SAMPLE REPORT

Practical 5: Preparation and evaluation of colloidal formulation

Aim:
To prepare and characterize colloidal formulation, and evaluate the physical stability of the
developed colloidal formulation.

Principle:
Colloid contains dispersed particles larger than the solute particles in true solutions. Colloids
are homogeneous mixtures that are intermediate between a true solution and heterogeneous
mixtures. Colloids contain "solute" particles that are many times larger than the solvent
molecules, but too small to be seen by an ordinary light microscope. Since it is not a true
solution, the terms solvent and solute are often not being used. Instead, the following terms
are used: dispersing medium (which is equivalent to solvent) and dispersed phase (equivalent
to solute).
Nanoemulsions, one of the example of colloidal formulations, are quaternary or
pseudoquaternary systems composed of oil, water and surfactant and/ or co-surfactant. These
are optically isotropic, transparent, homogeneous/heterogeneous systems at molecular scale
and are thermodynamically stable as compared to conventional emulsion formulations.
Nanoemulsion was also known to be a nano-sized dispersion of oil droplets in the continuous
phase of water, or vice versa, where the thin layer of surfactants (frequently used with a co-
surfactant) system stabilizes the nanostructures. It is also termed as microemulsion,
miniemulsion, fine-dispersed emulsion, submicron emulsion, ultrafine emulsion, translucent
emulsion etc. with an overlapping particle size within a nano range of 10 to 500 nm.
However, the widely reported particle or globule size of nanoemulsion was found to be
within a range of 10 to 200 nm.

Preparation of colloid (nanoemulsion):

In general, preparation of thermodynamically stable colloids is difficult. Therefore, special
methods are required in the development of colloids. There are different methods to develop
nanoemulsion, basically can be differentiated by low-energy emulsification and high-energy
emulsification. Reduction of size of the dispersed phases could be achieved by introduction
of external forces using high-speed homogenizer, ultra-sonication, etc.
Nanoemulsion manufacturing utilizing high energy emulsification method can highly be
monitored to get the desired nanoemulsion of required droplet size simply by changing in
components, which will further control the rheological property, formulation stability and the
color of the final emulsion. The size of the droplets of even within the range of around 1 nm
can be achieved by this method using high-pressure homogenizer/ piston homogenizer.
Despite such advantages of this methodology along with industrial scalability property, the
method is not suitable for thermo-labile components, viz: retinoids, and macromolecules,
such as peptides, nucleic acids and enzymes. However, incorporation of external energy
makes them thermodynamically unstable, and thus stability of the formulation get hampered.

Stability of hydrophobic colloids:

Stability of a nanoemulsion system is a major issue that needs scientific knowledge of colloid
chemistry. Judicious selection of oil, surfactant and co-surfactant ratio will ensure stable
formulation; or else phase separation may result. The small droplet size favors high
thermodynamic stability by preventing flocculation, aggregation, coalescence and Ostwald
ripening.

Principle on the effect of components:

The main components of nanoemulsion are oil, surfactants, co-surfactants, and aqueous
phases.

Oil:
The oil is a decent solvent system for the hydrophobic drug of interest to entrap its required
amount of loading by quick emulsification. Generation of emulsification region in phase
studies assist in the selection of oils, which is again influenced by the molecular volume and
the chain length of the oil. It has been reported that small emulsification region of vegetable
oils with triglycerides of long-chain fatty acids (viz: soybean oil) results in difficulties in
emulsification process. On the other hand, a decrease in the hydrophobicity of the oil phase
could affect emulsification region positively, although the decrease in hydrophobicity of oil
negatively effects on the solubility of hydrophobic drugs into it.

Surfactant:
Surfactants are an integral component of a nanoemulsion system. They are incorporated in
nanoemulsion to stabilize the emulsion system with retaining the globule sizes in nano range;
otherwise, individual globules merge to increase their size, which ultimately leads to phase
separation. The stability of the system depends on the choice of surfactant and its
concentration. Some of the desired criteria of a surfactant include- ability to reduce the
surface tension, rapid absorption onto dispersed phase globule to form a complete and
coherent film so that it does not coalesce and viscosity in the system to confer optimum
stability, and effective at low concentration.

Co-surfactants:
Co-surfactant in a nanoemulsion plays an important role as its combination with surfactant
provides required structural flexibility to the nanoemulsion system. Surfactant alone results in
a rigid surfactant film that cannot lower the oil-water interfacial tension sufficiently to yield
emulsification process. When a co-surfactant is combined with surfactant in a nanoemulsion
system, it penetrates the surfactant layer, disrupts the interfacial film, confers required
fluidity, lowers the interfacial tension, and finally help in the emulsification process.
Co-surfactants also play a role in modification of the curvature of the oil-water interface
based on the relative importance of their polar groups. It may also help solubilization of the
oil. Cosurfactants that can be used in nanoemulsion formulations include short-chain
alcohols, such as ethanol, propylene glycol, glycerol, benzyl alcohol, polyethylene glycol 400
and glycofurol.

