Biomaterials 21 (2000) 223}234

The phase diagram of the monoolein/water system:

metastability and equilibrium aspects
Hong Qiu, Martin Ca!rey*
Department of Chemistry, The Ohio State University, 100 W. 18th Avenue, Columbus, OH 43210, USA
Received 14 September 1998; accepted 28 June 1999
Abstract
Interest in the liquid crystal structure, transport and membrane protein crystallizing properties of the monoolein/water system has
grown in the recent past. Monoolein is also an important homolog in a series of monoacylglycerols used to decipher how lipid
molecular structure relates to liquid crystal phase behavior*information needed for rational design applications and for understand-
ing the origin of membrane lipid diversity. To make intelligent use of the monoolein/water system, a reliable and detailed
temperature}composition phase diagram is needed. The phase diagram of Briggs et al. (J Phys II France 1996;6:723}51) was
constructed for this purpose. However, we have established that the liquid crystal phases in the latter below ca. 203C are metastable.
By implementing a sub-zero degree (3C) sample incubation prior to data collection in the heating direction, we can reset the system
into the lamellar crystal phase which we assume represents equilibrium behavior. We have re-examined the low-temperature part of
the phase diagram and characterized structurally the new &equilibrium' phases by static and time-resolved low- and wide-angle X-ray
di!raction and by di!erential scanning calorimetry. A more complete phase diagram that incorporates the new equilibrium behavior
at low temperatures is reported. 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Cubic phase; Lipid bilayer; Liquid crystal; Mesophase; Phase diagram; Undercooling
1. Introduction
1-Monoolein is a monoacylglycerol with 9-cis-oc-
tadecenoic acid at the sn-1 position of glycerol. The
mesophase propensities and structure of monoolein dis-
persed in water are of interest in a number of areas
ranging from controlled uptake and release to cosmetic,
food and pharmaceutical formulations. Most recently,
the cubic phase of hydrated monoolein was reported to
serve as an environment in which to grow crystals of the
membrane protein, bacteriorhodopsin, for use in solving
its three-dimensional structure [2]. The tantalizing pros-
pect exists that the monoolein, and related hydrated
monoacylglycerol systems, might be used in crystallizing
other membrane proteins.
To make full use of the monoolein/water system in
such applications calls for a reliable and detailed temper-
ature}composition (}C) phase diagram and for a struc-
* Corresponding author. Tel.: #1-614-292-8437; fax: #1-614-292-
1532.
E-mail address: ca!rey@chemistry.ohio-state.edu (M. Ca!rey)
ture characterization of the phases formed. Over the
years, studies have been performed to realize these objec-
tives and the literature abounds with such information.
The most detailed phase characterization performed to
date in the temperature range from 0 to 1103C was
reported from this lab [1]. We have since established,
and report herein, that the liquid crystal phases existing
below 203C described in that study represent metastable
behavior. Equilibrium properties of the system have been
assessed and quanti"ed in the current study by imple-
menting an extended, low-temperature incubation of
samples prepared at room temperature before perform-
ing phase characterization in the heating direction.
Here, we describe the problems encountered in charac-
terizing the equilibrium phase behavior of a system hav-
ing phases with a pronounced tendency to undercool,
and our methods for solving them. The equilibrium}C
phase diagram of the monoolein/water system is de-
scribed in the temperature range from!15 to 1103C and
in the water concentration range from dry to full hy-
dration. In addition, structure characteristics of the as-
sorted phases (lamellar crystal, lamellar liquid crystal,
cubic (Pn3m, Ia3d), Fig. 1) found in the low-temperature
0142-9612/00/$- see front matter 1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 4 2 - 9 6 1 2 ( 9 9 ) 0 0 1 2 6 - X
Fig. 1. Schematic representation of the various crystal, liquid crystal
and #uid phases identi"ed in the temperature}composition phase dia-
gram of monoolein.
region of the phase diagram are reported based on X-ray
di!raction measurements. These are compared with
those of related hydrated monoacylglycerols. The objec-
tive here is to extend our understanding of the relation-
ship between lipid molecular structure and mesophase
properties for use in rational design. The information
should also prove useful in deciphering the membrane
lipid diversity enigma: why most biomembranes contain
such a bewildering array of lipid types.
2. Materials and methods
2.1. Materials
Monoolein was purchased from Nu Chek Prep Inc.
(Elysian, MN). It had a purity of*99%as determined by
thin layer chromatography [3] and was used without
further puri"cation. Thin layer chromatography was also
used to monitor monoolein purity change after the sam-
ples had been used in X-ray and DSC measurements. No
obvious degradation of the lipid was detected. It is
assumed that the isomerization equilibrium between
1-monoolein and 2-monoolein [4] is independent of tem-
perature in the range studied and that the lipid can be
treated as a single component. Water was puri"ed by
using a Milli-Q Water System (Millipore Corporation,
Bedford, MA) consisting of a carbon "lter cartridge, two
ion-exchange "lter cartridges and an organic removal
cartridge.
2.2. Sample preparation for X-ray diwraction
2.2.1. Standard preparation
Dry solid monoolein (ca. 20 mg) was mechanically
mixed with appropriate amounts of water (ca. 1}50 mg)
in a syringe-based mixing device as described [5] to
achieve the desired sample composition. Mixing was
carried out at room temperature (ca. 213C) while prepar-
ing the "xed hydration samples ranging in composition
from 0 to 50% (w/w) water. The homogeneously mixed
samples were transferred to 1 mm diameter quartz
capillaries (Charles Supper, Natick, MA), #ame-
sealed and glued with 5-min epoxy (Hardman Inc.,
Belleville, NJ), and were stored prior to data collection at
43C for a period ranging from a few days up to
4 weeks. The actual composition of the samples was
determined gravimetrically as described [6] with an
accuracy of better than 0.1% (w/w) water for low water
content (ca. 0}30% (w/w)) samples and 0.5% (w/w) water
for high water content ('30% (w/w)) samples,
using a microbalance (M3P-000V001, Sartorius Corp.,
Edgewood, NY) [5].
2.2.2. 03C preparation
Besides the standard room-temperature sample prep-
aration protocol described, samples were also prepared
at 03C. A monoolein sample with 32% (w/w) water was
prepared by mechanically mixing 21 mg dry monoolein
powder and 10 mg milliQ water at 03C on ice. A slow rate
of mechanical mixing as described [5] was used to min-
imize the likelihood of inadvertent sample frictional heat-
ing. The resultant mixture was placed in capillaries at
03C. Some of the "lled capillaries were stored sub-
sequently at 43C while others were held at room temper-
ature (ca. 213C). X-ray di!raction was used to monitor
the phase state of these samples as a function of time at
4 and 213C.
2.2.3. Passive swelling
The passive swelling technique of Briggs et al. [1] was
used to prepare fully hydrated samples from water-
stressed samples without mechanical mixing and thus,
the occurrence of possible inadvertent frictional heating.
This approach was used to minimize the likelihood of
producing an undercooled cubic phase as described [1].
For this purpose, an 18% (w/w) water sample was pre-
pared following the standard preparation method above
224 H. Qiu, M. Cawrey / Biomaterials 21 (2000) 223}234
and was subsequently stored at 43C for one week. The
capillary was then carefully broken at its glued end, and
an excess of milliQ water at 43C was placed in the
capillary in direct contact with the hydrated lipid. The
sample was subsequently equilibrated at 43C and
monitored visually and by X-ray di!raction at 43C.
2.3. X-ray source
Copper K

