ISEC2014 – International Solvent Extraction Conference September 7-11, 2014

Formation and extraction ability of reverse micelle by

intermolecular interaction between di(2-ethylhexyl)
phosphoric acid and ethanolamines

K. Ejima1, S. Kiyoyama2, T. Sana3, K. Shiomori*1

The formation of reverse micelle by intermolecular interaction between di (2-ethylhexyl) phosphoric
acid (DEHPA) and ethanolamines was investigated under various conditions. By the addition of
ethanolamines, such as diethanolamine, triethanolamine and tris(hydroxymethyl) amino methane, in
the aqueous phase, reverse micelles of DEHPA were formed with high extraction of water.
Furthermore the addition of octanol in the organic phase was drastically stabilized the reverse
micelles. When triethanolamine was added in the aqueous phase, the extraction of water was
enhanced at pH between 5.5 and 8.5. The ethanolamines added to the aqueous phase would be
extracted into the water pool of the reverse micelles by the electrostatic interaction with the head
group of DEHPA and enhance the ability to form the reverse micelles. The reverse micellar systemes
consisted of both DEHPA and the ethanolamines show high ability to extract cytchrome-c at pH values
which enhance the water extraction and lower than pI of the protein. The extracted protein could be
back-extracted at pH higher than its pI value.

Introduction
Reverse micelle are self-aggregates of surfactant molecules in a nonpolar
organic solvents, and water pool of nanometer-order are formed in their polar cores.
By this unique nature, reverse micelles have ability to extract proteins from aqueous
phase [1-4]. The mechanism of protein extraction has been explained by the
interaction between protein and reverse micelles, such as electrostatic, steric and
hydrophobic interactions. Extraction control of protein with the usual reverse micellar
system, such as sodium bis-2-ethylhexylsulphosuccinate (AOT), is carried out using
changes of the surface characteristics of proteins and the size of reverse micelles by
the pH value and the salt concentration, respectively. However, proteins are known to
suffer from denaturation, and the efficiency of protein separation using reverse
micelles decrease significantly when the interaction between proteins and reverse
micelles is too strong. If it possible to change the characteristics and structure of
reverse micelles by external stimulation, it will be possible to construct a more
effective separation process of protein with the reverse micelles. Some studies have
reported that the addition of alkyl amines to the AOT reverse micellar system was
found effective to control the formation of reverse micelles by pH change [5]. The
molecular interaction of ionic surfactants with counterionic molecules affects their
interfacial properties, formation of milecular assemblies and the extraction ability. In
this study, formation of reverse micelle by intermolecular interaction between di (2-
ethylhexyl) phosphoric acid and ethanolamines shown in Fig. 1 and extraction ability

*1 Corresponding Author, Dept. Applied Chem., Univ. Miyazaki, Gakuenkibanadai-nishi 1-1, Miyazaki 889-2192, Japan
2
Dept. Chem. Sci. Eng., Miyakonojyo Natnl. Collage Tech., Miyakonojyo 885-8567,Japan
3
Sansei Giken Co. Ltd., Yoshikawa 342-0008, Japan t
 ISEC2014 – International Solvent Extraction Conference September 7-11, 2014

of proteins with the reversed micellar

system developed have been () ( () (
investigated under various conditions.

Experimental
Monoethaolamine (MEA),
diethanolamine (DEA), triethanolamine () ( ) () () (
(TEA), and Tris(hydroxymethyl)
aminomethane (Tris) were used as the Fig. 1 Chemical structure of ethanolamines used
additives in the aqueous phase. DEHPA
as a surfactant and octanol as a co-solvent were dissolved in isooctane, in which was
used as an organic phase. Distilled water dissolving NaCl and the ethanolamines
was used as an aqueous phase, and then protein was dissolved in the aqueous
solution. Cytochrome c was used as a model protein. Equal volumes of the aqueous
and organic phases were mixed well in a screw-cap sample tubes with magnetic stir
in a water bath at 298 K for 30 min. Then, the solution was separated into two
phases by centrifugation at 259 K and 3,500rpm for 30 min. The concentration of
cytochrome c in the organic and aqueous phases was measured by the adsorption at
280 nm. The water concentration in the organic phase was determined by Karl-Fisher
titration. The molar ratio of water to surfactant, Wo=[H2O]/[DEHPA] was used to
characterize the reverse micellar formation.

Results and discussion

Effect of ethanol amines on the formation of reverse micelles of DEHPA
The effects of pH on the extraction of water in the DEHPA-ethanolamine
mixed systems with or without octanol in the organic phase are shown in Fig 2. In the
DEHPA alone system without both ethanolamines and octanol, few water were
extracted in the organic phase. When MEA, DEA or Tris was added in the aqueous

9
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31/ H 9
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DA A

