Food Research International 32 (1999) 249±259

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FT±IR studies on polymorphism of fats: molecular structures and

interactions
J. Yano, K. Sato*
Faculty of Applied Biological Science, Hiroshima University, Higash-Hiroshima 739-8528, Japan

Abstract
FT±IR analyses have been made on polymorphic structures of food fats, employing newly developed techniques such as attenuated
total re¯ection (ATR), micro-probe polarized, oblique transmission, re¯ection absorption spectroscopy (RAS), etc. Two fat crystals,
1,2-dipalmitoyl-3-myristoyl-sn-glycerol (PPM) and 1,3-distearoyl-2-oleoyl-sn-glycerol (SOS) were focused on: PPM is a 0 -stable fat
and SOS is the major component of cocoa butter. The stearoyl chains in SOS were fully deuterated, so that the FT±IR spectra of
the oleoyl and stearoyl chains were di€erentiated. As for 01 form of PPM, the conformations of three acyl chains with respect to the
glycerol group and the inclination of the acyl chains against the O? subcell axes and the lamellar plane were observed. In ®ve
polymorphs of SOS, it was found that the conformational ordering of stearoyl chains took place in a less stable form, form,
whereas the ordering of oleoyl chains occurred in a more stable form,  form. These results indicate that the FT±IR spectroscopic ana-
lyses are sensitive to molecular-level structures of the polymorphic forms of fats. # 1999 Canadian Institute of Food Science and
Technology. Published by Elsevier Science Ltd. All rights reserved.
Keywords: FT±IR; Fatty acids; Fats; Ole®nic conformation; Polymorphism; Triacylglycerols

1. Introduction are called ``mono-acid TAGs'' and those having more

Triacylglycerols (TAGs) are the main constituents of
fats present in foods, pharmaceuticals, cosmetics, and so
on. Physical properties of TAGs determine key processes
in the processing of fat products and fractionation of fats
and oils (Dickinson & McClements, 1995; Formo, 1979;
Precht, 1988; Sato, 1996). In analogy with other lipids
than two types of chains ``mixed-acid TAGs''. Natural
oils and fats contain many kinds of mixed-acid TAGs.
The physical properties of the mixed acid TAGs are more
complicated than those of the mono-acid TAGs, in par-
ticular when the acyl chains at sn-1 and sn-2 positions are
di€erent, and when a TAG molecule consists of unsatu-
rated acyl chains.
and long chain compounds, TAGs show complicated The basic polymorphs of TAGs are called ; 0 and
polymorphism (Hagemann, 1988; Small, 1986). Depending  (Larsson, 1966; Lutton, 1950; Malkin, 1954), which
on the environmental conditions (temperature, pressure, are identi®ed by subcell structure (Fig. 1). The  form
solvent, etc.) as well as thermal histories they have passed is the most stable and packed in T== ; 0 less stable
through, various crystalline states occur, resulting in var- packed in O?, and  with hexagonal subcell is the
ious functional physical properties of TAGs, in combi- least stable form. Depending on the acyl chain com-
nation with crystallization behavior and crystal particles position, other metastable phases and multiple 0 and
aggregation.  forms are observed in mixed acid TAGs. As to
The polymorphic properties of TAGs depend on the the structural properties, the crystal structure has
fatty acid compositions: e.g. saturated or unsaturated
acids, short or chain acids, even or odd carbon-num-
bered acids, straight branched chain acids, and so on
(Small). The TAGs having only one type of acyl chain
been determined only for the stable  forms (Goto,
Kodali, Small, Honda, Kozawa & Uchida, 1992;
Jensen & Mabis, 1966; Larsson, 1963, 1964). However,
many important problems have still been open to
question as noted in the following: (i) the structures of
meta-stable phases, in particular for 0 forms, (ii) the
* Corresponding author. Tel.: +81-824-24-7935; fax: +81-824-22-
structures of saturated±unsaturated mixed acid TAGs,
7062. and (iii) the structures of mixture phases of di€erent
E-mail address: kyosato@ipc.hiroshima-.ac.jp (K. Sato) TAGs.
0963-9969/99/$20.00 # 1999 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.
PII: S0963-9969(99)00079-4
250 J. Yano, K. Sato / Food Research International 32 (1999) 249±259

