Langmuir monolayer miscibility of single

Journal of Colloid and Interface Science 337 (2009) 201–210
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Journal of Colloid and Interface Science
www.elsevier.com/locate/jcis
Langmuir monolayer miscibility of single-chain partially fluorinated
amphiphiles with tetradecanoic acid
Hiromichi Nakahara a, Minami Tsuji b, Yukiko Sato b, Marie Pierre Krafft c,*, Osamu Shibata a,b,*
a
Department of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan
Division of Biointerfacial Science, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
c
Université de Strasbourg, Systèmes Organisés Fluorés à Finalités Thérapeutiques (SOFFT), Institut Charles Sadron (CNRS, UPR 22), 23 rue du Loess, BP 84047,
67034 Strasbourg Cedex 2, France
b
a r t i c l e
i n f o
Article history:
Received 20 March 2009
Accepted 7 May 2009
Available online 19 May 2009
Keywords:
Langmuir monolayer
Partially fluorinated amphiphile
Tetradecanoic acid
Surface pressure
Surface potential
Brewster angle microscopy (BAM)
Fluorescence microscopy (FM)
Atomic force microscopy (AFM)
a b s t r a c t
The surface pressure (p)–molecular area (A) and surface potential (DV)–A isotherms have been measured
for monolayers of tetradecanoic acid (myristic acid: MA), partially fluorinated amphiphiles [single-chain
(perfluorooctyl)pentanol (F8C5OH) and single-chain (perfluorooctyl)pentylphosphocholine (F8C5PC)],
and their two-component combinations in order to investigate their miscibility at the air/water interface.
The data for these systems were analyzed in terms of an additivity rule and excess Gibbs free energy. An
interaction parameter and an interaction energy between the two components were calculated from the
Joos equation, which allows description of collapse pressures of miscible monolayers. Two-dimensional
phase diagrams for the binary systems were constructed and found to be a positive azeotropic type. These
results indicate that the two-component MA/F8C5OH and MA/F8C5PC monolayers are miscible in the
monolayer state. To confirm their miscibility and phase behavior upon compression, morphological
observations with fluorescence microscopy (FM), Brewster angle microscopy (BAM), and atomic force
microscopy (AFM) have been performed. These observations show that the addition of F8C5OH or
F8C5PC to MA makes MA ordered domains in the monolayer region fluidize very effectively and that a
fern-like network is formed as a 3-D structure by over-compression beyond the monolayer collapse.
The present paper systematically clarifies the miscibility between MA and F8C5OH or F8C5PC within
the monolayer and indicates that these fluorinated chemicals may have a possibility of biomedical uses
and applications.
Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
A fluorinated amphiphile is characterized by unique properties
such as a stronger surface activity and a formation of micelles and
other self-assemblies at lower concentrations in comparison with
corresponding hydrogenated analogue [1,2]. Due to its high biological inertness, remarkable ability to solubilize oxygen, and extremely low solubility in water, highly fluorinated compounds have
been investigated for various biological applications [3–5]. Many
unusual features have been reported in these areas. In particular,
stimulating attention have been paid to their potential use as intravascular oxygen transport [6], contrast agent in ultrasound imaging [7], and efficient additive to pulmonary surfactants [8,9].
Perfluoroalkyl chains are considerably different from the normal
* Corresponding authors. Fax: +33 3 88 40 41 99 (M.P. Krafft), +81 956(20)5686
(O. Shibata).
E-mail addresses: [email protected] (M.P. Krafft), [email protected]
(O. Shibata).
URLs: http://www-ics.u-strasbg.fr/ (M.P. Krafft), http://www.niu.ac.jp/~pharm1/
lab/physchem/index.html (O. Shibata).
0021-9797/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.jcis.2009.05.022
alkyl chains in their bulkiness, helical conformation, and stiffness
[2]. Perfluorocarbon chains are much more hydrophobic than the
corresponding hydrocarbon ones and have a pronounced lipophobicity, which is estimated to be about one-third the free energy of
transfer of a methylene group from alkane to water [10]. Furthermore, the low polarizability of fluorine atoms results in very weak
van der Waals interactions between perfluorocarbon chains. The
combination of fluorinated and hydrogenated surfactants generates unusual and interesting phenomena such as miscibility, phase
separation, and compartmentalization for micelles, liposomes, and
Langmuir monolayers [11–13]. New self-organized films and
membranes, discrete objects, and interfaces have been obtained,
and novel applications have been explored in the medical area,
materials science, and other fields.
Langmuir monolayers at the air/liquid interface, which provide
two-dimensional (2-D) structural information, have been intensively adopted as experimental paradigms of a biological membrane for elucidating the interfacial properties and mechanisms
of surfactants [14–17]. The monolayer technique has an advantage
in simplifying physicochemical behavior compared with the method employed with three-dimensional (3-D) aggregates such as
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micelles, vesicles, and emulsions. In addition, the phase behavior of
monolayers at the interface under compression can be visualized at
the micro- and nanoscale by optical techniques such as Brewster
angle microscopy (BAM) [8,18,19], fluorescence microscopy (FM)
[20–22], and atomic force microscopy (AFM) [8,23,24]. Thus, many
researchers have used the monolayer techniques to understand the
interaction between surfactants, polymers, proteins, peptides, and
so on [25–27]. Recently, interesting studies on nucleation process
in collapsed monolayers of semifluorinated alkanes have been reported utilizing BAM [28]. When the monolayers of the alkanes
were transferred from 2-D to 3-D aggregates upon compression,
the different collapse types were observed depending on the magnitude of their fluorination; for example, long ribbon-like, spikelike, and plateau-like structures. Langmuir monolayer conversions
to 3-D collapsed aggregates play a key role in several biological
processes such as the mechanism of lipid bilayer formation [29]
and the hysteresis of compression–expansion cycles in the pulmonary function of lung surfactants [30–32].
