear need for more flexible, and potentially (Burton and Harding, 2001), micellar liquid chromatography
more efficient and cost-effective, large-scale bioseparation (Berthod and Garcı́a-Alvarez-Coque, 2000; Baltus et al.,
techniques (Lightfoot and Moscariello, 2004; Noble and 2002), and surfactant-aided size exclusion chromatography
Agrawal, 2005). Typically, similar species in dilute streams (Horneman et al., 2004).
have to be separated, like monomer –multimer protein sep- Another mechanism of separation involves phase
aration, and this should potentially include viral clearance addition or phase creation. This includes precipitation
(Aranha, 2001). Also in the analytical field, advanced (Tessier and Lenhoff, 2003; Lenhoff, 2003) and extraction.
new developments have been shown for the separation of In particular, micelle-mediated extraction (Pires et al.,
protein-polypeptide mixtures (Manabe, 2003). 1996), such as liquid –liquid extraction using reverse
For the separation of biomolecules a variety of interactions micelles (Quina and Hinze, 1999; Tani et al., 1997) and
and mechanisms is available. The main interactions include aqueous two-phase-systems (Walter et al., 1991; Liu
excluded-volume, hydrophobic, charge (or ion-exchange), et al., 1998), is used for protein separation. Excluded-
and affinity or ligand interactions (Aboul-Enein, 1999). A volume interactions are one of the main interactions used
convenient classification of separation systems is based on to partition proteins in aqueous nonionic micellar solutions
the mechanism of separation (Seader and Henley, 1998). (Nikas et al., 1992; Lazzara et al., 2000; White and Deen,
For the separation of biomolecules, the use of a barrier 2001) and size-exclusion chromatography (Albertsson,
1956; Barth et al., 1998; Wu, 1999).

Correspondence to: Dr L. J. P. van den Broeke, Process Development
Group (SPD), Department of Chemical Engineering and Chemistry, Eind- Micellar-Based Bioseparation
hoven University of Technology, PO Box 513, 5600 MB Eindhoven, The
Netherlands. In this work, a generalized concept to use aqueous
E-mail: l.j.p.van.den.broeke@tue.nl micellar two-phase systems for the partitioning of proteins

52 ROOSMALEN et al.

is presented. The main mechanism for the partitioning is Table 1. Overview of different methods for phase separation (M) and the
resulting two-phase systems.
based on excluded-volume interactions, which are induced
by the micellar phase. The idea is to construct two separate Method used for
phases, i.e., a size-selective micellar phase and an aqueous Phase I Phase II phase separation (M)
micelle-poor phase, based on surfactants forming cylindri-
AMTPS Micellar phase Aqueous phase Temperature
cally-shaped micelles. This means that if a solid fibrous AMGS Micellar phase Aqueous phase Ratio of gel pore size
phase is used, like a gel or a membrane, with pore dimen- outside gel inside gel to micellar size
sions smaller than the length of the micelles, the micelles AMMS Micellar phase Aqueous phase Ratio of membrane pore
will be excluded from the solid phase and in this way at feed side at permeate side size to micellar size
the two separate phases are formed. Clearly, the pore size
of the solid fibrous structure should be large enough to
accommodate the proteins.
In Figure 1 the principle of constructing different two- In the following, first the partitioning of single-
phase configurations is schematically depicted. In order component protein and binary protein solutions will be
to obtain a size-selective micellar phase, referred to as discussed for each of the three micellar systems. Then, a
phase I, which is physically separated from an aqueous comparison will be made between various results for the
phase, phase II, different methods for phase separation protein partitioning obtained for the three different micellar
(M) are applied. The methods of phase separation and the systems.
resulting two-phase systems, used in this work, are
summarized in Table 1. EXPERIMENTAL
In the aqueous micellar two-phase system (AMTPS) there
is no actual physical barrier between the micelle-rich and the All reagents were used as received. McIlvaine buffer
micelle-poor phase. Phase separation is induced by tempera- (McIlvaine, 1921) with a pH of 7.3 (87.2 mM Na2HPO4
ture, and the two phases are separated because there is a and 6.5 mM citric acid in deionized water) or diluted McIl-
small difference in density. In the case of the aqueous vaine buffer with a pH of 7.3 (16.4 mM Na2HPO4 and
micellar-gel systems (AMGS) the aqueous micelle-poor 1.82 mM citric acid in deionized water) was utilized for
phase is inside the gel beads. The properties of the gel are the protein and surfactant solutions.
chosen such that, in the absence of the micelles, the proteins
partition almost equally over the aqueous phase inside and Surfactants
outside the gel. In the aqueous micellar-membrane system
(AMMS) the micelles are ‘localized’ at the feed side of The nonionic surfactants were purchased from Nikko
the membrane. The micelle-poor phase is the permeate Chemicals (Tokyo, Japan). Nonionic n-decyl tetra(ethylene
side of the membrane. oxide) (C10E4) and n-dodecyl octa(ethylene oxide) (C12E8)
The main advantage of the AMGS and the AMMS over surfactants were used to form the micellar phase. C10E4 sur-
the traditional AMTPS is that the temperature and the factant molecules in aqueous solution form cylindrically-
volume fraction of the micelles are no longer coupled shaped micelles with a cross-sectional hydrodynamic
(Van Roosmalen et al., 2004). This means that different prop- radius of 1.78 nm and a minimum length of approximately
erties of the micellar phase can be changed in situ using 170 nm (Nikas et al., 1992). The cloud point of a C10E4
temperature, salt concentration and surfactant concentration surfactant solution is 19.08C. C12E8 surfactant molecules
simultaneously to optimize the bioseparation process. in aqueous solution form spherically-shaped micelles with
a hydrodynamic radius of about 3.0 nm (Tanford et al.,
1977; Brown et al., 1988), and the cloud point of this
solution is about 808C.
The micelle-rich and the micelle-poor phases have
densities of about 1  103 g L21. Surfactant weight percen-
tages can therefore simply be converted into micellar
volume fractions by dividing by 100. In a typical experi-
ment, the surfactant-rich phase has a micellar volume
fraction equal to 0.20.