recent development of FT±IR spectrometers, it has been

possible to obtain IR spectra from a small domain of
several tens of mms in size, and one can measure IR
spectra under various conditions of specimens with dif-
fuse re¯ection spectroscopy, attenuated total re¯ection
spectroscopy, re¯ection-absorption spectroscopy, and
so on. The IR data thus corrected were more informa-
tive compared to those obtained by conventional IR
methods (Chapman, 1964; Fichmeister, 1974; Ruig,
1977).
This paper summarizes recent results of FT±IR analyses
of two mixed-acid TAGs; 1,2-dipalmitoyl-3-myristoyl-sn-
glycerol (PPM) and 1,3-distearoyl-2-oleoyl-sn-glycerol
Fig. 1. Typical subcell structure of TAG polymorphs.
(SOS). PPM is a 0 -stable fat and SOS is the major
component of cocoa butter. The stearoyl chains in SOS
were fully deuterated, so that the FT±IR spectra of the
oleoyl and stearoyl chains were di€erentiated. After the
description of the new FT±IR techniques, the details of
the molecular conformation and chain inclination of the
0 form of PPM and the chain-chain interactions
As for the 0 structures, this polymorph is usually between the stearoyl and oleoyl acids of SOS poly-
most functional in fat products, due to its better crys- morphs will be presented.
tallinity having a tendency to make a minute crystalline
network with thin needle-shape morphology, which
gives proper softness in the fat products (Precht, 1988). 2. Experimental section
Therefore, the 0 !  transition often results in
deterioration of the end products. The 0 form is known 2.1. Sample preparation
to pack in the O? subcell, yet the molecular conforma-
tion of the acyl chains and the glycerol group, and the Fig. 2 shows the polymorphism of PPM (Kodali,
overall conformation are still a controversial matter Atkinson, & Small, 1990) and SOS (Sato et al., 1989),
(Hernqvist, 1988). where the most stable 01 , of PPM, and the all forms of
Concerning the mixed-acid TAGs involving the unsa- sub- through 1 forms of SOS were examined.
turated fatty acid moieties, serious complexity in the PPM was synthesized according to the method
polymorphism arises from the heterogeneous interac- described previously (Kodali, Atkinson, Redgrave &
tions between the saturated and unsaturated acyl chains
(Givon, Durant & Deroanne, 1986; Kodali, Atkinson,
Redgrave & Small 1987; Larsson, 1972; Lovegren,
Gray & Feuge, 1971; Lutton, 1950; Sato, Arishima,
Wang, Ojima, Sagi & Mori, 1989). In some speci®c sets
of saturated-unsaturated mixed acid TAGs, several
researchers have reported the formation of molecular
Small, 1984). Single-crystal specimens of 01 of PPM
were crystallized from acetonitrile solutions by slow
cooling. As to SOS, two samples were prepared.
Hydrogenated-SOS (h-SOS) was provided by Fuji Oil
Co. Ltd. Deuterated-SOS (d-SOS) was supplied from
D.R, Kodali, who synthesized it by adapting the meth-
ods described in the literature (Bentley & McCrae, 1970;
compounds due to chain-chain interactions (Engstrom, Kodali et al., 1987). Since the 2 -form crystal was not
1992, Kaneko, Yano & Sato, 1998; Koyano, Hachiya & obtained for d-SOS, we focused on the most stable 1
Sato, 1992, Minato, Ueno, Smith, Amemiya & Sato, 1997, form of SOS, which is described as  in this text. Single
Minato, Ueno, Yano et al., 1997, Rossel, 1967). Although crystals of the b-form were grown from an acetonitrile
the thermodynamic properties and structural character- solution by slow cooling.
istics of the mixed-acid TAGs have been studied, the
proposed structural models contain many ambiguities
on the conformation of saturated and unsaturated acyl
chains and glycerol groups.
The authors have recently applied Fourier transform
infra-red (FT±IR) spectroscopy to examine the mole-
cular structures and interactions of the fat polymorphs
(Yano et al., 1993; Yano, Kaneko, Kobayashi & Sato,
1997a; Yano, Kaneko, Kobayashi, Kodali et al., 1997;
Yano, Kaneko, Sato, Kodali & Small, 1999). With a Fig. 2. Polymorphic transformations in PPM and SOS.
 J. Yano, K. Sato / Food Research International 32 (1999) 249±259
2.2. FT±IR spectroscopy
The IR spectra were taken with a Perkin±Elmer spec-
trum 2000 spectrometer attached with a Perkin±Elmer
i-Series FT±IR microscope. An Oxford ¯ow type cryo-
stat CF1104 and an Oxford temperature controller ITC-
4 were used for the measurements at low temperatures.
The polarized IR spectra were measured with a MCT
detector and a wire-grid polarizer (Perkin±Elmer
PR500). The spectra without mention of the conditions
were taken at room temperature. To determine the
directions of transition moments three-dimensionally,
we employed the three methods as elaborated in the
following.
2.2.1. Special methods
2.2.1.1. Polarized IR transmission methods. Normal
incident transmission spectra and oblique transmission
spectra were taken in the present study (Kaneko, Shirai,
Miyamoto, Kobayashi & Suzuki, 1994; Kaneko, Ishi-
kawa, Kobayayashi & Suzuki, 1994). As for the former
case [Fig. 3(a)], the direction of IR radiation was nor-
mal to the (001) plane of the crystal. The direction of
the electric ®eld of the incident radiation was ®xed, and
the sample was rotated about the normal of the (001)
plane. In this study, we assumed that the b-axis of the
crystals is parallel to the 90 polarization direction. In this
Fig. 3. FT±IR set-ups of (a) normal incident transmission, (b) oblique
transmission method, (c) attenuated total re¯ection (ATR), and (d)
re¯ection absorption spectroscopy (RAS).
251
case, the information deduced from the polarization
dependence of the IR spectra concerns the projection of
the transition moments onto the (001) plane.
The oblique transmission method [Fig. 3(b)] has been
developed to overcome the above limit of the normal
incident method (Kaneko, Shirai et al., 1994; Kaneko,
Miyamoto & Kobayashi, 1996). The specimen was set at
a suitable position for the oblique measurement (per-
pendicular or parallel to the subcell axis) and then tilted
by an angle of . The spectra measured at  ˆ ‡30 and
ÿ30 were compared with the spectrum at  ˆ 0 to
obtain the bands having the transition moments per-
pendicular to the (001) plane.
2.2.1.2. Attenuated total reflection (ATR) method. The
transmission method cannot be applied for thick speci-
mens or strong absorption bands. In such a case, the
ATR method is useful (Kaneko et al., 1996). The ¯at
(001) plane of specimens was set on the sampling plane
of an internal re¯ection element (IRE) of ZnSe or Ge
whose wedge angle was 45 , and re¯ection spectra were
measured with p- or s-polarized incident radiation as
shown in Fig. 3(c). The p-polarized incident radiation
has the vibrational electric ®eld perpendicular to the
basal plane of IRE, whereas the electric ®eld of the s-
polarized one is parallel to it. With p-polarization, the
x- and z-components of a transition moment are
observed, and the y-component with s-polarization. In
the above conditions the z-component, which cannot be
observed with the normal incident transmission method,
is enhanced in the p-polarized spectra.
By the combination of the p- and s-polarization mea-
surements, three-dimensional structural information
can be obtained (Fraser, 1958a, 1958b; Higashiyama &
Takenaka, 1974). The analysis was done according to
the theory of Flournoy (Flournoy, 1966; Flournoy &
Scha€ers, 1966).
2.2.1.3. Reflection absorption spectroscopic (RAS)
method. Since a strong electric ®eld normal to the metal
surface can be generated in this method as shown in Fig.
3(d), the modes of this direction are observed selectively
(Allara & Swalen, 1982; Greenler, 1966; Rabolt, Bums,
Schlotter & Swalen, 1983). Samples were built up on a
metal surface as a thin ®lm where the lamellar interface
is parallel to the metal surface by developing chloro-
form solution. Accordingly, the spectral bands whose
transition moments are parallel to the chain axis are
enhanced.
2.2.2. Conformationally sensitive IR bands
2.2.2.1. CH2 progression bands. To study the con-
formational state of saturated acyl chains of mixed acid
TAGs, the information about the IR bands of mono-
acid TAGs are important as references. Therefore, the
assignments of the 3 progression bands in the region of
252 J. Yano, K. Sato / Food Research International 32 (1999) 249±259

