ft-ir studies on polymorphism of fats - molecular structures and interactions

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

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 fatand 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 theglycerol group and the inclination of the acyl chains against the O? subcell axes and the lamellar plane were observed. In ®vepolymorphs 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 andTechnology. Published by Elsevier Science Ltd. All rights reserved.

Keywords: FT±IR; Fatty acids; Fats; Ole®nic conformation; Polymorphism; Triacylglycerols

1. Introduction

Triacylglycerols (TAGs) are the main constituents offats present in foods, pharmaceuticals, cosmetics, and soon. Physical properties of TAGs determine key processesin the processing of fat products and fractionation of fatsand oils (Dickinson & McClements, 1995; Formo, 1979;Precht, 1988; Sato, 1996). In analogy with other lipidsand long chain compounds, TAGs show complicatedpolymorphism (Hagemann, 1988; Small, 1986). Dependingon the environmental conditions (temperature, pressure,solvent, etc.) as well as thermal histories they have passedthrough, various crystalline states occur, resulting in var-ious functional physical properties of TAGs, in combi-nation with crystallization behavior and crystal particlesaggregation.The polymorphic properties of TAGs depend on the

fatty acid compositions: e.g. saturated or unsaturatedacids, 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

are called ``mono-acid TAGs'' and those having morethan two types of chains ``mixed-acid TAGs''. Naturaloils and fats contain many kinds of mixed-acid TAGs.The physical properties of the mixed acid TAGs are morecomplicated than those of the mono-acid TAGs, in par-ticular when the acyl chains at sn-1 and sn-2 positions aredi�erent, and when a TAG molecule consists of unsatu-rated acyl chains.The basic polymorphs of TAGs are called �; �0 and

� (Larsson, 1966; Lutton, 1950; Malkin, 1954), whichare identi®ed by subcell structure (Fig. 1). The � formis the most stable and packed in T==; �

0 less stablepacked in O?, and � with hexagonal subcell is theleast stable form. Depending on the acyl chain com-position, other metastable phases and multiple �0 and� forms are observed in mixed acid TAGs. As tothe structural properties, the crystal structure hasbeen 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 toquestion as noted in the following: (i) the structures ofmeta-stable phases, in particular for �0 forms, (ii) thestructures of saturated±unsaturated mixed acid TAGs,and (iii) the structures of mixture phases of di�erentTAGs.

0963-9969/99/$20.00 # 1999 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.

PI I : S0963-9969(99 )00079-4

Food Research International 32 (1999) 249±259

www.elsevier.com/locate/foodres

* Corresponding author. Tel.: +81-824-24-7935; fax: +81-824-22-

7062.

E-mail address: [email protected] (K. Sato)

As for the �0 structures, this polymorph is usuallymost functional in fat products, due to its better crys-tallinity having a tendency to make a minute crystallinenetwork with thin needle-shape morphology, whichgives proper softness in the fat products (Precht, 1988).Therefore, the �0 ! � transition often results indeterioration of the end products. The �0 form is knownto pack in the O? subcell, yet the molecular conforma-tion of the acyl chains and the glycerol group, and theoverall conformation are still a controversial matter(Hernqvist, 1988).Concerning the mixed-acid TAGs involving the unsa-

turated fatty acid moieties, serious complexity in thepolymorphism arises from the heterogeneous interac-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 setsof saturated-unsaturated mixed acid TAGs, severalresearchers have reported the formation of molecularcompounds due to chain-chain interactions (Engstrom,1992, Kaneko, Yano & Sato, 1998; Koyano, Hachiya &Sato, 1992,Minato, Ueno, Smith, Amemiya & Sato, 1997,Minato, Ueno, Yano et al., 1997, Rossel, 1967). Althoughthe thermodynamic properties and structural character-istics of the mixed-acid TAGs have been studied, theproposed structural models contain many ambiguitieson the conformation of saturated and unsaturated acylchains 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

recent development of FT±IR spectrometers, it has beenpossible to obtain IR spectra from a small domain ofseveral tens of mms in size, and one can measure IRspectra under various conditions of specimens with dif-fuse re¯ection spectroscopy, attenuated total re¯ectionspectroscopy, re¯ection-absorption spectroscopy, andso on. The IR data thus corrected were more informa-tive compared to those obtained by conventional IRmethods (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(SOS). PPM is a �0-stable fat and SOS is the majorcomponent of cocoa butter. The stearoyl chains in SOSwere fully deuterated, so that the FT±IR spectra of theoleoyl and stearoyl chains were di�erentiated. After thedescription of the new FT±IR techniques, the details ofthe molecular conformation and chain inclination of the�0 form of PPM and the chain-chain interactionsbetween the stearoyl and oleoyl acids of SOS poly-morphs will be presented.

