Colloids and Surfaces A: Physicochem. Eng.

Aspects 288 (2006) 144150

Optimization of nano-emulsions prepared by low-energy emulsification

methods at constant temperature using a factorial design study
C.M. Pey a , A. Maestro a , I. Sole a,b , C. Gonzalez a , C. Solans b , J.M. Gutierrez a,
a

Departament dEnginyeria Qumica, Universitat de Barcelona, Mart i Franques 1, 08028 Barcelona, Spain
Departament Tecnologia de Tensioactius, Institut dInvestigacions Qumiques i Ambientals de Barcelona (IIQAB),
Consejo Superior de Investigaciones Cientficas (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain
Received 3 October 2005; received in revised form 3 February 2006; accepted 10 February 2006
Available online 6 March 2006

Abstract
The aim of this work is the study and optimization of composition and preparation method of nano-emulsions O/W by addition of one of the
components at constant temperature. Experimental design techniques have been used to carry out this study. A factorial design has been done in
order to investigate the effect of formulation and preparation variables over emulsion properties. The conclusion of this study is that emulsion
droplet size and polydispersity change with composition and preparation method. These variables have been optimized using a central composite
design obtaining response surfaces that describe this preparation method of nano-emulsions.
2006 Elsevier B.V. All rights reserved.
Keywords: Nano-emulsions; Emulsification method; O/W emulsion; Full factorial design; Response surface methodology

1. Introduction
Nano-emulsions, also referred to in the literature as
miniemulsions [13], ultrafine emulsions [4,5], emulsoids [6,7],
unstable microemulsions [8], submicrometer emulsions [9,10],
. . ., are a class of emulsions with very small and uniform
droplet size, typically in the range of 20500 nm. Due to their
small droplet size, they may appear transparent or translucent,
resembling microemulsions. However, in contrast to microemulsions, they are not thermodynamically stable, i.e. they are
not equilibrium phases and the size of the droplets tends
to increase with time, before phase separation. Nevertheless,
the small droplet size makes them stable for a long time
against sedimentation and creaming, hence offering increased
stability.
The characteristic properties of nano-emulsions are interesting for practical applications. Nano-emulsions are used in
cosmetics as personal-care formulations [1113], in agrochemicals for pesticide delivery [14,15], in chemical industry for the
preparation of latex particles [1618], etc. They can be used in

Corresponding author. Tel.: +93 402 12 92; fax: +93 402 12 91.
E-mail address: josemaria.gutierrez@ub.edu (J.M. Gutierrez).

0927-7757/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfa.2006.02.026

pharmaceutical field as drug delivery systems [1921] for parenteral, oral, ocular or transdermal administration.
A direct consequence of the thermodynamic instability of
nano-emulsions is the dependence of their properties on the
preparation method. Nano-emulsions can be achieved either by
high-energy emulsification methods (e.g., high-pressure homogenization) or by low-energy emulsification methods which
employ the physicochemical properties of the system [22]. These
methods make use of changing the spontaneous curvature of surfactant with temperature for nonionic surfactants called phase
inversion temperature (PIT) method and introduced by Shinoda
and Saito [23] or with the variation of the volume fraction of
one of the components, emulsion inversion point (EIP) method
[24].
In recent years, some studies have been carried out on
the mechanism of formation of nano-emulsions by low-energy
emulsification methods [2530]. These studies show the relationship between nano-emulsions properties and system components, some aspects of preparation method and influence of
phases which are present during the phase inversion. In the case
of O/W nano-emulsions, the main requirement for the formation
of bluish transparent or translucent emulsion is the presence of
bicontinuous microemulsion or lamellar liquid crystalline phase
during the emulsification process where a complete solubiliza-

