Journal of Dispersion Science and Technology

ISSN: 0193-2691 (Print) 1532-2351 (Online) Journal homepage: https://www.tandfonline.com/loi/ldis20

Effect of dilution on particle size analysis of w/o

emulsions by dynamic light scattering

Mathieu Balcaen, Lorenz De Neve, Koen Dewettinck & Paul Van der Meeren

To cite this article: Mathieu Balcaen, Lorenz De Neve, Koen Dewettinck & Paul Van der Meeren
(2020): Effect of dilution on particle size analysis of w/o emulsions by dynamic light scattering,
Journal of Dispersion Science and Technology, DOI: 10.1080/01932691.2020.1712216

To link to this article: https://doi.org/10.1080/01932691.2020.1712216

Published online: 11 Jan 2020.

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JOURNAL OF DISPERSION SCIENCE AND TECHNOLOGY
https://doi.org/10.1080/01932691.2020.1712216

Effect of dilution on particle size analysis of w/o emulsions by dynamic

light scattering
Mathieu Balcaena, Lorenz De Nevea, Koen Dewettinckb , and Paul Van der Meerena
a
Particle and Interfacial Technology Group, Department of Green Chemistry and Technology, Ghent University, Ghent, Belgium; bLaboratory
of Food Technology and Engineering, Department of Food Technology, Safety and Health, Ghent University, Ghent, Belgium

ABSTRACT ARTICLE HISTORY

Dynamic Light Scattering (DLS) is a well-known (sub)micron particle size analysis technique. As such, Received 13 December 2019
it should be a valuable technique for water droplet size determination in (sub)micron water-in-oil Accepted 30 December 2019
emulsions. In this study, the sample pretreatment conditions were optimized in order to enable accur-
KEYWORDS
ate droplet size determination by DLS. Whereas DLS was indeed capable of detecting differences
DLS; w/o emulsions;
between differently produced (sub)micron sized w/o emulsions, it was observed that the estimated droplet sizing
droplet size was highly influenced by the measurement conditions used. First of all, the experimen-
tally determined diffusion coefficient decreased with increasing water droplet concentration, which is
thought to be due to interaction effects between the water droplets. To minimize this effect, the used
concentration should be as low as possible. However, in doing so, the water droplets were frequently
observed to disappear during storage or even during analysis, due to their dissolution in the dilution
medium. The latter disturbing effect could be avoided by (sufficient) water saturation of the dilution
medium and preventing water evaporation by closing the diluted sample and by restricting the head
space. Summarizing, (sub)micron water droplets can be characterized using DLS. However, careful
selection of the sample preparation and measurement conditions is necessary to obtain reliable data.

GRAPHICAL ABSTRACT

1. Introduction diluted with water, which is an essential, but quite straightfor-

Emulsions are used in a wide variety of industrial applications,
such as food, cosmetics and pharmaceuticals. These two-phase
systems are most often a mixture of a polar and an apolar
liquid, whereby one phase is dispersed as droplets in the other
one. Hereby, the dispersed droplets are stabilized by emulsi-
fiers.[1] When the apolar liquid (e.g., a triglyceride oil) is dis-
persed in the polar liquid (e.g., water), an oil-in-water (o/w)
emulsion is obtained. Intravenous emulsions are typical sub-
micron o/w emulsions.[2] This emulsion type can easily be
ward sample pretreatment step in particle size analysis. On the
other hand, emulsions in which the polar phase is dispersed
into the apolar phase, so-called water-in-oil (w/o) emulsions,
are much less frequently studied.
Emulsion properties such as rheology, texture, appear-
ance, flavor, release rate of encapsulated compounds and
stability against sedimentation/creaming, droplet coalescence
and microbiological growth, are all influenced by the size of
the dispersed droplets.[1,3–5] The importance of droplet size