The proteins were purchased from Sigma-Aldrich. Myo-
globin (horse heart), ovalbumin (chicken egg), and bovine
serum albumin (BSA) have been studied. The protein mole-
cular weights are 17.2, 45.0 and 68.0 kDa, and the hydro-
dynamic radii are 2.0, 2.8 and 3.6 nm, respectively. For
the single-component systems the protein concentration
has been varied between 10  1026 and 800  1026 mol
protein kg21 solution. The individual protein partition
Figure 1. Schematic representation of the aqueous micellar systems
considered. Phase I is the aqueous size-selective micellar phase. Phase II coefficients or protein permeation fluxes in binary protein
is the aqueous micelle-poor phase. M represents the method for phase mixtures have been obtained for different mixture
separation. compositions.

Trans IChemE, Part C, Food and Bioproducts Processing, 2006, 84(C1): 51– 58

Gels The surfactant concentration in the aqueous solution was

determined by refractive index measurements (Euromex
The gel beads were purchased from Sigma-Aldrich. The
Microscopes Holland) at 16.08C.
two gels used are Sephacryl S-300 HR (S-300) and Sepha-
cryl S-500 HR (S-500). The gel phase is made up of a
matrix of fibres, which are randomly placed. Furthermore, THEORETICAL BACKGROUND
the fibres are considered to be rigid, and to be long and
thin. The fibres have a length to radius ratio, which is In the generalized concept of micellar two-phase sys-
much larger than unity and the pore dimension of the tems, the excluded-volume interactions result in protein
partitioning over the micelle-rich phase and the micelle-
S-300 gel is about 13 nm (Hagel et al., 1996). The pore
dimension of the S-500 gel is in the range of 75 –200 nm poor phase. In the AMTPS and the AMGS, the equilibrium
and the S-300 and S-500 gel beads have an average partition coefficient Ki of component i is defined as (Liu
et al., 1996):
diameter of about 50 mm (Hagel et al., 1989).
Ki ; (1)
Membrane Ciaq
The membrane was kindly provided by Microdyn-Nadir
(Wuppertal, Germany). The ultrafiltration membrane where Cmr i is the concentration of component i in the aqu-