C.M. Pey et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 288 (2006) 144150

tion of the oil phase exists [28,29]. Depending on the preparation

method, different droplet size distributions might be achieved,
explaining why the route of preparation can have an influence
on the emulsion stability.
However, despite the formation mechanism and the effect of
different variables on the nano-emulsions properties prepared
by low-energy emulsification methods have been studied before,
there are few systematic studies dealing with the effect of formation and composition variables and, in these, all of the variables
are held constant during test runs except the one being studied,
following the traditional method of experimentation evaluating
only one variable (or factor) at time. This type of experiment
reveals the effect of the chosen variable under set conditions
assuming that variables are independent and that the effect will
be the same at another level of the remaining variables. It does
not show what would happen if other variables are also changed.
In these cases, experimental design is an effective and efficient
optimization strategy to overcome these drawbacks which has
found widespread application in all branches. Some basic statistical methods are also developed to use in different techniques
of experimental design.
Experimental design allows to estimate the effects of several variables simultaneously. The significant factors affecting
the nano-emulsion properties can be deduced, in a screening
study, by applying a full factorial design, a powerful tool commonly used in exploratory studies. Response surface methodology (RSM), which consists in a group of mathematical and
statistical techniques, is useful in the modeling and analysis
of processes in which a response of interests, such as droplet
size, is simultaneously influenced by several significant variables [3133].
The aim of this work was to study by using experimental design methodology the influence of different variables and to evaluate simultaneous effect of more significant
variables on droplet size of O/W nano-emulsions of system
waterTween20/Span20liquid paraffin prepared by the stepwise addition of one component to the others at constant temperature. The experimental conditions selected were then applied
in order to obtain a minimum droplet diameter.
2. Experimental
2.1. Materials
Sorbitan monolaurate (Span20 (S20), NHLB = 8.6), and
polyoxyethylene sorbitan monolaurate (Tween20 (T20),
NHLB = 16.7), technical grade surfactants, were purchased
from Sigma-Aldrich. Liquid paraffin was obtained from Merck
KGaA, Darmstadt, Germany. It was a mixture of C18 to C40
linear chains, with a density 0.85 g/ml. All products were
used without further purification. Water was desionized and
further purified by Milli-Q filtration.

145

equilibrate during a time that depended on the viscosity of the

phases present (from a few hours to several weeks). Compositions are expressed in wt.% ratio between components and also
as w/w ratio. The boundary lines drawn on the phase diagram
lie equidistant between consecutive experimental measurements
on either side of the phase boundary. Anisotropic phases were
identified visually under polarized light.
2.3. Emulsion formation
Emulsions were prepared by continuous addition of water
with a dosing pump at 25 C to the surfactant-oil mixture previously homogenized and continuously mixed with a helix mixer.
The addition was carried out under controlled mixing and addition rates. The final water composition was always 70% w/w.
2.4. Droplet size and polydispersity
Emulsion droplet size and polydispersity (intensity based
size distributions) were measured by photon correlation spectroscopy (PCS) using Malvern Zetasizer ZS at 25 C. Samples
were diluted with water for the measurements.
2.5. Software
STATGRAPHICS (Statistical Institution Edition Version 4.1,
Statistical Graphics Co., Rockville, MD, USA) was used.
2.6. Variable screening
The influence of different variables and their interactions
on emulsion properties was checked by using experimental
design. In fast screening studies, linear or second order interaction models are common. Four variables were considered
addition time, mixing rate, O/S ratio and Tween20/Span20 relation (%Tween20). The droplet size was chosen as the main
response since it determines whether an emulsion is a nanoemulsion or not.
To ascertain the individual effects of these variables on emulsion droplet size, a full factorial design for four variables at two
levels was applied. This experimental design requires one experiment at all possible combinations between two levels of each
variable considered. In general, the necessary number of experiments is 2k , where k is the number of factors.
Different replicates were carried out in different days, preparing again each mixture from pure components, so a total 32 runs
were performed. The experimental variables considered and the
design matrix are shown in Table 1. All points shown represent
the average of measurements done at same experimental conditions. The design matrix was generated and results evaluated by
using Statgraphics V4.1 software.