2. Experimental section

2.1. Sample preparation

Fig. 2 shows the polymorphism of PPM (Kodali,Atkinson, & Small, 1990) and SOS (Sato et al., 1989),where the most stable �01, of PPM, and the all forms ofsub-� through �1 forms of SOS were examined.PPM was synthesized according to the method

described previously (Kodali, Atkinson, Redgrave &Small, 1984). Single-crystal specimens of �01 of PPMwere crystallized from acetonitrile solutions by slowcooling. As to SOS, two samples were prepared.Hydrogenated-SOS (h-SOS) was provided by Fuji OilCo. Ltd. Deuterated-SOS (d-SOS) was supplied fromD.R, Kodali, who synthesized it by adapting the meth-ods described in the literature (Bentley & McCrae, 1970;Kodali et al., 1987). Since the �2-form crystal was notobtained for d-SOS, we focused on the most stable �1form of SOS, which is described as � in this text. Singlecrystals of the b-form were grown from an acetonitrilesolution by slow cooling.

Fig. 1. Typical subcell structure of TAG polymorphs.

Fig. 2. Polymorphic transformations in PPM and SOS.

250 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±Elmeri-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 MCTdetector and a wire-grid polarizer (Perkin±ElmerPR500). The spectra without mention of the conditionswere taken at room temperature. To determine thedirections of transition moments three-dimensionally,we employed the three methods as elaborated in thefollowing.

2.2.1. Special methods

2.2.1.1. Polarized IR transmission methods. Normalincident transmission spectra and oblique transmissionspectra were taken in the present study (Kaneko, Shirai,Miyamoto, Kobayashi & Suzuki, 1994; Kaneko, Ishi-kawa, Kobayayashi & Suzuki, 1994). As for the formercase [Fig. 3(a)], the direction of IR radiation was nor-mal to the (001) plane of the crystal. The direction ofthe electric ®eld of the incident radiation was ®xed, andthe sample was rotated about the normal of the (001)plane. In this study, we assumed that the b-axis of thecrystals is parallel to the 90� polarization direction. In this

case, the information deduced from the polarizationdependence of the IR spectra concerns the projection ofthe transition moments onto the (001) plane.The oblique transmission method [Fig. 3(b)] has been

developed to overcome the above limit of the normalincident method (Kaneko, Shirai et al., 1994; Kaneko,Miyamoto & Kobayashi, 1996). The specimen was set ata suitable position for the oblique measurement (per-pendicular or parallel to the subcell axis) and then tiltedby an angle of �. The spectra measured at � � �30� andÿ30� were compared with the spectrum at � � 0� toobtain the bands having the transition moments per-pendicular to the (001) plane.

2.2.1.2. Attenuated total reflection (ATR) method. Thetransmission method cannot be applied for thick speci-mens or strong absorption bands. In such a case, theATR method is useful (Kaneko et al., 1996). The ¯at(001) plane of specimens was set on the sampling planeof an internal re¯ection element (IRE) of ZnSe or Gewhose wedge angle was 45�, and re¯ection spectra weremeasured with p- or s-polarized incident radiation asshown in Fig. 3(c). The p-polarized incident radiationhas the vibrational electric ®eld perpendicular to thebasal plane of IRE, whereas the electric ®eld of the s-polarized one is parallel to it. With p-polarization, thex- and z-components of a transition moment areobserved, and the y-component with s-polarization. Inthe above conditions the z-component, which cannot beobserved 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 informationcan be obtained (Fraser, 1958a, 1958b; Higashiyama &Takenaka, 1974). The analysis was done according tothe 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 metalsurface 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 ametal surface as a thin ®lm where the lamellar interfaceis parallel to the metal surface by developing chloro-form solution. Accordingly, the spectral bands whosetransition moments are parallel to the chain axis areenhanced.

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 acidTAGs, the information about the IR bands of mono-acid TAGs are important as references. Therefore, theassignments of the �3 progression bands in the region of

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).

