Review of Nanoemulsion Formulation and Characterization Techniques

Abstract

Nanoemulsions are colloidal dispersion systems that are thermodynamically stable, composed of two immiscible liquids mixed along with emulsifying agents (surfactants and co-surfactants) to form a single phase. Nanoemulsions have extensively been investigated as drug delivery systems. This review aims to provide consolidated information regarding various formulation and characterization techniques developed for nanoemulsions. Nanoemulsions are formulated using two different methods, the persuasion method and the Brute force method. Various characterization techniques for nanoemulsions include determination of entrapment efficiency, particle size, polydispersity index, zeta potential as well as characterization through differential scanning calorimetry, Fourier-transform infrared spectroscopy and transmission electron microscopy. Nanoemulsions are further evaluated by studying in vitro drug release, in vitro permeation, stability and thermodynamic stability, shelf life, dispersibility, viscosity, surface tension, friccohesity, refractive index, percent transmittance, pH and osmolarity.

Full text

September-October 2018 Indian Journal of Pharmaceutical Sciences 781
Review Article
Nanoemulsions, also known as submicron emulsions,
ultrane emulsions and miniemulsions, are submicron
sized colloidal particulate systems considered as
thermodynamically and kinetically stable isotropic
dispersions, which consist of two immiscible liquids
like water and oil, stabilized by an interfacial lm
consisting of a suitable surfactant and co-surfactant
to form a single phase. A number of surfactants with
diverse characteristics (ionic or non-ionic) had been
used with such nanoemulsions. Most widely used
among them were nonionic surfactants (sorbitan
esters, polysorbates), anionic surfactants (potassium
laurate, sodium lauryl sulphate), cationic surfactants
(quaternary ammonium halide) and zwitterions
surfactants (quaternary ammonium halide). Early
nanoemulsions were oil-in-water (O/W) type
emulsions with average droplet diameter ranging from
50 to 1000 nm. Nanoemulsions more recently are
classied into three categories such as O/W type (oil is
dispersed in aqueous phase), water-in-oil (W/O) type
(water is dispersed in oil phase), and bi-continuous
(microdomains of water and oil are interdispersed
within the system). Transformation among these three
types can be attained by altering the components of
the emulsions. Multiple emulsions are also a type of
nanoemulsions, where both O/W and W/O emulsions
present simultaneously in one system. For stabilizing
these two emulsions, both hydrophilic and lipophilic
surfactants are used simultaneously. Nanoemulsions
offer various advantages over other dosage forms and
these advantages are, (1) increased rate of absorption,
(2) reduced variability in absorption, (3) protection
from oxidation and hydrolysis in O/W nanoemulsions,
(4) delivery of lipophilic drugs after solubilisation,
(5) aqueous dosage form for water insoluble drugs,
(6) enhanced bioavailability for many drugs, (7) ability
to incorporate both lipophilic and hydrophilic drugs,
(8) delivery systems to enhance efcacy while reduce
total dose and side effects, (9) as non-toxic and non-
irritant vehicles for skin and mucous membrane delivery
and (10) release control by permeation of drug through
liquid lm, whose hydrophilicity or lipophilicity as
well as thickness can be precisely controlled.
Review of Nanoemulsion Formulation and
Characterization Techniques
K. GURPREET AND S. K. SINGH*
Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar, Haryana-125 001,
India
Gurpreet, et al.: Formulation and Characterization of Nanoemulsion
Nanoemulsions are colloidal dispersion systems that are thermodynamically stable, composed of two
immiscible liquids mixed along with emulsifying agents (surfactants and co-surfactants) to form a single
phase. Nanoemulsions have extensively been investigated as drug delivery systems. This review aims to
provide consolidated information regarding various formulation and characterization techniques developed
for nanoemulsions. Nanoemulsions are formulated using two different methods, the persuasion method and
the Brute force method. Various characterization techniques for nanoemulsions include determination of
entrapment efciency, particle size, polydispersity index, zeta potential as well as characterization through
differential scanning calorimetry, Fourier-transform infrared spectroscopy and transmission electron
microscopy. Nanoemulsions are further evaluated by studying in vitro drug release, in vitro permeation,
stability and thermodynamic stability, shelf life, dispersibility, viscosity, surface tension, friccohesity,
refractive index, percent transmittance, pH and osmolarity.