2.2. Phase diagrams

2.7. Simultaneous effect of the selected variables on the

emulsion properties

All components were weighted and mixed with a Heidolph

REAX Top vibromixer. Then, samples were kept at 25 C to

The preparation method is controlled by different variables

that can interact. One experimental approach is to apply the

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C.M. Pey et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 288 (2006) 144150

Table 1
Full factorial design matrix of screening experiments and mean droplet diameter
measured

Table 3
Experimental field for a design matrix: variables and emulsion properties
measured

Run

%Tween20

O/S

Addition
time (min)

Agitation
rate (rpm)

Droplet
diameter (nm)

Run

Addition rate
(ml/min)

Agitation
rate (rpm)

Droplet
diameter (nm)

Polydispersity

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

48.2
51.2
48.2
51.2
48.2
51.2
48.2
51.2
48.2
51.2
48.2
51.2
48.2
51.2
48.2
51.2

2.0
2.0
3.0
3.0
2.0
2.0
3.0
3.0
2.0
2.0
3.0
3.0
2.0
2.0
3.0
3.0

51.3
51.3
51.3
51.3
4.3
4.3
4.3
4.3
51.3
51.3
51.3
51.3
4.3
4.3
4.3
4.3

200
200
200
200
200
200
200
200
700
700
700
700
700
700
700
700

81.7
94.1
145.0
164.0
98.2
94.9
139.3
149.1
80.6
102.1
144.3
173.1
86.0
90.1
143.6
158.2

1
2
3
4
5
6
7
8
9
10
11

4.0
4.0
16.0
16.0
1.5
18.5
10.0
10.0
10.0
10.0
10.0

250
550
250
550
400
400
188
612
400
400
400

93.3
87.3
106.1
91.4
78.9
94.9
91.2
101.4
92.1
91.8
92.3

0.146
0.141
0.173
0.131
0.102
0.130
0.146
0.161
0.140
0.120
0.125

cated. Statgraphics software was used to obtain the combination

of values that draw the surface response.
3. Results and discussion

response surface methodology (RSM) for modeling emulsion

droplet size and polydispersity as a function of selected variables. Two different central composite design (CCD), with k = 2,
were used in order to generate 11 treatment combinations, one
with O/S ratio and Tween20/Span20 relation as independent
variables holding constant preparation conditions, an another
with mixing and addition rates as independent variables at a
constant composition. In this case, five levels of each variable
were studied.
In the statistical model, Y denotes different emulsion properties and x1 and x2 the two independent variables studied.
Tables 2 and 3 show the actual levels corresponding to the coded
settings, the treatment combinations and responses of two central
composite designs that have been done. This design is represented by a second-order polynomial regression model, Eq. (1),
to generate contour plots:
Y = b0 + b1 x1 + b2 x2 + b11 x12 + b22 x22 + b12 x1 x2 + .

(1)

The experiment at the centre of the experimental field was

performed three times and some more runs have been also repliTable 2
Experimental field for a design matrix: variables and emulsion properties
measured
Run

%Tween20

O/S

Droplet diameter (nm)

Polydispersity

1
2
3
4
5
6
7
8
9
10
11

45.1
45.1
51.2
51.2
43.8
52.5
48.2
48.2
48.2
48.2
48.2

2.00
3.00
2.00
3.00
2.50
2.50
1.79
3.21
2.50
2.50
2.50

121.6
174.5
97.0
160.6
148.0
141.6
76.0
148.2
111.0
111.1
108.8

0.174
0.302
0.117
0.117
0.225
0.134
0.092
0.143
0.131
0.100
0.126

The application of different experimental design techniques

allowed programming an experimental strategy to study and
optimize the preparation method of O/W nano-emulsions of
waterTween20/Span20liquid paraffin prepared by addition of
one component at constant temperature.
3.1. Factor screening
Some parameters were preliminary considered in order to
define the experimental field. Variables such as oil and surfactants ratio (O/S) and Tween20/Span20 relation (%Tween20)
were selected in order to obtain nano-emulsions stable enough
to have time to measure their droplet size and polydisersity. Mixing rate and addition time were selected using the same criteria,
taking into account that emulsion properties, as thermodynamic
unstable systems, depend on preparation method.
Once the variables and their experimental field were selected,
the influence of variables and their interactions on droplet size
was checked. Estimated effects and interactions can be seen
in Table 4, which also shows the standard error corresponding to each effect. Effects and interactions are defined as the
differences between the mean droplet sizes obtained at the low
level and the mean droplet size obtained at the high level of
each variable. It is necessary to determine which differences are
significantly different from zero and which are due to the experimental error. A statistical method is used based in analysis of
the variance (ANOVA) which determines the statistical signification of effects and interactions comparing the mean square
with an estimation of experimental error. Raw data have been
used to do the ANOVA analysis. In this case, five effects and
interactions have P-values less than 0.05, indicating that they
are significantly different from zero at the 95.0% confidence
level (Table 5).
Comparing the mean square against this estimation, only two
factors appeared to be significant: O/S ratio and %Tween20, both