We have investigated the miscibility behavior of perfluorocarboxylic acids (FCn, n = 12,14,16,18) with tetradecanoic acid (myristic acid: MA) [33] or dipalmitoylphosphatidylcholine (DPPC) [24]
in the monolayer state, which are components in biological membranes. FCn molecules are partially miscible with the lipids
depending on compositions and chain lengths, although perfluorocarbons do not commonly mix with normal hydrocarbons in the
bulk state. The interfacial behavior of two-component monolayers
has been studied for dipalmitoylphosphatidylethanolamine (DPPE)
and semifluorinated alkanes without headgroups, where it was
found that miscible monolayers and vertically phase-separated
bilayers were formed depending on compositions and surface pressures [34]. The driving force in monolayer miscibility is induced by
attractive forces between headgroups. Other researchers have
extensively examined the surface behavior and miscibility between partially fluorinated compounds and hydrogenated ones
[35–38]. In the previous studies, the phase behavior and interaction have been systematically reported for two-component monolayers of DPPC, the partially fluorinated n-alcohol (F8C5OH), and
alkylphosphocholine (F8C5PC) at the air/water interface [8,39]. In
that case, both F8C5OH and F8C5PC are completely miscible with
DPPC within a monolayer in spite of the difference in chain length
by three methylene groups. In addition, the DPPC/F8C5OH mixtures at a distinct mole fraction showed a triskelion morphology
in BAM and FM micrographs differently from the DPPC/F8C5PC
systems. However, F8C5PC liquefied more a liquid-condensed
(LC) domain of DPPC. The species of headgroups are thought to
contribute to the morphological variation. However, the reasons
and mechanisms for the outcome have not been made clear yet because of the difference in chain length and number (single or double) of hydrophobic parts between DPPC and the fluorinated
compounds. Moreover, there are still few reports on the miscibility
behavior and the interaction mode between partially fluorinated
amphiphiles and biomembrane components, which provide helpful information to industries, biomedical fields, and environmental
sciences.
In the present work, our attention is focused on the effect of polar headgroup and hydrophobic parts interactions on the monolayer miscibility. Thus, myristic acid (MA) was chosen as one of
biomembrane components. For example, the myristic acid has a
sufficiently high hydrophobicity to become incorporated into the
fatty acyl core of the phospholipid bilayer of the plasma membrane
of the eukaryotic cell. In this way, myristic acid acts as a lipid anchor in biomembranes. MA has a single chain (C14:0) within a molecule and forms a stable monolayer with temperature-sensitive
phase transition at the air/water interface. The MA monolayer
behavior has been widely studied using various methods [40–42]
and the phase behavior is well known to the researchers employ-
ing the monolayer technique. The use of MA as one component allows us to make it easy to interpret the phase variations induced
by others. Therefore, to investigate such the interfacial behavior,
we selected a binary MA/F8C5OH and MA/F8C5PC systems.
F8C5OH and F8C5PC have identical hydrophobic chains. However,
the headgroup of F8C5PC is more bulky than its chains. In addition,
the three compounds have essentially the same single hydrophobic
chain lengths. The aims of this study are to clarify the miscibility of
MA with F8C5OH or F8C5PC, the miscibility effect of the fluorinated amphiphiles on the LC domains of MA, and the nucleation
mode in their collapsed monolayers. Surface pressure (p)–molecular area (A) and surface potential (DV)–A isotherms of the binary
monolayers were measured on 0.15 M NaCl (pH 2.0) at 298.2 K.
The phase behavior was examined using the additivity rule for
the mean A and DV as well as an excess Gibbs free energy of mixing. Furthermore, the Langmuir monolayers and Langmuir–Blodgett (LB) films were also morphologically investigated using BAM,
FM, and AFM.
2. Experimental section
2.1. Materials
(Perfluorooctyl)pentanol (F8C5OH) and (perfluorooctyl)pentylphosphocholine (F8C5PC) were synthesized as reported previously [43]. Tetradecanoic acid (Myristic acid, MA, 99–100%) was
purchased from Sigma (St. Louis, MO) and was used without further purification. Octadecyl rhodamine B chloride (R18) was purchased from Molecular Probes, Inc. (Eugene, OR) as a fluorescent
probe. n-Hexane (>98.5%) was came from Cica-Merck (Uvasol, Tokyo, Japan) and ethanol (99.5%) was purchased from nacalai tesque (Kyoto, Japan). The n-hexane/ethanol (9/1, v/v) mixtures were
used as a spreading solvent for each sample. Sodium chloride (nacalai tesque) was roasted at 1023 K for 24 h to remove all surfaceactive organic impurities. Hydrochloric acid (HCl) of ultra fine
grade for the preparation of a subphase was purchased from nacalai tesque. The substrate solution was prepared using thrice distilled water (the surface tension = 72.0 mN m1 at 298.2 K and
the electrical resistivity = 18 MX cm).
2.2. Methods
2.2.1. Surface pressure–molecular area isotherms
The surface pressure (p) of monolayers was measured using an
automated homemade Wilhelmy balance. The surface pressure
balance (Mettler Toledo, AG245) had a resolution of 0.01 mN m1.
The pressure-measuring system was equipped with a filter paper
(Whatman 541, periphery = 4.0 cm). The trough was made from
Teflon-coated brass (area = 720 cm2), and Teflon-made barriers
(both hydrophobic and lipophobic) were used in this study. Surface
pressure (p)–molecular area (A) isotherms were recorded at
298.2 K within ±0.1 K. Stock solutions (1.0 mM) of MA, F8C5OH,
and F8C5PC were prepared in n-hexane/ethanol (9/1, v/v). The
spreading solvents were allowed to evaporate for 15 min prior to
compression. The monolayer was compressed at a speed of
0.10 nm2 molecule1 min1. The standard deviations for molecular surface area and surface pressure were 0.01 nm2 and
0.1 mN m1, respectively. The subphase pH of a 0.15 M NaCl solution was adjusted to 2.0 with an adequate amount of HCl.