1380±1150 cmÿ1, which sensitively re¯ect the stem these modes appear as single bands in the crystal having
length of the trans zigzag chain, has been made for  forms the T== subcell, in which polymethylene chains are par-
of three TAGs; tristearin (SSS), tripalmitin (PPP), and tri- allel to each other.
myristin (MMM), as summarized shortly (Yano, Kaneko,
Kobayashi, & Sato, 1997). Two series of progression
bands overlap in this region, one is due to the 3 branch 3. Structural analysis of PPM 01 form
modes (CH2 wagging: 1300±1150 cmÿ1) and the other
due to the 7 branch modes (CH2 twisting-rocking: 3.1. Crystal morphology and subcell orientation
1300±1150 cmÿ1). The 3 progression bands were iso-
lated clearly, and could be used as reference bands to The shape of the 01 crystal is a parallelogram whose
analyze the conformational state of the acyl chains of acute angle is 57 . The  (CH2) and r(CH2) bands of the
PPM and SOS. O? subcell show clear dichroism for the single crystals
2.2.2.2. CH2 scissoring and rocking modes. The vibra-
tional spectra of CH2 groups re¯ect the crystal struc-
tures as well. The relationships between the frequencies
of bands and subcell packing are listed in Table 1. In the
crystal having the O? subcell, the CH2 scissoring and
rocking modes split into two components, due to the
intermolecular interaction of the two chains in the unit
cell (Kobayashi, 1988; Tasumi & Krimm, 1967; Tasumi
& Shimanouchi, 1965). One component is polarized
along the as-axis, and the other one along the bs-axis of
the O? subcell. The splitting width of two components
depends on the intermolecular force constant. By contrast,
of 01 (Fig. 4) (Kobayashi, 1988; Tasumi & Krimm,
1967; Tasumi & Shimanouchi, 1965), where the crystal
morphology and the polarization of the incident radia-
tion of E are depicted. The as-components, 730 cmÿ1
(ras(CH2)) and 1473 cmÿ1 (as (CH2)) bands, appear
most intense at  ˆ 0 and vanish at  ˆ 90 , while the
intensi®es of the bs-components, 720 cmÿ1 (rbs(CH2))
and 1463 cmÿ1 (bs (CH2)) bands, become maximum at