CONTACT Paul Van der Meeren Paul.VanderMeeren@UGent.be Particle and Interfacial Technology Group, Department of Green Chemistry and
Technology, Ghent University, Ghent, Belgium
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ldis.
ß 2020 Taylor & Francis Group, LLC
2 M. BALCAEN ET AL.
with respect to a range of emulsion properties renders
accurate droplet size determination techniques indispensable.
As most droplet size analysis techniques rely on laser light
scattering, dilution is mostly an essential sample pretreat-
ment step.[1,6] Whereas PFG-NMR diffusometry does not
require dilution of the sample, we showed in previous
research that it cannot be used to determine the droplet size
of w/o emulsions in case (sub)micron sized water droplets
are present.[7]
Dynamic light scattering (DLS) is an interesting tech-
nique to characterize (sub)micron sized w/o emulsions as it
only requires knowledge of the viscosity and refractive index
of the continuous phase.[8] Due to the fact that the
Brownian motion of droplets is followed during a DLS-
measurement, less viscous dilution media than the oil phase
of the w/o emulsion itself are frequently preferred for DLS
sample pretreatment. Typical examples include hexadecane,
dodecane and dodecane containing 0.5 wt% sorbitan mono-
oleate.[9–12] In this context, it should be mentioned that the
used dilution medium has an influence on the interactions
between water droplets. According to Leal-Calderon et al.
(1997), changing the continuous phase can lead to non-
flocculated w/o emulsions turning into flocculated ones or
vice versa.[13] This property can also be used as an advan-
tage: as light scattering techniques will measure the size of
the flocs if present, diluting a flocculated sample into an
appropriate dilution liquid might enable the determination
of the size of the individual droplets.[14] Actually, in order
to dilute without affecting the composition of the continu-
ous and dispersed phase, the dilution liquid should not only
contain the apolar liquid, but should be in equilibrium with
the polar liquid and contain emulsifier in the same concen-
tration as the continuous phase of the emulsion; the latter is
lower than the added concentration due to adsorption at the
droplet interface. As mentioned by Bahtz et al., spontaneous
emulsification occurs when an oil phase containing PGPR is
brought into contact with a water phase.[15] Thus, also tak-
ing into account the selective adsorption of certain species
present in commercial emulsifier mixtures, it is impossible
to formulate an ideal dilution liquid. The dilution liquid
should therefore be best prepared from the emulsion itself
by removal of the dispersed phase.
In the current study, the possibility of the DLS technique
to accurately determine the droplet size of w/o emulsions
was checked. To this end, the influence of different sample
pretreatment conditions on the droplet size determination
by DLS was investigated. First of all, we examined whether
the limited solubility of water in the oil phase influenced the
measurement when emulsions were diluted in the continu-
ous oil phase.[16]
Subsequently, the influence of the used dilution medium
was checked by comparing 1-bromododecane to the con-
tinuous oil phase.
Finally, the optimal measurement conditions were used
in the determination of the droplet size distribution of three
w/o emulsions which were prepared using a varying shear
intensity, thus having a different droplet size distribution.
2. Materials and methods
2.1. Materials
The lipophilic emulsifier polyglycerol polyricinoleate (PGPR
4175) was kindly provided by Palsgaard A/S (Juelsminde,
Denmark) and high oleic sunflower oil (Hoso; Iodine Value
¼ 87; 82% C18:1) was acquired from Contined BV
(Wageningen, The Netherlands). The water phase of the w/o
emulsions contained NaCl (0.1 M) (VWR Chemicals: BDH
Prolabo, Leuven, Belgium) and the anti-microbial agent
NaN3 (0.02 wt%) (Sigma-Aldrich, Steinheim, Germany).
As dilution media, high oleic sunflower oil (Contined
BV, Wageningen, The Netherlands), 1-bromododecane (Alfa
Aesar, Kandel, Germany) and dodecane (mixture of isomers,
Acros Organics, Geel, Belgium) were used.
2.2. Preparation of the w/o emulsions
PGPR 4175 (10 wt%) was added to Hoso after which this
phase was heated to 60  C and stirred to obtain a homoge-
neous solution. The water phase was also heated to 60  C
after which the emulsions were prepared.
In a first production scheme, the 50/50 (m m1) emul-
sions were prepared using a rotor-stator homogenizer
(Ultra-Turrax: type S25-10G, IKAV R -Werke, Germany).
Hereby, the water phase was added gradually during mixing.
Two different mixing intensities were used namely 6500 and
24,000 rpm. These emulsions are referred to as UT 6500 and
UT 24000, respectively.
In a second production scheme, a premix was prepared
using the same rotor stator homogenizer (Ultra-Turrax: type
S25-10G, IKAV R -Werke, Germany). Hereby, the water phase
was added gradually during mixing at 24,000 rpm.
Afterwards, the emulsion was further processed using 1 pass
through a Microfluidizer (type M110S, Microfluidics) at
840 bar during which the temperature was kept at 60  C
using a heating bath. This emulsion is referred to as
MF 840.
2.3. Water saturation of dilution media
The dilution media were saturated with water by adding dis-
tilled water (20% (v v1)) to the respective dilution media
(80% (v v1)) and end-over-end rotating this mixture using
a Stuart SB3 rotator (Cole-Palmer, Staffordshire, UK) for at
least 4 hours at 20  C and 2 rpm. Both phases were subse-
quently separated through centrifugation using a Sigma 3-
16 P device (Osterode am Harz, Germany) at 2000 g for
5 minutes. Most of the water was removed while a small vol-
ume was left to keep the dilution medium water saturated
during storage at 20  C.
To saturate the dilution medium at 30  C, distilled water
(20% (v v1)) was shaken with the respective dilution media
(80% (v v1)) and placed in a water bath at 30  C. The
phases were shaken at least once an hour and kept in this
state for at least 4 hours after which they were separated
using centrifugation at 2000 g for 5 minutes; most of the
 JOURNAL OF DISPERSION SCIENCE AND TECHNOLOGY 3