Method:
1. Measure the required volume of oil and transfer it in a test tube.
2. Add the measured volume of surfactant and co-surfactant mixture dropwise to the oil in
the test tube.
3. Mix them properly using vortex mixer.
4. Measure the water required to prepare the colloidal nanoemulsion. Add measured volume
of water dropwise to the above mixture and continue mixing. The mixture should be
mixed vigorously for 10 minutes.
5. Observe the preparation for its appearance and record in Table 1.
6. Measure the droplet size and polydispersity index (PDI) using Malvern Zeta sizer
instrument and note down the obtained result in Table 1.
7. To evaluate thermodynamic stability, centrifuge the developed formulation at 5000 rpm
for 15 minutes and repeat the steps described in 5 and 6.
8. Discuss the findings and conclude.
 Formula
Formulation 1 Formulation 2 Formulation 3 Formulation 4
Ingredients
%v/v %v/v %v/v %v/v

Capryol 90 3.5 6 8.5 11

Oil
Vit E 4 4 4 4
Surfactant %
25 25 25 25
(tween 20)
Co-surfactant %
5 5 5 5
(Transcutol P)

Table 1: Observations and inference to predict the stability of colloidal nanoemulsion

Initial observations Final observations
Transparent/ Transparent/
Test tube
translucent/ Observed data translucent/ Observed data
white white
Formulation 1 Transparent Size: 37.92 d.nm Transparent Size: 63.95
with a faint with a faint d.nm
yellow color PDI: 0.371 yellow color
PDI: 0.147
Formulation 2 Transparent Size: 90.78 d.nm
with a faint
yellow color PDI: 0.352

Formulation Size: 183.4nm Size: 169.3nm

Translucent Translucent
Centrifugation

3 with a faint PDI: 0.450 PDI: 0.534

yellow
color
Formulation Size: 161.4nm
Translucent - -
4 with a faint PDI: 0.526
yellow
color

Results and discussion:

A colloid may be defined as a heterogeneous (two phase system consisting of minute
particulate of 1nm- 100nm) substance dispersed into a continuous phase or dispersion
medium. Stability of colloids is accomplished by two means: lyophilic colloids and lyophobic
colloids. In addition, the stability of the colloid also affects the optical, kinetic, and electrical
properties of the colloid.

The polydispersity index (PDI) is a parameter that describes the width or spread of the
particle size distribution, oftentimes used in the evaluation of nanosuspensions. The
maximum PDI value is arbitrarily limited to 1.0, whereby a PDI of 1.0 indicates that the
sample has a very broad size distribution and may contain large particles or aggregates that
could sediment slowly. As such, it can be noted in the observations that both colloidal
formulations prepared have a relatively narrow particle size distribution with Formulation 1
having a slightly wider range at 0.371 than Formulation 2 at 0.352.

The Z-average refers to the mean or average particle size of the formulation. As such, it can
be seen in the observations that Formulation 1 has a smaller average particle size at 37.92
d.nm than Formulation 2 at 90.78 d.nm. It can be noted that the PDI decreases as the Z-
average increases, as seen when the PDI of Formulation 1 decreases from 0.371 to 0.147 as
the Z-average particle size increases from 37.92 d.nm to 63.95 d.nm after centrifugation.

Formulation 1 shows relatively good stability as cracking and separation of phases is not
observed after centrifugation. The formulation also remains clear after centrifugation and the
Z-average of the formulation remain in the nano range of the zetasizer with a value of 63.95
d.nm and the PDI indicates a narrower particle size distribution range at 0.147. Based on the
Table 1, the nanoparticle size of formulation 3 is 183.4nm, however, after centrifuge the
coated nano particle size decreased to 169.3nm. Formulation 3 had a PDI value of 0.450
before centrifugation. The PDI value became higher after centrifugation, at 0.534.

Besides, the average particle size of formulation 3 before centrifugation was 183.4 nm, while
the particle size of formulation 4 was 161.4 nm. However, the PDI value of formulation 4
(0.526) was higher than that of formulation 3 (0.450) before centrifugation. This is due to the
concentration of Capryol 90 in formulation 4 is higher than formulation 3.

PDI is basically a representation of the distribution of size populations within a given sample.
The numerical value of PDI ranges from 0.0 (for a perfectly uniform sample with respect to
the particle size) to 1.0 (for a highly polydisperse sample with multiple particle size
populations). Before centrifugation all the formulations had the values of PDI that denotes the
high polydisperse with multiple particle size. PDI value of Formulation 1 and 2 after
centrifugation shows that they are having uniform particle size, whereas Formulation 3 and 4
shows that they possess high polydisperse with multiple particle size. Stability of the
formulations can be compared using the zetapotential obtained. The zeta potential measures
the potential difference between the surface charges and those of opposite sign deriving from
the medium that are arranged around the particle. More positive or negative is the zeta
potential, larger is the colloidal stability. Generally, zeta potentials of +/- 40-50 mV are
sufficient to ensure good colloidal stability.
Conclusion:

Formulation 1 shows good stability, as no cracking is observed after centrifugation and the
formulation remains transparent. The Z-average of the colloid remain in the nano range of the
zetasizer and the PDI indicates a narrow particle size distribution range.