C.M. Pey et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 288 (2006) 144150

147

Table 4
Effects corresponding to factors and interactions obtained by full factorial design
Factor/interaction

Effect

Average
A: %Tween20
B: O/S relation
C: addition time
D: agitation rate
AB
AC
AD
BC
BD
CD
ABC
ABD
ACD
BCD
ABCD

122.8
13.3
62.4
2.2
2.9
5.9
10.6
5.0
7.0
7.6
3.8
0.3
1.3
2.0
3.9
3.2

1.4
2.8
2.8
2.8
2.8
2.8
2.8
2.8
2.8
2.8
2.8
2.8
2.8
2.8
2.8
2.8

Fig. 1. Droplet diameter function of addition time and mixing rate for two different compositions.

having a positive influence on the response, which was not completely clear in Table 1, especially for %Tween20. So, when
doubts exist, this full factorial analysis is capable to clarify if
one effect influences the response or not. The study also shows
that there were significant interactions between some factors:
%Tween20 and addition time (BC), O/S ratio and addition time
(BD), O/S ratio and mixing rate (AC). Therefore, this analysis concludes that the rest of effects and interactions are due to
experimental error. So preparation variables seemed to have no
influence on emulsion properties. Instead of these results, significant differences are observed between emulsions prepared on
different experimental conditions. However, this effect is dependent on the composition studied, as it is shown in Fig. 1. As an
example, Fig. 1 shows that addition time, that has no influence
according to ANOVA analysis, has a negative influence when
working at 48.2% Tween and O/S 2:1 (a smaller droplet size
is obtained). However, it has a positive influence when samples

with 51.2% Tween20 and O/S 3:1 (composition that gives less
stable emulsions) are used, because if the addition time is very
long these relatively unstable samples start to destabilize. Consequently, addition time have contrary effects depending on the
composition of the samples, that are compensated in the ANOVA
analysis, and it seems that it does not influence the properties of
the nano-emulsions. This is a limitation of ANOVA, and results
cannot be taken blindly, without being accurately analyzed.
The results from this first step led to study and optimize
firstly the formulation variables at constant preparation conditions. Then the study and optimization of preparation variables
on emulsion properties has been done for one optimal composition.
3.2. Study and optimization of formulation variables
A central composite design has been done in order to study
and optimize the final composition of O/W emulsions for the system waterTween20/Span20liquid paraffin at constant preparation conditions and final continuous phase (400 rpm, 33 min,

Table 5
ANOVA on the data obtained using full factorial design
Sum of squares

d.f.

A: %Tween20
B: O/S relation
C: addition time
D: agitation rate
AB
AC
AD
BC
BD
CD
ABC
ABD
ACD
BCD
ABCD

924.2
20365.8
25.0
43.6
184.1
589.2
130.9
255.7
298.5
73.5
0.4
8.3
21.7
77.5
53.9

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

Total error
Total (correlation)

360.936
28182.5

9
24

Mean square

F-ratio

P-value

924.167
20365.8
25.0441
43.6182
184.051
589.212
130.925
255.707
298.479
73.4837
0.368767
8.26905
21.6635
77.5385
53.8878

23.04
507.82
0.62
1.09
4.59
14.69
3.26
6.38
7.44
1.83
0.01
0.21
0.54
1.93
1.34

0.001
0.000
0.450
0.324
0.061
0.004
0.104
0.033
0.023
0.209
0.926
0.661
0.481
0.198
0.276

40.104

R2 = 0.9872; R2 (ad) = 0.9658; standard error of estimation = 6.33277; mean absolute error = 2.18777; F-ratio: MS factor/MS error.