Table 1. Density, viscosity, water solubility and refractive index of the dilution media and water.
Density Viscosity Water solubility Refractive
Dilution medium Temperature ( C) (kg m3) (mPa.s) (mg ml1) index (-)
Hoso 20 911[17] 82.58 0.9[17] 1.47
1-Bromododecane 20 1040[18] 3.70 0.058[19],a 1.46
30 1032[20] 3.02 1.46
Dodecane (mixture of isomers) 20 750[21] 1.53 0.065[22],b 1.42
Water 20 998[23] 1.00[23] 1.33[23]
30 996[23] 0.80[23] 1.33[23]
a
Measurement temperature was not specified.
b
Determined at 25  C.

water was removed after which the water saturated dilution
media were stored at 30  C.
2.4. Dynamic light scattering
Unless stated otherwise, DLS measurements were conducted
at 20  C with a Zetasizer 3000HS (Malvern Instruments,
UK) device. The extent of dilution of the different emulsions
measured via DLS is expressed as the water concentration in
the diluted samples related to water droplets in case no dis-
solution takes place. An initial concentration (0.2% (m v1))
of water in the dilution medium was prepared by diluting
the w/o emulsions (24 mg) in the dilution medium (6 mL)
after which subsequent dilutions were made by diluting 1–1
(v v1) with the same dilution medium. Unless stated other-
wise, a cap was put on top of the cuvette. For every concen-
tration, 3 subsequent measurements were conducted. Both
the average value and standard deviation on these measure-
ments are presented in figures throughout this study. If no
error bar is visible for a data point shown in a figure, the
standard deviation is smaller than the used symbol.
The average scattered intensity at 90 is expressed as kilo-
counts (kcounts) per second whereby the scattering intensity
of the pure dilution medium (3–5 kcounts s1) is subtracted
from the measured value. The correlogram was fitted using
the monomodal option (cumulant analysis) whereby at least
25 kcounts per second were necessary in order to obtain
repeatable measurements within an acceptable measurement
time. The measurement duration was automatically adjusted
by the instrument.
2.5. Characterization of the dilution media
In order to conduct a DLS-measurement, the viscosity and
refractive index of the dilution medium are needed. These
values were determined using a Brookfield LV-DV-II þ Pro
viscometer with small sample adapter and spindle SC4-18
and a Officine Galileo 51510 refractometer, respectively.
Saturation of the dilution medium with water did not influ-
ence the measured parameters which are shown in Table 1.
2.6. Cryo-SEM
Before analysis, the undiluted w/o emulsions were vitrified
in liquid nitrogen and transferred to a PP3000T device
(Quorum Technologies, East Sussex, UK) at 140  C. The
samples were sublimated for 45 min at 70  C to remove
the water from the water droplets. As the best images are
obtained at horizontally cut surfaces, a top layer was
removed from the sample using a knife before sublimation.
Before transferring the sample from the cryo-preparation
to the SEM chamber, a thin layer of conductive material
(Pt) was applied to the samples using argon gas. This sputter
coating process prevents charging of the samples by the
electron beam.
The droplets in the samples were visualized using a JSM-
7100 F TTLS LV TFEG-SEM (JEOL Ltd, Tokyo, Japan)
under high vacuum and a voltage of 3 keV.
2.7. Statistical analysis
The scattered light intensity (kcounts s1) as a function of
water concentration (%) was analyzed using linear regression
analysis which was performed using the Analysis ToolPak in
Excel 2010.
3. Results and discussion
As the main goal of this study was to determine whether
DLS can be used to accurately determine the droplet size of
(submicron) w/o emulsions, samples were first prepared at
different shear intensities. Cryo-SEM was used to verify
whether these w/o emulsions (UT 6500, UT 24000 and MF
840) indeed differed in water droplet size. Sample UT 24000
was used in the initial experiments to evaluate the influence
of the sample pretreatment conditions on the DLS-results.
Afterwards, the optimal measurement conditions were used
to evaluate the droplet size distribution of the differ-
ent samples.
3.1. Cryo-SEM of emulsions prepared at different shear
intensities
Figure 1 shows cryo-SEM images of the three w/o emulsions
with the same composition but prepared with different shear
intensity. Figure 1a shows a lot of droplets with a diameter
of about 1 mm in the sample prepared with an Ultra-Turrax
at a speed of 6500 rpm (UT 6500) while the biggest droplets
had a diameter of about 10 mm. Figure 1b shows that the
biggest droplets found in the sample prepared at 24,000 rpm
(UT 24000) were slightly bigger than 1 mm. Finally, Figure
1c clearly shows that the biggest droplets found in the sam-
ple which was prepared by microfluidisation at a pressure of
840 bar (MF 840) had a diameter of about 1 mm with most
4 M. BALCAEN ET AL.

