* Corresponding author. E-mail: toshi@chem.sci.osaka-u.ac.jp.

Journal of Crystal Growth 198/199 (1999) 13521359

Polymorphic transformations during crystallization processes
of fatty acids studied with FT-IR spectroscopy
Fumitoshi Kaneko*, Kohji Tashiro, Masamichi Kobayashi
Department of Macromolecular Science, Faculty of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
Abstract
Polymorphic crystallization processes of fatty acids have been studied with FT-IR spectroscopy, which is highly
sensitive to structural dierences in functional groups of a molecule. It has been found that structural changes take place
during the crystallization of fatty acids. In the solution crystallization of stearic acid, single crystals of the E form
transformto the B form. This transformation starts with a heterogeneous nucleation of the B form on the 0 0 1 faces of
the single crystal of E, and the B domains spread to the whole region of the single crystal through a solid-state phase
transition. In addition, the polytypic transformation froma single-layer to a double-layer structure takes place during the
crystallization of the E form. In the melt crystallization of the [ phase of oleic acid, the growth mechanism greatly
changes with increasing supercooling (A). In the condition of A(1.5C, the crystal edge grows as [

, while in the case

of A'1.5C, the growth edge grew as [

initially, then changed gradually to [

through a solid-state phase transition.

It was assumed that the lattice imperfections present in the single crystals and the relaxation rate of structural changes at
the growing faces are predominant factors for the polymorphic transformations to occur during crystallization. 1999
Elsevier Science B.V. All rights reserved.
Keywords: Fatty acid; FT-IR spectroscopy; Attenuated total reection (ATR) spectroscopy; Chemical etching; Solid-
state phase transition; Heterogeneous nucleation; Polymorphism
1. Introduction
One of the important characteristics of lipids,
and other related long-chain compounds, is diver-
sity in the occurrence of dierent structures. The
hydrocarbon moieties of these compounds can re-
veal various molecular conformations and lateral
packing, which results in a diversity of aggregation
state. Depending on crystallization condition and
thermal history, a variety of crystal structures ap-
pear [15].
Fatty acids form a major class of common lipids
and they are building blocks of all complex lipids
such as phospholipids [3]. They are abundantly
present in biological organs; the hydrophobic
portion of biomembranes consists of the acyl chains
of phospholipids. The aggregation state and dy-
namic property of acyl chains have signicant eects
on functional activities of biomembranes [3].
0022-0248/99/$ see front matter 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 2 - 0 2 4 8 ( 9 8 ) 0 1 0 1 1 - 2
Fig. 1. Crystal structures of stearic acid B(Mon), B(Orth II),
E(Mon) and E(Orth II).
Furthermore, fatty acids have been widely used in
many industrial products, such as foods, cosmetics,
detergents and lubricants, where polymorphism of
fatty acids is of fundamental importance. Fatty
acids show a remarkable diversity of crystal struc-
ture, depending on the length, parity and unsatur-
ation of acyl chains [3,5].
We have studied the polymorphismof two major
classes of fatty acids, saturated and monoun-
saturated fatty acids [1,5,6]. In these materials, it
was found that some specic polymorphs would
change their structures during crystallization. The
structural changes, however, were so small that
their identications were hardly made with conven-
tional experimental methods, such as X-ray dirac-
tion and optical observation.
To resolve this diculty, we have applied
Fourier-transform infrared (FT-IR) spectros-
copy with novel techniques, which enable to
take infrared spectra of a small single-crystal
specimen [7]. What is more, IR spectroscopy is
highly sensitive to local structural changes of
functional groups, such as methyl and carboxyl
groups. It was conrmed that a specic poly-
morph of fatty acids transforms to another forms,
keeping single crystallinity and morphology. In this
paper, we describe the cases of stearic acid (oc-
tadecanoic acid) and oleic acid (cis-9-octadecenoic
acid), which are the most representative fatty acids
in biotissue.
2. Experimentals
High pure samples of stearic, oleic and elaidic
acids ('99%) were prepared by a combination of
urea adducts, acid soap and fractional distillation
methods. n-hexane ('99% purity) was used as sol-
vent for growing single crystals.
Infrared spectra were taken with a Jasco FT-IR
8300 and a Jasco Janssen Micro FT-IR spectrom-
eter equipped with a MCT (mercurycadmiumtel-
luride) detector. For the measurement of tiny single
crystals, a specimen was sandwiched in a pair of
KBr plates with a small amount of hexa-
chlorobutadiene in order to suppress the scattering
of the incident radiation. For ATR (attenuated to-
tal reection) and oblique transmission measure-
ments, a JASCO ATR 500/M attachment and an
original homemade sample stage were used.
3. Results
3.1. Polymorphic and polytypic transformations
of stearic acid
3.1.1. EB polymorphic transition
Stearic acid has three polymorphs having an
orthorhombic subcell, B, C and E [811]. The
C form is stable above 32C, and the B form is
stable below that temperature [12,13]. The E form
is metastable in the whole temperature region. The
existence of the E form was disregarded in many
studies of n-fatty acids, owing to the lack of in-
formation about its occurrence conditions and
crystal structures, which was unveiled rather re-
cently [1419]. The obtained structure suggested
the close relationship between the B and E forms.
Contrary to the all-trans conformation of the
E form, the B form has a gauche bond near the
COOHgroup. Despite this dierence, their crystal
structures are quite similar to each other; the acyl
chain inclines towards the b