(C100F, nominal molecular weight cut-off 100 kDa) con- eous micelle-rich phase and Caqi refers to the concentration

sists of regenerated cellulose, and has a pure water flux of component i in the aqueous micelle-poor phase. In this
in the range of 200 – 400 L m22 h21 (data supplied by the work the AMTPS and the AMGS properties are chosen
manufacturer). such that the volume of the micelle-rich phase equals the
volume of the micelle-poor phase. Since in the AMGS
the aqueous phase inside the gel beads is not experimen-
Experimental Methods tally accessible, the concentrations of the various compo-
nents in the aqueous gel-bead phase are determined by
For the AMTPS experiments, the surfactant and the mass.
protein solution buffer were put together in a beaker. Typi- The general expression for the equilibrium partition
cally, the total volume was about 8 mL. Phase separation coefficient Ki of component i, induced by excluded-
was induced by a constant temperature of 19.08C, using a volume interactions, is given by (Ogston, 1958; Lazzara
water bath. The system was stirred for 1.5 h and sub- et al., 2000; Van Roosmalen et al., 2004):
sequently left to equilibrate overnight (about 16 h). Both
phases were separately collected and analysed.
Ki ¼ exp½fmr (1 þ Ri =Rmic )m þ faq (1 þ Ri =Rf )n  (2)
For the AMGS experiments, the gel-beads were washed
with buffer as described by Van Roosmalen et al. (2004).
The gel beads, the surfactant, and the protein solution where fmr is the micellar volume fraction in the micelle-
buffer were put together in a beaker. Typically the total rich phase, faq is the micellar or gel-fibre volume fraction
volume was about 8 mL. To avoid phase separation the of in the micelle-poor phase, Ri is the hydrodynamic radius
system was kept at a constant temperature of 16.08C, of the spherically-shaped component i, where the subscript
using a water bath. The system was stirred for 1.5 h and i refers to spherically-shaped proteins or micelles, and Rmic
subsequently left to equilibrate overnight (16 h). After and Rf are the (cross-sectional) radius of the micelle and
protein partitioning, the aqueous surfactant rich-phase the gel-fibre, respectively. The exponent m equals 2 in
was collected. To check the protein mass balance, the the case of cylindrically-shaped micelles and m equals 3
remaining fraction was also collected. in the case of spherically-shaped micelles. The same
For the AMMS experiments, a dialysis setup was used holds for the exponent n, which refers to the fibrous struc-
where at the feed side a solution of proteins and micelles ture. Typically, cylindrically-shaped gel fibres are being
of 11.5 g was used and at the permeate side a buffer used, and in that case n is equal to 2.
solution of 115 g was used. To avoid phase separation
the system was kept at a constant temperature of 16.08C, RESULTS AND DISCUSSION
using a water bath. Both phases were continuously stirred.
At least twice a day a sample of 0.3 mL was taken from Aqueous Micellar Two-Phase System
the permeate for 3 –5 days. To check the protein mass The AMTPS is obtained by bringing an aqueous surfac-
balance, the retentate was analysed at the end of the tant solution above its cloud point. The two phases
experiment. are macroscopically separated by a small density difference
that results in a micelle-rich (top) phase and a micelle-poor
(bottom) phase. The compositions of each of the two phases
Experimental Analysis
are thermodynamically connected through the tie line in the
The protein concentrations were determined using HPLC phase diagram. A small variation in temperature, say less
(Shimadzu SCL-10A Class-VP system, Supelco Nucleosil than 0.58C, results in a considerable difference in compo-
C18 column, and water-acetonitrile-TFA gradient eluent). sition of each of the two phases, as shown schematically
For each protein-surfactant system separate calibrations in Figure 2. The cloud point of the surfactant solution
were used to determine the protein concentrations. Typi- (Blankschtein et al., 1986) and the denaturation tempera-
cally, the resulting protein recovery was within 85 and ture of the proteins determine the temperature range of
115%. the system. This means that due to the limited length

Trans IChemE, Part C, Food and Bioproducts Processing, 2006, 84(C1): 51– 58
54 ROOSMALEN et al.

