Advances in Nanoparticles, 2013, 2, 366-371
Published Online November 2013 (http://www.scirp.org/journal/anp)
http://dx.doi.org/10.4236/anp.2013.24050
Open Access ANP
Formation and Solubilization Property of Water-in-Oil
Microemulsions of Alkyl Glucosides
Hidetaka Noritomi1*, Yuki Ishida1, Tomokazu Yama da 1, Hiroaki Saito2, Satoru Kato1
1Department of Applied Chemistry, Tokyo Metropolitan University, Tokyo, Japan
2GUN EI Chemical Industry CO., LTD., Takasaki-shi, Japan
Received August 27, 2013; revised October 8, 2013; accepted October 27, 2013
Copyright © 2013 Hidetaka Noritomi et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The dependence of solubilization properties on the alkyl chain length of alkyl glucosides (AG) in AG/isooctane/
n-butanol/water system was investigated. The stable Winsor II system consisting of a water-in-oil (w/o) microemulsion
phase and an aqueous phase was formed in AG/isooctane/n-butanol/water system. The apparent critical micelle concen-
tration of AG reverse micelles in organic phases was markedly dependent upon the alkyl chain length of AG. The lim-
iting amount of solubilized water increased with an increase in the alkyl chain length of AG. The solubilization capacity
of methyl orange (MO) was superior to that of methylene blue (MB), and the solubilization capacities of MO and MB
tended to increase with increasing the alkyl chain length of AG. Reverse micelles of dodecyl glucoside (AG12) exhib-
ited the significant solubilization capacities of cytochrome c and lysozyme, while ribonuclease A was not solubilized by
AG12 reverse micelles.
Keywords: Microemulsion; Reverse Micelle; Winsor II; Alkyl Glucoside; Solubilization; Organic Dye; Protein
1. Introduction
Microemulsions are so-called soft matter, and are ther-
modynamically stable isotropic dispersions of oil and
water containing domains of nanometer dimensions sta-
bilized by the interfacial film of surface active agents [1].
Especially, water-in-oil (w/o) microemulsions have at-
tracted increasing attention as tools of nanoparticle
preparation, protein separation, protein refolding, en-
zyme-catalyzed conversion, and so on [2]. The w/o mi-
croemulsions containing a small amount of water in their
centers are formed in a hydrophobic organic phase like
octane by reverse micelles, which are thermodynamically
stable nanometer-sized aggregates of surfactant mole-
cules. In order to form reverse micelles exhibiting the
sufficient solubilization efficiency, ionic surfactants such
as cetyltrimethylammonium bromide (CTAB) and so-
dium bis (2-ethylhexyl) sulfosuccinate (AOT) have
mainly been used [3-6]. However, as ionic surfactants
strongly interact with proteins through the electrostatic
force, the structure of proteins is sometimes destabilized
and denatured. Furthermore, when the separation of pro-
teins is carried out by the reverse micellar extraction, it is
often difficult to recover proteins extracted from a feed
aqueous phase to a reverse micellar organic phase. On
the other hand, those surfactants have several types of
toxicity to aquatic organisms and pollute the environment
[7,8]. Thus, the development in reverse micellar systems
having not only sufficient solubilization efficiency but
also excellent biocompatibility and biodegradability has
been desired.
In our previous work, we have reported that reverse
micelles of sucrose fatty acid esters can effectively solu-
bilize proteins, refold denatured proteins, and disperse
highly concentrated metal nanoparticles synthesized in
reverse micelles [9-16]. Sucrose fatty acid esters are
commercial food grade nonionic surfactants, and are
biodegradable and nonhazardous to the environment [17].
Likewise, we have found out that it is possible for re-
verse micelles of dodecyl glucoside (AG12) to solubilize
water-miscible organic dyes, and to prepare silver nano-
particles in reverse micelles [18,19]. Alkyl glucosides
such as AG12 have biodegradability and biocompatibil-
ity, and are widely used as a detergent for dishes and a
shampoo. In the present work, we furthermore investi-
gated the effect of alkyl chain length of alkyl glucosides
*Corresponding author.

H. NORITOMI ET AL. 367
on the solubilization capacities of water, organic dyes,
and proteins in organic solvents.