X-rays (1.542 As , nickel (0.025 mm-thick)

"ltered) were produced using a two-beam port Rigaku
RU-300 rotating anode X-ray generator (18 kW, Rigaku
USA Inc., Danvers, MA), operated at 40 kV and 200 mA,
and a horizontally positioned 0.5 mm;10 mm "lament.
The beam was focused horizontally and vertically by
a pair of 6 cm;2 cm (length;width) nickel-coated glass
mirrors (Charles Supper, Natick, MA) placed vertically
and horizontally ca. 20 cm downstream of the anode,
giving a focused beam measuring 1.5 mm;1 mm at the
detector which was positioned ca. 25 cm from the down-
stream mirror. This beam was used for both static and
time-resolved X-ray di!raction measurements.
2.4. X-ray diwraction data collection
Static X-ray di!raction patterns were collected in the
temperature range from!15 to 553C in increments of
1}53C in the heating direction. Up to seven samples can
be accommodated at one time in a temperature-control-
led beryllium, multiple sample holder. Seven di!raction
patterns measuring 1 in;8 in (covering a real space
range from ca. 2 to 160 As at a sample-to-detector dis-
tance of 20}25 cm) were collected side-by-side behind
a 1 in wide lead slit on an 8 in;10 in image plate (Fuji
HR-IIIN, Fuji Photo Film Co. Ltd., Japan).
Before making di!raction measurements, samples were
incubated at !103C or lower for at least 2 h to facilitate
full development of the L

phase. This is what we refer to

as the standard &sub-zero degree' (Celsius) incubation.
Subsequently, temperature was increased and samples
were equilibrated at a particular measurement temper-
ature for anywhere from 3 to 12 h before making the
15 min}1.5 h exposure at a sample-to-detector distance
of ca. 25 cm.
Time-resolved X-ray di!raction measurements were
made following temperature-jump perturbations to the
sample. The sample was initially incubated at 253C for at
least 30 min at which point temperature dropped to
!103C within 5 min. Mesophase properties of the
sample before, during and for 30 min after the temper-
ature jump were monitored continuously in the streak
mode with the image plate translating at a rate of
1.5 mm/min while positioned approximately 5 mm be-
hind a 3 mm wide lead slit at a 21.3 cm sample-to-
detector distance (see [23] for details).
2.5. Image analysis
A phosphorimage scanner (Storm 840, Molecular Dy-
namics Inc., Sunnyvale, CA) operating at a resolution of
100 m/pixel and a dynamic range of 10 was used to
read images recorded on the image plates. The pattern
was stored as a .gel "le and subsequently converted to .tif
"le for viewing and analysis using Adobe Photoshop (v
4.0, Adobe Systems Inc., San Jose, CA) and Matlab (v 5,
The Mathworks Inc., Natick, MA). An image analysis
program, written in Matlab, was used to locate the center
and to circularly average the di!raction patterns. Origin
(v 5.0, The Microcal Software Inc., Northampton, MA)
was used to "t the averaged intensity versus scattering
angle pro"les and to determine peak position. Sample-
to-detector distance was calculated using silver behenate
(sample provided by Dr. Blanton, Analytical Technology
Division, Eastman Kodak Company, Rochester, NY)
and 58.4 As as the lattice parameter [7].
2.6. Calorimetry measurements
Di!erential scanning calorimetry (DSC) measurements
were made using a Hart DSC-II (CSC4100, Calorimetry
Sciences Corp., Provo, UT). Calorimetry was used to
evaluate the e!ect di!erent sample thermal histories had
on phase behavior.
Appropriate amounts (tens of milligrams) of mono-
olein and water were mechanically mixed at room tem-
perature as stated in Section 2.2. Three samples with
di!erent concentrations were made, and a portion of
each was placed in two DSC ampoules. In this way, two
sets of three samples containing 14.5, 37.3 and 68.5%
(w/w) water were prepared. Each set was held at 373C for
10 min to induce the formation of the L

