JOURNAL FOR THE ADVANCEMENT OF SCIENCE & ARTS, VOL. 3, NO. 1, 2012
Density of Lecithin and Its Effective
Molecular Volume in Lecithin-Water
Dispersions
1
P. K. Yeap, 1,a K. O. Lim , 2C. S. Chong, 3T. T. Teng, 4A. R. Mohamed
1, 2
School of Physics, Universiti Sains Malaysia, 11800 Penang, Malaysia
3
School of Industrial Technology, Universiti Sains Malaysia, 11800 Penang, Malaysia
4
School of Chemical Engineering, Universiti Sains Malaysia, 14300 Penang, Malaysia
a
Corresponding author. Tel.: 604-6534111; fax: 604-6579150. Email: [email protected] (K.O.
Lim).
_____________________________________________________________________
Abstract
Liposomes are of major interest as model membrane systems and their potential applications. As the packing
structure of lipid molecules in the liposomes will vary with different liposome sizes, it is expected that the
density of the lipid in the dispersions will also vary, albeit minutely. The main objective of this study is to
obtain accurate density measurements of lipid-water dispersions, and to relate the results to the molecular
packing structure of the liposomes. Lipid-water dispersions were prepared by means of sonication. The average
sizes of liposomes were estimated via turbidity measurements, while the densities of the lipid dispersions were
determined using a digital density meter. The density of the lecithin in the dispersions was calculated and the
value found was comparable to the value in the dry state. A thermotropic phase transition at about 40 °C - 45 °C
was observed. The results for the density of the lecithin in the dispersions are 1.0579 g cm-3 at 25 °C and 0.9961
g cm-3 at 50 °C. However, the average values of the effective molecular volume of lecithin in the dispersions are
1.152E-21 cm3 at 25 °C and 1.224E-21 cm3 at 50 °C.
Keywords: Lecithin; Density; Effective Molecular Volume; Dispersion
______________________________________________________________
1. INTRODUCTION
Liposomes are quasi-spherical structures composed of lipid bilayers that encapsulate an
aqueous space [1]. Liposomes form spontaneously when lipids are dispersed in an aqueous
media, giving rise to a population of liposomes with various sizes. They can be prepared in
the laboratory by various methods, such as organic solvent injection [2, 3], sonication [4 - 7],
reverse phase evaporation [8] or extrusion [9 - 12].
The study of liposomes is very essential because of their similarity to biological membranes
and their medical value as delivery agents for enzymes, drugs, and in genetic manipulation
and diagnostic imaging [13 - 16]. Hence, liposomes are of particular interest.
Various investigations have addressed the biological aspects of liposomes, both in vivo and
in vitro. The relationship between the structure of the simple lipid membrane and that of the
lipid phase of biological membranes has been a matter of some concern. As is well known,
the polymorphism of the bilayer arises from alterations in the packing arrangements of lipid
hydrocarbon chains, order or disorder isomerizations in intramolecules, and hydrophobic or
hydrophilic interactions between water and lipid. Consequently, considerable interest
underlying the subject of the molecular packing structure of liposomes in lipid-water systems
has been generated.
1
The occurrence of a thermotropic phase transition is characteristic of lipid-water systems.
The study of thermotropic lipid phase transitions in both natural and model membranes has
proven to be a productive approach towards the understanding of the structure, organization
and interactions present in lipid bilayer assemblies. Generally, the lipid bilayer composed of
pure one-component phospholipids, in excess water, can undergo multiple thermotropic
phase transitions upon heating [17]. Of these several transitions, the chain-melting transition
or the gel to liquid-crystalline phase transition is the main phase transition, which is
accompanied by the largest entropy change. A variety of physical techniques have been
applied in the study of phase transitions, including differential thermal analysis [18, 19],
Raman spectroscopy [20, 21] as well as infrared spectroscopy [22, 23].
The main objective of this study is to obtain accurate density measurements of lipid-water
dispersions, and to relate the results to the molecular packing structure of the liposomes. The
lipid-water dispersions were prepared by means of sonication to produce liposomes of minute
sizes. Measurements of the turbidity as a function of wavelength, range from 400 nm to 800
nm, using a spectrophotometer, provided a means of evaluating the average size of the
liposomes in the dispersions. The densities of the lipid-water dispersions were measured
using a digital density meter, in the temperature range from 25 °C to 55 °C. A theoretical
approach, based on the precise density measurements, was formulated to estimate the
effective volume of lipid molecules packed in the liposomes in the dispersions.