Key words: Nanoemulsions, nanoemulsion characterization, entrapment efciency, droplet size
*Address for correspondence
E-mail: sksingh_gju@rediffmail.com
This is an open access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
allows others to remix, tweak, and build upon the work non-commercially,
as long as the author is credited and the new creations are licensed under
the identical terms
Accepted 02 July 2018
Revised 26 May 2017
Received 24 January 2017
Indian J Pharm Sci 2018;80(5):781-789

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Indian Journal of Pharmaceutical Sciences
782
FORMULATION OF MICROEMULSIONS
A number of techniques had been adopted for
formulation of nanoemulsions such as high pressure
homogenization, microudization, phase inversion,
spontaneous emulsication, solvent evaporation and
hydrogel formation[1-4]. Multiple emulsions are usually
prepared using the double emulsion-solvent evaporation
technique. A variety of techniques had been utilized
for characterization of such nanoemulsions used as
drug delivery systems. Nanoemulsions are formulated
mainly using two primary methods, (a) the persuasion
method and (b) the Brute force method.
Persuasion method/phase inversion technique:
Nanoemulsion preparation by persuasion method
doesn’t require any external force, but instead it
involves formation of ne dispersions when phase
transitions occur by changing either the temperature
or composition while keeping the alternate parameter
constant. Persuasion method can be broadly categorised
as, (i) phase transition from near-optimum state via
change in single variable, which includes altering one
variable of formulation such as temperature or salinity
close to optimal value. Hydrophilic-lipophilic deviation
(HLD) for optimal value is close to centre level for a
system, for example, employing higher temperature
to microemulsion. (ii) Phase transition from near-
optimal state via change in multiple variables, meaning
altering more than one variable of formulation. For
example, employing higher temperature and including
an additional salt in a microemulsion. (iii) Catastrophic
inversion, an inversion of low internal phase emulsion
so that the internal phase converts to external phase.
(iv) Phase transition stabilized by liquid crystal
formation, which includes nanodroplets stabilization
from a state close to HLD-0 by liquid crystal formation.
Brute force method:
This method includes utilization of brute forces
for breaking the oil droplets into the nano range.
Instruments that have been utilized for formulation of
nanomeulsions include high pressure homogenizer,
high speed mixer, small pore membrane and high
frequency ultrasonic device. Nanoemulsion properties
like its small size, optical transparency and high kinetic
stability is not only dependent upon the composition
of variables but also on the processing variables like
emulsication time, degree of mixing, energy input
and emulsifying path. High-pressure homogenization
and microuidization methods are employed at both
industrial and laboratory scale for attaining very
small size of nanoemulsion by utilizing high pressure
equipment. Various other methods are also being
employed for preparation of nanoemulsion such as
ultrasonication and in situ emulsication. Various
techniques employed for preparation of nanoemulsion
are shown in Table 1[5-21].
High pressure homogenization:
Nanoemulsion preparation required high shear force,
therefore in this strategy high-pressure homogenizer
or piston homogenizer is utilized for production of
nanoemulsions with very small particle size (up to
1 nm). In this technique, a mixture is forced to pass
through an orice at a very high pressure ranging
from 500 to 5000 psi. The resultant product is further
subjected to intense turbulence and hydraulic shear
resulting into emulsion with extremely ne particles.
This has been proved to be the most efcient method
for nanoemulsion preparation but the only drawback
associated with this technique is high energy
consumption and rise in temperature of emulsion
during processing. For obtaining smaller particle
size, it also requires larger runs of homogenization
cycles. Yilmaz et al. formulated phytosphingosine
O/W nanoemulsions by employing high pressure
homogenization method and found out that droplet size
was decreased after 8 homogenisation cycles and such
nanoemulsion was stable for over 6 mo[22].