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C.M. Pey et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 288 (2006) 144150

Fig. 2. Response surface: droplet diameter as a function of formulation variables.

70%W). In this case, the range of %Tween and O/S has been
slightly extended (Table 2). The results of this study show that
experimental response as a function of O/S ratio and %Tween20
can be approximated by a quadratic equation, the term (O/S)2
having no significance. The crossed term (Tween20)(O/S),
which was found no significant when experiments on Table 1
were analyzed, was found significant in this case, when range
was extended. It indicates that results are only valid into the
experimental range. The equation which describes droplet size
in terms of the significant variables (i.e. effects with P-values
lower than 0.05) is as follows:


droplet diameter (nm) = a + b (%Tween20) + c O
S
+ d (%Tween20)2


+ e (Tween20) O
S

(2)

Fig. 3. Droplet size as a function of %Tween20 for one OS ratio. Quadratic

model.

Fig. 4. Droplet size as a function of OS ratio for one %Tween20. Quadratic

model.

a = 5893.3 1055.9; b = 231.6 47.0;

c = 176.8 157.6; d = 2.3 0.5; e = 4.7 3.2.
The R2 of this equation is 99.21%, indicating that the fitting
is really good. Variances of every factor and their importance
can be obtained by the F-test method showing only significant parameters at 95% of confidence level. Fig. 2 shows
response surface developed by the model for O/S ratio and
Tween20/Span20 relation. These graphs offer a visual means of
understanding how factors influence the measurement system.
In this case, Tween20/Span20 relation has a non-lineal behavior (a quadratic dependence) showing an optimal relation which
minimizes the droplet size (Fig. 2). This optimal relation, i.e.
the minimum is dependent on the O/S ratio due to the existence
of a light interaction between these two variables. Fig. 3 shows
an example of a cross sectional cut of the Fig. 2 where O/S
ratio was maintained constant, and here the optimum can also
be observed. Referring to O/S ratio, an almost lineal behavior
was observed denoting that the smaller O/S ratio, the smaller
diameter is obtained (Fig. 2). An example of a cross section of
Fig. 2 where the %Tween20 was maintained constant can be seen
in Fig. 4. This figure shows that points can be nearly described
by a straight line.
In order to validate the empiric model, some more experimental data were obtained and added to the Figs. 3 and 4. Results
denote that the empiric model describes quite well the behaviour

of the system because most of experimental data are between the

confidence levels of the model at 95%.
Some studies of phase transitions during emulsification process have been done to explain these results. Fig. 5 shows an
example of a partial phase diagram at constant %Tween20,
where the phases that are crossed during emulsification process

Fig. 5. Phase diagram. Emulsification paths.

C.M. Pey et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 288 (2006) 144150

149

for different emulsification paths at this relation have been determined. The diagram shows the presence of a two-phase region
near the 0% water line, as these surfactants are insoluble in paraffin. When a small amount of water is added (around 23%) a
water-in-oil microemulsion appears (Om ). Higher amounts of
water cause the appearance of a multiphase region where liquid
crystalline phases have been identified under polarized light.
This region is wide and extends to relatively high concentrations of water at the highest concentrations of surfactants tested.
When the O/S ratio is increased the multiphase area progressively narrows, and disappears at 3:1 O/S. When more water
is added, a two-phase region (oil-in-water microemulsion Wm
and free oil O), appears. Is into this zone, at 70% water, where
nano-emulsions are formed. Best results (small droplet size and
low polydispersity) were obtained when the multiphase area was
present at higher percentage of water, i.e., at the highest concentrations of surfactants. If one tries to extend the O/S ratio further
than 3:1, nano-emulsions cannot be formed, as any liquid crystal
is not crossed along the emulsification path, and it seems to be
necessary [22,29].

surface developed by the model for mixing and addition rate.