2.2.2. Surface potential–molecular area isotherms
The surface potential (DV) was simultaneously recorded together with the surface pressure while the monolayer was compressed at the air/water interface. It was monitored by using an
ionizing 241Am electrode at 1–2 mm above the interface while a
H. Nakahara et al. / Journal of Colloid and Interface Science 337 (2009) 201–210
reference electrode was dipped in the subphase. The electrometer
(Keithley 6517) was used to measure the surface potential. The
standard deviation for the surface potential was 5 mV [8,44–46].
2.2.3. Brewster angle microscopy (BAM)
The monolayer was directly visualized by a Brewster angle
microscope (KSV Optrel BAM 300, KSVInstruments Ltd., Finland)
coupled to a commercially available film balance system (KSV
Minitrough, KSV Instruments Ltd., Finland). The application of a
20 mW He–Ne laser emitting p-polarized light of 632.8 nm wavelength and a 10 objective lens allowed a lateral resolution of
2 lm. The angle of the incident beam to the air/water interface
was fixed to the Brewster angle (53.1°) at 298.2 K. The reflected
beam was recorded with a high grade charge coupled device
(CCD) camera (EHDkamPro02, EHD Imaging GmbH, Germany),
and then the BAM images were digitally saved to the computer
hard disk.
2.2.4. Fluorescence microscopy (FM)
The film balance system (KSV Minitrough) was mounted onto the
stage of the Olympus microscope BX51WI (Tokyo, Japan) equipped
with a 100-W mercury lamp (USH-1030L), an objective lens
(LMPlanFL20, working distance = 12 mm), and a 3CCD camera
with a camera control unit (IKTU51CU, Toshiba, Japan). The z-directional focus on the monolayer was exactly adjusted using an automation controller (MAC 5000, Ludl Electronic Products Ltd., NY).
FM observations and compression isotherm measurements were
carried out simultaneously. A spreading solution of the samples
was prepared as a mixed solution doped with 1 mol% of a fluorescence probe (R18). The excitation (546 nm) and emission (590 nm)
wavelengths were selected by a mirror unit (U-MWIG3). FM micrographs were directly recorded with the hard disk via an online image
processor (DVgate Plus, Sony Corp., Japan) connected to the microscope. Image processing and analysis were carried out by using the
software, Scion Image Beta 4.02 for Windows (Scion Corp., Frederick,
MD). The total amount of ordered domains (dark contrast regions)
was evaluated and expressed as a percentage per frame by dividing
the respective frame into dark and bright regions.
2.2.5. Atomic force microscopy (AFM)
Langmuir–Blodgett (LB) film preparations were carried out with
the KSV Minitrough. Freshly cleaved mica (Okenshoji Co., Tokyo,
Japan) was used as a supporting solid substrate for film deposition.
At selected surface pressures, a transfer velocity of 5 mm min1
was used for film-forming materials on a 0.15 M NaCl (pH 2.0) at
298.2 K. LB films with deposition ratio of 1 were used in the
experiments. AFM images were obtained using an SPA 400 instrument (Seiko Instruments Co., Chiba, Japan) at room temperature in
the tapping mode, which provided both a topographical image and
a phase contrast image. The tapping mode images (256 or 512
points per line) were collected with scan rates of 1 Hz, using silicon
tips (Olympus Co., Japan) with a nominal spring constant of
1.8 N m1 under normal atmosphere [8,47]. The lateral and vertical
resolutions were 0.2 and 0.1 nm, respectively. The transferred samples were checked for possible tip-induced deformation by zooming out after a region had been scanned.
3. Results and discussion
3.1. Compression isotherms for pure systems
Under the present subphase condition of pH 2.0, the hydroxyl
group of F8C5OH is clearly undissociated and F8C5OH is neutral
within a molecule. F8C5PC possesses the PC headgroup, which is
in the zwitterionic form in the subphase pH range of 3.5 to 11.5
203
[48], and is thus charged positively; 98–100% of the phosphate
group are expected to be protonated using the pKa value of DPPC
(pKa = 3.8–5.0 [48–50]). In the case of MA molecules (pKa = 4.9
[51]), it is found that the carboxyl group is almost undissociated
(0.1% dissociated) even under consideration of the surface pH value (pHs) using the Gouy–Chapman approach [52,53]. The following equations were employed above procedure. The monolayer
ionization degree (a) is determined by the surface proton concentration [H+]s and the surface equilibrium constant K for the acid
group dissociation (Ks):
a
1a
¼
Ks
½Hþ s
ð1Þ
The surface proton concentration is calculated by using Boltzmann
equation, if the proton concentration in the bulk [H+]b is known,
zeu 0
½Hþ s ¼ ½Hþ b exp kT
ð2Þ
An important application of Eq. (2) can be made by calculating the
pH near a surface. If we take pH ¼ log cHþ , Eq. (2) leads to
pHs ¼ pHb þ
eu0 2:303kT
ð3Þ
where pHs is the surface pH and pHb is that in the bulk solution
[54,55]. The Gouy–Chapman approach is then used to relate u0
the charge density at the interface,
"
#
2kT
r
1
u0 ¼
sinh
ze
ð2eeV n0 kTÞ1=2
ð4Þ
where u0 refers to the electrostatic potential at the interface relative
to the adjacent conducting phase (mV), k is the Boltzmann constant,
T is the absolute temperature, e is electrostatic unit charge, z is
valency of electrolyte, r is surface charge density (Cm2), e is the
dielectric constant (78.3 at 298.2 K), ev is vacuum permittivity
(8.854 1012 J1 C2 1), and n0 is the number density of charges
in the system. On the contrary, pKs for monolayer is
pHs ¼ pK s þ log
a
ð5Þ
1a
Combining Eqs. (3) and (5), one can obtain the following equation.