2. Experimental
2.1. Materials
As a series of alkyl glucoside (AG) surfactants, n-heptyl
glucoside (AG7), n-octyl glucoside (AG8L), 2-ethyl-
hexyl glucoside (AG8B), n-nonyl glucoside (AG9),
n-decyl glucoside (AG10), n-undecyl glucoside (AG11),
and n-dodecyl glucoside (AG12) were supplied from
GUN EI Chemical Industry (Gunma, Japan). The hydro-
philic group of AG consisted of monoglucoside (75%),
diglucoside (20%), and triglucoside (5%). Figure 1
shows the structure of AG. The surfactant was used
without further purification. Isooctane and n-butanol
were from Kanto Chemicals (Tokyo, Japan), and were of
analytical grade. Cytochrome c from bovine heart (MW
= 12,327, pI = 10.5), lysozyme from chicken egg-white
(MW = 14,300, pI = 11.1), and ribonuclease A from bo-
vine pancreas (MW = 13,700, pI = 9.6) were purchased
from Sigma-Aldrich Co. Methyl orange and methylene
blue were obtained from Kanto Chemicals (Tokyo, Ja-
pan).
2.2. Measurement of Limiting Amount of
Solubilized Water
The limiting amount of water solubilized by AG in an
organic phase was measured as follows. Isooctane/n-
butanol (7:3 (v/v)) containing a certain amount of AG
and 0.01 M phosphate buffer solution at pH 7 were con-
tacted in a 1:1 volume ratio at 25˚C and 120 rpm for 1 h.
After the incubation, the organic phase was centrifuged
at 4000 rpm for 30 min. The water concentration of the
resultant organic phase after centrifugation was deter-
mined by the optimized Karl Fisher potentiometric titra-
tion using a Hiranuma AQ-6 aquacounter.
2.3. Measurement of Solubilized Organic Dyes
The extraction of methyl orange or methylene blue was
mainly performed by contacting isooctane/n-butanol (7:3
(v/v)) containing 50 g/L AG and 0.01 M phosphate
buffer solution at pH 7 containing 50 μM methyl orange
OH H
O
H
HO
OH H
O
HH
CH
2
OH
C
n
H
2n+1
(n=7～12)
m(m=1～3)
Figure 1. Structure of alkyl glucoside.
or 50 μM methylene blue in a 1:1 volume ratio at 25˚C
and 120 rpm for 2 h. After extraction, the organic phase
was centrifuged at 4000 rpm for 30 min., and the con-
centration of methyl orange or methylene blue in the or-
ganic phase was measured spectrophotometrically by a
UV/vis spectrophotometer (Ubest-55, Japan Spectro-
scopic Co. Ltd.) as mentioned in our previous report
[18].
2.4. Measurement of Solubilized Proteins
The extraction of cytochrome c, lysozyme, or ribonucle-
ase A was mainly performed by contacting isooc-
tane/n-butanol (7:3 (v/v)) containing 0.12 M AG12 and
0.01 M phosphate buffer solution at pH 7 containing 5
μM cytochrome c, 10 μM lysozyme, or 10 μM ribonu-
clease A in a 1:1 volume ratio at 25˚C and 120 rpm for 2
h. After extraction, the organic phase was centrifuged at
4000 rpm for 30 min., and the concentration of cyto-
chrome c, lysozyme, or ribonuclease A in the organic
phase was measured at 280 nm spectrophotometrically
by a UV/vis spectrophotometer (Ubest-55, Japan Spec-
troscopic Co. Ltd.).
3. Results and Discussion
3.1. Formation Ability of Reverse Micelles by
AG
In order to examine the formation ability of reverse mi-
celles by AG in organic solvents, we have measured the
water concentration solubilized by AG having a different
alkyl chain length as a function of the concentration of
AG at 25˚C. For instance, Figure 2 shows the plot of
water concentration against AG7 concentration in the or-
ganic phase. The water concentration increased abruptly
at 59 mM AG7, followed by a linear increase. Such an-
abrupt increase in the water concentration may be due to
the fact that reverse micelles are formed above 59 mM
AG7 in the organic phase. Thus, the apparent critical
0
500
1000
1500
2000
2500
3000
050100 150 200
Concentration of Water (mM)
Concentration of AG7 (mM)
Figure 2. Dependence of water concentration in the organic
phase containing AG7 on the concentration of AG7. The
incubation was carried out at 25˚C and 120 rpm for 1 h.
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H. NORITOMI ET AL.
368
micelle concentration (cmc) of AG7 is considered to ex-
ist around 59 mM, since the organic phase measured in
the range from 0 to 180 mM AG7 was transparent. Simi-
larly, the apparent critical micelle concentration (cmc) of
AG having a different alkyl chain length was measured.
