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Current Pharmaceutical Analysis, 2015, 11, 11-15 11

Application of Droplet Size Analysis for the Determination of the Required

HLB of Lemon Oil in O/W Emulsion

N.A. Niczinger, N. Kllai-Szab, J. Dredn, L. Budai, M. Hajd and I. Antal*

Department of Pharmaceutics, Semmelweis University, 1092 Budapest, Hungary

Abstract: Hydrophile-lipophile balance method is one of the requirements which can complete the existing guidelines,
thereby making the most stable emulsion. The aim of present work was to determine the required hydrophile-lipophile
balance of lemon essential oil in oil-in-water emulsions. Paraffin oil and its known required hydrophile-lipophile balance
were used as a standard. Span 80 and Tween 80 or Gelucire 44/14 blend were applied as emulsifying agents. Emulsions
were evaluated by droplet size distribution and turbidity measurements. Based on the estimated stability of emulsion se-
ries and according to the droplet size analysis with lower variations, the required hydrophile-lipophile balance of lemon
oil was measured approximately 12 in oil-in-water systems.
Keywords: Droplet size distribution, oil-in-water emulsion, paraffin oil standard, required HLB, surface active agents,
turbidity.

1. INTRODUCTION of emulsion physical stability and HLB. In the last decade,

Recently pharmaceutical emulsion systems have im-
mensely attracted the interests of researchers due to their
variable stabilities depending on the applied surface active
agents with both hydrophilic and lipophilic groups. In the
1940s, William C. Griffin defined the Hydrophile-Lipophile
Balance (HLB) as a balance of the size and strengths of these
two opposing groups and introduced the HLB scale (between
0 and 20 with the exception of some extreme cases) which
can help choosing the appropriate emulsifier [1]. It is well
known that surface active agents with lower HLB are typi-
cally lipophilic and with higher HLB have hydrophilic prop-
erties [2, 3]. In addition, the blends of emulsifiers are consid-
ered as more efficient than using one surfactant [4]. For de-
scription of the HLB system, various experimental and theo-
retical methods were used in order to determine the HLB
values of commonly used surface active agents [5]. Although
Griffin has already mentioned about the required HLB val-
ues for oils and waxes but currently such data are less well
known than the HLB values of the emulsifiers [6]. The sig-
nificance of HLB is also demonstrated by the introduction of
new synthesized surface active agents which are character-
ized to offer the proper use in the practice [7].
The required HLB value is a characteristic attribute of
pharmaceutical substances. It corresponds to the HLB value
of the combined emulsifying agents which are able to form
an oil-in-water (O/W) or water-in-oil (W/O) stable emulsion
with the substance. Several indirect methods are applied for
the analysis of required HLB which are based practically on
the study of emulsion stability. Droplet size analysis and
turbidimetry are the most often used test methods demon-
strating a minimum or a maximum value in the relationship
*Address correspondence to this author at the Department of Pharmaceutics,
Semmelweis University, 1092 Budapest, Hungary Tel: +36/1-2317-0914;
Fax: +36/1-2317-0914; E-mail: antal.istvan@pharma.semmelweis-univ.hu
investigations focused on the required HLB values of several
oils and essential oils are summarized in Table 1 [6, 8, 9, 10,
11, 12, 13].
Essential oils are made up from plants and are volatile
and fragrant. Their importance is increasing due to their sig-
nificant therapeutic effects [14] therefore several experi-
ments focus on their further applicability [6, 15, 16, 17].
Lemon essential oil is described in the European Pharmaco-
poeia 7 (Limonis aetheroleum Citrus limon (L). Burman
fil.).
The objective of the present work was to determine the
required HLB of the lemon oil in oil-in-water emulsion sys-
tems in order to prepare more stable emulsion systems with
this essential oil.
2. MATERIAL AND METHODS
2.1. Materials
Sorbitan oleate (Span 80, HLB 4.3), Polysorbate 80
(Tween 80, HLB 15.0) and liquid paraffin were purchased
from the Sigma-Aldrich Ltd. (St. Louis, USA). Lemon oil
was obtained from the Hungaropharma Ltd. (Hungary). Ge-
lucire 44/14 (lauroyl macrogol-32 glyceride with HLB
value of 14) was donated from the Gattefoss Corp. (France).
All materials were of European Pharmacopoeia grade.
Demineralized water ( < 3 S/cm) was used as an aqueous
phase.
2.2. Methods
2.2.1. Preparation of Emulsions
Emulsion series were made according to the method used
by Orafidiya and Oladimeji [6]. Three different emulsion
compositions (3x8=24 formulas) were prepared according to
Table 2. Lemon oil emulsions contained a reduced ratio of

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12 Current Pharmaceutical Analysis, 2015, Vol. 11, No. 1 Niczinger et al.