Table 1
Characteristic vibrational modes of a polymethylene chain and crys-
tals

Subcell

Vibrational Polymethylene as Component bs Component

modes chain

2 …† O?
(CH2 scissoring)

8 …†
(CH2 rocking)

Fig. 4. Polarized FT±IR spectra of PPM b'1 in CH2 scissoring and

CH2 rocking regions.
 J. Yano, K. Sato / Food Research International 32 (1999) 249±259 253

 ˆ 90 and minimum at  ˆ 0 (see, Table 1). There-

fore, we can conclude that the projections of the as- and
bs-axes of the O-? subcell are almost perpendicular and
parallel to the long axis of the single-crystal, respec-
tively.

3.2. Acyl chain conformation

The acyl chains of PPM are composed of two palmi-

toyl chains and one myristoyl chain. When these chains
take a chain conformation with one chain pointing in
one direction opposite to the others, two possible acyl
chain conformations are considered: (a) the two palmi-
toyl chains have the straight (all-trans) conformation
and the myristoyl chain has the bent conformation in
which one gauche bond is inserted near the ester bond,
or (b) one palmitoyl and one myristoyl chain have the
all-trans conformation and the second palmitoyl chain
has the bent conformation. Using the 3 progression
bands appearing in the 1350±1150 cmÿ1 region, we
examined the validity of the postulated acyl chain con-
®gurations (Yano, Kaneko, Kobayashi, Kodali et al.,
1997).
The conformation (a) must provide the progression
bands arising from the straight palmitoyl chains toge-
ther with bent myristoyl chain. On the other hand, the
conformation (b) may consist of the bands from straight
palmitoyl and myristoyl chains and one bent palmitoyl
chain. The 3 bands of the straight chains mainly appear
in RAS due to the transition dipole moment parallel to
the chain axis. On the other hand, those of the bent
chains, which involve a perpendicular component to the
chain axis due to the bent conformation, strongly
appear in transmission spectra. Taking into account the
di€erence in the polarization direction of the 3 bands
from the two types of chains, we assigned the 3 bands
of 01 of PPM based on the results of MMM and PPP 
forms (Yano, Kaneko, Kobayashi & Sato, 1997).
Fig. 5(A) shows the normal incident transmission
spectra taken at 87 K and RAS taken at 297 K. In the
specimen for RAS, the lamellar interface is parallel to
the metal surface, and thereby the chain axes are almost
normal to it. Accordingly, the bands whose transition
moments are parallel to the chain axis are emphasized
with a strong electron ®eld normal to the metal surface.
The comparison in the range of 1360±1150 cmÿ1 indi-
cated that the marked bands with  in the transmission
spectra correspond to the bands observed in RAS,
meaning that the transition moments of these bands
mainly involve the components parallel to the chain
axis. Therefore, these bands were assigned to the 3
progression bands due to the palmitoyl and/or myristoyl
chain. In order to distinguish the contributions from the
Fig. 5. Assignments of the 3 progression bands of PPM 01 : (A) nor-
two chains, the frequencies of RAS bands were com- mal incident transmission method and RAS, (B) RAS of PPM 01 , PPP
pared with those of the  forms of PPP and MMM [Fig. , and MMM , and (C) polarized transmission spectra of PPM 01 ,
5(B)]. The bands marked with closed circles of PPP and PPP , and MMM .
254 J. Yano, K. Sato / Food Research International 32 (1999) 249±259