Figure 1. Cryo-SEM images of the UT 6500 (a), the UT 24000 (b), and the MF 840 (c) w/o emulsions at 10,000 times magnification.

Figure 2. Scattered intensity (a) and inverse of the apparent Z-average diameter (b) as a function of water concentration after dilution of the UT 24000 emulsion in
water saturated (squares) or non-saturated (diamonds) Hoso.

droplets being submicron in size. The cryo-SEM images
thus clearly indicated that these 3 samples differed in drop-
let size (distribution).
3.2. Influence of sample pretreatment on DLS
measurements
3.2.1. Influence of water saturation of the dilu-
tion medium
In order to prevent dilution-induced effects, dilution is pref-
erentially performed using the continuous phase liquid, i.e.,
high oleic sunflower oil for the w/o emulsions considered.
Figure 2a shows the importance of water saturating high
oleic sunflower oil when diluting the w/o emulsions.
Considering the same degree of dilution, the scattered inten-
sity was higher when the dilution medium was saturated
with water, indicating the partial dissolution of water drop-
lets upon dilution in water-free sunflower oil. The linear
relation between the scattering intensity and the water con-
centration, for scattering intensities higher than 25 kcounts
per second, indicates that even at the highest water concen-
tration (i.e., lowest dilution) no multiple scattering was
taking place.[24]