J. Yano, K. Sato / Food Research International 32 (1999) 249±259 251

1380±1150 cmÿ1, which sensitively re¯ect the stemlength of the trans zigzag chain, has beenmade for � formsof three TAGs; tristearin (SSS), tripalmitin (PPP), and tri-myristin (MMM), as summarized shortly (Yano, Kaneko,Kobayashi, & Sato, 1997). Two series of progressionbands overlap in this region, one is due to the �3 branchmodes (CH2 wagging: 1300±1150 cmÿ1) and the otherdue to the �7 branch modes (CH2 twisting-rocking:1300±1150 cmÿ1). The �3 progression bands were iso-lated clearly, and could be used as reference bands toanalyze the conformational state of the acyl chains ofPPM and SOS.

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 frequenciesof bands and subcell packing are listed in Table 1. In thecrystal having the O? subcell, the CH2 scissoring androcking modes split into two components, due to theintermolecular interaction of the two chains in the unitcell (Kobayashi, 1988; Tasumi & Krimm, 1967; Tasumi& Shimanouchi, 1965). One component is polarizedalong the as-axis, and the other one along the bs-axis ofthe O? subcell. The splitting width of two componentsdepends on the intermolecular force constant. By contrast,

these modes appear as single bands in the crystal havingthe T== subcell, in which polymethylene chains are par-allel to each other.

3. Structural analysis of PPM �01 form

3.1. Crystal morphology and subcell orientation

The shape of the �01 crystal is a parallelogram whoseacute angle is 57�. The � (CH2) and r(CH2) bands of theO? subcell show clear dichroism for the single crystalsof �01 (Fig. 4) (Kobayashi, 1988; Tasumi & Krimm,1967; Tasumi & Shimanouchi, 1965), where the crystalmorphology 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, appearmost intense at � � 0� and vanish at � � 90�, while theintensi®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

modes

Polymethylene

chain

as Component bs Component

�2��(CH2 scissoring)

O?

�8��(CH2 rocking)

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

CH2 rocking regions.


� � 90� and minimum at � � 0� (see, Table 1). There-fore, we can conclude that the projections of the as- andbs-axes of the O-? subcell are almost perpendicular andparallel 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 chainstake a chain conformation with one chain pointing inone direction opposite to the others, two possible acylchain conformations are considered: (a) the two palmi-toyl chains have the straight (all-trans) conformationand the myristoyl chain has the bent conformation inwhich one gauche bond is inserted near the ester bond,or (b) one palmitoyl and one myristoyl chain have theall-trans conformation and the second palmitoyl chainhas the bent conformation. Using the �3 progressionbands appearing in the 1350±1150 cmÿ1 region, weexamined 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, theconformation (b) may consist of the bands from straightpalmitoyl and myristoyl chains and one bent palmitoylchain. The �3 bands of the straight chains mainly appearin RAS due to the transition dipole moment parallel tothe chain axis. On the other hand, those of the bentchains, which involve a perpendicular component to thechain axis due to the bent conformation, stronglyappear in transmission spectra. Taking into account thedi�erence in the polarization direction of the �3 bandsfrom the two types of chains, we assigned the �3 bandsof �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 thespecimen for RAS, the lamellar interface is parallel tothe metal surface, and thereby the chain axes are almostnormal to it. Accordingly, the bands whose transitionmoments are parallel to the chain axis are emphasizedwith 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 transmissionspectra correspond to the bands observed in RAS,meaning that the transition moments of these bandsmainly involve the components parallel to the chainaxis. Therefore, these bands were assigned to the �3progression bands due to the palmitoyl and/or myristoylchain. In order to distinguish the contributions from thetwo chains, the frequencies of RAS bands were com-pared with those of the � forms of PPP and MMM [Fig.5(B)]. The bands marked with closed circles of PPP and

Fig. 5. Assignments of the �3 progression bands of PPM �01: (A) nor-

mal incident transmission method and RAS, (B) RAS of PPM �01, PPP�, and MMM �, and (C) polarized transmission spectra of PPM �01,PPP �, and MMM �.