Microuidization:
This method employed a device known as
microuidizer that utilizes high pressure positive
displacement pump (500-20 000 psi) that pushes the
product out through the interaction chamber consisting
of stainless steel microchannels on the impingement
area resulting into formation of very small particles of
sub-micron range. The mixture is repeatedly circulated
through the microuidizer until the required particle
size is achieved. Resultant product is also passed
through the lter to separate smaller droplets from
larger ones and to obtain a uniform nanoemulsion.
Uluata et al. fabricated octadecane O/W nanoemulsions
using a microuidizer and observed that on increasing
the number of passes and homogenization pressure,
the droplet size decreased[23]. Goh et al. prepared
tocotrienol-rich fraction nanoemulsions by two step
homogenization where a primary coarse emulsion was
prepared by using a stirrer, which was further processed
using a microuidizer. They reported that the droplet

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size reduced from 120 to 65.1 nm after passing through
10 homogenization cycles at an increased pressure[24].
Ultrasonication:
In this technique premixed emulsion is exposed to
agitation at ultrasonic frequency of 20 kHz reducing the
droplets to nanodroplets size. The resultant emulsion
is then passed through high shear region to form
droplets with uniform size distribution. Water jacket is
employed in this technique to regulate the temperature.
Sonotrodes also known as sonicator probe consisted
of piezoelectric quartz crystals as the energy providers
during ultrasonic emulsication. On application of
alternating electric voltage, these sonotrodes contract
and expand. Mechanical vibrations are produced
when the sonicator tip contacted the liquid resulting in
cavitation, which leads to collapse of vapour cavities
formed within the liquid. This technique is mainly
adopted when droplet size less than 0.2 μ is required.
Shi et al. formulated emodin-loaded nanoemulsion by
using ultrasonic emulsication method at a frequency
of 25 kHz and achieved mean diameter of emodin-
loaded nanoemulsion was found to be in the range of
10-30 nm[25].
Spontaneous emulsication:
This technique involved preparation of nanoemulsion
in 3 stages. The rst stage included formation of an
organic solution, comprising of oil and lipophilic
surfactant in water miscible solvent and hydrophilic
surfactant and then the O/W emulsion is formed
by injecting this organic phase into the aqueous
Technique Formulation Conclusions References
High pressure homogenization Oral lipid nanoemulsion
(primaquine)
Enhanced oral bioavailability, 10-200 nm
particle size [5]
Pseudoternary phase
diagram+spontaneous
emulsication method
Ramipril nanoemulsion Increased bioavailability, droplet size
80.9 nm [6]
High pressure homogenization O/W nanoemulsions Improved skin hydration and elasticity [7]
Spontaneous emulsication O/W nanoemulsion
(aceclofenac)
Nanoemulsion with potential for
transdermal delivery of aceclofenac [8]
Spontaneous emulsication Celecoxib nanoemulsion Enhanced physical and chemical stability
of celecoxib in nanoemulsion [9]
High pressure homogenization Lecithin-based nanoemulsions
(progesterone)
Improved permeation rates of progesterone
with long-term stability [10]
High pressure homogenization Prednicarbate nanoemulsion Increased chemical stability of the drug in
formulation [11]
Phase inversion temperature
method
Acyclovir-loaded multiple
W/O/W nanoemulsions
Excellent physicochemical stability for 6
mo at RT, mean droplet size of 100 nm [12]
Spontaneous
nanoemulsication method Clotrimazole nanoemulsion Improved solubility of clotrimazole, mean
globule size <25 nm [13]
Ultrasonic emulsication
method Basil oil nanoemulsion Nanoemulsions with droplet size of 29.6
nm, for food preservation [14]
Phase inversion composition
method Efavirenz nanoemulsion Enhanced bioavailability, globule size <30
nm [34]
High-pressure homogenizer Dimethyl silicone dry
nanoemulsion inhalation
Effective in acute lung injury, particle size
of 19.8 nm [15]
High-pressure homogenizer
Parenteral lecithin-based
nanoemulsions
(risperidone)
Enhanced brain availability of risperidone
with a mean particle size of 160 nm [28]
Microuidization method Pitavastatin-containing
nanoemulsions Enhanced permeation [16]
High-pressure
homogenization+ultrasound Nanoemulsion Reduced energy demand for emulsication,
low particle dimensions and higher stability [17]
Sonication method Saponin-stabilized quercetin-
loaded o/w nanoemulsion
Stable for 45 d at RT, mean particle size of
52±10 nm [18]
High-pressure homogenization Paclitaxel-baicalein
nanoemulsion Strategy to overcome multidrug resistance [19]
Nanoemulsion templating PLGA nanoparticles Imaging agents for biomedical purposes [20]
Spontaneous emulsication
method
Chitosan lms with
cinnamaldehyde nanoemulsions Good UV barrier properties [21]
TABLE 1: TECHNIQUES EMPLOYED FOR PREPARATION OF NANOEMULSIONS

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phase under magnetic stirring. The organic solvent
was then removed in the third stage by evaporation.