or cubic phases.
Afterwards, the "rst set (Set 1) was incubated at !103C
for 1 h while Set 2 was incubated at 03C for 6 h before
recording the thermograms up to 603C at a heating rate
of 153C/h.
3. Results
3.1. Temperature}composition phase diagram
In this study, great care has been taken to ensure that
equilibrium phase behavior prevails in the temper-
ature}composition (}C) phase diagram reported for the
monoolein/water system (Fig. 2). This includes incuba-
tion of all samples at !133C for at least 2 h before data
collection performed in the heating direction and long
equilibration times (3}12 h) at each measurement tem-
perature. The phases identi"ed by X-ray di!raction and
their location in temperature}composition space are
shown in Fig. 2A. Fig. 2B shows the phase boundaries
and coexistence regions as identi"ed by the di!raction
JBMT 1216
H. Qiu, M. Cawrey / Biomaterials 21 (2000) 223}234 225
Fig. 2. (A) Identity and location in temperature}composition space of each phase and coexisting phases in the monoolein/water system as determined
by X-ray di!raction in the heating direction in the!15}553C temperature and 0}50% (w/w) water composition ranges. The identity of the phases is as
follows: () ice, () L

, (;) L

, (*) cubic-Ia3d, () cubic-Pn3m and (#) FI. (B) Temperature}composition phase diagram of the monoolein/water
system based on an interpretation of the data in Fig. 2A, and the hydration data as in Fig. 3B (location of hydration boundary). The excess water
boundary of the L

phase is at about 4% (w/w) water, a best estimate as described in the text, and is indicated by a dashed line. (C) Composite
temperature}composition phase diagram of the monoolein/water system that combines Fig. 2B of the current work covering the range of !15}553C
and Fig. 5B of Briggs et al. [1] in the region above 303C.
data. It was drawn to conform to both the experimental
data and the Gibbs' phase rule.
The pure phases found in this system include the
L

phase, the L

phase, two inverted bicontinuous cubic

phases belonging to space groups Ia3d and Pn3m, and
the #uid isotropic (FI) phase. The description of the
phase diagram that follows is based on that presented in
Fig. 2B. The pure L

phase exists only at low water

concentration below ca. 343C. However, the excess water
boundary for the L

phase has not been determined with

accuracy. The latter is shown at ca. 4% (w/w) water
(dashed straight line, Fig. 2B) based upon the following
observations: (i) the lattice parameter of the L

phase
does not change with hydration above 4% (w/w) (see
Fig. 3B) and (ii) ice formation was seen in samples with
'4% (w/w) water but not in samples of lower hydration.
At least two L

polymorphs exist as identi"ed by their

disparate wide-angle di!raction patterns (Fig. 4). How-
ever, this work is not concerned with crystal polymor-
phism and this issue was not explored in the current
study.
Fusion of dry monoolein crystals to the melt occurs
between 31 and 363C. The FI phase occupies the high-
temperature part of the diagram at all hydration levels
and its low-temperature limit reaches a minimum of ca.
263C in the vicinity of 4% (w/w) water. In the temper-
ature range from 20 to 503C, the series of pure liquid
crystal phases that form with increasing hydration are
the L

, the cubic-Ia3d and the cubic-Pn3m phases.

Phases that exist in equilibrium with excess water in the
0}503C range include the L

and the Pn3m phases.

Phase behavior involving L

at temperatures below its

melting point is relatively complicated (see Fig. 2B).
Special care was taken to decipher the phase sequence in
this lower temperature region by making measurements
in increments as small as 0.53C and by implementing
226 H. Qiu, M. Cawrey / Biomaterials 21 (2000) 223}234
Fig. 3. Temperature- and composition-dependence of the structure parameters of the phases found in the monoolein/water system at the indicated
sample compositions in units of % (w/w) water and at selected temperatures. The structure parameter values reported are accurate to $0.1 As for all of
the phases, with the exception of the FI phase (characterized by di!use scatter) and the cubic phases when coexisting with the L

phase (where
scattering angle is small and di!raction is weak). The identity of the phases is as follows: () ice (the re#ection at 3.9 As is used in the plot), () L

, (;)
L

, (*) cubic-Ia3d, () cubic-Pn3m, and (#) FI.

long incubation times prior to data collection. The data
(Fig. 2A) are consistent with L