Table 1. Experimentally measured required HLB values of oils.

Oil/essential oil (Name of the plant) required HLB References

Eucalyptus oil (Eucalyptus sp.) 9.9 [6]

Lippia oil (Lippia multiflora) 12.0 [6]

Peppermint oil (Mentha sp.) 12.4 [6]

Marigold oil (Calendula officinalis) 6 [8]

Andiroba oil (Carapa guianensis) 16.7 [9]

Jojoba oil (Simmondsia chinensis) 12.5 [10]

Rosmary oil (Rosmarinus officinalis) 15 [11]

Rosmary oil (Rosmarinus officinalis) 16.5 [12]

Illicium verum essential oil 16.7 [12]

Licuri oil (Syagrus coronate) 11.8 [13]

Table 2. HLB values and compositions of the emulsion series.

Formulation HLB value 0.2 Tween 80 (%) Gelucire 44/14 (%) Span 80 (%) Liquid paraffin (%) Lemon oil (%) Water (%)

I/1 6.0 15.9 - 84.1 20.0 - 75.0

I/2 8.0 34.6 - 65.4 20.0 - 75.0

I/3 10.0 53.3 - 46.7 20.0 - 75.0

I/4 11.0 62.6 - 37.4 20.0 - 75.0

I/5 12.0 71.9 - 28.1 20.0 - 75.0

I/6 15.0 100.0 - 0.0 20.0 - 75.0

II/7 6.0 - 17.5 82.5 20.0 - 75.0

II/8 8.0 - 38.1 61.9 20.0 - 75.0

II/9 10.0 - 58.8 41.2 20.0 - 75.0

II/10 11.0 - 69.1 30.9 20.0 - 75.0

II/11 12.0 - 79.4 20.6 20.0 - 75.0

II/12 14.0 - 100.0 0.0 20.0 - 75.0

III/13 4.3 0.0 - 100.0 - 5.0 92.5

III/14 5.0 6.5 - 93.5 - 5.0 92.5

III/15 6.0 15.9 - 84.1 - 5.0 92.5

III/16 7.0 25.2 - 74.8 - 5.0 92.5

III/17 8.0 34.6 - 65.4 - 5.0 92.5

III/18 9.0 43.9 - 56.1 - 5.0 92.5

III/19 10.0 53.3 - 46.7 - 5.0 92.5

III/20 11.0 62.6 - 37.4 - 5.0 92.5

III/21 12.0 71.9 - 28.1 - 5.0 92.5

III/22 13.0 81.3 - 18.7 - 5.0 92.5

III/23 14.0 90.6 - 9.4 - 5.0 92.5

III/24 15.0 100.0 - 0.0 - 5.0 92.5

Application of Droplet Size Analysis for the Determination
lemon oil in the oil phase due to the diverse chemical proper-
ties compared to the paraffin oil. Surface active agents were
used in various ratios to reach the desired HLB values. The
required amount was calculated with equation used by
Rodrguez-Rojo et al. [11]. Systems contain Span and
Tween or Gelucire in different ratio as emulgents.
The refrigerated (2-6 C) samples were examined after 1
week storage.
2.2.2. Macroscopic Appearance of the Emulsions
Macroscopic observations were carried out to find any
changes for the emulsions structure [8, 12]. Refrigerated
samples were examined after 1 week storage. During the
storage period, emulsions were kept in glass tubes sealed
with rubber stopper and parafilm. Photos were taken by the
digital camera (EX-FH100, Casio, Japan) and analyzed the
differences appearing on the interfaces of the upper and
lower phases.
2.2.3. Droplet Size Analysis
Prepared emulsions on day 0 and after 1 week storage
were characterized with Mastersizer 2000 (Malvern In-
struments Ltd., UK) in the dispersion unit Hydro SM
(1500 rpm). Triplicate samples were analyzed and the
measurements were carried out within 5 minutes. Since a
correlation was found earlier between droplet size and
HLB value [6], D 0.9 value was evaluated as a main charac-
teristic parameter meaning the diameter (m) where 10%
Fig. (1). Emulsion series after 1 week: Formulation I (1a) and III (1b, 1c).
Current Pharmaceutical Analysis, 2015, Vol. 11, No. 1 13
of the distribution is above and 90% is below than the
given value.
2.2.4. Turbidimetric Measurement
The procedure and calculation are based on earlier works
[6, 9, 10, 12]. It is a well known and useful technique to de-
termine which system is the most stable. The percentage
transmission (%T) was measured with a UV/VIS spectropho-
tometer (UV2, Unicam Ltd., UK).
3. RESULTS AND DISCUSSION
3.1. Macroscopic Appearance of the Emulsions
After 1 week storage of paraffin O/W emulsions (Formu-