Sugumar et al. formulated stable eucalyptus oil
nanoemulsion by adopting spontaneous emulsication
and the mean droplet size was found to be in the range
of 50-100 nm[26].
Solvent evaporation technique/hydrogel method:
In this technique, drug solution is prepared and
emulsied into another liquid (non-solvent for drug)
and then solvent is evaporated, which led to drug
precipitation. High speed stirrer can be employed for
regulating the crystal growth and particle aggregation.
Hydrogel method is very similar the solvent
evaporation method. The only difference from the
solvent evaporation method is that the drug solution in
this case is miscible with the drug antisolvent.
CHARACTERIZATION OF NANOEMULSIONS
Determination of encapsulation efciency:
For determining the amount of drug entrapped in
the formulation, weighed amount of formulation is
dispersed in organic solvent by ultrasonication
and the drug is extracted into suitable buffer.
Drug content is estimated by analysing the extract
spectrophotometrically at λmax of drug after making
suitable dilutions against suitable blank. The entrapment
efciency (EE) and loading efciency (LE) of the
drug can be calculated by using the following Eqns.
[27], drug EE = drug content in the product obtained
(mg)/total amount of drug added (mg)×100 and drug
LE = drug content in the product obtained (mg)/total
product weight (mg)×100. Drug content could also
be determined using reverse phase high-performance
liquid chromatography (HPLC) techniques. Singh et
al. employed this technique for nding primaquine
concentration and reported 95 % encapsulation
efciency of formulated nanoemulsion[5].
Determination of particle size and polydispersity
index (PDI):
The particle size and PDI of nanoemulsions are
analysed employing photon correlation spectroscopy
(PCS) using Malvern Zetasizer, which monitors the
variation in light scattering because of Brownian motion
of particles as function of time. PCS is based on the
principle that the particles with small size travels with
higher velocity as compared to particles with large size.
The laser beam gets diffracted by sub-micron particles
present in solution. Due to diffusion of particles, rapid
uctuations in laser scattering intensity occur around
a mean value at a xed angle and this is dependent
upon particle size. The calculated photoelectron time-
correlation function generates a histogram of the line
width distribution that can be related to the size of
particle. For measuring particle size, weighed amount
of formulation is dispersed in double-distilled water for
obtaining homogenous dispersion and that has to be
used instantly for measuring the particle size and PDI.
The PDI can range from 0 to 1, where 0 (zero) stands for
monodisperse system and 1 for a polydisperse particle
dispersion[28]. Đorđević et al. evaluated the particle
size and PDI of risperidone nanoemulsion by using
this method and reported mean particle size around
160 nm with mean size distribution less than 0.15[29].
Singh et al. has also adopted the same technique and
reported particle size of primaquine nanoemulsion in
the range of 20-200 nm[5].
Determination of zeta potential:
The zeta potential is a method for measuring surface
charge of particles when it is placed in liquid. Zeta
potential is used for predicting dispersion stability
and its value depends on physicochemical property
of drug, polymer, vehicle, presence of electrolytes
and their adsorption. It is measured by Malvern
Zetasizer instrument. For measuring zeta potential,
nanoemulsion is diluted and its value is estimated
from the electrophoretic mobility of oil droplets. Zeta
potential of ±30 mV is believed to be sufcient for
ensuring physical stability of nanoemulsion. Đorđević
et al. obtained zeta potential around –50 mV by using
Malvern Zetasizer for risperidone nanoemulsion[29].