coexisting with ice below

03C, L

plus water coexistence from 03C to ca. 83C,

L

plus cubic-Pn3m coexistence from 8 to 173C, and

L

plus cubic-Ia3d from 17 to 183C. The L

phase co-
exists with L

between 18 and 263C. Coexistence of

L

and FI was detected at 0% (w/w) water at 22 and

313C. In going from low to high temperature, the hy-
dration range associated with each of these coexistence
"elds decreases.
A detailed examination of the transition occurring in
the vicinity of 03C was not undertaken. Accordingly, the
line drawn along the 03C isotherm in Fig. 2B and C could
represent a polytectic transition if it occurs at exactly
03C. On the other hand, if it is isothermal below 03C,
then a eutectic transition is implied. The poor solubility
of monoolein in water would place the latter just
below 03C.
The current data refer to the phase behavior of the
monoolein/water system in the !15}553C range and
from the dry state to the condition of full hydration. The
phase diagram of Briggs et al. [1] covers much the same
composition range, extends to considerably higher tem-
peratures and includes metastable phase behavior in the
low temperature region. By way of producing a more
complete representation of what we consider to be equi-
librium phase behavior over the full temperature range
from!15 to 1103C, we have combined the two phase
diagrams as shown in Fig. 2C. Thus, Fig. 2C combines
the '303C data in Fig. 2B of Briggs et al. [1] and the
data in Fig. 2B of the current study. We consider the
phase diagrampresented in Fig. 2Cto faithfully represent
JBMT 1216
H. Qiu, M. Cawrey / Biomaterials 21 (2000) 223}234 227
Fig. 4. X-Ray di!raction patterns of monoolein with 25% (w/w) water in the!15}313C range. The patterns were collected on an image plate at the
Advanced Photon Source (line 1-BM, Argonne National Lab) with an exposure time of 3 s. X-ray wavelength was 1.377 As and the sample-to-detector
distance was 373 mm. (a) !14.53C, coexistence of the L

phase and ice; (b) 2.63C, coexistence of the L

phase and water; (c) 15.13C, coexistence of the

L

and Pn3m phases; (d) 17.43C, coexistence of the L

and Ia3d phases; (e) 19.73C, coexistence of the L

and Ia3d phases; (f ) 31.13C, pure Ia3d phase.

Di!erent polymorphs of the L

phase were observed (compare wide-angle pattern in (a), (b) and (c).
the equilibrium phase properties of the monoolein/water
systemin the temperature and composition range shown.
3.2. Structure parameter temperature- and
composition-dependence
The temperature- and composition-dependence of the
structure parameter of the di!erent mesophases are
shown in Fig. 3. The monoolein/water system exhibits
typical liquid crystal thermal and composition expansivi-
ties to within the limits of measurement accuracy. Speci"-
cally, the structure parameter of all mesophases decreases
with increasing temperature (Fig. 3A), while the structure
parameter of all pure phases increases with sample hy-
dration (Fig. 3B).
The composition dependence of the lattice parameter
(Fig. 3B) shows that the monoolein/water system exhibits
thermodynamic invariance with respect to composition.
This is expected for a binary system where two phases
coexist at a "xed temperature (and pressure). Thus, the
composition of the two coexisting phases remains con-
stant while the relative amounts of the two phases change
as the overall composition is varied isothermally. This
means that in any two phase coexistence region, the
lattice parameter of each phase should remain constant
and insensitive to overall sample composition. Thus, for
example, at 183C, the L

phase coexists with Ia3d in the

concentration range from 18.4 to 39% (w/w) water
(Fig. 3B). In this composition range, the lattice parameter
of the L

phase is constant at 49.3$0.3 A

s
, while that of
Ia3d remains "xed at 173$2.2 A
s
. The relatively large
error associated with the Ia3d lattice parameter arises
from the fact that the phase does not scatter X-rays
particularly strongly, which exacerbates the problem
when the cubic phase is not the only phase in the system.
On top of this, the scattering angle is low which adds to
the uncertainty in peak scatter determination. The same
holds for the Pn3m phase in coexistence with the
L

phase.
3.3. Calorimetry measurements
DSC measurements were used to determine the e!ect
of two di!erent pre-measurement incubation protocols
on the phase properties of the monoolein/water system.
For this part of the study, duplicate sets of samples with
14.5, 38.5 and 87% (w/w) water were prepared in sealed
calorimetry ampoules. Following a 10 min incubation at
228 H. Qiu, M. Cawrey / Biomaterials 21 (2000) 223}234
Fig. 5. Di!erential scanning calorimetry thermograms of monoolein/water mixtures recorded in the heating direction at 153C/h. Each set consists of
three samples: (a) 14.5% (w/w) water, (b) 38.5% (w/w) water, and (c). 87% (w/w) water. Set 1 was incubated at 03C for 6 h before performing the scan.
Set 2 was incubated at!103C for 1 h before performing the scan. The y-axis scale in Set 1 is 10-times that in Set 2. What looks like a peak in the vicinity
of 33C in Set 1 thermograms arises from sample-reference mismatch. The same e!ect is present in Set 2 but is less obvious because of the di!erent scale
settings. The phases encountered during the heating scans are noted and were identi"ed based on X-ray measurements.
373C to induce the existence of the L

and/or cubic
phases, Set 1 samples were held at 03C for 6 h while those
in Set 2 were incubated at !103C for 1 h before record-
ing the heating thermograms (Fig. 5). The protocol im-
plemented with Set 1 samples mimics the conditions used
by Briggs et al. [1] where metastability was observed.
The so-called sub-zero degree incubation conditions used
with Set 2 were chosen to avoid undercooling.
The primary di!erence between the two sets of samples
in terms of calorimetric behavior is that a major en-
dotherm is seen in Set 2 between 5 and 203C that is
absent in the Set 1 thermograms (compare Fig. 5(1) and
(2)). Referring to the phase diagramshown in Fig. 2B, it is
obvious that the endothermic feature in Set 2 arises from
a series of transformations involving the L

phase. The
nature of these changes depends on sample composition
as dictated by the data in Fig. 2B. In stark contrast, no
such endothermic event occurs in Set 1 samples sugges-
ting that the L

phase has not formed under these condi-

tions and that the phases seen below ca. 203C are
undercooled. This is precisely what was reported in the
original paper by Briggs et al. [1].
The heat change associated with the L

phase trans-
formation is highly endothermic regardless of the liquid
crystal phase to which it converts (Fig. 5(2)). The corre-
sponding enthalpy changes (H) are as follows: ca.
20 cal/g of mixture (8 kcal/mol of lipid) for the L