2. MATERIALS AND METHODS
2.1 Materials
Synthetic DL-α Phosphatidylcholine, dipalmitoyl (which hereafter abbreviated to DPPC or
simply called lecithin) of 99 % purity was purchased from Sigma Chemical Company and
was used without further purification. Ultra pure water was used throughout the experiments.
2.2 Preparation of samples
Dispersions of lecithin were prepared by sonicating the lecithin in 25 ml of water using an
Artek Sonic Dismembrator 150 with a titanium probe operated at a fixed power setting of 70
% of maximum intensity for 35 min. During the sonication, the probe was maintained at a
constant depth of about 1 mm beneath the surface of the sample held in the beaker, which
was immersed in a water bath at room temperature (~25 °C). The concentrations of lecithin in
the dispersions were 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml and 3 mg/ml (i.e. mass of
lecithin in 25 ml of water).
2.3 Turbidity measurements
Measurements of turbidity of the lecithin-water dispersions were carried out using a UVVisible Spectrophotometer (Shimadzu UV-1601 PC), at the wavelength scan that extended
from 400 nm to 800 nm. From the turbidity measurements of the dispersions, the average
diameters of the liposomes were estimated according to the approach of Pozharski et al. [24].
2.4 Density measurements
2
The densities of the lecithin-water dispersions were measured using a vibrating-tube Anton
Paar Density Meter DMA 58, in the temperature range from 25 °C to 55 °C. The density
meter is utilized to determine the oscillating periods that are automatically converted to liquid
densities after calibrations. The proper calibrations of the density meter were achieved at
each working temperature, with air and water as references. The accuracy of the density
meter has been reported to be 1 part per 100,000 by Salleh et al. [25] and Maham et al. [26].
3. THEORETICAL CONSIDERATIONS
The density of the dispersion,  is expressed as
wl  ww
w
 

(1)
v
vl  v w
where w is the value of total mass and v is the value of total volume; subscript ‘ l ’
represents the lecithin and subscript ‘ w ’ represents the water. To express the density of the
dispersion in terms of more directly measurable parameters, Equation (1) can be written as
 w  1  wr 
 
(2)
1  vr
ww
wl
vl
where  w 
is the density of the water, wr 
and v r 
are the mass ratio and
vw
ww
vw
volume ratio of the lecithin to the water respectively. wr is constant for a fixed mass of water
and lecithin used. The density of the dispersion therefore, is dependent on the value of v r .
The larger is the v r , the smaller the density of the dispersion.
From Equation (2), the volume ratio can be written as
 w  1  wr   
vr 
(3)

Thus, the value of v r can be calculated from the experimental value of density,  , and
known values of  w and wr . The volume of lecithin in the dispersion is given by
vl  v r  v w
(4)
This value can hence be calculated using the value of v r from Equation (3) and the volume
of the water, v w .
The total number of lecithin molecules, N l present in the dispersion is given by
wl
Nl 
 NA
(5)
Ml
where M l is the molecular weight of the lecithin and N A is the Avogadro’s number.
Knowing the value of N l , the expression for the effective volume of a lecithin molecule
packed in the liposomes in the dispersion can finally be expressed as
vl
veff 
(6)
Nl
which is dependent on the volume of lecithin in the dispersion. When the volume of lecithin
changes, the effective volume occupied by a lecithin molecule in the dispersion would
therefore change, while wl is held constant.
Assuming that the above considerations are applicable, the density of the lecithin in
the dispersion may also be derived from the relationship
3
wl
(7)
vl
where  l is the density of the lecithin and is dependent on the experimental parameters of wl
and v l .
l

4. RESULTS AND DISCUSSION
4.1 Estimation of liposome sizes
Table 1 summarizes the average sizes of the liposomes generated in dispersions with different
concentrations of lecithin. The estimate of 90 nm was found for the average diameters of the
liposomes generated in dispersions with concentration of 1 mg/ml and 1.5 mg/ml. The
average liposome diameter, however, increased to 120 nm as the concentration of lecithin in
the dispersion was increased to 2 mg/ml. Further increases of concentration of lecithin in the
dispersion to 2.5 mg/ml and 3 mg/ml caused a more pronounced increase of the average
liposome diameter to 170 nm. This variation may be attributed to insufficient sonication
process for dispersions with higher concentrations of lecithin.