Fourier-transform infrared spectroscopy (FTIR)
spectral analysis:
FTIR analysis can be carried out for the assessment
of drug excipient interaction, polymerization,
crosslinking as well as drug loading in the formulation.
It is also used for identifying the functional groups
with their means of attachment and the ngerprint of
the molecule. At low temperature a molecule exists in
ground state and on absorbing the radiant energy, they
get excited to higher energy states. IR spectroscopy
is based on determining this energy difference (∆E)
between the excited and ground states of the molecule.
For performing FTIR, sample can be prepared by
employing suitable method such as potassium bromide
pellet method, Nujol mulls and then sample is scanned
in FTIR at moderate scanning speed between 4000-
400 cm-1. Srilatha et al. conducted FTIR studies on pure
drug and glipizide nanoemulsion and reported absence

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of drug excipient interactions (hence compatibility of
drug and excipient) as all the characteristics peaks of
drug appeared at same point in formulation[30].
Morphological study of nanoemulsion:
The morphological study of nanoemulsion is carried
by using transmission electron microscopy (TEM).
In TEM, a beam of electron is incident on a thin foil
specimen and passed through it. On interacting with
the specimen, these incident electrons transform into
unscattered electrons, elastically scattered electrons
or inelastically scattered electrons. The distance
among the objective lens and the specimen and among
the objective lens and its image plane regulates the
magnication. The electromagnetic lenses concerted
the unscattered or scattered electrons and cast them
onto a screen that produce amplitude-contrast picture, a
phase-contrast image, electron diffraction, or a phantom
picture of distinct darkness, which is dependent upon
the density of unscattered electrons. Bright eld
imaging at increasing magnication in combination
with diffraction modes used for disclosing the size
and form of nanoemulsion droplets. For performing
TEM, few drops of nanoemulsion or a suspension
of lyophilized nanoparticles is prepared in double-
distilled water and are placed onto holey lm grid and
immobilized. Excess solution has to be wicked off from
the grid following immobilization and stained. The
stained nanoparticles are then examined at particular
voltage[31]. Singh et al. studied surface morphology
characteristics of primaquine nanoemulsion by TEM
analysis and reported spherical shape of primaquine
nanoemulsion with smooth surface[5].
Atomic force microscope (AFM):
AFM is comparatively a new technique being used
these days for exploring the surface morphology of
nanoemulsion formulations. AFM is carried out by
diluting nanoemulsions with water followed by drop
coating of the diluted nanoemulsion on a glass slide.
Further the coated drops are dried in oven and scanned
at of 100 mV/s[32]. Drais et al. performed AFM study
on carvedilol nanoemulsion and found that the size
varied from 42 to 83 nm with good stability of the
formulation[33].
In vitro drug release study:
In vitro drug release studies help to estimate the in vivo
performance of drug formulation. The in vitro release
rate of a drug is usually studied on a USP dissolution
apparatus. Nanoemulsion or dried nanoparticles
containing drug equivalent to 10 mg were dispersed in
buffer and then it is introduced into dialysis membrane
pouches and placed in a ask containing buffer. This
study is carried out at 37±0.5° and a stirring speed of
50 rpm. Sample are withdrawn at periodic intervals
and each time replaced by the same volume of
fresh dissolution medium. Samples are then diluted
suitably and the absorbance of sample is measured
spectrophotometrically at a particular wavelength.
Absorbance of the collected sample is used for
calculating % drug release at different time intervals
using calibration curve[31]. Kotta et al. studied the
in vitro drug release prole of antiHIV drug
nanoemulsion using dissolution apparatus type-II and
reported 80 % drug release in 6 h[34].
In vitro skin permeation studies:
Keshary Chien-diffusion cell is used for investigating
in vitro and ex vivo permeation studies. For performing
permeation studies, abdominal skin of adult male rats
weighing 250±10 g is usually employed. The rat skin is
positioned between the donor and the receiver chambers
of diffusion cells. Temperature of receiver chambers
containing fresh water with 20 % ethanol is xed at
37° and the contents of the chamber are continuously
stirred at 300 rpm. The formulations are kept in the
donor chamber. At specic time intervals such as 2, 4,
6, 8 h, a certain amount (0.5 ml) of the solution from
the receiver chamber was removed for performing gas
chromatographic analysis and each time replaced with
an equivalent volume of fresh solution immediately.