pHb ¼ pK s þ log
a
1a
eu0
2:303kT
ð6Þ
The surface pressure (p)–molecular area (A) and surface potential (DV)–A isotherms of pure MA, F8C5OH, and F8C5PC monolayers have been measured on 0.15 M NaCl at 298.2 K. The subphase
pH was adjusted to 2.0 throughout the experiment to prevent
the dissociation of carboxyl group in MA molecules and their dissolution into the subphase. The transition can be more precisely
determined using DV–A isotherms due to better sensitivity of DV
to the variation of phase behavior, although the transition pressure
is conventionally determined only from the p–A isotherms. Thus,
the transition and collapse pressures obtained here were determined in the manner shown in Fig. 1(c) [14,45,47]. As shown in
Fig. 1(a), F8C5OH monolayers are stable up to 46 mN m1 with
a transition from a disordered to an ordered state at 9 mN m1
(0.33 nm2), which is indicated by arrows (Fig. 1(c)). The p–A isotherm of F8C5OH at 293.2 K shows a clear kink point corresponding to the transition pressure of 4.5 mN m1 [39]. Unfortunately, in
the present condition, the kink becomes unclear due to the temperature increase. On the other hand, F8C5PC collapses from a disordered state at 47 mN m1. As for the MA monolayer, the
plateau at 22 mN m1 indicated by an arrow in Fig. 1(a) represents the liquid-expanded/liquid-condensed (LE/LC) coexistence
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H. Nakahara et al. / Journal of Colloid and Interface Science 337 (2009) 201–210
monolayer orientation of F8C5PC upon compression [8,39]. Furthermore, under the present condition, the positive charge of F8C5PC
may contribute to the smaller value. In contrast, the DV values of
MA monolayers are positive and increase from 0 to 365 mV upon
compression. A positive variation is typical for most surfactants with
hydrocarbon chains, although the DV value is, in common, strongly
affected by a polar headgroup and a subphase condition [25,56].
3.2. Ideal mixing of the binary monolayers
Fig. 1. (a) Surface pressure (p)–molecular area (A) isotherms and (b) surface
potential (DV)–A isotherms of MA (curve 1), F8C5OH (curve 2), and F8C5PC (curve
3) on 0.15 M NaCl (pH 2.0) at 298.2 K. (c) Superimposed p–A and DV–A isotherms of
F8C5OH. The inset (c) shows how to determine the disordered/ordered transition
pressure.
region, and then the monolayer collapses at 27 mN m1 upon further compression [33].
The DV values of F8C5OH and F8C5PC monolayers are always
negative and reach minimum values of 700 and 140 mV at their
collapse pressures, respectively. This negative sign is commonly observed for fluorinated monolayers and is considered to be caused by
the strongly electronegative fluorine atoms [8,21,24,39,44]. The
smaller value of F8C5PC is accounted for the evidence that the spatial bulkiness of the PC headgroup disturbs the enhancement in
3.2.1. Isotherm behavior
The binary MA/F8C5OH and MA/F8C5PC monolayers have been
investigated to clarify the effects of polar headgroups in the fluorinated compounds on their interaction and miscibility within a
monolayer. The p–A and DV–A isotherms of the binary mixtures
were measured for various molar fractions of XF8C5OH or XF8C5PC at
298.2 K on 0.15 M NaCl (pH 2.0). In the case of the MA/F8C5OH systems (Fig. 2A), all of the p–A isotherms are located on the similar
molecular area at low surface pressures less than 20 mN m1. The
p–A isotherms display a phase transition pressure that changes from
XF8C5OH = 0 to 0.2 and the collapse pressure varies against XF8C5OH
(Figures S1 and 4(a)). The change in transition and collapse pressure
with XF8C5OH provides the first evidence of the miscibility between
the two components in the monolayer state. The value of transition
pressures was also confirmed by Brewster angle microscopy (BAM)
and fluorescence microscopy (FM) in the later section. In the
MA/F8C5PC system (Fig. 2B), the p–A isotherms clearly have the
transition pressure at 0 6 XF8C5PC 6 0.1 and the values increase with
increasing XF8C5PC. Correspondingly, the collapse pressure changes
similarly to the MA/F8C5OH monolayers as shown in Fig. 4. These
variations on p–A isotherms imply that MA is, at least, partially miscible with both F8C5OH and F8C5PC in the monolayer state.
All of the DV–A isotherms for the two systems regularly shift to
negative DV values with increasing XF8C5OH or XF8C5PC. The magnitude of variation in DV values against the molar fractions in the
MA/F8C5OH system is larger than that in the MA/F8C5PC system.
The difference is resulted from the fact that the polar headgroup
of F8C5PC is more bulky than that of F8C5OH and then the orientation of F8C5OH cannot be improved upon compression differ-
Fig. 2. p–A and DV–A isotherms of the binary (A) MA/F8C5OH and (B) MA/F8C5PC systems on 0.15 M NaCl (pH 2.0) at 298.2 K for different XF8C5OH and XF8C5PC ratios,
respectively.
H. Nakahara et al. / Journal of Colloid and Interface Science 337 (2009) 201–210
ently from F8C5OH. These variations effectually reflect the structural difference of headgroups between F8C5OH and F8C5PC.
3.2.2. Excess Gibbs free energy
The miscibility of MA with F8C5OH or F8C5PC can be analyzed
in terms of the additivity rule for molecular area (A) and surface
potential (DV) (see Figures S2 and S3) and of the excess Gibbs free
energy of mixing DGexc. The DGexc value is evaluated by integration
of the p–A isotherms from zero to the selected surface pressure
(Eq. (7)) [59],
exc
DG
¼
Z p
ðA12 X 1 A1 X 2 A2 Þdp
both the systems, the transition pressures change positively with
an increase in molar fraction of the fluorinated compounds. In
addition, the collapse pressures irregularly vary against the molar
fractions. The components of the two binary systems are miscible
judging from such continuous change of the transition pressures
upon composition. The coexistence phase boundary between the
monolayer phase and bulk phase can be theoretically simulated
from the Joos equation [61],
1 ¼ xs1 expfðpcm pc1 Þx1 =kTg expfnðxs2 Þ2 g
þ xs2 expfðpcm pc2 Þx2 =kTg expfnðxs1 Þ2 g
ð7Þ
0
where Ai and Xi are the molecular area and the molar fraction of
component i, respectively, and A12 is the mean molecular area in
the mixture. In the absence of interactions between components,
DGexc = 0 [57,58]. Negative or positive deviation from ideality indicates more attractive or less attractive interaction, respectively. The
variation of DGexc for the MA/F8C5OH and MA/F8C5PC monolayers
against molar fractions of the fluorinated components at three surface pressures (5, 15, and 25 mN m1) is shown in Fig. 3. For the
MA/F8C5OH system (Fig. 3(a)), the DGexc XF8C5OH curves can be
divided into two regions; the negative values for 0 6 XF8C5OH 6 0.3
and the positive values for 0.3 < XF8C5OH 6 1. In the former region,
MA attractively interacts with F8C5OH in the monolayer state.