Figure 2. Schematic temperature versus surfactant volume fraction phase

diagram for a typical aqueous nonionic micellar system. The coexistence
or cloud-point curve separates the two-phase region at elevated tempera- Figure 3. Protein partition coefficient as a function of the ratio of the cross-
ture from the one-phase region at lower temperature. Tc is the temperature sectional radii of the protein and the micelle for an AMTPS. The system
corresponding to the critical point C. The tie line at To connects the consists of a 0.10 micellar volume fraction difference between the
micelle-poor phase composition, fmp, at point A with the micelle-rich micelle-rich and the micelle-poor phase. The protein partition coefficient
phase composition, fmr, at point B. is calculated using equation (3) for cylindrically-shaped micelles (solid
line) and for spherically-shaped micelles (dashed line).

of the tie line, the micellar volume fraction in the micelle-

rich phase is fixed to a limited range, typically up to 0.08 Table 2. Properties of the spherically-shaped and the cylindrically-shaped
(Nikas et al., 1992).
For diluted protein solutions, the partition coefficient Phase separating Hydrodynamic Micellar
given by equation (2) reduces to the well-known relation temperature (8C) radius (nm) length (nm)
for cylindrically-shaped obstacles (Ogston, 1958; Nikas C10E4 19 1.78 170
et al., 1992): C12E8 80 3.0 —

KiAMTPS ¼ exp½(fmr  faq )(1 þ Ri =Rmic )2  (3) Aqueous Micellar – Gel System
In the AMGS, the micellar phase is physically separated
from the gel phase, because the cylindrically-shaped C10E4
Based on excluded-volume interactions there are three micelles are too large to enter the pores of the gel. This
ways to improve the partition behaviour of spherically- results in a micelle-rich phase outside the gel beads and
shaped proteins. First, the ratio of the protein radius to an aqueous micelle-poor phase inside the gel beads. For
the cross-sectional radius of the cylindrically-shaped this system the partition coefficient is given by (Lazzara
micelle can be increased. This means that smaller micelles et al., 2000; Van Roosmalen et al., 2004):
should be used. Second, if spherically-shaped micelles are
being used, replacing the exponent 2 by 3 in equation (3), KiAMGS ¼ exp½fmr (1 þ Ri =Rmic )2
a more extreme protein partitioning behaviour is obtained.
Third and finally, the difference between the micellar þ fgel (1 þ Ri =Rf )2  (4)
volume fractions of the two coexisting phases can be
increased. A more extreme protein partition coefficient Again there is a number of ways to optimize the partition
would be obtained in the case of a higher micellar concen- behaviour of spherically-shaped proteins. First of all, the par-
tration in the surfactant-rich phase and lower micellar con- tition coefficient is determined by the ratio of the protein
centration in the aqueous phase. radius to the cross-sectional radius of the cylindrically-
In Figure 3 a comparison is made between the protein shaped micelle and the ratio of the protein radius to the radius
partition coefficient calculated for a micellar system with of the gel-fibre. Second, if spherically-shaped micelles were
spherically-shaped or cylindrically-shaped micelles, using used, the exponent of the first term on the right-hand-side of
equation (3) and the data given in Table 2. The protein par- equation (4) would become equal to 3. However, in general
tition coefficient decreases with an increase in ratio of the spherically-shaped micelles have a diameter that is much
protein radius to the cross-sectional radius of the micelles. smaller than the length of cylindrically-shaped micelles
It is noted that only a limited number of CiEj surfactants in (Brown et al., 1988; Tanford et al., 1977), and this would
solution form long, cylindrically-shaped micelles. In gen- imply that the spherically-shaped micelles could diffuse into
eral, nonionic CiEj surfactants in solution form globular the gel beads.
micelles and these solutions generally phase separate at Finally, in the AMGS it is possible to independently
elevated temperatures (Van Os et al., 1993; Huibers increase the micellar volume fraction, and to simultaneously
et al., 1997). minimalize the gel-fibre fraction, without being limited