Droplet size decreases when addition rate of water over the
mixture of oil and surfactants is slow. Emulsions with small
droplet size are obtained if, during the emulsification path, the
equilibrium is reached in a zone with liquid crystal phases or
bicontinuous phases, with as much of oil as possible solubilized in [22,29]. Then, when the nano-emulsion is formed, the
oil is intimate mixed with the rest of components and it has
just to be redistributed. However, the presence of liquid crystal phases confers more viscosity to the system due to the
presence of an organized structure, and it means that a good
mixture necessary for equilibrium requires enough time and
high mixing rate. So, high mixing rates are useful in order
to obtain these phases transitions. Otherwise, when the multiphase zone is crossed and we enter into the Wm + O zone,
where emulsion droplets are formed, the viscosity of the system is drastically decreased. Then, if the mixing rate is too
high, it could promote some destabilization mechanisms like
coalescence, sedimentation, . . . resulting a higher final droplet
size.

3.3. Study and optimization of the effect of preparation

variables on emulsion properties

4. Conclusions

A central composite design has been done to study and optimize the preparation variables of emulsions O/W at a constant
composition (48.2% Tween20, 2:1 O/S ratio). In this case, two
preparation variables are studied: addition and mixing rate. For
the experimental data (Table 3), regression technique was used
to fit the parameters to a response surface. The Eq. (2) describes
the droplet size in terms of preparation variables, only showing
significant parameters (with P-values lower than 0.05) at 95%
of confidence level:
droplet diameter (nm) = a + b (addition rate) + c (mixing rate)
+ d (mixing rate)2

(3)

a = 117.922 20.208; b = 0.823 0.423;

c = 0.150 0.104; d = 1.54 104 1.28 104 .
The fitting of this equation is not very good (R2 = 80.99%).
However, it is capable to qualitatively explain the dependence of
droplet size on formation variables. Fig. 6 shows the response

Experimental design is a good tool to study and optimize

nano-emulsion properties prepared by low-energy emulsification methods. Eq. (2) fits experimental results related to composition variables. A linear dependence of the droplet size with
O/S was obtained, according to the fact that smaller droplet
sizes were obtained when a higher amount of surfactant was
present, since the multiphase zone with liquid crystal crossed
during emulsification was extended to higher concentrations of
water. A quadratic dependence of droplet size on %Tween20
was found, according to an optimal Tween20/Span20 relation
(related to an optimal HLB number, NHLB ). Eq. (2) predicts
a slight interaction between OS and %Tween, and it can be
explained because the oil present preferentially solubilizes the
Span20 and, as a result, changes the effective Tween20/Span20
relation.
Although Eq. (3) does not fit results so well as Eq. (2), it
qualitatively predicts experimental results, according to emulsification mechanism. A linear dependence of droplet size on
addition rate was obtained, the best nano-emulsions found at
the lowest addition rates. At lower addition rates the liquid
crystalline phases with oil solubilized, necessary for a good
emulsification, have more time to be appropriately formed. A
quadratic dependence of droplet size on mixing rate was found,
related to an optimal mixing rate. Small droplet size were,
then, obtained when mixing rate was the adequate to obtain liquid crystalline phases without promoting some destabilization
mechanisms when emulsion droplets were formed during the
emulsification process.
Acknowledgment

Fig. 6. Response surface: droplet diameter as a function of preparation variables.

This work was funded under Spanish MCYT Project No.

PPQ2002-04514-C03-02.

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References
[1] J. Ugelstad, M.S. El-Aasser, J.W. Vanderhoff, J. Polym. Sci. Polym.
Lett. 11 (1973) 503513.
[2] M.S. El-Aasser, C.D. Lack, Y.T. Choi, J. Colloids Surf. 12 (1984) 7997.
[3] M.S. El-Aasser, C.D. Lack, J.W. Vanderhoff, F. Fowkes, J. Colloids Surf.
29 (1988) 103118.
[4] H. Nakajima, in: C. Solans, H. Kunieda (Eds.), Industrial Applications of Microemulsions, 66, Marcel Dekker, New York, 1997,
pp. 175197.
[5] H. Nakajima, S. Tomomasa, M. Okabe, Premier Congres Mondial de