-to-L

transition at 14.5% (w/w) water; ca. 16 cal/g of mixture

(9 kcal/mol of lipid) for the L

-to-Ia3d transition at
38.5% (w/w) water; and ca. 4 cal/g of mixture (11 kcal/
mole of lipid) for the L

-to-Pn3m transition at 87% (w/w)

water.
The thermograms also reveal weakly endothermic
transitions such as those associated with the L

-to-Ia3d
phase change at 503C (scan rate 153C/h, H"
100 mcal/g of mixture (40 cal/mol lipid) Fig. 5(1a)
and the Ia3d-to-Pn3m transition at 403C (scan rate
153C/h, H"10}20 mcal/g of mixture (5}10 cal/mol lipid)
Fig. 5(1b)). Hyde et al. [15] estimated that the latter
transition should have an associated H of &less than
about 0.01 kJ/mol' (about 10 mcal/g of mixture) when
viewed in the context that the interconverting cubic
phases incorporate in"nite periodic minimal surfaces and
that the phase transition involves a change in lipid bi-
layer structure but no change in curvature or breakage of
the bilayer itself. The experimental and estimated values
are in remarkably good agreement. A similar value has
been measured for the Ia3d-to-Pn3m transition in hy-
drated monovaccenin, a monoacylglycerol di!ering from
monoolein in that the double bond is at C11 (unpub-
lished data from this lab).
Given our focus on metastability and undercooling,
it is interesting to note that water in the hydrated
monoolein samples readily undercools, but it does so
JBMT 1216
H. Qiu, M. Cawrey / Biomaterials 21 (2000) 223}234 229
intermittently. The thermograms in Fig. 5(2) illustrate
this point in that the ice melting endotherm at 03C is seen
in just one of the three samples that had been incubated
for 1 h at!103C. In this and related hydrated monoacyl-
glycerol systems, we "nd that water will undercool read-
ily to!173C.
4. Discussion
4.1.
A
as the equilibrium phase at low temperature
In what follows, we present the evidence assembled to
date in support of the claim that in the low-temperature
region of the monoolein/water phase diagram, the
L

phase represents equilibrium behavior.

4.1.1. Sub-zero degree preincubation
The "rst piece of evidence is presented in Fig. 2 where
L

exists pure or in coexistence with ice, water, or

a variety of lipidic liquid crystal and liquid phases. The
phase diagram was constructed using samples that had
been subjected to a 2 h incubation at !133C prior to
data collection in the heating direction. This so-called
sub-zero degree preincubation sets the system into the
equilibrium L

phase from which the other liquid and

liquid crystal phases emerge upon heating. Omitting the
sub-zero degree preincubation with samples hydrated at
temperatures *203C allows for undercooling and per-
sistence of the liquid crystal phases down to 03C at least
as observed by Briggs et al. [1] and in the calorimetry
part of the current study.
4.1.2. Passive swelling
The idea we are following here is as follows. Fully
hydrated monoolein prepared at room temperature
spontaneously forms the cubic phase. When such sam-
ples are cooled to 03C, for example, in preparation for
phase studies to be performed in the heating direction
beginning at 03C, they are prone to undercool. Thus, the
phase observed in the 0}203C range very likely does not
represent equilibriumfor the system. The question is how
to avoid or to by-pass the cubic phase as an initial phase
in preparing a sample that is fully hydrated at 03C and in
its equilibrium state? Our approach here was to prepare
a hydrated monoolein sample at 18% (w/w) water where
it exists in the L

phase at room temperature and to set it

into the equilibrium L

phase following the protocol

described in Section 2.4. It was subsequently brought
from!13 to 43C and incubated in direct contact with an
excess of MilliQ water. The sample was monitored at 43C
by X-ray di!raction to follow any changes in phase state
and/or hydration with time. None was observed and the
sample remained in the original L

phase for a period of

at least 5 months. While not proof positive that the
L

phase represents equilibrium, this result is consistent

with it being the equilibrium phase.
Particularly intriguing is the observation that a similar
passive swelling experiment performed at 43C by Briggs
et al. [1] produced the cubic-Pn3m phase from an under-
cooled L

phase. This requires the system to remain

undercooled while the L

phase imbibes water, trans-

forms "rst to the Ia3d and then to the Pn3m phase. This
highlights the enigmatic nature of metastability in that
phases behave structurally (see below) and undergo
phase transitions just like systems at equilibrium all the
while being metastable. A similar observation has been
made in the N-dodecanoyl-N-methylglucamine}water
system [8].
4.1.3. Low-temperature sample preparation and incubation
In this experiment, a sample of monoolein at 32%
(w/w) was prepared by mechanically mixing the two
components, water and monoolein, on ice at 03C. The
L

phase forms under these conditions as demonstrated

by X-ray di!raction performed on the sample at 03C.
Temperature was adjusted subsequently to 43C and the
samples were held at 43C for a period of at least
5 months. By X-ray di!raction, the L

phase persisted
throughout the entire incubation. This protocol avoids
all liquid crystal phases during sample preparation and
thus, the possibility of the system becoming trapped in
a metastable L

or cubic phase.
4.1.4. Calorimetry
The thermograms in Fig. 5 demonstrate clearly the
existence of metastable phases in the monoolein/water
system when sub-zero degree incubation conditions are
not implemented prior to data collection in the heating
direction. Preincubation at !103C for 1 h is su$cient to
destabilize the undercooled liquid crystal phases in favor
of the equilibrium L

phase.
The phase diagram in Fig. 2B includes several regions
where coexistence with the L