If the dilution medium was saturated with water, meas-
urements of the Z-average diameter were possible down to a
water concentration of about 0.0125%, whereas the lower
limit was found at about 0.05% when the dilution medium
was not saturated with water. As the extent of dilution of
the emulsions is expressed as water concentration in the
diluted samples, the solubility of water in the dilution
medium can be derived from the intercept with the X-axis
of a linear regression through the data of the scattering
intensity in function of the water concentration. The inter-
section with the X-axis follows from the absolute value of
the intercept, divided by the slope. As, however, only 3 data
points could be considered in the linear regression analysis
of the curve of scattered intensity as a function of water
concentration for non-saturated Hoso (Figure 2a), broad
confidence intervals were obtained for the slope and inter-
cept, and hence for the intersection with the X-axis. Overall,
the water solubility seems to be slightly lower than the water
solubility in glycerol trioleate at 20  C (i.e., 0.11 wt%).[16]
However, the dispersed phase of the emulsions used con-
tained 0.1 M NaCl while pure water was considered by
Hilder (1968).[16] Moreover, the oil used for dilution was
not dried prior to use and might have contained traces
of water.
Droplet size determination by DLS is based on the
Brownian motion of the droplets. As such, this technique
actually determines the droplet diffusion coefficient, which
is not only influenced by the size of the droplets, and the
viscosity of the continuous phase, but also by interactions
between droplets. As the latter effect is not corrected for by
the software of the instrument, the reported droplet diam-
eter can deviate from the actual droplet size. It should thus
be noted that the droplet sizes mentioned in the remainder
of this study are apparent values.
Figure 2b shows that due to dissolution of the water
droplets in the continuous phase, the droplet diffusion
(inversely related to the apparent Z-average diameter)[25] at
a given degree of dilution was faster in case the continuous
phase was not saturated with water. However, even when
the dilution medium was saturated, the scattered intensity
versus water content graph (Figure 2a) did not cross the ori-
gin, indicating that the dilution liquid still had a limited
solubility for water droplets. This effect may be ascribed to
the small size of the droplets, which increases their solubil-
ity, which is known as the Kelvin effect.[26]
Figure 2b shows that the water concentration had a big
influence on the droplet diffusion. The observed linear rela-
tion between the scattering intensity and water concentra-
tion within the studied concentration range (for scattering
intensities higher than 25 kcounts per second) (Figure 2a),
indicated that the influence of the water concentration on
the droplet diffusion cannot be ascribed to multiple scatter-
ing. In fact, multiple scattering is known to give rise to a
decrease in apparent size, which is opposite to the trend
observed.[27] The slower droplet diffusion at higher water
concentration thus suggests interaction effects between the
water droplets. Mettu et al. recently also measured and
described the occurrence of an attractive interaction between
JOURNAL OF DISPERSION SCIENCE AND TECHNOLOGY 5
PGPR-stabilized water droplets. When retracting one water
droplet from the other, they measured an attractive min-
imum which they ascribed to short-range attractive interac-
tions due to PGPR bridging between droplets when they are
in close contact.[28] Bridging between droplets can occur in
case their surface is not fully covered by emulsifier.
Therefore, bridging is usually only observed in cases where
a low emulsifier concentration is used. Nollet et al. deter-
mined the monolayer concentration of PGPR at the w/o
interface of a system containing PGPR in sunflower oil and
a water phase containing 0.1 M NaCl. They calculated that
1.67 mmol m2 of PGPR molecules are necessary in order to
form a complete monolayer.[29] As such, the surface area
per polar head group was calculated to be equal to about
100 Å2. Considering a molecular weight of 3000 g mol1
(MWPGPR), the Sauter mean diameter D32 of the water drop-
lets in the w/o emulsions can be determined when all PGPR
is adsorbed to the w/o interface using Equation (1).
6  MWPGPR  1:67E  6 mol m2  CH2O
D32 ¼
(1)
qH2O 106 g m3  CPGPR
In Equation (1), CPGPR represents the PGPR concentra-
tion expressed per unit mass of emulsion; in our experi-
ments, it was equal to 0.05 g of PGPR per g of emulsion.
CH2O represents the water mass fraction in the emulsion
(i.e., 0.5 g of water per g of emulsion in our experiments).
The estimated D32 amounted to 300 nm, which indicates
that enough PGPR was present in the prepared w/o emul-
sions to cover droplets of 300 nm with a complete mono-
layer. As shown by Cryo-SEM, even the droplets present in
the emulsion prepared at the highest shear intensity had a
diameter of at least a few hundreds of nm with the largest
droplets being only slightly smaller than 1 mm (Figure 1c).
As such, unless desorption of PGPR occurred due to dilu-
tion of the w/o emulsions, the droplets in the w/o emulsions
which were prepared in this study can be expected to be
fully covered by PGPR-molecules. Another possible explan-
ation for the observed attraction between water droplets is a
stronger hydrophobic interaction between the polyricinoleate
tails than between these tails and the continuous phase. In
any case, this attractive energy only decreased the diffusion
speed of the water droplets and did not lead to aggregation,
as observed using light microscopy on water-in-sunflower
oil emulsions stabilized using PGPR.[7] This implies that the
thermal energy of the droplets is sufficient to overcome the
attractive energy. The measurement of higher apparent
diameters (and related lower diffusion coefficients) due to
interaction effects between water droplets in water-in-oil
emulsions has also been observed by other researchers.[30,31]
However, in these works, micro-emulsions were considered
and the interaction effects were less pronounced as com-
pared to what we observed. Both research works note that