lEmulsion,
Paris, France, 1993, Paper No. 1-11-162.
[6] F. Lachampt, R.M. Vila, Parfum. Cosmet. Savons 10 (1967) 372382.
[7] F. Lachampt, R.M. Vila, Parfum. Cosmet. Savons 12 (1969) 239251.
[8] H.L. Rosano, T. Lan, A. Weiss, J.H. Whittam, W.E.F. Gerbacia, J. Phys.
Chem. 85 (1981) 468473.
[9] S. Benita, M. Levy, J. Pharm. Sci. 82 (1993) 10691079.
[10] S. Benita, in: A.T. Florence, G. Gregoriadis (Eds.), Submicron Emulsions in Drug Targeting and Delivery, 9, Harwood Academic Publishers,
Amsterdam, 1998.
[11] H. Sagitani, J. Am. Oil. Chem. Soc. 58 (1981) 738.
[12] S. Restle, D. Cauwet-Martin, Eur. Patent Appl. EP 842652 A1, 1998.
[13] H. Lorenz, H.R. Wagner, A. Kawamata, Ger. Offen. DE 19735851 A1,
1999.
[14] G.W.J. Lee, Th.F. Tadros, Colloids Surf. A 5 (1982) 105115.
[15] D.I. Jon, D.I. Prettypaul, M.J. Benning, R.M. Narayanan, Ianiello, Int.
Patent Appl. WO 9919256 A2, 1999.
[16] P.I. Blyte, A. Kleim, J.A. Phillips, E.L. Sudol, M.S. El-Aasser, J. Polym.
Sci. A: Polym. Chem. 37 (1999) 4449.
[17] M. Antonietti, K. Landfester, Ger. Offen. DE 19852784 A1, 2000.
[18] S.T. Wang, F.J. Schork, G.W. Poelhein, J.W. Gooch, J. Appl. Polym.
Sci. 60 (1996) 2069.

[19] P. Calvo, J. Remuna n-Lopez, J.L. Vila-Jato, M.J. Alonso, Colloid Polym.
Sci. 275 (1997) 46.
[20] M. Baluom, D.I. Friedman, A. Rubinstein, Int. J. Pharm. 154 (1997)
235.
[21] M. Sznitowska, K. Zurowska-Pryczkowska, E. Dabrowska, S. Janicki,
Int. J. Pharm. 202 (2000) 161.
[22] C. Solans, J. Esquena, A. Forgiarini, P. Izquierdo, D. Morales, N. Uson,
N. Azemar, M.J. Garcia-Celma, Surfactants in solution: fundamentals
and applications, in: K.L. Mittal, D. Shah (Eds.), Surfactant Science
Series, Marcel Dekker, New York, 2002.
[23] K. Shinoda, H. Saito, J. Colloid Interf. Sci. 26 (1968) 70.
[24] L. Marshall, Nonionic surfactants, in: M.J. Shick (Ed.), in: Surfactant
Sciences Series, vol. 23, Marcel Dekker, New York, 1987, pp. 493547.
[25] P. Izquierdo, J. Feng, J. Esquena, Th.F. Tadros, C. Dederen, M.J. Garcia,
N. Azemar, C. Solans, J. Colloid Interf. Sci. 285 (1) (2005) 388394.
[26] P. Izquierdo, J. Esquena, Th.F. Tadros, C. Dederen, M.J. Garcia, N.
Azemar, C. Solans, Langmuir 18 (2002) 2630.
[27] A. Forgiarini, J. Esquena, C. Gonzalez, C. Solans, Prog. Colloid Polym.
Sci. 115 (2000) 3640.
[28] A. Forgiarini, J. Esquena, C. Gonzalez, C. Solans, Langmuir 17 (2001)
20762083.
[29] D. Morales, J.M. Gutierrez, M.J. Garca-Celma, C. Solans, Langmuir
19 (2003) 7196.
[30] N. Uson, M.J. Garcia, C. Solans, Colloids Surf. A: Physicochem. Eng.
Aspects 250 (2004) 415421.
[31] G.E.P. Box, J.S. Hunter, Estadstica para Investigadores, Introduccion al
diseno de experimentos, analisis de datos y construccion de modelos,
1999, Ed. Reverte.
[32] D.C. Montgomery, Design and Analysis of Experiments, fifth ed., Willey,
2001.
[33] A. Prat, X. Tort-Martorell, P. Grima, L. Pozueta, Metodos Estadsticos,
Control y mejora de la calidad, 1997, Ed. UPC.