phase is observed and

where the lever rule should and does apply as presented
under Section 3. At "rst pass, this might be taken as
evidence that the L

phase is behaving as an equilibrium

phase. However, the data in the paper by Briggs et al.
([1], Fig. 7A, 0}303C) where metastable L

and cubic-
Ia3d phases coexist also show this same behavior. Thus,
adherence to the lever rule cannot be used as evidence for
equilibrium.
We have implemented a sub-zero degree preincubation
protocol as a means for reliably setting monoolein/water
samples into the equilibrium L

phase. We must now

address the possible e!ect that ice formation, which can
accompany such low-temperature treatment, has on the
phase properties of the system. Thus, it is possible that ice
formation triggers the production of the L

phase as
a result of sample dehydration and that the L

phase is
really an artifact of the sample preincubation protocol.
230 H. Qiu, M. Cawrey / Biomaterials 21 (2000) 223}234
Fig. 6. Phase changes in hydrated monoolein following a jump in
temperature. Shown in (A) is an example of portion of a streak low- and
wide-angle X-ray di!raction pattern collected before, during and after
the temperature jump from 253C at t"5 min where the hydrated lipid
is in Ia3d cubic mesophase, to!103C where coexistence of the L

phase
and ice prevails. Sample composition was 32% (w/w) water. The image
plate was translated behind a 5 mm wide slit at a constant rate of
1.5 mm/min. The identity of the di!erent phases is indicated at the top
of the "gure. The d-spacing of the L

(0 0 1) and Ia3d (2 1 1) re#ections

is indicated. The re#ections at 3.9, 3.7 and 3.5 As are from ice. The
horizontal axis corresponds to elapsed time as indicated in (B). The
vertical axis represents scattering angle (2) with 2"0 at and increas-
ing in either direction above and below the asterisk. The time course of
in-sample temperature shown in (B) was measured with a T-type
thermocouple (0.41 mm in diameter) read by Physitemp BAT-12
(Physitemp Instruments Inc., Clifton, NJ). In this temperature-jump
study, two L

polymorphs (L

1, L

2) emerge during cooling. The

number of L

polymorphs observed in such measurements is variable.

In some cases, a single polymorph is seen. In others, as in this case, two
polymorphs are observed. What is consistently observed however is
that L

formation precedes the water/ice transition.

Our time-resolved X-ray di!raction measurements dur-
ing the course of a temperature jump from 25 to!103C
demonstrate unequivocally that this is not the case
(Fig. 6). What is seen is the emergence of the L

phase, at
the expense of the liquid crystal phase, that precedes ice
formation. The calorimetry data in Fig. 5(2) also support
this claim. As noted, ice formation upon cooling to
!103C is intermittent. Thus, in two of the three thermo-
grams in Fig. 5(2), ice fails to form but the highly en-
dothermic transition below 203C that is characteristic of
the L

phase, is present in all three.

4.2. Comparison of monoolein/water phase diagrams
An overview of the literature concerning monoolein/
water miscibility suggests a three-stage development in
our understanding of the system. The "rst stage is repre-
sented by the work of Lutton in 1965 [9] where &consist-
ency' and polarized light microscopy were used to
identify the major phases (L

, cubic, inverted hexagonal

(H
''
), FI). The second stage involves further phase identi-
"cation and structure characterization by means of X-ray
di!raction and NMR-based di!usion measurements
[10}14]. It was in this period that the di!erent cubic
phases were identi"ed. The third stage heralds in more
detailed studies of miscibility properties and an attempt
to characterize true equilibrium behavior of the system
by a myriad of methodologies [1,15}19]. At temper-
atures above ca. 303C, the reported phase diagrams are in
reasonable agreement. However, signi"cant di!erences
are observed below this temperature. In particular, the
diagram of Briggs et al. [1] shows liquid crystal phases
prevailing down to 03C (Fig. 7A) while that of Aomori
et al. [17] has liquid crystal phases stable to approxi-
mately 83C and &crystal#water' in the region from 8 to
!403C (Fig. 7B). The phase diagram reported in the
current study (Fig. 2B) is at variance with both of these.
We now know that the disparity between the current
work and that of Briggs et al. is due to inadvertent
undercooling of the liquid crystal phases below 203C
encountered in the latter work. The cause of di!erences
with the Aomori et al. phase diagram is a little more
di$cult to identify but could in part be attributed to the
fact that the latter study was performed using relatively
impure lipid; speci"cally, commercially available food
additive containing 97.5% monoolein [17].
Our conclusion is that the phase diagram in Fig. 2C
represents equilibrium over the full temperature and
composition range shown and that phase boundary loca-
tions are reliable to $23C and $2% (w/w) water. The
diagram reported by Briggs et al. [1] in the temperature
range below 203C represents metastable behavior of
a system that is kinetically stable. Following the protocol
for sample preparation as described in that work will,
with high probability, produce the corresponding meta-
stable behavior. Indeed, this same metastability might be
used to advantage in certain applications where the
structural and phase properties of the undercooled hy-
drated monoolein satisfy a particular need that cannot be
met by another monoacylglyceride.
4.3. Correlation between molecular structure
and phase behavior
In the long term, we wish to use phase behavior studies
of the monoacylglycerols to understand the relationship
between lipid molecular structure and mesophase
propensity and microscopic structure. For example, by
JBMT 1216
H. Qiu, M. Cawrey / Biomaterials 21 (2000) 223}234 231
Fig. 7. Temperature}composition phase diagrams of the monoolein/water system redrawn from (A) Briggs et al. [1] and (B) Aomori et al. [17].
comparing the members of a series of monoacylglycerol
homologs, we seek to establish the e!ect that fatty acyl
chain length and double bond position has on phase
behavior.
Such a comparison is particularly appropriate in the
case of monoolein and monovaccenin, two monoacyl-
glycerols that di!er structurally only in the position of
the cis double bond. In the case of monoolein, the site of
unsaturation is at C9. In monovaccenin, it is at C11.
A comprehensive comparison of the two has been made
in the temperature range above 303C [3]. Because they
are positional isomers with very similar e!ective lipid
chain lengths, they exhibit the same phases in the same
order with respect to temperature and hydration and
have a similar structure parameter temperature- and
composition-dependence as expected. However, there are
slight di!erences in phase boundaries that were at-
tributed to the disparate molecular shapes arising from
the di!erent location along the chain of the double bond
kink. The current study that focuses on phase behavior of
the monoolein/water system in the !15}553C temper-
ature range shows that the two systems also exhibit
qualitatively very similar phase behavior in this low-
temperature region. There are notable di!erences how-
ever in that the transition temperatures associated with
the L