the magnitude of the interaction effects increases with
increasing size of the droplets. This might again be linked to
the higher thermal energy of smaller compared to bigger
droplets. Finally, it can be noted that Lim et al. observed a
similar increase of the apparent diameter (and related
decrease in droplet diffusion) as ours with increasing droplet
6 M. BALCAEN ET AL.
concentration. These researchers were able to dilute their
samples to a concentration at which the interaction effects
were not influencing the diffusion anymore and a maximum
concentration independent size of about 100 nm was
obtained.[25] In our measurements, further dilution was not
feasible due to the limited difference in refractive index
between continuous and dispersed phase, which led to a low
scattered intensity and a higher minimal concentration of
droplets to perform a DLS-measurement. This means that
we could not sufficiently dilute our emulsions to reach the
region in which the measured droplet size became concen-
tration independent. Hou et al. determined the diffusion
coefficient of water droplets without interaction effects (i.e.,
the intrinsic diffusion coefficient) by measuring the diffusion
coefficient of the droplets at different degrees of dilution
and extrapolating to infinite dilution.[31] As the diffusion
coefficient is inversely proportional to the Z-average diam-
eter,[25] extrapolation of the inverse of the Z-average diam-
eter as a function of the water concentration to infinite
dilution is expected to provide an estimate of the Z-average
diameter without interaction effects. However, the linear
part of the curve, at high dilution, has to be used in order
to obtain the intercept at infinite dilution. Figure 2b shows
that the curve only becomes linear at water concentrations
below 0.05%. This means that only 3 points could be con-
sidered in the regression analysis and very broad confidence
intervals were obtained. Indeed, regression analysis revealed
that with 95% certainty, the Z-average diameter (at infinite
dilution) was between 176 and 308 nm. In order to obtain a
more accurate estimate, more measurements should be con-
ducted in the concentration range between 0.01 and 0.05%.
This was not done in the current study as the goal was not
an accurate droplet size determination of one sample, but
rather the determination of the influence of different sample
preparation conditions.
It should also be noted that the calculated Z-average
diameter at infinite dilution (222 nm) was only slightly
smaller than the Z-average diameter which was obtained
when the DLS-measurement was conducted using a water
concentration of 0.0125% (256 nm). Therefore, measuring at
0.0125% is a quick way to obtain a reliable estimate of the
Z-average diameter.
Figure 2b shows that water saturation of the dilution
medium mainly caused a shift in the curve, corresponding
to the (difference in) water solubility of the droplets.
Plotting the measured apparent Z-average diameter as a
function of the scattered intensity indeed ensured that the
data obtained upon dilution in oil that was either water-
saturated or not coincided (Figure 3).
Considering the apparent Z-average diameter as a func-
tion of scattered intensity (with scattered intensity below
1200 kcounts s1) for both dilution liquids, a linear relation
was found with determination coefficient of 0.99. The Z-
average diameter leveled off at higher scattered intensities.
These results show that the dissolution of part of the water
droplets as such did not influence the measured droplet size
distribution. Instead, an indirect effect was observed due to
a difference in concentration of water droplets at the same
Figure 3. Apparent Z-average diameter of the UT 24000 emulsion as a function
of the scattered intensity for Hoso (diamonds) and water saturated Hoso
(squares) as dilution liquids. Linear regression analysis was applied on the com-
bined datasets of both dilution liquids with scattered intensities below 1200
kcounts s1.
degree of dilution. Hereby, it should be mentioned that the
presence of salt in the water phase probably avoided that
the smallest droplets dissolved first, as would be expected
from their higher curvature.[26]
In a second series of measurements, 1-bromododecane was
selected as dilution medium. The latter solvent was selected
because 1-bromododecane has a similar polarity to oil (i.e.,
emulsion continuous phase).[21] Furthermore, it has a density
similar to water (Table 1), which limits gravitational effects.
The lower viscosity of 1-bromododecane compared to oil
(Table 1) also facilitates emulsion dilution. Figure 4a shows
that water saturation was less crucial when 1-bromododecane
was used as the dilution medium. This observation can be
linked to the lower solubility of water in 1-bromododecane
(Table 1). On the other hand, Figure 4b clearly shows a similar
big influence of concentration on the measured apparent Z-
average droplet diameter for 1-bromododecane. As the inverse
apparent Z-average diameters in function of the water concen-
tration showed no linear relation between more than two
points, it was not possible to determine the Z-average diameter
without interaction effects (i.e., at infinite dilution) by linear
extrapolation.
3.2.2. Influence of water evaporation
Figure 5a shows a pronounced decrease of the measured scat-
tered intensity as a function of time if the cuvette containing a
sample with a water concentration of 0.025% in water saturated
1-bromododecane was not closed. As this effect did not occur in
a closed cuvette, evaporation of water from the continuous phase
and subsequent dissolution of water droplets into the unsatur-
ated 1-bromododecane was thought to be the reason for this
observation. As shown in Figure 5b, dissolution of water drop-
lets in the continuous phase also led to a decrease of the meas-
ured droplet size. These measurements therefore show the
importance of preventing the dissolution of water droplets in the
continuous phase. Hereby, it is essential to keep the cuvette
closed as well as to keep the headspace volume as small as pos-
sible in order to prevent evaporation of water from the dilution
medium which induces further dissolution of the water droplets.
 JOURNAL OF DISPERSION SCIENCE AND TECHNOLOGY 7

