Formation and Solubilization Property of Water-in-Oil Microemulsions of Alkyl Glucosides

doi:10.4236/anp.2013.24050

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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

Email: *[email protected]

Received August 27, 2013; revised October 8, 2013; accepted October 27, 2013

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

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

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

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.

Open Access ANP

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

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

AG7AG8LAG8BAG9AG10 AG11 AG12

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

0.00005

0.0001

0.00015

0.0002

0.00025

0.0003

AG7 AG8LAG8BAG9 AG10AG11AG12

[Organic dye]/[AG] (-)

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-

0.000005

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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