MMM arise from the 3 bands of the straight palmitoyl 01 of PPM. The complete decision, however, of the four
and myristoyl chains. It is noted that the bands in 01 possible structure displayed in Fig. 6 must await X-ray
totally correspond to the 3 bands of the straight chains structural determination.
of PPP and MMM.
As for the composition of the bent chain, Fig. 5(C) 3.3. Chain inclination behavior
shows the comparison of the transmission spectra of
PPM 01 with those of MMM and PPP, connected by The chain inclination behavior in the 0 forms of
the dotted lines. No band series of the bent conforma- TAGs has been of high interest, since it is related to the
tion of the myristoyl moieties, whose band positions are 0 ÿ  transition mechanism (Hernqvist, 1988; Precht &
indicated by the broken lines, was observed in the 01 Frede, 1977). The present FT±IR studies provided the
form of PPM. For example, the strongest 3 band of the information of the inclination direction of the acyl chain
bent myristoyl chain at 1276 cmÿ1 was not observed in with respect to the subcell axes and the lamellar inter-
the spectra of PPM 01 . face in 01 of PPM, using the oblique transmission, RAS,
Thus, the preferred acyl chain conformation of the and ATR methods. Since the subcell orientation is
PPM 01 form was assumed in the following: one
straight palmitoyl and one straight myristoyl chain, and
one bent palmitoyl chain. Then the next problem arises;
which chain, myristoyl or palmitoyl, is adjacent to the
bent palmitoyl chain and which glycerol con®guration,
sn-1,2 (or sn-2,3) or sn-1,3 con®guration, is most con-
directly related to the inclination direction of the acyl-
chains, (CH2) and r(CH2) bands having the transition
dipole moments perpendicular to the molecular chain
axis were employed as the reference bands of the chain
inclination direction.
Fig. 7 shows the oblique transmission spectra of the
ceivable, as exhibited in four possible con®gurations
shown in Fig. 6.
Detailed band assignments of the carbonyl stretch
bands [(CˆO)] may be most determinative to solve this
problem, since the frequencies and polarization direc-
tions of these bands are directly related to the glycerol
01 of PPM. In Fig. 7(a), the electric vector of the inci-
dent radiation and the as-axis of the O? subcell are in
the same plane, and the angle between the as-axis and
the electric vector is varied by tilting the specimen (as-
inclination). By contrast, the spectra in Fig. 7(b) were
taken with the inclination in the plane involving the bs-
con®guration. A comparison of the PPM 01 with those axis and the electric vector (bs-inclination). With varia-
of the PPP  form in the region of 1800±1600 cmÿ1
indicated the homology of the two crystal structures
(Yano, Kaneko, Kobayashi, Kodali et al., 1997).
Although the (CˆO) bands are very sensitive to the
glycerol conformation, these two forms have common
characteristics in the polarization direction and the
tion of  values in the as-inclination, the intensity of
as …CH2 † band signi®cantly changed, being strongest at
 ˆ 0 . It is notable that an increase in the changes of
the band intensity due to the inclination angles of +30
and ÿ30 are not symmetric, namely the former is larger
than the latter. This implies that the as-axis is slightly
band frequencies. In the mono-acid TAGs, the 0 form inclined with respect to the (001) plane as depicted in
transforms to the  form in a solid state, and the gly-
cerol con®guration of  takes the sn-1,2 con®guration
having the glycerol structure of type (c) in Fig. 6, as
shown by the X-ray crystal analysis (Jensen & Mabis,
1966; Larsson, 1964). Therefore, it is reasonable to
assume that the same con®guration may also be applied
to the present case of 01 of PPM. Consequently, type (c)
in Fig. 6 is the most plausible glycerol con®guration for