Figure 4. Scattered intensity (a) and inverse of the apparent Z-average diameter (b) as a function of water concentration after dilution of the UT 24000 emulsion in
water saturated (squares) or non-saturated (diamonds) 1-bromododecane.

Figure 5. Scattered intensity (a) and apparent Z-average diameter (b) of 0.025 % (w v1) UT 24000 in water saturated 1-bromododecane as a function of time with
an open (diamonds) or closed (squares) cuvette.

Figure 6. Scattered intensity (a) and apparent Z-average diameter (b) as a function of water concentration after dilution of the UT 24000 emulsion in water satu-
rated dodecane (diamonds), Hoso (squares) or 1-bromododecane (triangles).

3.2.3. Influence of the used dilution medium
Figure 6a clearly shows that the scattered intensity as a func-
tion of concentration for water saturated Hoso and water
saturated bromododecane was similar. Indeed, the 95% con-
fidence intervals of their slopes (10,954–12,350 and
10,067–11,900 (kcounts s1) (% w v1)1 for water saturated
Hoso and water saturated bromododecane, respectively)
overlap. However, when water saturated dodecane was used
as the dilution medium, the scattered intensity was much
lower when the same concentration of droplets was consid-
ered. This effect can mainly be attributed to the smaller
difference in refractive index between dodecane and water
(Table 1), which decreases the scattered light intensity. This
effect, however, also led to the fact that the minimal water
concentration limit, at which reproducible DLS-measure-
ments could be conducted, corresponding to a scattered
light intensity of at least 25 kcounts s1, was found at about
0.025% for dodecane while for bromododecane and Hoso,
about 0.0125% was sufficient.
Figure 6b shows that the measured apparent droplet size
increased in function of concentration for all three used
dilution media (i.e., dodecane, Hoso and 1-bromododecane)
8 M. BALCAEN ET AL.
Figure 7. Scattered intensity (a) and apparent Z-average diameter (b) as a function of water concentration after dilution of the UT 24000 emulsion in water satu-
rated 1-bromododecane at 20 (diamonds) or 30  C (squares).
within the studied water concentration range. The data
could be well described by a second order polynomial.
Similar droplet sizes are expected to be obtained for all three
dilution liquids in case the emulsions could be infinitely
diluted; the 95% confidence intervals of the intercept of
these second order polynomials with the y-axis indeed over-
lapped (i.e., 11–319, 195–326 and 4–245 nm for dodecane,
1-bromododecane and Hoso, respectively). Within the
studied water concentration range, clearly higher apparent
droplet sizes were observed for emulsions diluted in dodec-
ane compared to 1-bromododecane or Hoso. This stronger
concentration dependency might indicate a stronger inter-
action between water droplets when the emulsions are
diluted in dodecane compared to 1-bromododecane or
Hoso. This is an important issue; dilution can indeed lead
to either flocculation or deflocculation depending on the
used dilution liquid.[13] This implies that the measured
droplet diffusion (and derived droplet diameter) will be a
function of the used emulsifier, concentration of droplets
and dilution medium. As such, reported W/O diameters
obtained by DLS are hardly useful whenever the sample pre-
treatment conditions (including the dilution liquid, as well
as the degree of dilution) are not specified.
Based on our results it is concluded that, out of the three
tested dilution media (i.e., water saturated dodecane, Hoso
and 1-bromododecane), water saturated 1-bromododecane is
the most optimal dilution medium. As it has a density closer
to water (Dq ¼ 42 kg m3 at 20  C) compared to Hoso
(Dq ¼ 87 kg m3 at 20  C) and dodecane (Dq ¼ 248 kg m3
at 20  C), gravitational effects are limited. As mentioned in
section 2.1, the UT 6500 contained water droplets with a
diameter up to 10 mm. In case dodecane would be used as
dilution medium, Stokes’ law indicates that water droplets of
10 mm would sediment over a distance of about 5 mm in
10 min. This sedimentation process could influence the
determined droplet diffusion coefficient (and derived diam-
eter) in case of longer measurements. In case 1-bromodode-
cane is used as dilution medium, water droplet creaming is
limited to 0.4 mm in 10 min. The density of Hoso is also
close to water, thus also limiting gravitational effects.
However, 1-bromododecane is preferred over Hoso due to
its lower viscosity (Table 1), thus facilitating emulsion
dilution in the dilution medium. Furthermore, the lower
solubility of water in 1-bromododecane compared to Hoso
reduces the necessity to water saturate the dilution medium,
which speeds up the sample preparation procedure.
3.2.4. Influence of temperature
As shown in Figure 7a, the curve for scattered intensity ver-
sus water concentration is similar at 20 and at 30  C. The
small differences in scattering intensity may be due to differ-
ences in water solubility or a slightly smaller difference in
refractive index between the oil and water phase. Table 1
suggests no temperature effect on the refractive indices
larger than 0.005. As such, if the difference in refractive
index between oil and water does change in function of
temperature, this effect is expected to be small.
Figure 7b shows a smaller concentration dependency of
the apparent droplet size at higher temperature, which might
indicate less interaction between the water droplets. The
data could again be well described by a second order poly-
nomial, whereby quite similar intercepts with the y-axis are
obtained, which represents the Z-average diameter at infinite
dilution. The aforementioned observations imply that the
necessary degree of dilution to eliminate interaction effects
was reduced by increasing the measurement temperature.
However, the scattered intensity for a certain degree of dilu-
tion was lower when the temperature was increased, imply-
ing the need of a higher concentration in order to conduct a
reproducible DLS experiment. Moreover, the risk of evap-
orative losses of water increases at higher temperature. It is
therefore more convenient to measure at lower
temperatures.
3.3. Measurement of droplet size of samples prepared
at different shear intensities
In the previous paragraphs, it was shown that measuring in
water saturated 1-bromododecane at 20  C in closed cuvette
leads to satisfactory droplet size determinations by DLS.
Hereby, the concentration was varied using subsequent dilu-
tions and the most reliable results were obtained at the
highest possible dilution.