Trans IChemE, Part C, Food and Bioproducts Processing, 2006, 84(C1): 51– 58

by the phase diagram of the surfactant. A more extreme

protein partition coefficient is obtained in the case of a
higher concentration of micelles in the micelle-rich phase.
In Figure 4 a comparison is made between the results for
the partition coefficients of BSA as a function of the overall
volume fraction of C10E4 or C12E8 micelles, using the
S-500 gel. Indeed, almost no partitioning of BSA takes
place in the case of the spherically-shaped C12E8 micelles,
because the C12E8 micelles also partition over the aqueous
phase inside and outside the gel-bead phase. Furthermore, it
is seen that for the cylindrically-shaped C10E4 micelles the
BSA partition coefficients decrease with an increase in the
micellar volume fraction.
In Figure 5 results for the (individual) protein partition
coefficients of binary mixtures of myoglobin and BSA are
given for different protein compositions, for an overall
C10E4 micellar volume fraction of 0.10. The partition co-
efficient of myoglobin is plotted as a function of the overall
myoglobin concentration in the mixture, and the partition
coefficient of BSA is plotted as a function of the overall
BSA concentration in the mixture. A comparison is made Figure 5. Myoglobin and BSA partition coefficients for binary protein
between the partition coefficients of myoglobin and BSA mixtures as a function of the individual protein concentration using the
S-500 gel at 16.08C. Myoglobin (open symbols) and BSA (solid symbols)
in the protein mixtures and the protein partition coefficients partition coefficients obtained for an overall volume fraction of 0.10 C10E4
obtained for the single-component protein systems. The micelles. Each experiment was numbered and the protein concentrations of
protein concentrations for the mixtures are given in each mixture are given in Table 3. The lines are based on the results for the
Table 3. For both proteins, the partition coefficients single-component myoglobin (solid line) and BSA (dashed line) partition
coefficient as a function of protein concentration.
obtained in the mixtures are comparable to the partition
coefficients obtained for the single-component systems.
This indicates that mixture effects are absent; the partition-
ing of each protein in the mixture is predominately deter- AMGS, the C10E4 micelles are too large to enter the mem-
mined by the C10E4 micellar volume fraction in the brane. The protein flux is given by:
micellar phase.
Ciperm  M perm
JiAMMS ¼ (mol m2 h1 ) (5)
Amemb  t
Aqueous Micellar –Membrane System
In the AMMS, the micellar phase is localized at the feed where JAMMS
i the flux of protein i through the membrane,
side of a membrane, and the aqueous micelle-poor phase is Cperm
i is the concentration of protein i at the permeate
at the permeate side of the membrane. Similar as for the side, M perm is the mass of the permeate side of the mem-
brane, A memb is membrane surface area, and t is the time.
There are a number of ways to enhance the flux of
spherically-shaped proteins through the membrane. First,
as in all aqueous micellar systems, the ratio of the protein
radius to the cross-sectional radius of the cylindrically-
shaped micelle can be increased to obtain a more extreme
excluded-volume effect. Second, in the AMMS it is poss-
ible to independently increase the micellar volume fraction,

Table 3. Protein concentrations in 1026 mol kg21 of the mix-

tures presented in Figure 5, with an overall volume fraction of
0.10 C10E4 micelles present.

Figure 5 (with C10E4 surfactant)

Experiment # Myoglobin BSA

1 8.9 9.1
2 9.4 84
3 8.0 202
4 79 10
5 84 87
Figure 4. BSA partition coefficient, in an AMGS, as a function of the 6 85 204
overall volume fraction cylindrically-shaped C10E4 micelles (solid 7 200 9.1
squares) and the spherically-shaped C12E8 micelles (solid circles) at 8 191 82
16.08C. Results are for a S-500 gel and an overall BSA concentration of 9 190 195
180  1026 mol kg21.

Trans IChemE, Part C, Food and Bioproducts Processing, 2006, 84(C1): 51– 58
56 ROOSMALEN et al.