-axis of the orthor-

hombic subcell by 27 as shown in Fig. 1, where the
setting of the a

, b

- and c

-axis is made in accord-

ance with the orthorhombic polyethylene [20].
1353 F. Kaneko et al. / Journal of Crystal Growth 198/199 (1999) 13521359
Furthermore, the two polymorphs occur only by
solution crystallization.
Because of their structural similarity, the E and
B forms exhibit greatly intense X-ray reections at
almost the same 20 angles showing the same crystal
morphology: a lozenge plate with an acute in-
teredge angle of 74. Therefore, the identication of
the E form by optical observation and powder
X-ray diraction studies is impossible. Thus, it
might happen to consider E and B as one poly-
morph in the previous polymorphic studies. How-
ever, there is a marked distinction in IR spectra
between B and E, due to the dierence in molecular
conformation. For example, the OCO in-plane de-
formation band appears at 648 cm\ in B and
688 cm\ in E [1]. A careful examination was
made to dierentiate E and B for the single crystals
grown from a solution, by cooling the super-
saturated solution, and by keeping it at the same
temperature to induce solvent-mediated trans-
formations [21]. The crystals, occurred within 1 h
after the crystallization initiated, exhibited the IR
spectral feature of E. Then, some single crystals
began to showthe spectral features both of B and of
E. Here, we refer this type of crystal as BE type.
After the occurrence of the BE type crystal, single
crystals of the B form increased in its number in the
solution, and after 1 day, the single crystals present
in the solution were all of B.
Fig. 2a shows the ATR-IR spectra taken at the
0 0 1 faces of the BE type crystal. The front face
exhibited the spectral feature of B, whereas, the
reverse face showed those of E. Namely, the B and
E regions formed a layered structure in this crystal.
The BE type crystal gradually transformed to the
B form through the solid-state phase transition
(Fig. 2b and c). However, pure single crystals of the
E form did not transform spontaneously. It means
that heterogeneous nucleation of the B form on the
0 0 1 faces of the E crystal is prerequisite to the
EB solid-state transition. Once the B domain nu-
cleated on the single crystal of E in solution, the
transition proceeded irrespective of the ambience;
the single crystal of the BE type transformed to the
B form, in solution as well as out of solution.
From the spectral changes measured with the
oblique transmission method, it was claried that
only the portion near carboxyl group changed its
arrangement during this transition. Thus, a specu-
lation can be made that the EB transition pro-
ceeds in a bimolecular layer through the succession
of this structure change from an all-trans to a bent
conformation at the phase boundary between
B and E, as depicted in Fig. 2d. However, the inter-
lamellar interactions are so weak, compared with
the intralamellar interactions, that the structure
change on the crystal surface may not induce the
phase transition in the interior. An in situ observa-
tion of the growth processes claried that not all
single crystals transformed to the B form [22]. In
particular, most crystals generated at relatively
moderate conditions remained as the E form.
Chemical etching showed a striking contrast be-
tween the two types of single crystals. The single
crystal, which had speedily transformed to B, ex-
hibited many lozenge-shaped etch pits on the (0 0 1)
plane, some of which penetrated through the whole
crystal, while very few pits were observed in the
crystal that did not convert to B.
The observation of growth spirals on the 0 0 1
faces of fatty acid crystals suggests that the growth
of the 0 0 1 faces was principally controlled by
the spiral growth mechanism [2327]. Taking this
fact into consideration, one may explain the rela-
tionship of the striking contrast in chemical etching
to the EB transition, as follows. (1) Growth spiral
steps due to screw dislocations are extremely long
and durable, compared with the steps occurring in
a two-dimensional nucleation growth mechanism;
the long steps may catalyze the heterogeneous nu-
cleation of the B form. (2) Since bimolecular layers
formspiral planes that are analogous to the oor of
a modern multistoried parking garage with a slight
upward gradient [28], the EB transition is able to
travel from a 0 0 1 face to the interior without
interruption by driving on the ramp in a circle
about a dislocation line.
3.2. Orth II-Mon polytypic transformation
It has been found that the crystallization of the
B and E forms involves another kind of structure
change, a polytypic transformation [22]. Poly-
typism means a higher order structural dierence
caused by a dierent stacking sequence of layers
[29]. In the long-chain compounds, polytypismhas
F. Kaneko et al. / Journal of Crystal Growth 198/199 (1999) 13521359 1354
Fig. 2. IR spectra and schematic representation of the solid-state EB transition. (a) p-polarized ATR IR spectra taken on the 0 0 1
faces, (b) IR spectral changes due to solid-state EB transition, (c) process of the EB transition, and (d) a model of schematic
representation of EB transition.
been observed in n-alkanes, n-alcohols, and fatty
acids [3034]. The B and E forms of stearic acid
exhibit two polytypic structures: single-layer struc-
ture, Mon, and double-layer structure, Orth II
(Fig. 1) [35,36]. Contrary to the xed chain
inclination direction in Mon, the inclination direc-
tion is inverted at every stacking in Orth II. It has
been found that the IR bands due to methyl CH
1355 F. Kaneko et al. / Journal of Crystal Growth 198/199 (1999) 13521359
Fig. 3. An increase of the IRbands due to Orth II type structure
growing during the crystallization of stearic acid E form. The
two arrows indicate the IR bands assigned to antisymmetric and
symmetric o(CH