Fig. 7. (a) and (b) Oblique transmission spectra, (c) and (d) schematic
representations of the inclination directions of the acyl chains of PPM
01 . The tilting directions of the specimen are (a) parallel to the as-axis
Fig. 6. Four possible structures of PPM 01 . and (b) parallel to the bs-axis.
 J. Yano, K. Sato / Food Research International 32 (1999) 249±259
Fig. 7(c). On the contrary, no remarkable changes were
observed in the absorption intensity of the bs-compo-
nent, as (CH2), in the bs-inclination. This behavior sug-
gested the two possibilities: the bs-axis is parallel to the
(001) plane, or it inclines alternately against the (001)
plane. The results obtained from RAS and ATR spectra
supported the second possibility as shown Fig. 7(d).
Since the component of a transition moment perpendi-
cular to the (001) plane is only measurable with RAS, the
perpendicular arrangement of the acyl chain exhibits no
(CH2) and r(CH2) bands. However, the bs- and as-com-
ponents of (CH2) appeared in RAS and the bs-compo-
nent was stronger in intensity than the as-component. This
indicated that both the as- and bs-axes are inclined, yet
the bs-axis is more inclined than the as-axis [Fig. 7(c),(d)].
Therefore, we infer that the unchanged intensi®es of the
bs-components shown in Fig. 7(b) are ascribed to such
an inclination behavior that the bs-axis inclines toward
the opposite directions alternately along the successive
layer [Fig. 7(d)] (Kaneko, Ishikawa, et al., 1994;
Kaneko, Shirai, et al., 1994). In this type of stacking
mode, the bs-components of (CH2) mode due to the A-
lea¯et in Fig. 7(d) become weak at  ˆ ‡30 , while they
increase at  ˆ ÿ30 . On the other hand, the bs-com-
ponents due to the B-lea¯et increase in intensity at
 ˆ ‡30 , while they decrease at  ˆ ÿ30 . Therefore, it
255
for confectionery science and technology. The primary
concerns are, as recently reviewed (Sato, 1996), precise
information of polymorphic structures, kinetics of
polymorphic crystallization, crystal morphology and
network, etc., all must be elucidated in relation to poly-
morphic forms of cocoa butter (Wille & Lutton, 1966).
The present FT±IR studies highlighted molecular-level
information of conformation and chain-chain interactions
in SOS, which are tightly related to structural stabilization
of the polymorphism of SOS (Yano et al., 1993,1999).
4.1. Acyl chain conformation and subcell packing
The changes in the acyl chain conformations during the
complex phase transitions of SOS were assessed by the
FT±IR spectra in the progression bands, polymethylene
rocking and scissoring bands, ole®nic and glycerol group
bands. The basic properties of the conformational states
of stearoyl and oleoyl groups in the ®ve polymorphs of
SOS, shown in Fig. 8, are illustrated as follows. In  and
sub- forms, stearoyl chains mostly take all-trans con-
formation, yet they contain conformational disorder to a
certain extent. The oleoyl group is conformationally dis-
ordered. There is no clear-cut conformational di€erence
between the two stearoyl chains at sn-1 and 3 position. In
the
and 0 forms, one of stearoyl chains changes into a
results in the unchanged intensity, by cancelling out, of bent conformation and the other stearoyl chain takes the
the bs (CH2) band with the alternation of  values. The all-trans form. The oleoyl group is still in a disordered
polarized ATR spectra provided the inclination angles
toward the as- and bs-axes, 82.3 and 73.4 , respectively
(Yano, Kaneko, Kobayashi, Kodali, et al., 1997). These
(ˆCÿH) at
values imply that the inclination is slightly enhanced in
the bs-axis compared with the as-axis. Assuming that the
molecular chain length is 4.3 nm [in the case of Fig. 6(c)],
we roughly estimated the inclination angle of the mole-
cular axis with respect to the (001) plane from the
results of the d(001) spacing (4.02 nm). The  value of
20.8 obtained from the XRD method satisfactorily
agrees with the results obtained from the ATR method.
Consequently, it was con®rmed that the acyl chains in
state. Finally, in the  form the oleoyl group takes an
ordered conformation, as con®rmed with a strong IR
band of ˆCÿH out-of-plane deformation
688 cmÿ1 (Yano et al., 1993). This also indicated that
both the methyl-side (the chain segment between an ole-
®nic group and a CH3 group) and glycerol-side oleoyl
chains (the segment between an ole®nic group and a gly-
cerol group) have the all-trans conformation.
The di€erentiation of the conformational changes of
the stearoyl and oleoyl chains has been made by using
duetrated SOS samples. Infrared CH2 scissoring and
CH2 rocking regions are good indicators of lateral
01 of PPM are stacked in a unidirectional manner
toward the as-axis, while they are inclined alternately
along the successive layer toward the bs-axis. However, we
could not judge the two possibilities of the stacking modes
which were proposed by previous researchers, either the
chain tilt direction alternates at the glycerol group region
(Precht & Frede, 1977), or at the methyl end terrace
(Hernqvist, 1988). The precise determination of the layer-
packing, i.e. subcell packing (Abrahamsson, Dahlen,
Lofgren & Pascher, 1978). However, the bands of oleoyl
groups overlap with those of stearoyl groups for the
usual hydrogenated specimens. To overcome this, par-
tial deuteration has been attempted. Deuterated acyl
stacking mode in the 0 forms needs further work.

4. Chain-chain interactions in polymorphic transforma-

tion of SOS

Since SOS is the major fat component of cocoa but-

ter, the polymorphism of SOS has critical implications Fig. 8. Molecular models of polymorphic transitions in SOS.
256 J. Yano, K. Sato / Food Research International 32 (1999) 249±259