Each sample is performed three times. Cumulative
corrections are done for obtaining total amount of
drug permeated through rat skins at each time interval
and are plotted against function of time. Slope of
plot is used for calculating the permeation rates of
drug at a steady-state[35]. Harwansh et al. used Franz
diffusion cell for assessing transdermal permeability of
glycyrrhizin through human cadaver skin and reported
increased permeability with nanoemulsion formulation
as compared to conventional gel[36].
Stability studies:
Stability studies are performed for assessing stability
of the drug substance under the inuence of a various
environmental factors like temperature, humidity
and light. The stability studies of nanoemulsion are
carried out after storing the formulation for 24 mo in
dispersed and freeze-dried state as per International
Conference on Harmonisation guidelines. The storage
conditions followed are ambient (25±2°/60±5 % RH),

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refrigeration (5±3°) and freeze (–20±5°). The requisite
volume of nanoemulsion is stored in glass bottles and
is tightly sealed. Samples are withdrawn at predened
time interval and analysed for the characteristics such
as particle size, loading and EE and in vitro drug
release prole[26]. Singh et al. performed stability
studies on nanoemulsion and observed that no change
in viscosity, drug content and particle size when the
formulation was stored for 3 mo at 25°/60 % RH and
30°/65 % RH[5].
Shelf life determination:
For determining shelf life of a nanoemulsion,
accelerated stability studies are performed. The
formulations are stored at three distinct temperatures
and ambient humidity conditions (30°, 40° and 50±0.5°)
for almost 3 mo. After a particular time interval (0,
30, 60 and 90 d) samples are withdrawn and analysed
using HPLC at λmax for estimating the remaining drug
content. Samples withdrawn at zero time are used as
controls. The order of the reaction is determined by
this and after that the reaction rate constant (K) for the
degradation is calculated from the slope of the lines by
using following equation at each elevated temperature:
slope = −K/2.303, the logarithm values of K are plotted
at different elevated temperatures against the reciprocal
of absolute temperature (Arrhenius plot). From this
plot value of K at 25° is determined and it is further
used for calculating shelf life by putting the value in
following Eqn.: t0.9=0.1052/K25. Where t0.9 stands for
time required for 10 % degradation of the drug and it is
termed as shelf life[31]. Ali et al. determined the shelf life
of clobetasol propionate-loaded nanoemulsion around
2.18 y at room temperature (25°) and concluded that
the stability of clobetasol propionate can be augmented
by incorporating in a nanoemulsion[37]. Parveen et al.
reported that the shelf life of a silymarin nanoemulsion
to be around 3.8 y when stored in a refrigerator[38].
Thermodynamic stability studies:
Thermodynamic stability studies are usually carried
out in three steps. Firstly heating-cooling cycle, which
is performed for observing any effect on the stability
of nanoemulsion by varying temperature conditions.
Nanoemulsion is exposed to six cycles between 4°
(refrigeration temperature) and 40° by storing the
formulation at each temperature for not less than
48 h. The formulations which are stable at these
temperatures are further chosen for centrifugation
studies. Secondly, centrifugation study in which
the formulated nanoemulsions are centrifuged at
5000 rpm for 30 min and observed for phase separation
or creaming or cracking. Those which did not show any
sign of instability are subjected to freeze thaw cycle.
Thirdly, the freeze-thaw cycle, in which nanoemulsion
formulations are exposed to three freeze-thaw cycles
with temperature varying between –21° and +25°.
Formulations that show no signs of instability pass
this test and deemed to have good stability[6]. These
formulations are then subjected to dispersibility studies
for evaluating the efciency of self-emulsication.
Srilatha et al. performed thermodynamic studies on
glipizide nanoemulsion by subjecting it to three cycles
of stability and reported good physical stability of
nanoemulsion with no appearance of phase separation,
creaming or cracking[30].