When a more amount of F8C5OH is added to MA, the interaction becomes weaker. The DGexc XF8C5OH curves are quite similar in spite
of an increase in surface pressure, although DGexc values should become larger in most cases [15,60]. This result indicates that the mutual interaction is kept constant despite a reduction in the
intermolecular distance between MA and F8C5OH. In the case of
the binary MA/F8C5PC system, the similar behavior is observed in
Fig. 3(b). However, compared to the MA/F8C5OH system, the
DGexc XF8C5PC curve at each surface pressure almost exists in the
negative region. Furthermore, the amount of DGexc increases more
negatively with increasing surface pressure. These results demonstrate that the disordered state of F8C5PC is more miscible with MA.
3.2.3. Two-dimensional phase diagrams
Two-dimensional phase diagrams for the binary MA/F8C5OH
and MA/F8C5PC mixtures were constructed by plotting the transition pressure and collapse pressure values at the various molar
fractions. The phase diagrams at 298.2 K are shown in Fig. 4. For
Fig. 3. Excess Gibbs free energy of mixing (DGexc) of (a) MA/F8C5OH and (b) MA/
F8C5PC mixtures as a function of XF8C5OH and XF8C5PC at three different pressures (5,
15, and 25 mN m1) on 0.15 M NaCl (pH 2.0) at 298.2 K, respectively. The solid lines
represent values calculated from Eq. (7).
205
ð8Þ
where xs1 and xs2 denote the respective mole fraction in the twocomponent monolayers of components 1 and 2, and pc1 and pc2 are
the respective collapse pressure of components 1 and 2, pcm is the
collapse pressure of the two-component monolayer at a given composition of xs1 and xs2 ; x1 and x2 are the corresponding limiting area
at the collapse points, n is the interaction parameter, and kT is the
product of the Boltzmann constant and the Kelvin temperature.
The solid curve obtained by adjusting the interaction parameter in
the above equation (Eq. (8)) coincides with the experimental values.
It is noteworthy that the both systems exhibit negative interaction
parameters and have two different parameters in the two regions.
The negative interaction parameter implies an interaction energy
between two kinds of molecules which is higher than the mean energy of interaction among the same molecules. The new finding here
is that the MA/F8C5OH system shows two interaction parameters:
n = 1.5 for 0 6 XF8C5OH 6 0.3 and n = 0.60 for 0.3 < XF8C5OH 6 1.
The MA/F8C5PC system also indicates two interaction parameters:
n = 1.2 for 0 6 XF8C5PC 6 0.3 and n = 0.89 for 0.3 < XF8C5PC 6 1. At
the molar fraction of 0.3, the interaction parameter becomes n = 0.
These signs resemble to the variations of DGexc against molar
fractions of the fluorinated compounds (Fig. 3). The interaction energies (De = nRT/6) were calculated to be 620 J mol1 (n = 1.5) and
248 J mol1 (n = 0.60) for the MA/F8C5OH system. On the other
hand, the MA/F8C5PC system has interaction energies of 496 J mol1
(n = 1.2) and 368 J mol1 (n = 0.89). That is, they are completely
miscible, because the interaction energy De < 2RT (=4958.7
J mol1). The two components are miscible each other in both the disordered and ordered states, and the disordered and disordered states.
Fig. 4. Phase diagrams from the variation of the transition pressure (peq: open
circle) and collapse pressure (pc: solid circle) on 0.15 M NaCl (pH 2.0) at 298.2 K, as
a function of composition: (a) MA/F8C5OH and (b) MA/F8C5PC systems. The dashed
lines were calculated according to Eq. (8) for n = 0. The solid line represents
experimental values for collapse pressures. M. indicates a mixed monolayer formed
by MA, F8C5OH, and F8C5PC species, whereas Bulk denotes a solid phase of MA,
F8C5OH, and F8C5PC (‘‘bulk phase” may be called ‘‘solid phase”).
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H. Nakahara et al. / Journal of Colloid and Interface Science 337 (2009) 201–210
This system is likely to be the positive azeotropic type, and the phase
diagram is completely constructed.