Figure 3 shows the apparent critical micelle concentra-
tion of AG having a different alkyl chain length. The
sequence of the apparent critical micelle concentration
went as follows: AG8B > AG7 > AG8L > AG9 > AG10
> AG11 > AG12. From the result, the longer alkyl
straight-chain length AG has, the easier reverse micelles
is formed. The formation of AG having alkyl straight-
chain was superior to that of AG having alkyl branched-
chain, since the apparent critical micelle concentration of
AG8L was much smaller than that of AG8B. Thus, it
appears that the bulkiness in the hydrophobic group of
AG8B is not beneficial for the formation of reverse mi-
celles in the present system. The formation and stability
of micelles are due to the structure and/or hydrophile-
lipophile balance (HLB) of surfactants [20-22]. The HLB
of AG decreases with an increase in its alkyl chain length,
and thereby AG becomes more hydrophobic. In general,
lipophilic ionic surfactants such as AOT and CTAB are
used to form stable reverse micelles. The apparent criti-
cal micelle concentration of AOT in cyclohexane ob-
tained from the relationship of the concentration of solu-
bilized water with AOT concentration is 8 mmol·kg−1
[23]. Likewise, concerning nonionic surfactants, the ap-
parent critical micelle concentrations of pentaethylene
glycol monododecyl ether (C12E5) and nonylphenol eth-
oxylate (NP-6) in cyclohexane are 75 and 40 mmol·kg−1,
respectively [24]. The apparent critical micelle concen-
tration of sucrose fatty acid ester (DK-F-110) in isooc-
tane/n-butanol (7:3 (v/v)) is 14 mM [9]. The result indi-
cates that AG has the sufficient formation ability of re-
verse micelles. On the other hand, the extraction system
with reverse micelles has been carried out on the basis of
the formation of the Winsor II system consisting of a w/o
microemulsion phase and an aqueous phase [9]. In the
0
20
40
60
80
100
AG7AG8L AG8BAG9AG10 AG11 AG12
CMC (mM )
Figure 3. Effect of alkyl chain length of AG on the apparent
critical micelle concentration. The incubation was carried
out at 25˚C and 120 rpm for 1 h.
present system, a stable Winsor II system was found to
be formed when an organic solution containing AG and
an aqueous solution was contacted.
3.2. Solubilization Capacity of Water
In order to examine the effect of alkyl chain length of
AG on the solubilization capacity of water by AG, we
have measured the limiting amount of solubilized water
by reverse micelles of AG having a different alkyl chain
length at 25˚C. Figure 4 shows the molar ratio of the
limiting amount of solubilized water to the amount of
AG ([H2O]/[AG]) having a different alkyl chain length.
The [H2O]/[AG] value refers to the solubilization capac-
ity of water by AG. The [H2O]/[AG] value tended to in-
crease with increasing the alkyl chain length of AG ex-
cept AG8B. Particularly, the abrupt increase in the
[H2O]/[AG] value was induced by switching from AG7
to AG8. As seen in Figures 3 and 4, the decrease in the
cmc value corresponds to the increase in the [H2O]/[AG]
value. This indicates that the solubilization capacity of
water by AG is attributable to the formation ability of
reverse micelles by AG.
3.3. Solubilization Capacity of Organic Dyes
A stable Winsor II system is useful for the applications to
the extraction of water miscible compounds such as pro-
teins, DNA, and organic dyes from the feed aqueous
phase and pollutant water [2]. We have reported that
AG12 exhibits the high extraction efficiency of organic
dyes such as methyl orange and methylene blue. In order
to examine the effect of the alkyl chain length of AG on
the solubilization capacity of organic dyes, we have
measured the amount of organic dyes solubilized in the
organic phase of reverse micelles of AG having a differ-
ent alkyl chain length at 25˚C. Figure 5 shows the molar
ratio of the amount of solubilized methyl orange (MO) or
methylene blue (MB) to the amount of AG ([Organic
0
2
4
6
8
10
12
14
16
18
20
AG7AG8LAG8BAG9AG10 AG11 AG12
[H
2
O]/[AG] (-)
Figure 4. Effect of alkyl chain length of AG on the molar
ratio of the limiting amount of solubilized water to the
amount of AG ([H2O]/[AG]). The incubation was carried
out at 25˚C and 120 rpm for 1 h.