lation I/1-6; Fig. (1a)) a distinct line appeared between the
separated phases. In accordance with literature data - as men-
tioned above - the required HLB of the paraffin oil was
found to be 11-12. Similar results were obtained in emul-
sions prepared with Gelucire 44/14 as a hydrophilic emul-
gent (Formulation II/7-12). However, it was not possible to
identify macroscopically an exact interval for required HLB
value of the lemon essential oil emulsions (Formulation
III/13-24; Fig. (1b and 1c)).
3.2. Droplet Size Distribution and Turbidity Analysis in
Paraffin Oil Emulsions
Knowing the fact that the most stable emulsion has the
lowest droplet size distribution and the highest turbidity,
14 Current Pharmaceutical Analysis, 2015, Vol. 11, No. 1 Niczinger et al.

Fig. (2). Droplet size distribution (solid line) and turbidity (dotted line) as a function of HLB for paraffin oil emulsion series (n=3; SD): -
Tween 80-Span 80, - Gelucire 44/14-Span 80.

Fig. (3). Droplet size distribution of lemon oil emulsions characterized by D0.9 on day 0 (dotted line) and after 1 week (solid line) (3a) and by
cumulative distribution curves after 1 week (3b) (n=3; SD): * - HLB 5, - HLB 6, + - HLB 7, HLB 8, x HLB 9, HLB 10, -
HLB 11, HLB 12, HLB 13, HLB 14, - HLB 15.

makes possible the determination of required HLB values for
various emulsions [6, 10, 11, 12, 13]. Plotting the average
droplet size as a function of HLB value, a minimum point
can be observed for each curve. On the other hand, a maxi-
mum point can be seen in case of plotting the turbidity as a
function of HLB value in (Fig. 2). The two above mentioned
parameters, droplet size and turbidity were changed similarly
allowing the precise determination of the required HLB
value. The results of the same procedure using paraffin oil
were correlated with earlier data.
3.3. Droplet Size Distribution in the Lemon Oil Emul-
sions
Droplet size distribution is shown on (Fig. 3a and 3b).
Plotting the D0.9 values as a function of HLB values (Fig. 3a)
a minimum can be seen at 12 value which serves for the de-
termination of the required HLB. Because of the strong me-
chanical impact, the same tendency can be observed even
after 1 week storage (Fig. 3a). In addition, the comparison of
the cumulative distribution curves of lemon oil after 1 week
storage allows the same conclusions (Fig. 3b).
3.4. Turbidity Analysis in Lemon Oil Emulsions
As mentioned above, in emulsions after 1 week storage
phase separation can be observed. The lower (aqueous)
phases were tested by turbidimetric method. As shown in
(Fig. 4) the plots of the turbidity measurements have a maxi-
mum point at HLB 11 value.
CONCLUSION
In this study, lemon essential oil emulsions were pre-
pared and the required HLB was determined in oil-in-water
Application of Droplet Size Analysis for the Determination
Fig. (4). Turbidity for lemon oil series (n=3; SD).
systems compared to paraffin oil emulsion standards. Since
the results obtained by turbidimetry were characterized with
higher variations, the droplet size distribution measurements
were preferred and the required HLB value in case of lemon
essential oil was found to be close to 12. This experimentally
determined value of the required HLB of lemon oil may
serve as a characteristic parameter for the pharmaceutical
substance. Its knowledge gives hand in the formulation of
stable oil-in-water emulsion systems as well as in the selec-
tion of the emulsifier blend.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
Declared none.
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Accepted: November 05, 2014