and to simultaneously increase the membrane pore size. For

the larger protein a relatively high protein flux is obtained
in the case of a higher concentration of micelles in the
surfactant-rich phase.
In Figure 6 a comparison is made between the results for
the single component flux of ovalbumin and the flux of
BSA as a function of the C10E4 volume fraction at the
feed side. The protein flux is determined from the protein
concentration at the permeate side, which increases linearly
with time. The resulting values for the protein flux given in
Figure 6 are obtained with equation (5). With an increase in
micellar volume fraction both the BSA and the ovalbumin
flux increase. For the same C10E4 micellar volume fraction,
the flux of ovalbumin (the smaller biomolecule) is always
higher than the flux of BSA. The protein fluxes can be nor-
malized on the protein flux obtained without micelles pres-
ent. With an increase in the micellar volume fraction from
0 to 0.20, the ovalbumin flux increases by a factor of 2.3,
Figure 7. Protein fluxes for binary mixtures of ovalbumin and BSA (with
and the BSA flux increases by a factor of 5.6. This is due different compositions) as a function of the ovalbumin feed concentration,
to the excluded-volume interactions exerted by the micelles using the C100F membrane. Ovalbumin flux (solid triangles) and BSA flux
on the proteins. (solid squares) obtained for an initial total protein concentration of
In Figure 7 the individual protein fluxes for different 600  1026 mol kg21 feed, with a volume fraction of 0.20 C10E4 micelles
in the feed. The solid lines are a fit of the single-component flux values.
binary mixtures are shown as a function of the ovalbumin
feed concentration, using the C100F membrane and a
C10E4 micellar volume fraction of 0.20 in the feed. The
Comparison of the Different Configurations
binary results are for fixed total concentration of
600  1026 mol kg21 feed, with different ratios of oval- Optimal separation of mixtures of biomolecules using
bumin and BSA. For ovalbumin the flux increases linearly size exclusion chromatography, requires for each different
with an increase in ovalbumin concentration. The flux for target biomolecule a different selectivity of the gel or an
BSA decreases linearly with an increase in the ovalbumin adjustment of the operating conditions, such as pH, temp-
concentration in the feed. For comaprison also the result erature, and flow rate (Jennings et al., 1995). The main
for the single-compoent flux of ovalbumin and BSA are advantage of using a size-selective micellar phase is that
given, showing reasonable agreement with the results for the micellar properties can be adjusted in situ to the
the fluxes in the mixture. It can be concluded that for the target molecule to be separated.
binary protein solutions a similar dependence of both indi- In Figure 8 a comparison is made between partitioning
vidual protein fluxes on the protein concentration is results obtained for different proteins in the AMGS using
observed as for the single-component protein solutions. two different buffer solutions. Protein partitioning was
achieved using an overall micellar volume fraction of
0.10 in the McIlvaine buffer and using an overall micellar
volume fraction of about 0.035 in the diluted McIlvaine
buffer. The diameter of the micelles with the buffer solution
with the lowest salt concentration are smaller than the
diameter of the micelles with the buffer solution with the
highest salt concentration. As a result, for the situation
with the smaller micelles somewhat more extreme protein
partition coefficients are obtained, despite the fact that the
micellar volume fraction is considerably smaller.
In Figure 9 the BSA partition coefficient obtained with
the AMTPS and the AMGS are compared, both using a
C10E4 micellar volume fraction of 0.20. The smallest,
most extreme partition coefficient is obtained for the
AMTPS. The addition of the gel makes that the protein par-
tition coefficients in the AMGS are intrinsically larger than
the partition coefficients obtained in the AMTPS. This is a
result of the extra term, second term on the right-hand-side,
in equation (4), as compared to equation (3). This means
that because of the presence of the gel that for the same
micellar volume fraction the proetin partition coefficient
in the AMGS will always be higher than the protein
partition coefficient for the AMTPS.
Figure 6. Ovalbumin (solid triangles) and BSA (solid squares) fluxes in an
AMMS as a function of the volume fraction of C10E4 micelles in the feed.
From a comparison between the results obtained for the
Permeation results are obtained for an initial protein concentration of AMGS and the AMMS it can be concluded that generally
150  1026 mol kg21 feed, using the C100F membrane. the behaviour of the individual proteins in the binary