) modes of E(Orth II), respectively.

Fig. 4. Crystal structure of the [ form of oleic acid. Two
crystallographically independent molecules, A and B, in an
asymmetric unit.
deformation o(CH

) modes are sensitive to the al-

ternative chain stacking sequence [34]. Reecting
the dierence in the CH

CH

stacking behavior at
lamellar interfaces, Orth II showed its character-
istic IR bands at 1382 and 1447 cm\, which were
assigned to symmetric and antisymmetric methyl
CH deformation modes, o

(CH

) and o

(CH

).
Using the two marker IR bands, we monitored
the polytypic conversion during the solution
growth of the E form of stearic acid. Fig. 3 shows
an example, in which a saturated solution at 20C
was cooled to 16C, where crystallization was in-
itiated by stirring and kept quiescent for hours. The

(CH

) and

(CH

) bands were not found at the

inception of crystallization, but gradually increased
in intensity. Contrary to this spectral change, the
spectral features of the E form in the other regions
did not change. Namely, the rst occurring single
crystals were of Mon-type, and then gradually
changed to the Orth II type. This transformation is
due to the relative stability between polytypes.
Orth II is thermodynamically more stable than
Mon [11,13,34]. At the high supersaturation, the
metastable Mon occurs for kinetic reasons. As the
growth rate of Mon falls with decreasing solute
concentration, the growth rate of Orth II surpasses
that of Mon near the saturated concentration with
respect to Mon. Accordingly, the single crystals
begin to grow with the Orth II-type stacking.
3.3. [

transition of oleic acid

Oleic acid is the most abundant fatty acid in
nature; it ranges in various biological organs and
promotes the functional activity of biomembranes
[3]. Oleic acid has three polymorphs (:, [ and ) in
which a reversible solid-state transition occurs
from to :, revealing conformational disordering
[3740]. The crystal structure of the [ phase is
quite unique [41]. Most of the fatty acids form
a lamellar structure whose lamellar interface con-
sists of only CH

terminals. However, in oleic acid

[, two crystallographically independent molecules
in an asymmetric unit, A and B, make up an inter-
digitated structure, where the CH

group of mol-
ecule A and the COOH group of molecule B (or
vice versa) are located in the same plane (Fig. 4a).
F. Kaneko et al. / Journal of Crystal Growth 198/199 (1999) 13521359 1356
Fig. 6. Polarization dependence of IR C"O stretch bands in
single crystal specimens of oleic acid [