phase are lower and the hydration boundary shifts

to lower water contents in the case of the monoolein
system. For example, the temperature where the L

phase
"rst transforms to a liquid crystal phase is ca. 83C in the
case of monoolein and ca. 163C for monovaccenin. Fur-
ther, the (L

#Pn3m)-to-(L

#Ia3d) transition occurs at

ca. 173C and ca. 203C in the case of monoolein and
monovaccenin, respectively. These di!erences will be in-
terpreted in the context of the variation in molecular
shapes of the two molecules as described quantitatively
by means of the so-called shape factor [20].
The shape factor or lipid packing parameter, , is
a simple and quantitative way of describing molecular
shape in a self-assembled aggregate such as the assorted
lyotropic liquid crystal phases encountered in lipidic sys-
tems. In the bicontinuous cubic phase, can be calculated
based on the in"nite periodic minimal surface model as
follows:
"
<
A(l )l
"

J
a
2l(A

a#2l)
, (1)
where < is the lipid molecular volume, A(l) is the lipid
headgroup area, l is the lipid length, (l ) is the volume
fraction of lipid in the unit cell (approximated as the
weight fraction of lipid in the sample based on the as-
sumption that lipid and water density are equal), a is the
cubic phase lattice parameter, and A

and are the ratio

of the minimal surface in the unit cell to the quantity (unit
cell volume) and the Euler}Poincare characteristic,
respectively. In the case of the Pn3m phase, A

"1.919
and "!2 [21]. For both monoolein and monovac-
cenin, '1 [1,3] and its temperature dependence is
shown in Fig. 8. Over the entire temperature range, the
-value for monoolein is greater than that for monovac-
cenin indicating that monoolein has a more pronounced
wedge shape at a "xed temperature given that both
molecules have virtually identical values of A(l) and l.
One consequence of a larger is that the lipid/water
interface is more highly curved and the phase can accom-
modate less water. This is borne out in the relative
positions of the excess water boundaries of the Pn3m
phase of the two monoacylglycerols where it is shifted to
higher water contents in the case of monovaccenin. For
example, the Pn3m phase hydration boundary at 403C is
at 46% (w/w) in the monovaccenin system, but only 36%
(w/w) water in the monoolein system. Similarly, at 803C,
the boundary position is at 30 and 26% (w/w) water in
the monovaccenin and monoolein systems, respectively.
While is a characteristic of an existing phase and does
not provide a direct measure of the relative stability of
232 H. Qiu, M. Cawrey / Biomaterials 21 (2000) 223}234
Fig. 8. Temperature dependence of the Pn3m cubic phase shape factor
for (A) monoolein (C18:1c9, ), (B) monovaccenin (C18:1c11, ) [3],
and (C) monoheptadecenoin (C17:1c10, ) [22].
mesophases, we consider the fact that monoolein has
a larger -value than monovaccenin as suggesting
a greater driving force for the former to access the highly
curved cubic phase. This in turn could be re#ected in the
lower temperature at which the L

phase of the mono-

olein transforms to the cubic phase regardless of hy-
dration level.
On a related topic, the phase diagram of the mono-
heptadecenoin (C17:1c10)/water system has been re-
ported [22] that includes the L

phase below ca. 203C in

the composition range from 0 to 52% (w/w) water [22].
However, the details of the interconversions involving
the L

and liquid crystal phases in this system have not

yet been deciphered. We expect that these will mimic
more closely those of monovaccenin than monoolein
because of the similarity in -values (Fig. 8). Part of our
future plans involves testing this prediction.
5. Conclusions
In this paper, we report the temperature}composition
phase diagram of the monoolein/water system in the
!15}553C range using X-ray di!raction for purposes of
phase identi"cation and structure characterization. The
existence and equilibrium nature of the L