3.3. Morphological observations
3.3.1. Brewster angle microscopy
BAM is an optimal method to visualize the morphologic differences of the condensed (ordered) phase domains without a dye
such as a fluorescent probe. In comparison to FM and AFM, however, BAM generally has lower resolution and magnification. Systematic observations with BAM have been performed for the
binary systems against molar fractions of the fluorinated compounds and surface pressures. Unfortunately, the resultant BAM
images could not provide better understanding of the phase behavior upon compression due to the above-mentioned defects and the
narrow range between two phase coexistence states in the binary
monolayer systems. Detail phase behavior with surface pressure is
presented in the later sections (FM and AFM). Here our attention is
focused on the ordered domains at each transition pressure for the
binary MA/F8C5OH and MA/F8C5PC monolayers. The BAM images
of pure MA, MA/F8C5OH (XF8C5OH = 0.05, 0.1, and 0.2), and
MA/F8C5PC (XF8C5PC = 0.05 and 0.1) monolayers spread on 0.15 M
NaCl (pH 2.0) at respective transition pressures are shown in
Fig. 5. The BAM image of pure MA monolayers (Fig. 5(a)) shows a
characteristic large ordered domain (bright) like a flower and
agrees well with that reported previously [62]. When a small
amount of F8C5OH is added to MA monolayer, the ordered domains become smaller in size and the branched arms tend to be
thicker and shorter as shown in Fig. 5(b)–(d). The condensed domain shape is commonly controlled by line tension of phase
boundary and long range dipole interaction. In this system, the
flower-like ordered domains shift to the circular domains with
increasing XF8C5OH. This means that the mixing of F8C5OH improves the line-tension control over the domain shape due to the
miscibility between MA and F8C5OH. The similar phase behavior
is also observed in the MA/F8C5PC system (Fig. 5(e) and (f)). However, the degree of reducing the size and rounding the shape by the
addition of F8C5PC is larger compared with that for the MA/
F8C5OH mixtures. This indicates that the mixing of F8C5PC exerts
stronger fluidizing effects on the ordered domains. It is demonstrated that the bulkiness in the polar headgroups of fluorinated
compounds is important for the fluidization of fatty acid as well
as phospholipid monolayers [8].
3.3.2. Fluorescence microscopy
A fluorescent probe is required for in situ FM observations to
visualize the monolayer phase behavior. FM provides powerful
information on the phase growth and variation with higher resolution and magnification compared with BAM, although the
probe may act as an impurity for samples. There is a worry of
the possibility that the probe affects the original phase behavior
of monolayers. However, the doping of 1 mol% R18 as a fluorescent dye has no influence on the original p–A and DV–A isotherms and domain shapes in BAM images. Differences in the
dye solubility in between disordered (or LE) and ordered (or
LC) phases generate a color contrast. FM images of the binary
MA/F8C5OH (XF8C5OH = 0 (MA), 0.05, 0.1, and 0.2) monolayers
are shown in Fig. 6 at various surface pressures. FM images at
the molar fractions are homogeneously bright below the respective transition pressures (data not shown). In the vicinity of the
transitions, two different phases can be visualized clearly as seen
in each lower row of the images. The percentage (%) in the FM
images indicates the ratio of the ordered (LC) domain in the
micrograph. In pure MA monolayers, domain nucleation occurs
at the kink (22 mN m1) on the p–A isotherm (Fig. 1). The individual nucleation starts at the same time. During an increase of
1 mN m1 after the nucleation, LC domains drastically grow in
size and number of dendritic arms at 23 mN m1. They do not
fuse upon compression due to the repulsive interactions of their
dipoles. Then, as the monolayer is further compressed to the collapse pressure (27 mN m1), the LC domains are packed laterally and the LE phases reduce in area, which results in an
increase in LC ratio from 52% to 82%. When small amounts of
F8C5OH are added to MA (XF8C5OH = 0.05–0.2), both the transition
Fig. 5. Representative BAM images of (a) pure MA at 23 mN m1, (b) MA/F8C5OH (XF8C5OH = 0.05) at 25 mN m1, (c) MA/F8C5OH (XF8C5OH = 0.1) at 27 mN m1, (d)
MA/F8C5OH (XF8C5OH = 0.2) at 33 mN m1, (e) MA/F8C5PC (XF8C5PC = 0.05) at 24 mN m1, and (f) MA/F8C5PC (XF8C5PC = 0.1) monolayers at 28 mN m1 observed on 0.15 M
NaCl (pH 2.0) at 298.2 K. The scale bar represents 50 lm.
H. Nakahara et al. / Journal of Colloid and Interface Science 337 (2009) 201–210
207
Fig. 6. Typical FM images of the MA/F8C5OH monolayers for XF8C5OH = 0 (MA), 0.05, 0.1, and 0.2 on 0.15 M NaCl (pH 2.0) at 298.2 K. Numerical characters in higher-right
corner of each micrograph denote surface pressures (mN m1). In the coexistent phases, the percentage (%) in lower-left corner refers to the ordered domains in the
micrograph. The monolayer contained 1 mol% fluorescent probe. The scale bar represents 200 lm.
and collapse pressures increase more and more. For the mixtures
of XF8C5OH = 0.05 and 0.1, the similar phase behavior to single MA
monolayers is observed in the monolayer region. However, the
addition of F8C5OH leads to slower nucleation of ordered domains upon compression as shown in the image of XF8C5OH = 0.1
(27 mN m1). Compared with MA alone, the ordered domains
shrink in size and their percentage also reduces with increasing
XF8C5OH, where their shapes are retained. The further addition of
F8C5OH brings about a drastic influence on the phase morphology. In XF8C5OH = 0.2, the ordered domains are finely dispersed
and become a small circular shape with a diameter of 10 lm.
No fusion of each domain occurs in spite of lateral compression
due to the repulsive dipole interactions. The homogeneous circular domains are attributed to the domination of line tension in
the phase boundaries. Above XF8C5OH = 0.2, the FM images are
homogeneously bright (disordered phases) independently of an
increase in surface pressure (data not shown).
As for the MA/F8C5PC system in Fig. 7, the disordered/ordered
coexistence phase appears between XF8C5PC = 0 and 0.1. When the
content of F8C5PC increases further, FM images become homogeneously bright contrasts as shown in the images at XF8C5PC = 0.2
regardless of increasing surface pressures and molar fraction of
F8C5PC. In XF8C5PC = 0.05, the addition of F8C5PC make the size of
ordered domains shrink, where their shapes are almost held at
each surface pressure. The size and percentage of ordered domains
are smaller compared with those at the same fraction in the
MA/F8C5OH system. Moreover, small circular domains with a
diameter of 10–30 lm appear in XF8C5PC = 0.1, although the emergence of the circular domains requires the larger molar amount
(XF8C5OH = 0.2) in the MA/F8C5OH mixture. These results indicate
the stronger interaction of F8C5PC with MA. These phase variations
demonstrate that the partially fluorinated amphiphiles investigated here fluidize the MA monolayer. In particular, F8C5PC
achieves the stronger fluidizing and miscibility effect on the MA
monolayer compared to F8C5OH.