Figure 8. Scattered intensity (a) and apparent Z-average diameter (b) as a function of water concentration after dilution of the UT 6500 (diamonds), UT 24000
(squares) and MF 840 (triangles) emulsions in water saturated 1-bromododecane at 20  C.
Figure 9. Intensity weighted distributions of the UT 6500 (dotted line), UT
24000 (dashed line) and MF 840 (full line) emulsions, measured at 20  C at a
concentration of 0.0125 % (w v1) in water saturated 1-bromododecane.
This optimized method was used for the determination
of the droplet size distribution of the emulsions prepared
with a different shear intensity (i.e., UT 6500, UT 24000 and
MF 840).
Figure 8a shows that the scattered intensity at a certain
water concentration was higher when the shear intensity
during preparation was higher and hence when smaller
droplets were present in the sample. Whereas the Rayleigh
theory (which holds for droplets much smaller compared to
the wavelength of the used light source) predicts an
increased scattering for bigger droplets, the Mie theory
(which holds for droplets with a similar size as the wave-
length used) does not allow a general trend, but rather
describes a complex relationship between scattered intensity
and droplet size, which is frequently characterized by a max-
imum scattering intensity for droplets of some hundreds of
nm in diameter. Considering the fact that the used DLS-
device has a laser which emits light at 632.8 nm, the size of
the droplets is in the range of the wavelength and their light
scattering can be best predicted by the latter theory.
For the sample which contained the smallest droplets
(MF 840), the increase of the scattered light intensity with
increasing water concentration flattens, which points to mul-
tiple scattering of light by the droplets at these higher water
concentrations.[24]
JOURNAL OF DISPERSION SCIENCE AND TECHNOLOGY 9
Looking at Figure 8b, it can be seen that for all samples, the
measured apparent Z-average diameter was again concentra-
tion dependent. Hereby, the concentration dependency was
lower for smaller droplets, which might be due to less inter-
action effects between these droplets. This observation corre-
sponds to what was noted by Hamilton et al. (1990) and Hou
et al. (1988).[30,31] The decreasing slope in Figure 8b at higher
concentrations for the MF 840 samples is in line with the mul-
tiple scattering effect as deduced from the scattered light inten-
sity (Figure 8a), as this effect gives rise to an underestimation
of the droplet size. No linear relation could be determined
between the inverse of the apparent Z-average diameter and
the water concentration based on more than two points for
any of the samples. As such, a determination of the Z-average
diameter at infinite dilution (i.e., without interaction effects)
by linear extrapolation was not possible. As the data of the
apparent Z-average diameter in function of the water droplet
concentration could again be well described by a second order
polynomial, the Z-average diameter at infinite dilution might
be approximated by the intercept of these polynomials with
the y-axis. These intercepts amounted to 213 nm, 260 nm and
534 nm for the samples MF 840, UT 24000 and UT 6500,
respectively. This approximation of the Z-average diameter
without interaction effects requires the determination of the
apparent Z-average diameter of w/o emulsions at different
degrees of dilution. On the other hand, a good approximation
of the Z-average diameter could also be obtained in a much
shorter time by considering the value at the lowest measurable
water concentration (i.e., 0.0125%), which amounted to
231 nm, 353 nm and 580 nm for the samples MF 840, UT
24000 and UT 6500, respectively.
Figure 9 shows the droplet size distributions which were
obtained at a water concentration of 0.0125%. This shows that
the droplet size distributions mainly became more narrow with
increasing shear intensity while the size of the smallest droplets
remained fairly constant. This observation agrees with the cryo-
SEM images taken of the different samples (Figure 1).
4. Conclusions
We showed that several aspects have to be taken into
account to ensure the accurate measurement of the droplet
10 M. BALCAEN ET AL.
size distribution of (sub)micron sized liquid w/o emulsions
using dynamic light scattering. DLS-measurements typically
require a high degree of dilution of concentrated samples.
Hereby, the solubility of water in the dilution medium has
to be taken into account; water saturation of the continuous
phase largely prevented the dissolution of water droplets.
Moreover, also evaporation of water from the dilution
medium should be avoided as this shifts the equilibrium and
leads to further dissolution of water droplets into the dilu-
tion medium during sample storage. Hence, the headspace
should be as small as possible, vials should be closed and
the temperature has to be chosen in a way that minimizes
evaporation.
Moreover, it was also shown that the used dilution medium
had a big influence on the measured droplet size. For every
dilution medium, an inverse relation was observed between
the measured diameter and the degree of dilution. Hereby, the
use of 1-bromododecane led to a similar behavior as Hoso.
However, the solubility of water in 1-bromododecane
appeared to be significantly lower than in Hoso, which
reduced the need to presaturate the dilution medium and also
reduced the error when the dilution medium was not suffi-
ciently saturated. In contrast to the aforementioned dilution
media, dodecane increased the apparent diameter which was
measured using DLS, which pointed toward an increased
interaction between PGPR-stabilized water droplets.
Finally, the droplet size distribution of three experimental
w/o emulsions was determined. Hereby, a less pronounced
concentration dependence of the apparent droplet size was
seen for smaller droplets. The particle size distributions
obtained via DLS-measurements of the samples after dilution
to about 0.01% (w v1) in water saturated 1-bromododecane
at 20  C in a closed cuvette corresponded to the cryo-SEM
images indicating the reliability of the optimized DLS method.
Disclosure statement
The authors declare no conflict of interest.
Funding
The Hercules foundation is acknowledged for its financial support in
the acquisition of the scanning electron microscope JEOL JSM-7100F
equipped with the cryo-transfer system Quorum PP3010T [grant no.
AUGE-09-029] used in this research.
ORCID
Koen Dewettinck https://orcid.org/0000-0001-5050-2269
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