Dispersibility studies:
Dispersibility studies for evaluating the efciency of
self-emulsication of nanoemulsion are carried out
by using a standard USP XXII dissolution apparatus
2.1 ml of each formulation is incorporated into 500 ml
of distilled water maintained at 37±0.5°. A standard
stainless steel dissolution paddle rotates at 50 rpm for
providing gentle agitation. In vitro performance of the
nanoemulsion formulations is evaluated visually by
using a grading system described below[6]. Grade A
nanoemulsions form rapidly within 1 min and appear to
be clear or bluish. Grade B nanoemulsions form rapidly
but are slightly less clear emulsions appear to be bluish-
white. Grade C nanoemulsions are ne milky emulsion
that form within 2 min. Grade D are those dull, greyish-
white emulsions that has a little oily appearance and
are slower to form (>2 min). Grade E nanoemulsions
display either poor or negligible emulsication with
large oil globules present on the surface.
Determination of viscosity:
Viscosity assessment is an important parameter for
physicochemical characterization of nanoemulsion.
Various instruments are employed for measuring
viscosity such as Ostwald viscometer, Hoeppler falling
ball viscometer, Stormer viscometer, Brookeld
viscometer and Ferranti-Shirley viscometer. Among
all these viscometer, Brookeld is the preferred
one for measuring the viscosity of nanoemulsion.
Determination of viscosities afrms whether the
system is O/W or W/O emulsion. Low viscosity of
systems shows that it is O/W type and high viscosity
shows that it is water in oil type system[28]. However,
currently survismeter has been the most widely
employed equipment as it measures surface tension,

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viscosity, interfacial tension, contact angle, dipole
moment and particle size and hydrodynamic volumes
of the nanoemulsions[39]. Shaq et al. has determined
viscosity of ramipril nanoemulsion formulations by
using Brookeld cone and plate rheometer and reported
the viscosity of formulations as less than 21 cP with the
minimum viscosity of 10.68 cP[6].
Refractive index:
Refractive index tells how light propagates through
the medium and transparency of nanoemulsion.
Refractive index (n) of medium can be dened as ratio
of speed of wave (c) in reference medium to the phase
speed of wave (vp) in medium: n=c/vp. Refractive
index of the nanoemulsion can be determined by
Abbes type refractometer at 25±0.5° by placing a
drop of nanoemulsion on slide and comparing it with
refractive index of water (1.333). If refractive index
of nanoemulsion has equal refractive index as that of
water, then the nanoemulsion is considered to have
transparent nature[2,28]. Harika et al. measured the
refractive index of amphotericin B nanoemulsion by
Abbe refractometer and the value of refractive index
of the formulation was found to be similar to that of
the water[40].
Percent transmittance:
Percent transmittance of a formulated nanoemulsion is
estimated using UV spectrophotometer at a particular
wavelength with distilled water as a blank. If percent
transmittance of a nanoemulsion is found to be greater
than 99 %, then it is considered as transparent in
nature[31]. Harika et al. reported percent transmittance
of >97 % for a amphotericin B nanoemulsion
formulated[40].
pH and osmolarity measurements:
The pH meter is used for measuring the pH of a
nanoemulsion and microosmometer is used for
determining the osmolarity of emulsion, which is
based upon freezing point method. For performing this,
100 µl of nanoemulsion is transferred in microtube and
measurements are taken[41]. Morsi et al. measured the
pH of the acetazolamide nanoemulsion by pH meter
and found pH in the range of 4.9 to 5.5 thus claiming
it to be adequate and non-irritant for application to the
eye[42].
Dye solubilisation:
A water soluble dye is dispersible in an O/W globule
whereas it is soluble in the aqueous phase of the W/O
globule. Similarly an oil soluble dye is dispersible in
the W/O globule but soluble in the oily phase of the
O/W globule[3]. On adding water soluble dye to O/W
nanoemulsion, it will evenly takes up the colour
whereas if it is a W/O emulsion, dye will remain in
dispersed phase only and the colour will not spread
evenly. This can be seen with microscopic examination
of emulsion[4]. Laxmi et al. carried out this test on
artemether nanoemulsion by adding eosin yellow, a
water soluble dye to the formulation and examined it
under a microscope. They discovered that the aqueous
continuous phase was labelled with dye while the
oily dispersed phase remained unlabelled therefore
conrming the formed nanoemulsion as O/W type[43].