The incorporation of the partially fluorinated amphiphiles treated here also generates a drastic effect on the ordered domains of
DPPC monolayers [8]. The fluorinated amphiphiles achieve fluidization of the DPPC monolayers. F8C5PC has the potential of fluidizing the DPPC ordered domains more effectively than F8C5OH.
These trends are also observed in the present systems, too. However, especially in the DPPC/F8C5OH system, the shape of the DPPC
ordered domains considerably changes by the addition of F8C5OH
(XF8C5OH = 0.1). That is, the domains transform into a triskelionshaped domain like a whirlpool with an anticlockwise branch.
Then, the further F8C5OH addition to DPPC varies the domain
shape from the triskelion to circular features. On the contrary,
the domain shape does not change at smaller molar fractions in
the present systems. Considering that the elongation of ordered
domains is strongly influenced by the long range dipole interaction, the partially fluorinated chains interact with double hydrocarbon chains (DPPC) rather than single ones (MA) within the
smaller molar fraction range. If the composition of the DPPC/
F8C5OH (or F8C5PC) systems is recalculated as the molar fraction
per single chain; e.g., XF8C5OH = 0.1 (per molecule) corresponds to
XF8C5OH 0.05 (per single chain), a quite smaller addition of the
fluorinated amphiphiles results in variation of the LC domain shape
of DPPC.
Shown in Fig. 8 are the FM images of the MA/F8C5OH and
MA/F8C5PC systems above the collapse pressures. When the
monolayers of both systems (XF8C5OH and XF8C5PC P 0.2) are compressed beyond the respective collapse pressures, a large organization like a fern leaf abruptly emerges and grows up in size,
which is observed in BAM and FM images. Considering that the
large organization does not contain the FM prove and is visualized as a dark contrast, it is a rigid 3-D aggregate (or collapsed
material) induced by over-compression of monolayers. Pure components of MA, F8C5OH, and F8C5PC do not undergo such the 3-D
aggregates formation under the over-compression. In the case of
the MA/F8C5OH system (Fig. 8(A)), the large organization
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H. Nakahara et al. / Journal of Colloid and Interface Science 337 (2009) 201–210
Fig. 7. Typical FM images of the MA/F8C5PC monolayers for XF8C5PC = 0 (MA), 0.05, 0.1, and 0.2 on 0.15 M NaCl (pH 2.0) at 298.2 K. Numerical characters in higher-right corner
of each micrograph denote surface pressures (mN m1). In the coexistent phases, the percentage (%) in lower-left corner refers to the ordered domains in the micrograph. The
monolayer contained 1 mol% fluorescent probe. The scale bar represents 200 lm.
Fig. 8. Typical FM images of the (A) MA/F8C5OH and (B) MA/F8C5PC monolayers for XF8C5OH (or XF8C5PC) = 0.2 and 0.3 above the collapse pressure on 0.15 M NaCl (pH 2.0) at
298.2 K. The monolayer contained 1 mol% fluorescent probe. Numerical characters in higher-right corner of each micrograph denote approximate surface pressures (mN m1)
beyond the monolayer collapse. The scale bar represents 200 lm.
corrodes monolayer regions of disordered/ordered states (the FM
image of XF8C5OH = 0.2). The collapsed organization becomes smaller in size with increasing XF8C5OH to 0.3 and its shape resembles
that of the LC domain of pure MA monolayers apart from their
size. The behavior suggests that the collapsed aggregate might
be made mainly of MA. As for the MA/F8C5PC system
(Fig. 8(B)), on the other hand, the collapsed aggregates are
smaller in size and possess less complex branches in comparison
to those for the MA/F8C5OH system. This is resulted from a difference in condition surrounding MA between the F8C5OH and
F8C5PC monolayers, which resembles a difference in crystal form
induced by solvent effect. In addition, the degree and trend of
H. Nakahara et al. / Journal of Colloid and Interface Science 337 (2009) 201–210
collapsed-domain dispersions correspond to those of ordered-domain dispersions for the monolayer state (Figs. 6 and 7). Similarly
to Fig. 8(A), an increase in XF8C5PC leads to reduction in size of the
collapsed aggregates (Fig. 8(B)). It is worth noting that some collapsed aggregates have a vanishing contrast, which is indicated
by white arrows in the FM image of XF8C5PC = 0.3. This means that
the aggregates with a vanishing contrast are formed below the
monolayers. With increasing XF8C5PC from 0.2 to 0.3, the translucent aggregates increase in number or amount. Some mechanisms
on forming direction (toward the air or into the subphase) of collapsed aggregates have been proposed. It is suggested that the
collapse process depends on surface pressure [63], compression
rate [64], and subphase condition (i.e., ions and pH) [65]. However, these factors have been determined for pure components.
In the present study, we are focusing on the binary systems,
and thus the FM images in Fig. 8 are observed under the almost
same conditions except for the sample compositions. Considering
no formation of collapsed aggregates for pure components, it is
supposed that the fluorinated chain generate an important driving force of the formation. In addition, the phenomenon of forming the aggregates into subphase (Fig. 8(B)) indicates that the
affinity between headgroups (–COOH and -PC) becomes larger
than that between hydrophobic chains with an increase in XF8C5PC.
In particular, the steric interaction between headgroups (COOH/
PC for MA/F8C5PC and COOH/OH for MA/F8C5OH) is found to
contribute to the formation (vanishing contrast) into subphase.
Recently, mathematical and phenomenological studies on
monolayer collapse, which is the spontaneous transition from 2-D
to 3-D phases, have been performed [65–68]. In particular, pure
components except for isomeric molecular species containing
enantiomer and diastereoisomer have been extensively examined
to obtain a thorough understanding of the collapse mechanism.