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H. NORITOMI ET AL. 369
0
0.00005
0.0001
0.00015
0.0002
0.00025
0.0003
AG7 AG8LAG8BAG9 AG10AG11AG12
[Organic dye]/[AG] (-)
MO
MB
Figure 5. Effect of alkyl chain length of AG on the molar
ratio of the amount of solubilized methyl orange (MO) or
methylene blue (MB) to the amount of AG ([Organic
dye]/[AG]). The incubation was carried out at 25˚C and 120
rpm for 2 h.
dye]/[AG]) having a different alkyl chain length. Re-
garding MO and MB, the [Organic dye]/[AG] value in-
creased with an increase in the alkyl chain length of AG
except AG8B, similar to the case of water. The [Organic
dye]/[AG] value of MO is much greater than that of MB.
MO solubilized in AG reverse micelles tend to be located
in the vicinity of the interface of reverse micelles due to
an amphiphilic form, while MB is mainly solubilized in
the free water of the water pool due to its ionic character
[18]. This indicates that the solubilization of MO is in-
fluenced by the interfacial chemical properties of surfac-
tants, compared to that of MB. Consequently, the differ-
ence of [Organic dye]/[AG] between AG8L and AG8B
for MO was larger than that for MB.
Similarly, it is considered that the formation ability of
reverse micelles affects the dispersibility of solid in or-
ganic solvents. When Ag nanoparticles were synthesized
in reverse micelles of AG having the linear alkyl chain
length except AG7, the resultant Ag nanoparticles were
sufficiently dispersed without the formation of aggrega-
tion, while the aggregation of Ag nanoparticles was im-
mediately observed in reverse micelles of AG7 or AG8B,
as soon as the synthesis started [19].
3.4. Solubilization Capacity of Proteins
As mentioned above, AG12 exhibited the high formation
ability of reverse micelles and the sufficient solubiliza-
tion capacity of solutes, compared to other AG surfac-
tants used in the present work. In order to examine the
solubilization capacity of proteins by AG12, we have
measured the amount of proteins solubilized in the or-
ganic phase of reverse micelles of AG12 at pH 7 and
25˚C. Figure 6 shows the molar ratio of the amount of
solubilized cytochrome c, lysozyme, or ribonuclease A to
the amount of AG12 ([Protein]/[AG12]). The [Pro-
tein]/[AG12] value of cytochrome c was superior to that
of lysozyme. On the other hand, ribonuclease A was not
solubilized. The molecular weights of cytochrome c, ly-
0
0.000005
0.00001
0.000015
0.00002
0.000025
CytochromecLysozyme RibonucleaseA
[P ro te in ]/[AG12]
Figure 6. Effect of kind of proteins on the molar ratio of the
amount of solubilized proteins to the amount of AG12
([Protein]/[AG12]). The incubation was carried out at 25˚C
and 120 rpm for 2 h.
sozyme, and ribonuclease A are almost similar, and the
net charges of those proteins are positive at pH 7, since
pH 7 is less than the isoelectric point (pI) of those pro-
teins, as mentioned in 2.1. Materials. On the other hand,
nonpolar side-chain frequency (NPS) of cytochrome c,
lysozyme, and ribonuclease A are 0.27, 0.26, and 0.23 as
a hydrophobic parameter, respectively [25]. Thus, cyto-
chrome c and lysozyme are hydrophobic, compared to
ribonuclease A. In general, when proteins are solubilized
in the reverse micellar system, cytochrome c and ly-
sozyme are located around the interface between the
layer of surfactant molecules and the water pool of re-
verse micelles, while ribonuclease A is located in the
vicinity of the center of water pools [1,26,27]. The result
indicates that the driving force for the solubilization of
proteins is attributable to the interaction of proteins with
surfactant molecules of AG12 reverse micelles. Thus, the
solubilization capacity of proteins is due to the solubili-
zation site of proteins in reverse micelles, similar to the
case of organic dyes. It is possible that solutes are selec-
tively extracted from feed aqueous solutions on the basis
of the difference of the solubilization site of solutes in
AG reverse micelles.
4. Conclusion
We have demonstrated that AG can form stable Winsor
II system, consisting of a w/o microemulsion phase and
an aqueous phase, and is available for the solubilization
of water-miscible organic dyes and proteins. The alkyl
chain length of AG markedly affected the micelle forma-
tion and solubilization capacity. On the other hand,
branched-chain alkyl group of AG was inferior to its
straight-chain alkyl group concerning the formation of
reverse micelles and the solubilization. AG exhibited the
significant solubilization capacity of MO, compared to
that of MB. Moreover, AG12 showed the difference of
solubilization capacity by a kind of proteins. These re-
sults would be encouraging for its choice in industrial
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H. NORITOMI ET AL.
370
application such as the extraction.
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