4.2 Temperature dependence of density
The densities of the dispersions as a function of concentration of lecithin in the dispersion at
different temperatures are shown in Table 2 and Figure 1. The densities of the dispersions
show only a slight variation with concentration of lecithin. However, the densities of the
dispersions show a more significant decrease with increase in temperature. It can be seen that
the densities of the dispersions are only a little higher than that of the water. Since there is
only a small change in density, the experimental density data for different lecithin dispersions
will thus be presented in terms of a more appropriate parameter, which is the fractional
change of density, given as
Density of lecithin dispersion  Density of water
Fractional changeof density 
(8)
Density of water
Temperature dependence of the fractional change of density for dispersions with different
concentrations of lecithin is illustrated in Figure 2. It can be seen that a rather abrupt decrease
occurred in the temperature range of 40 °C – 45 °C, which was observed as the gel to liquidcrystalline phase transition for the lecithin [19]. This reduction in density at the phase
transition region may be attributed to the increase in the effective volume of the lecithin
molecules in the liposomes in the dispersions. Note that the higher the concentration of
lecithin in the dispersion, the greater the degree of the fractional change of density. This
relationship is rather linear particularly for a temperature that is well below the transition
temperature (see Figure 3). However, the results for a temperature beyond the transition
temperature, the variation with different concentrations of lecithin in the dispersions is not so
significant. This may imply that at higher temperatures, the liposomes are larger in size as a
result of a more rigorous vibrational motion of the lecithin molecules in the dispersions. The
effective volume of the lecithin molecules has increased to the extent such that the density of
the lecithin itself is now only about the same as water.
4.3 Theoretical calculation of effective molecular volume
4
Figure 4 illustrates the volume of lecithin in the dispersions with different concentrations of
lecithin over a temperature range of 25 °C to 55 °C. It can be seen that the volume of lecithin
in the dispersion increases when the temperature is increased gradually through the phase
transition. An obvious feature is a slight increase of the volume of lecithin in the dispersion
near the transition temperature of 42 °C. These results are in agreement with the observation
reported by Trauble and Haynes [27] that a volume change does accompany the phase
transition. Further, as the concentration of lecithin in the dispersion is increased, the volume
of lecithin in the dispersion significantly increases. This relationship is linear for
temperatures below and beyond the transition temperature (sees Figure 5).
The effective molecular volume of lecithin generated in the dispersions with different
concentrations of lecithin is summarized in Table 3, which indicates that the effective
molecular volume generally increases with increasing temperature. These results also show
that the liposomes have undergone an expansion at the phase transition. This expansion can
be attributed to a conformational change of the lecithin molecules packed in the liposomes in
the dispersion [17]. As the temperature increases, the packing arrangement of the lecithin
molecules in the liposomes becomes loosened gradually. Because of the trans to gauche
conformational change of the lecithin molecules packed in the liposomes occurring at the
phase transition, the effective molecular volume would be larger at temperatures beyond the
phase transition than those below the phase transition. Another observation is that the values
of the effective molecular volume are generally smaller for dispersions of higher
concentrations of lecithin, particularly at a given temperature below the transition
temperature. This implies the molecular packing of lecithin is closer for these dispersions
with larger liposomes.
From the results of the above calculations, the density of the lecithin in the dispersion,  l can
be determined from Equation (7), as the values of the mass and the volume of the lecithin in
the dispersions are known. Table 4 summarizes the densities of the sonicated lecithin at
temperatures below and beyond the transition temperature. The calculated density data at a
room temperature of 25 °C are comparable to that obtained by Sheetz and Chan [6] as well as
Chong and Colbow [7]. Nevertheless, slightly higher values are obtained for dispersions of
higher concentrations of lecithin. This may imply that liposomes generated in these cases are
larger and more closely packed. As expected, the density at the post-transition is only slightly
higher than that of water.
From the results tabulated in Table 3 and Table 4, the average values of the density of the
lecithin and the effective molecular volume of lecithin in the dispersions at two temperatures
are found to be as follows:
 l (at 25 °C) = (1.0579 ± 0.0014) g cm-3
 l (at 50 °C) = (0.9961 ± 0.0009) g cm-3
veff (at 25 °C) = (1.152 ± 0.002) E-21 cm3
veff (at 50 °C) = 1.224 ± 0.001) E-21 cm3
5. ACKNOWLEDGMENTS
The authors would like to acknowledge the short term research grant provided by Universiti
Sains Malaysia.
5
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7
Figure 1: Density of lecithin dispersion as a function of concentration of lecithin in dispersion at different
temperatures.
1.001
25 oC
30 oC
35 oC
40 oC
45 oC
50 oC
1.000
0.999
0.998
Density of Lecithin Dispersion (g cm-3)
0.997
0.996
0.995
0.994
0.993
0.992
0.991
0.990
0.989
0.988
0.987
0.986
0.985
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Concentration of Lecithin in Dispersion (mg/ml)
8
Figure 2: Temperature dependence of the fractional change of density for dispersions with different
concentrations of lecithin.