phase at low
temperatures has been demonstrated based upon static
and time-resolved low- and wide-angle X-ray di!raction
and di!erential scanning calorimetry. These results high-
light the need for caution when reporting phase diagrams
that include liquid crystal phases as the low-temperature
state. Such mesophases are notorious in their ability to
undercool. Even more troublesome is the fact that the
response of these undercooled systems to temperature
and composition can be perfectly &normal', mimicking
equilibrium phase behavior. Thus, special care must be
taken to ensure that the system is set into its equilibrium
state before making phase characterization measure-
ments in the heating direction.
The phase diagram reported herein has been combined
with the high-temperature portion of the diagram de-
scribed by Briggs et al. [1]. We consider this composite
phase diagram to represent the equilibrium miscibility
properties of the monoolein/water system in the temper-
ature range from!15 to 1103C and in the composition
range from dry monoolein to full hydration.
Structure characteristics of the phases observed in the
!15}553C range are reported and are compared to those
of the related monoacylglycerol, monovaccenin, by way
of extending our understanding of the molecular struc-
ture}mesophase behavior relationship.
Acknowledgements
Thanks go to members of our research group for
valuable input on this manuscript. We also thank D.
Sellis for helping with part of the X-ray data collection
and the image analysis software development, and Drs. J.
Wang and V. Cherezov for assisting with the static X-ray
di!raction data collection at the Advanced Photon
Source. This work was supported by grants from the
National Institutes of Health (DK 36849, DK 45295, GM
56969) and the National Science Foundation (DIR
9016683).
References
[1] Briggs J, Chung H, Ca!rey M. The temperature}composition
phase diagram and mesophase structure characterization of the
monoolein/water system. J Phys II France 1996;6:723}51.
[2] Pebay-Peyroula E, Rummel G, Rosenbusch JP, Landau EM.
X-ray structure of bacteriorhodopsin at 2.5 As from microcrystals
grown in lipidic cubic phases. Science 1997;277:1676}81.
[3] Qiu H, Ca!rey M. Lyotropic and thermotropic phase behavior of
hydrated monoacylglycerols: structure characterization of mono-
vaccenin. J Phys Chem B 1998;102:4819}29.
[4] Ljusberg-Wahren H, Hersloef M, Larsson K. A comparison of the
phase behavior of the monoolein isomers in excess water. Chem
Phys Lipids 1983;33:211}4.
[5] Cheng A, Hummel B, Qiu H, Ca!rey M. A simple mechanical
mixer for small viscous samples. Chem Phys Lipids 1998;
95:11}21.
[6] Briggs J, Ca!rey M. The temperature}composition phase dia-
gram of monomyristolein in water: equilibrium and metastability
aspects. Biophys J 1994;66:573}87.
[7] Blanton TN, et al. JCPDS-International Centre for di!raction
data round robin study of silver behenate. A possible low-angle
X-ray di!raction calibration standard. Powder Di!r 1995;
10:91}5.
[8] Laughlin RG, Fu YC, Wireko FC, Scheibel JJ, Munyon RL. The
physical science of N-dodecanoyl-N-methylglucamine and its
aqueous mixtures. In: Holmberg K, editor. Surfactant science
series, vol. 74. New York: Marcel Dekker Inc., 1998. p. 1}30.
[9] Lutton ES. Phase behavior of aqueous systems of monoglycer-
ides. J Am Oil Chem Soc 1965;42:1068}70.
[10] Luzzati V. X-ray di!raction studies of lipid}water systems. In:
Chapman D, editor. Biological membranes, vol. 1. New York:
Academic Press, 1968. p. 71}123.
JBMT 1216
H. Qiu, M. Cawrey / Biomaterials 21 (2000) 223}234 233
[11] Larsson K, Fontell K, Krogh N. Structural relationships between
lamellar, cubic and hexagonal phases in monoglyceride}water
systems. Possibility of cubic structures in biological systems.
Chem Phys Lipids 1980;27:321}8.
[12] Lindblom G, Larsson K, Johansson L, Fontell K, ForseH n S. The cubic
phase of monoglyceride}water systems. Arguments for a structure based
upon lamellar bilayer units. J Am Chem Soc 1979;101:5465}70.
[13] Longley W, McIntosh TJ. A bicontinuous tetrahedral structure in
a liquid}crystalline lipid. Nature 1983;303:612}4.
[14] Larsson K. Two cubic phases in monoolein}water system. Nature
1983;304:664.
[15] Hyde S, Andersson S, Ericsson B, Larsson K. A cubic structure
consisting of a lipid bilayer forming an in"nite periodic minimum
surface of the gyroid type in the glycerolmonooleat}water system.
Z Kristallogr 1984;168:213}9.
[16] Ca!rey M. A lyotropic gradient method for liquid crystal temper-
ature}composition}mesomorph diagram construction using
time-resolved X-ray di!raction. Biophys J 1989;55:47}52.
[17] Aomori H, Ishiguro T, Kuwata K, Kaneko T, Ogino K. Study on
thermal and structural behavior of monoacylglycerol}water
systems. II. The phase behavior of monooleoylglycerol}water
systems. Yukagaku 1995;44:1004}11.
[18] EngstroK m S, Landh T, Ljunger G. The e!ect of lidocaine on the
phase behavior of the mono-olein/water system. Congr Int Tech-
nol Pharm 1989;3:432}8.
[19] Laughlin RG. Phase science of emulsi"ers: signi"cance and recent
developments in methodology. In: Wang PJ, editor. Food emul-
sions and foams: theory and practice, vol. 86. New York: Ameri-
can Institute of Chemical Engineers, 1990. p. 7}15.
[20] Israelachvili JN, Mitchell DJ, Ninham BW. Theory of self-assem-
bly of hydrocarbon amphiphiles into micells and bilayers. J Chem
Soc Faraday Trans II 1976;72:1525}68.
[21] Anderson DM, Gruner SM, Leibler S. Geometrical aspects of the
frustration in the cubic phases of lyotropic liquid crystals. Proc
Natl Acad Sci USA 1988;85:5364}8.
[22] Briggs J. Ph.D. Dissertation, The Ohio State University, Colum-
bus, OH, 1994.
[23] Chung H, Ca!rey M. Polymorphism, mesomorphism and meta-
stability of monoelaidin in excess water. Biophys J 1995;
69:1951}63.
234 H. Qiu, M. Cawrey / Biomaterials 21 (2000) 223}234