DA A

01/ D H DA 9
72 48D

-
72 48

1" 2" 3" 4" 5" 6" 7" 8" 9" 10" 11" 12" 13" 14" 1" 2" 3" 4" 5" 6" 7" 8" 9" 10" 11" 12" 13" 14"
E2 E2

9
0125/ 61/ H H 9 0125/ 6 H
9
61/
H 9
61/ D H DA 9
DA A

DA A

6
M 6 D H DA 9
72 48

72 48

( ) , - . ( ) 1" 2" 3" 4" 5" 6" 7" 8" 9" 10" 11" 12" 13" 14"
E2
E2

Fig. 2 Effect of pH in the aqueous phase on the extraction of water in the DEHPA-ethanolamine
mixed systems with or without the attition of octanol in the organic phase.
ISEC2014 – International Solvent Extraction Conference September 7-11, 2014

phase, quite large amount of water in the organic phase was extracted at narrow pH
range, which was pH 6.5-9.0 for MEA, pH 5.5-6.5 for DEA and pH 5.5-6.0 for Tris,
respectively. In the cases of TEA and Tris, the third phase was formed between the
organic and aqueous phases and at that time the extraction of water in the organic
phase was quite low. These phenomena can be explained by formations of the
anionic phosphoric head group of DEHPA and the cationic ammonium ions of the
ethanolamines with pH change. The phosphoric head group of DEHPA dissociates at
pH higher than 5.5 to form its anionic form. On the other hand, the ethanolamines
form their cationic ammonium ions at lower pH depending on their pKa values. The
cationic ammonium ions of the ethanolamines would be interacted with the anionic
phosphoric head group of DEHPA and formed the intermolecular ion complexes. The
complexes have high surface activities and form the reverse micelles or the third
phase depending on the properties of the ethanolamine, the cases of MEA, DEA and
Tris formed the reverse micelles and the case of TEA formed the third phase,
respectively.
The effects of octanol on the extraction of water in the DEHPA-ethanolamines
system are also shown in Fig. 2. In the cases of the DEHPA-MEA and DEA systems,
the amount of water extracted into the organic phase reduced by the addition of
octanol in the organic phase, the water extraction increased with an increase in pH
higher than 5.5, and reach constant values. On the other hand, in the cases of the
DEHPA-TEA and Tris systems, the amount of water in the organic phase increased
with an increase in pH value higher than 5.5, reached the maximum values and then
decreased until the almost same value with the MEA and DEA systems. These pH
range with large water extraction would be In reverse micellar system, co-solvent
plays two roles that it can increase the solubility of the surfactant in the organic phase
and it seems to buffer the strong repulsive ion-ion interaction between the surfactant
head groups, thus allowing their close packing to form the inner core of a reversed
micelle. Furthermore, when octanol and TEA were added into the organic phase, the
amount of water extracted was larger, and the pH range that water extracted was
wider than in the case of adding octanol alone. It seems that the repulsive interaction
between the surfactant became weaker by the electrostatic interaction between TEA
and DEHPA.
Extraction property of cytochrome c with the reverse micelles of DEHPA-TEA system
The effect of pH on the extraction of cytochrome c in the mixed and the
DEHPA single systems with octanol is shown in Fig. 3. In the DEHPA single system,
cytochrome c extraction occurred at a narrow pH range around neutral pH. In the pH
range lower than 7, the extracted fraction of cytochrome c into the organic phase, Ef,
was decreased and large amount of aggregates was observed at the interface and in
the aqueous phase. On the other hand, the extraction of cytochrome c in the TEA-
DEHPA mixed system occurred at wider pH range than that in the DEHPA system. In
this pH range, Wo values were also quite high compared with in the DEHPA system.
Ef was decreased with pH values lower than 5.5 which is lower than that in the
ISEC2014 – International Solvent Extraction Conference September 7-11, 2014

DEHPA system. It is consider that the

interaction between DEHPA and TEA
became weaker by the increase in the
nondissociative head group of DEHPA at
this pH range, thus the reverse micelles
were not formed and cytochrome c was
not extracted.
Back-extraction of cytochrome c from the
reverse micelles of TEA-DEHPA system
The back-extraction of cytochrome
c, which was extracted in the DEHPA-
TEA mixed system, was carried out by
contacting with new aqueous phase at
various pH. The effect of pH on the back-
extraction of cytochrome c is shown in
Fig. 4. The back-extraction of cytochrome
c was achieved at pH higher than its pI
value (pI=10.1). Cytochrome c, which has
anionic surface at pH higher than pI,
would be back-extracted by electrostatic Fig. 3 Effect of pH on extraction of cytochrome c
and water in DEHPA –TEA mixed system
repulsion with the anionic head group of
DEHPA. On the other hand, large amount
[DEHPA]=50mM
of aggregated was formed at pH lower [NaCl]=0.1mM
than 9. Cytochrome c would be strongly [TEA]=100mM
[octanol]=0.1M
denatured by strong electrostatic
interaction with DEHPA at the pH range.

Conclusion
The extraction of water and Fig. 4 Effect of pH on back extraction of
cytochrome c using the DEHPA reverse cytochrome c and water in DEHPA –TEA
mixed system
micelles with ethanolamines was
investigated. The formation of reverse micelles was affected by the pH value and the
structure of the ethanolamines. The addition of octanol in the DEHPA-TEA and Tris
systems enhanced the formation and the stability of the reverse micelles. The
DEHPA-TEA mixed system with octanol could extract cytochrome c at wider pH
range compared with that in the DEHPA system. Cytochrome c extracted with the
DEHPA-TEA system could be successfully back-extracted at pH higher than its pI
value.
ISEC2014 – International Solvent Extraction Conference September 7-11, 2014

Reference
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183 (1999)