groups show the CD2 scissoring (CD2) and CD2 rock-

ing r(CD2) bands around 1090 and 525 cmÿ1, instead of
the (CH2) and r(CH2) bands around 1460 and 720
cmÿ1, respectively. Here presented are the spectral
changes of the (CH2) and (CD2) regions (Figs. 9 and
10). Here, the symbols of (CH2)o and (CD2)s, mean
the (CH2) mode of hydrogenated oleoyl chains and the
(CD2) of deuterated stearoyl chains in partially deut-
erated SOS specimens.
In the  form, a single (CH2) band appeared at 1467
cmÿ1, indicating the hexagonal subcell (Fig. 9). The
(CH2) band split into two components by cooling, and
the splitting width increased as temperature decreased.
At 87 K the two components were observed at 1474 and
1463 cmÿ1. In the  form of -SOS, a single band was
observed at 1467 cmÿ1 for (CH2)o and at 1089 cmÿ1
for (CD2)s (Fig. 10), corresponding to the hexagonal
subcell. On the transformation from  to sub-, the
(CD2)s band split into two components at 1093 and
1084 cmÿ1 while no marked changes occurred in the
(CH2)o band. The splitting of the (CD2)s band is
attributed to the inter-chain vibrational coupling
between laterally adjoining predeuterated chains: the in-
phase and the out-of-phase vibrations of the two chains
(Kobayashi, 1988). Such a splitting of the (CH2) band
has been observed in many systems of long-chain mole-
cules where hydrocarbon chains form a perpendicular
Fig. 9. FT±IR (CH2) bands of hydrogenated-SOS. No-polarized
type subcell to a certain extent. We inferred that the spectra were taken for the sub- and  forms. For the
and 0 forms,
orientational changes of stearoyl chains containing polarized spectra were taken by the well-oriented specimens. For the 
somewhat conformational disorders from the hexagonal form, spectra were taken by the single crystal.
to the pseudo-hexagonal packing take place on the 
and sub- transition.
The previous study by powder X-ray di€raction
method revealed that the
form takes a trilayer struc-
ture, which means that the stearoyl and oleoyl moieties
make their own lea¯et (Sato et al., 1989). The (CH2)
band had two components (Fig. 9) for h-SOS; a strong
band at 1472 cmÿ1 and the other weak band at 1467
cmÿ1. Since the former band disappeared in the polar-
ized spectra of d-SOS, we assigned this band to the
stearoyl scissoring mode and the latter band to the
oleoyl scissoring mode (Fig. 10). A sharp single band of
(CD2)s appeared at 1092 cmÿ1, while a broad band of
(CH2)o. was observed at 1468 cmÿ1. It is suggested that
the stearoyl groups form a parallel packing and the
oleoyl moiety packs in the hexagonal subcell.
In the 0 form, the (CH2) band had two components,
1473 and 1465 cmÿ1 (Fig. 9) and the (CD2)s, mode split
into two components at 1091 and 1088 cmÿ1 (Fig. 10).
Although the polarization directions of the (CD2)s
bands were not clear due to the overlapping of other
modes, they showed the following characteristics: the
bands at 1092 and 1086 cmÿ1 became maximum at the 0
and 90 polarization directions, respectively. These
spectral data indicate the O? subcell of the stearoyl
lea¯ets. This splitting width of the (CD2)s bands (6.5
Fig. 10. FT±IR (CH2) and d(CD2)s bands of deuterated-SOS. The
spectra of each polymorph were measured under the same conditions
stated in Fig. 9.
cmÿ1) was smaller than that of the typical O? subcell
(7.8 cmÿ1) of deuterated n-alkanes, suggesting that the
packing of the stearoyl chains are not so tight as that of
n-alkanes (Snyder, Goh, Srivatsavoy, Strauss & Dorset,
1992). The glycerol backbone probably hinders stearoyl
 J. Yano, K. Sato / Food Research International 32 (1999) 249±259 257

groups being packed tightly. As to the oleoyl lea¯et, the having trilayer structure. The molecular conformation
(CH2)o mode became slightly sharper in 0 than in
, of the stearoyl chains in
takes the same ordered con-
but its frequency was still 1468 cmÿ1, suggesting a lat- formation as those of more stable 0 and  forms in
eral packing similar to the hexagonal subcell in . which one chain extends to form the all-trans conforma-
In the  form of h-SOS, the single (CH2) band tion, whereas the other takes a bent conformation in the
appeared at 1470 cmÿ1 with a clear polarization nearly neighborhood of the ester bond. These stearoyl chains
parallel to the b-axis (Fig. 9); maximum at  ˆ 70 and take a parallel type subcell in
and change to O? sub-
minimum at  ˆ 160 . The (CH2)o and (CD2)s bands cell in 0 , but the oleoyl lea¯et maintains the hexagonal
of d-SOS were also observed as single bands at 1471 and packing in
and 0 . Although the subcell structure is
1092 cmÿ1 with a clear polarization (Fig. 10). The the same in  through 0 , the molecular conformations
(CH2)o bands appeared at a higher frequency than in of the oleoyl chain are di€erent, judging from the spec-
the 0 form. These results suggested that both the stear- tral features of the oleoyl progression bands in the
oyl and oleoyl moieties were packed in T== subcell. 1350±1150 cmÿ1 region. In addition, the NMR studies
The FT±IR analyses using the d-SOS samples also high- showed that the methyl-side and the glycerol-side of the
lighted the molecular aspects of the reversible sub- and  oleoyl chain in SOS are not conformationally equivalent
transition occurring in the bilayered structure, where the in
and 0 unlike in  (Arishima, Sugimoto, Kiwata,
oleoyl and stearoyl chains are packed in the same lea¯ets Mori & Sato, 1996). We infer that the gauche bonds in
(Yano et al., 1999). Although not represented here, it was the oleoyl chain are concentrated in the vicinity of the
found that the orientational ordering of the stearoyl chains methyl-side chain in the
and 0 phases and are rela-
with decreasing temperature in the sub  form results in the tively delocated in the  and sub  phases.
reduction of the entropy factor, and thereby the enantio- The structural di€erence of the oleoyl chain between