Dilutability test:
The rationale of dilution test is that continuous phase
can be added in larger proportion into a nanoemulsion
without causing any problem in its stability. Thus
O/W nanoemulsions are dilutable with water but W/O
nanoemulsions are not and go through a phase inversion
into O/W nanoemulsion. The W/O nanoemulsion can
be diluted with oil only[3,4]. Laxmi et al. performed
dilutability test on nanoemulsion by diluting it with
water and observed no sign of phase inversion and
precipitation thus claiming their nanoemulsion
formulation to be stable[43].
Conductance measurement:
The O/W nanoemulsions are highly conducting
because they have water in external phase whereas
W/O nanoemulsions are not conducting as they
have water in internal or dispersal phase. Electrical
conductivity measurements are very much benecial
for determining the nature of the continuous phase
and for detecting phase inversion phenomena. At
low volume fractions, increase in conductivity of
certain W/O nanoemulsion systems was observed
and such kind of behaviour is deduced as an indicator
of a percolative behaviour or ions exchange among
droplets prior to the development of bicontinuous
structures. Dielectric measurements are a great means
of exploring the structural and dynamic features of
nanoemulsion systems[3]. Conductometer is employed
for determining the conductance of nanoemulsion.
For carrying out conductance measurement, a pair of
electrodes is attached to a lamp and an electric source is
immersed into an emulsion. When the emulsion is O/W
type then water will conduct the current and lamp will
glow because of passage of current among connecting

www.ijpsonline.com
September-October 2018
Indian Journal of Pharmaceutical Sciences
788
electrodes. The lamp will not glow if it is water in oil
emulsion as oil in external phase does not conduct
the current[4]. Harika et al. performed conductivity
test on amphotericin B nanoemulsion using an
electroconductometer. They reported conductivity of
the formulations in the range of 454.2-552.3 µS/cm
and concluded the system to be O/W on the basis of
electroconductivity study[40].
Interfacial tension:
By measuring the interfacial tension, the formation and
the properties of nanoemulsion can be investigated.
Ultra low values of interfacial tension corresponds to
phase behaviour, mainly the coexistence of surfactant
phase or middle-phase nanoemulsions with aqueous
and oil phases in equilibrium. For determining ultra-
low interfacial tension spinning-drop apparatus is
used. Interfacial tensions are obtained by measuring
the shape of a drop of the low-density phase, rotating it
in cylindrical capillary lled with high-density phase[3].
Fluorescence test:
There are numerous oils that show uorescence under
UV light. If a W/O nanoemulsion is subjected to a
uorescence light under a microscope, the whole eld
will uoresces and if it is an O/W the uorescence will
be in spots[4].
In vivo studies:
In vivo studies can be performed by adopting suitable
animal model according to the activity chosen.
Srilatha et al. has performed antidiabetic activity on
glipizide nanoemulsion by choosing hyperglycaemia
model in which they rst induce diabetes in rats by
intraperitoneal injection of streptozotocin solution
and then the formulation was given to diabetic rats
and the pharmacodynamic studies were performed on
them. They reported the reduction in blood glucose
levels for up to 12 h[30]. Chouksey et al. has evaluated
in vivo performance of atorvastatin nanoemulsion by
performing pharmacokinetic studies on nanoemulsion
and they reported better bioavailability of nanoemulsion
formulation as compared to pure drug[44].
Nanoemulsions hold great potential as an efcient
drug delivery tool that could be effectively harnessed
to realise the complete potential. Quality assurance and
quality control shall be of paramount importance with
such a precise delivery system and hence the evaluation
tests are to be performed rigorously.
Acknowledgements:
Authors thanks UGC, New Delhi, for awarding Rajiv
Gandhi National Fellowship.
Conicts of interest:
There are no conicts of interest among the authors.
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