(top) and [

(bottom).
The observation site for b2 is the hatched area at the growing
crystal edge. The [

form shows a clear band splitting of two

components having dierent polarizations at 0"60 and 110.
Fig. 5. Temperature dependence of crystal growth rate of the
[ form and its crystal morphology.
This interdigitated structure has been observed in
the crystals of mixed-chain triglycerides involving
the oleic acid chains, in which the oleoyl groups is
considered to form the interdigitated structure in
solid states [42,43].
The melt-crystallization behavior of the [ phase
revealed the following uniqueness: (a) its growth
rate is quite low, e.g., at A"0.5C, the growth
rate of [ is about 10\ times lower than that of :,
and (b) the crystal shape drastically changed when
A increased, e.g., A(2.3C: hexagonal shape,
2.3(A(3.3C: needle shape, and A'3.3C:
curved bril shape.
The crystal growth rate of the hexagonal-shape
crystal gradually increased below A"1.5C, but
drastically increased above A"1.5C as shown
in Fig. 5. Its melting behavior also changed in the
following; (a) a hexagonal-shape crystals grown in
the region of A(1.5C melted at 16.3C, (b) the
crystals grown in the condition of 1.5(A(
2.3C exhibited two melting points, and (c) the
central region melted at 16.3C and the other re-
gion melted at 16.0C. The two types of regions,
indicated in (c), revealed basically the same IR
spectra, suggesting that the crystal structures of the
two regions are very similar each other. However,
several spectral dierences were noted; the most
conspicuous dierence was observed in the IR
bands due to CO stretch v(CO) mode (Fig. 6).
The domain with the melting temperature (

) at
16.3C showed a clear band splitting at 1701 and
1712 cm\, which exhibited maximum intensity
with dierent polarization: 0"60 and 110. On
the other hand, such a clear band splitting was not
observed in the domain with

"16.0C. This
spectral dierence suggested subtle dierences in
COOH groups between the two domains with

"16.3C and

"16.0C which were named

[

and [

, respectively. Since the frequency of

v(CO) mode is sensitive to the hydrogen-bond
strength of dimerized COOH groups [44], the
splitting in [

suggests the dierence in hydrogen-

bond strength between the two molecules in the
asymmetric unit. Taking this molecular arrange-
ments into consideration, we assigned the bands at
1701 and 1712 cm\ to the v(CO) mode of mol-
ecule A and that of molecule B, respectively.
Through microprobe FT-IR measurement at grow-
ing crystal edges, it was claried that a change in
crystal growth mechanism took place around
A"1.5C; the crystal edge grew as [

at
1357 F. Kaneko et al. / Journal of Crystal Growth 198/199 (1999) 13521359
A(1.5C, while at A'1.5C the crystal edge
grew as [

initially and then changed gradually to

[

via the solid-state phase transition.

The change in growth mechanism of the two
[ forms is interpreted as follows. The molecules in
the [ form forming the interdigitated lamellar
seems to be less mobile, because a CH

terminal is
surrounded by four hydrogen bonded COOH
groups. Therefore, it is rather dicult for a molecu-
le to be incorporated into the growing crystal faces
to settle itself in the most stable position. As A
increase, the rearrangement falls behind the mo-
lecular incorporation and the structure of a grow-
ing face begins to change. As a result, the crystal
edge starts to grow as a metastable form of [

.
4. Discussion
Since stearic acid and oleic acid are quite com-
mon saturated and cis-unsaturated fatty acids, it is
highly anticipated that similar phenomena may
take place in the other long-chain compounds. In-
deed, we have already conrmed a polymorphic
transformation in palmitic and eicosanoic acids,
eicosanedioic acid (a dicarboxylic acid) and elaidic
acid (a trans-unsaturated fatty acid). The polytypic
transformation was also observed in petroselinic
acid (a cis-unsaturated fatty acid) [45]. The essen-
tial phenomena commonly observed in these ma-
terials are that the metastable forms that appear as
the rst-occurring forms, transform to more stable
forms. The crystal morphology of long-chain com-
pounds is mainly determined by the inclination and
lateral packing of hydrocarbon chains. In case that
the structural changes are revealed to be localized,
the crystal shape and unit cell parameters remain
almost unchanged, which brings a diculty to de-
tect the structural changes during the concurrent
events of crystallization and transformation. In this
paper, we showed usefulness of vibrational spectro-
scopic techniques to unveil these complicated
transformation behavior.
Various types of grown-in defects present in crys-
tal aect many of its physical properties [45]. The
EB transition mechanism unveiled in the present
study suggested that imperfection incorporated in
the crystals, which occurred at the early stages of
crystallization, has signicant inuences on the
structural changes in the later stages of crystalliza-
tion. The grown-in defects may provide a eld for
the emergence of the product phase and a channel
for its solid-state transition.
The study on the [ form of oleic acid suggests
that the rate of the structure relaxation at growing
crystal faces is another important factor to let
a metastable form occur in the rst. It is usually
considered that the molecules trapped into the
growing face take immediately the same structure
as the molecules in the inner part. However, if the
relaxation to the stable structure is too slow to
follow the incorporation of molecules at a growing
face, molecules start to form a metastable structure
at the growing faces.
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