tropic transformation occurs between  and sub . Such and 0 is probably due to the di€erence in the con-
enantiotropic transitions observed in the higher entropy
phases may be due to the characteristics common to the
highly disordered hydrocarbon chain assemblies such as the
rotator phases of n-alkanes (Doucet, Denicolo & Craie-
vich, 1981; Sirota, King, Singer & Shao, 1993; Snyder,
Maroncelli & Strauss, 1983; Ungar, 1983). From the
biological view point, it is interesting that the ¯uctua-
tion of the oleoyl chains is retained even at lower tem-
peratures due to the steric hindrance of the ole®nic
bond. This must be the important character of unsatu-
rated acyl moieties to maintain the ¯uidity in biomem-
brane lipids (Kaneko et al., 1998).
4.2. Transition properties from a through b forms
The layered structures and the subcell packing of the
stearoyl and oleoyl moieties in the polymorphic forms
of SOS are summarized in Table 2.
In SOS, the  to
transition is initiated by the stabi- subcell structure. The interfacial energy of the stable
lization of the stearoyl moieties. Correspondingly, the  polymorph is larger than those of the least stable or
form having bilayer structure transforms to the
form metastable forms. Namely, the crystallization rates are
Table 2
Chain-length structures and subcell packing of SOS polmorphs
Polymorphic Chain-length Subcell
form structure structure
Stearoyl-lea¯et Oleoyl-lea¯et
centration of gauche bonds. Finally, the 0 to  transi-
tion corresponds to the stabilization of the oleoyl lea¯et
where the methyl-side and glycerol-side chains take an
extended structure with the skew-cis-skew' ole®nic con-
formation (Yano et al., 1993).
The present ®ndings provided molecular level infor-
mation of steric hindrance between the saturated and
unsaturated acyl chain in fats and lipids having layered
structures. This steric hindrance results in the occur-
rence of various metastable phases and complicated
polymorphic transformations. In the TAG polymorph-
ism having the monotropic characteristics, the transi-
tion from the least stable form through to the most
stable one can be regarded as a process of the crystal
growth in a solid state. The saturated acyl chains trans-
form to a stable molecular conformation at the ®rst
stage of polymorphism and then the unsaturated moi-
eties are stabilized successively with the changes of the
always higher in the less stable forms and the formation
of the stable molecular conformation from melt is steri-
cally hindered.
5. Summary

 Double 4.83 (nm) Hexagonal In this paper, we focused on the usefulness of the

Triple 7.05 //-type Hexagonal vibrational spectroscopy for the structural study of
0 Triple 7.00 O? Hexagonal TAG crystals, especially for the metastable phases and
…2 †a Triple 6.75 T// T// or O0 //
tiny crystals which cannot be used for the X-ray struc-
1 Triple 6.60 T// T//
tural analysis. In the ®eld of the physicochemical studies
a
The results from Yano et al., 1993. of fats, further studies will be needed to resolve the
258 J. Yano, K. Sato / Food Research International 32 (1999) 249±259
problems: (1) the molecular structure of the inter-mole-
cular compounds which are formed in the binary sys-
tems of saturated±unsaturated mixed acid TAGs, and
(2) time-resolved feature of the crystallization and tran-
sition mechanisms governing the occurrence of di€erent
polymorphs.
Acknowledgements
The authors are indebted to Dr. F. Kaneko, Osaka
University, Dr. S. Ueno, Hiroshima University, Prof.
D. M. Small, Boston University and Dr. D. R. Kodali,
Cargill Research for their cooperative work and valuable
discussion. Thanks are also expressed to Prof. A. Mar-
angoni, University of Guelph for inviting us to submit this
paper to the present Journal.
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