Over-compression above the monolayer collapse pressure produces
a remarkably rich variety of 3-D structures such as vesicle [69],
giant folds [64], fiber-like networks [70], and so on. In the present
study, the both mixtures of MA/F8C5OH and MA/F8C5PC generate
fern-like networks as 3-D structures. The fern-like networks resemble fiber-like networks, which is formed on single monolayers of 2azido-4-fluoro-3-hydroxystearates (EAFHSs) [70] in their growth
and shape, although the present systems contain two components.
Considering no appearance of 3-D collapsed structures for pure
components investigated, the formation of the structures as observed in Fig. 8 collaterally supports the binary miscibility of MA
with F8C5OH and with F8C5PC in the monolayer state.
209
3.3.3. Atomic force microscopy
AFM observations of Langmuir–Blodgett (LB) films transferred onto mica have been performed to confirm the miscibility of the present systems on the nanometer level. The films
transferred at selected surface pressures were examined in the
tapping mode, providing simultaneously topography and phase
contrast images [8,47]. Notice that there are few differences
in chain lengths between MA and F8C5OH or F8C5PC, which induces the defect in detection limit and resolution under the
AFM measurements. In the disordered states below the respective transition pressures (see Fig. 4), AFM topographies and
phase contrasts for the two binary systems were homogeneous
images within height difference of 0.2 nm. Therefore, an experimental attention was focused on the disordered/ordered coexistence regions between transition pressure and collapse one.
Fig. 9 shows typical AFM height images for the MA/F8C5OH
and MA/F8C5PC systems at 30 or 35 mN m1. For the MA/
F8C5OH system, the AFM image at XF8C5OH = 0.1 (Fig. 9(a))
exhibits a larger and higher island than the surroundings. The
island is the ordered domain made mainly of MA as observed
in the BAM and FM images. However, a lot of lower defects
with height difference of 0.3 ± 0.1 nm exist in the islands (see
the cross-sectional image). It means that F8C5OH disperses
the ordered domains into the disordered phases in the shape
of a worm-eaten spot. When more amounts of F8C5OH are
added (Fig. 9(b)), the islands become smaller in size and are
dispersed in the same manner; the height difference within
the islands is 0.4 ± 0.1 nm. As shown in Fig. 9(c), on the other
hand, the AFM image for the MA/F8C5PC system (XF8C5PC = 0.1)
indicates the similar behavior to that for the MA/F8C5OH mixture. However, it is noted that the island is more finely dispersed by the F8C5PC addition compared with the case of
F8C5OH (Fig. 9(a)). The height difference in the island
(0.5 ± 0.1 nm) is slightly larger than that in Fig. 9(a). This is
attributed to the looser molecular orientation of F8C5PC
induced by its bulky headgroup. From the point of view of fluidizing lipids, it is possible to say that the fluorinated amphiphiles structurally require the spatially bulky moiety such as
phosphorcholine (or phosphatidylcholine) headgroup. The AFM
observations demonstrated that the mode of miscibility of MA
with F8C5OH and with F8C5PC is strongly dominated by the
fluidizing or dispersing effect of the fluorinated compounds on
the ordered domain of MA monolayers.
Fig. 9. Representative AFM topographic images of (a) MA/F8C5OH (XF8C5OH = 0.1, 30 mN m1), (b) MA/F8C5OH (XF8C5OH = 0.2, 35 mN m1), and (c) MA/F8C5PC (XF8C5PC = 0.1,
30 mN m1) on 0.15 M NaCl (pH 2.0) at 298.2 K. The scan area is 10 10 lm and the scale bar in the lower-right corner (c) represents 200 nm. The cross-sectional profiles
along the scanning line (white line) are drawn below the each image. The height difference between the arrowheads is indicated in the cross sectional profile.
210
H. Nakahara et al. / Journal of Colloid and Interface Science 337 (2009) 201–210
4. Conclusions
It was proved that MA is miscible with F8C5OH or F8C5PC in the
disordered as well as in the ordered monolayer state on 0.15 M
NaCl (pH 2.0) subphase. The 2-D phase diagrams were constructed
on the basis of transition and collapse pressures. Both of the
MA/F8C5OH and MA/F8C5PC systems were classified into a positive azeotropic type. Assuming a regular surface mixture, an interaction parameter (n) and interaction energy were calculated using
the Joos equation. The binary systems separately have two interaction parameters which change at XF8C5OH (or XF8C5PC) = 0.3: the
MA/F8C5OH (or/F8C5PC) systems have n = 1.5 (1.2) for
0 6 X 6 0.3 and n = 0.6 (0.89) for 0.3 6 X 6 1, respectively. The
parameters reveal the presence of relatively stronger interactions
between MA and F8C5PC under compression. Morphological
observations with BAM, FM, and AFM collaterally support the
two-component miscibility. A new finding in this study is that
the both binary mixtures investigated here generate fern-like networks as 3-D structures in the monolayer collapse state. The difference in shape of the collapsed structures between the two systems
may be responsible for a difference in crystal form which is often
induced by solvent effect. Furthermore, these observations suggest
that the miscibility behavior is strongly dominated by the fluidizing effect of the fluorinated compounds on the ordered domain
of MA monolayers. In particular, F8C5PC exerts the stronger fluidizing effect, indicating a significance of bulkiness of polar headgroup in fluorinated compounds for the lipid fluidization.
Focused on the shape of ordered domains, however, the present
systems did not show the large variation in shape differently from
the previous DPPC systems, where the domain shape transformed
to triskelion at the specific molar fraction. Above all, these results
indicate that F8C5OH and F8C5PC liquefied MA and DPPC monolayers in different extent, which is useful to develop their innovative applications to the biomedical field such as selective drug
delivery and lung surfactant additive.
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research 20500414 from the Japan Society for the Promotion of Science (JSPS). This work was also supported by a Grant-in-Aid for
Young Scientists 20810041 from JSPS (H.N.).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jcis.2009.05.022.
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