Trans IChemE, Part C, Food and Bioproducts Processing, 2006, 84(C1): 51– 58

solid phase. The key parameter for protein separations

based on micellar systems using excluded-volume inter-
actions is the ratio of the size of the proteins to be separated
to the characteristic dimensions of the nonionic micellar
phase. First of all, the hydrodynamic radius of globular
hydrophilic proteins typically ranges from 1 to 5 nm
(Freifelder, 1982). For practical protein separations, the
temperature range at which the target protein does not
denature, say 108C to 508C, determines the actual aqueous
micellar system that could be used.
Besides the temperature, the size and shape of the non-
ionic CiEj micelles is also a function of the surfactant con-
centration and the ratio of the length of the alkyl tail (Ci) to
the number of ethylene oxide groups (Ej) in the surfactant
head (Brown et al., 1988). Small differences in the
number of carbon atoms in the tail, or in the number of
ethylene oxide blocks in the head, will result in a consider-
Figure 8. Myoglobin (17.2 kDa), ovalbumin (45.0 kDa), and BSA able difference in the properties of the micelles. However,
(68.0 kDa) partition coefficients as a function of protein molecular weight in general nonionic CiEj surfactants form globular micelles
(kDa) for two different buffers in an AMGS. An overall protein concen- with a hydrodynamic radius smaller than 15 nm (Brown
tration of 100  1026 to 200  1026 mol kg21 was used. For the McIlvaine et al., 1988; Tanford et al., 1977) in the temperature
buffer (solid triangles), protein partitioning was achieved using the S-500
gel, a temperature of 16.08C, and cylindrically-shaped C10E4 micelles
range of 108C to 508C. This indicates that the possibility
with an overall micellar volume fraction of 0.10. For the diluted McIlvaine to improve protein partitioning by adjusting the shape of
buffer (solid circles), protein partitioning was achieved using the S-300 gel, the micelles is limited, because only a limited number of
a temperature of 15.08C, and cylindrically-shaped C10E4 micelles with an CiEj surfactants form large cylindrically-shaped micelles.
overall micellar volume fraction of about 0.035. Besides separation based on size using excluded-volume
interactions induced by the micelles, it is also possible to
use micellar systems where separation is achieved by
electrostatic interactions (charge), affinity, or solubility
(Bordier, 1981). These mechanisms can be used in addition
to the excluded-volume interactions to enhance partitioning
of the targeted biomolecule. Excluded-volume interactions
combined with electrostatic interactions have been used to
study the partitioning of the enzyme glucose-6-phosphate
dehydrogenase in an aqueous mixed nonionic-ionic micel-
lar system, consisting of C10E4 and CnTAB surfactants
(Rangel-Yagui et al., 2003). Affinity-enhanced partitioning
has been studied for an affinity-tagged protein in an
AMTPS formed using the nonionic surfactant n-decyl-b-
D-glucopyranoside (Lam et al., 2005).
It is also possible to modify the properties of the solid
phase by changing, to some extent, the pore dimensions
of the gel or the membrane. For example, the pore structure
of cross-linked gels can be changed by polymerization of
the gel using micellar templates (Rill et al., 1998). After
Figure 9. Myoglobin (17.2 kDa), ovalbumin (45.0 kDa), and BSA removal of the template, a gel with pores of the approxi-
(68.0 kDa) partition coefficients as a function of protein molecular mate shape and dimensions of the micellar templates will
weight (kDa) in an AMTPS and in an AMGS. An overall protein concen- be available. Another, more straightforward approach
tration of 100  1026 to 200  1026 mol kg21 and cylindrically-shaped would be to use gels or membranes with a different pore
C10E4 micelles with an overall micellar volume fraction of 0.10 were
used. In the AMTPS (solid diamonds), protein partitioning was achieved
size. With respect to the Sephacryl gels, these are available
using a temperature of 20.08C. In the AMGS (solid triangles), protein par- with a pore dimension of 6.6 nm (S-100 HR) to over 31 nm
titioning was achieved using the S-500 gel and a temperature of 16.08C. (S-500 HR) (Hagel et al., 1996).

solutions show a similar dependence on the protein concen-
tration as observed for the single-component protein Protein partitioning from a nonionic micellar solution in
solutions. three different systems has been studied. Results for the
proteins myoglobin, ovalbumin, and BSA from a solution
with cylindrically-shaped nonionic n-decyl tetra(ethylene
oxide) C10E4 micelles clearly show that protein partitioning
Potential Adjustments of the Different Configurations
is enhanced in the presence of the micelles. It is concluded
In the generalized concept it is possible to adjust the that the enhanced protein partitioning is induced by
properties of both the micelle-rich phase and the fibrous excluded-volume interactions in the micellar C10E4 phase.

Trans IChemE, Part C, Food and Bioproducts Processing, 2006, 84(C1): 51– 58
58 ROOSMALEN et al.

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