0.00020
1.0 mg/ml
1.5 mg/ml
2.0 mg/ml
2.5 mg/ml
3.0 mg/ml
0.00018
0.00016
Fractional Change of Density
0.00014
0.00012
0.00010
0.00008
0.00006
0.00004
0.00002
0.00000
20
25
30
35
40
45
50
55
60
o
Temperature ( C)
9
Figure 3: Fractional change of density as a function of concentration of lecithin in dispersion at 25 °C and 50
°C.
0.00022
25 oC
50 oC
0.00020
0.00018
Fractional Change of Density
0.00016
0.00014
0.00012
0.00010
0.00008
0.00006
0.00004
0.00002
0.00000
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Concentration of Lecithin in Dispersion (mg/ml)
10
Figure 4: Volume of lecithin in dispersion as a function of temperature for dispersions with different
concentrations of lecithin.
0.090
1.0 mg/ml
1.5 mg/ml
2.0 mg/ml
2.5 mg/ml
3.0 mg/ml
0.085
0.080
Volume of Lecithin in Dispersion (cm3)
0.075
0.070
0.065
0.060
0.055
0.050
0.045
0.040
0.035
0.030
0.025
0.020
20
25
30
35
40
45
50
55
60
Temperature (oC)
11
Figure 5: Volume of lecithin in dispersion as a function of concentration of lecithin in dispersion at 25 °C and
50 °C.
0.090
25 oC
50 oC
0.080
Volume of Lecithin in Dispersion (cm3)
0.070
0.060
0.050
0.040
0.030
0.020
0.010
0.000
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Concentration of Lecithin in Dispersion (mg/ml)
12
Table 1: Size estimation of the liposomes generated in dispersions with different concentrations of lecithin
Concentration of Lecithin in Dispersion
Average Diameter
(mg/ml)
(nm)
1.0
90
1.5
90
2.0
120
2.5
170
3.0
170
Table 2: Densities of lecithin dispersions at different temperatures
Concentration of
Density of Lecithin Dispersion (g cm-3)
Lecithin in Dispersion
(mg/ml)
25 °C
30 °C
35 °C
40 °C
45 °C
0.0*
0.997044 0.995647 0.994031 0.992215 0.990213
1.0
0.997097 0.995689 0.994059 0.992252 0.990221
1.5
0.997131 0.995720 0.994093 0.992279 0.990225
2.0
0.997163 0.995752 0.994120 0.992325 0.990248
2.5
0.997186 0.995774 0.994139 0.992334 0.990243
3.0
0.997223 0.995810 0.994168 0.992365 0.990239
* Density of water as a comparison.
50 °C
0.988036
0.988042
0.988046
0.988058
0.988058
0.988058
Table 3: Effective molecular volume of lecithin generated in dispersions with different concentrations of
lecithin
Concentration of
Effective Molecular Volume of Lecithin (cm3)
Lecithin in Dispersion
(mg/ml)
25 °C
30 °C
35 °C
40 °C
1.0
1.158E-21
1.173E-21
1.192E-21
1.183E-21
1.5
1.152E-21
1.164E-21
1.175E-21
1.176E-21
2.0
1.150E-21
1.160E-21
1.171E-21
1.160E-21
2.5
1.153E-21
1.162E-21
1.173E-21
1.170E-21
3.0
1.149E-21
1.157E-21
1.170E-21
1.167E-21
42 °C
45 °C
50 °C
55 °C
1.0
1.215E-21
1.221E-21
1.226E-21
1.212E-21
1.5
1.214E-21
1.221E-21
1.225E-21
1.224E-21
2.0
1.203E-21
1.209E-21
1.220E-21
1.209E-21
2.5
1.211E-21
1.216E-21
1.223E-21
1.217E-21
3.0
1.216E-21
1.220E-21
1.224E-21
1.219E-21
Table 4: Densities of sonicated lecithin at 25 °C and 50 °C
Concentration of
25 °C
50 °C
Lecithin in Dispersion
(mg/ml)
1.0
1.5
2.0
2.5
3.0
l
vl
3
(cm )
0.02374
0.03543
0.04716
0.05912
0.07072
l
vl
-3
(g cm )
1.0529
1.0585
1.0602
1.0572
1.0605
3
(cm )
0.02515
0.03770
0.05004
0.06269
0.07535
(g cm-3)
0.9941
0.9947
0.9991
0.9969
0.9954
13