MMM arise from the �3 bands of the straight palmitoyland myristoyl chains. It is noted that the bands in �01totally correspond to the �3 bands of the straight chainsof PPP and MMM.As for the composition of the bent chain, Fig. 5(C)

shows the comparison of the transmission spectra ofPPM �01 with those of MMM and PPP, connected bythe dotted lines. No band series of the bent conforma-tion of the myristoyl moieties, whose band positions areindicated by the broken lines, was observed in the �01form of PPM. For example, the strongest �3 band of thebent myristoyl chain at 1276 cmÿ1 was not observed inthe spectra of PPM �01.Thus, the preferred acyl chain conformation of the

PPM �01 form was assumed in the following: onestraight palmitoyl and one straight myristoyl chain, andone bent palmitoyl chain. Then the next problem arises;which chain, myristoyl or palmitoyl, is adjacent to thebent palmitoyl chain and which glycerol con®guration,sn-1,2 (or sn-2,3) or sn-1,3 con®guration, is most con-ceivable, as exhibited in four possible con®gurationsshown in Fig. 6.Detailed band assignments of the carbonyl stretch

bands [�(C�O)] may be most determinative to solve thisproblem, since the frequencies and polarization direc-tions of these bands are directly related to the glycerolcon®guration. A comparison of the PPM �01 with thoseof 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 �(C�O) bands are very sensitive to theglycerol conformation, these two forms have commoncharacteristics in the polarization direction and theband frequencies. In the mono-acid TAGs, the �0 formtransforms to the � form in a solid state, and the gly-cerol con®guration of � takes the sn-1,2 con®gurationhaving the glycerol structure of type (c) in Fig. 6, asshown by the X-ray crystal analysis (Jensen & Mabis,1966; Larsson, 1964). Therefore, it is reasonable toassume that the same con®guration may also be appliedto the present case of �01 of PPM. Consequently, type (c)in Fig. 6 is the most plausible glycerol con®guration for

�01 of PPM. The complete decision, however, of the fourpossible structure displayed in Fig. 6 must await X-raystructural determination.

3.3. Chain inclination behavior

The chain inclination behavior in the �0 forms ofTAGs has been of high interest, since it is related to the�0 ÿ � transition mechanism (Hernqvist, 1988; Precht &Frede, 1977). The present FT±IR studies provided theinformation of the inclination direction of the acyl chainwith respect to the subcell axes and the lamellar inter-face in �01 of PPM, using the oblique transmission, RAS,and ATR methods. Since the subcell orientation isdirectly related to the inclination direction of the acyl-chains, �(CH2) and r(CH2) bands having the transitiondipole moments perpendicular to the molecular chainaxis were employed as the reference bands of the chaininclination direction.Fig. 7 shows the oblique transmission spectra of the

�01 of PPM. In Fig. 7(a), the electric vector of the inci-dent radiation and the as-axis of the O? subcell are inthe same plane, and the angle between the as-axis andthe electric vector is varied by tilting the specimen (as-inclination). By contrast, the spectra in Fig. 7(b) weretaken with the inclination in the plane involving the bs-axis and the electric vector (bs-inclination). With varia-tion of � values in the as-inclination, the intensity of�as�CH2� band signi®cantly changed, being strongest at� � 0�. It is notable that an increase in the changes ofthe band intensity due to the inclination angles of +30�

and ÿ30� are not symmetric, namely the former is largerthan the latter. This implies that the as-axis is slightlyinclined with respect to the (001) plane as depicted in

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

and (b) parallel to the bs-axis.Fig. 6. Four possible structures of PPM �01.


Fig. 7(c). On the contrary, no remarkable changes wereobserved 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 spectrasupported 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, theperpendicular 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. Thisindicated that both the as- and bs-axes are inclined, yetthe bs-axis is more inclined than the as-axis [Fig. 7(c),(d)].Therefore, we infer that the unchanged intensi®es of thebs-components shown in Fig. 7(b) are ascribed to suchan inclination behavior that the bs-axis inclines towardthe opposite directions alternately along the successivelayer [Fig. 7(d)] (Kaneko, Ishikawa, et al., 1994;Kaneko, Shirai, et al., 1994). In this type of stackingmode, the bs-components of �(CH2) mode due to the A-lea¯et in Fig. 7(d) become weak at � � �30�, while theyincrease at � � ÿ30�. On the other hand, the bs-com-ponents due to the B-lea¯et increase in intensity at� � �30�, while they decrease at � � ÿ30�. Therefore, itresults in the unchanged intensity, by cancelling out, ofthe �bs(CH2) band with the alternation of � values. Thepolarized ATR spectra provided the inclination anglestoward the as- and bs-axes, 82.3 and 73.4�, respectively(Yano, Kaneko, Kobayashi, Kodali, et al., 1997). Thesevalues imply that the inclination is slightly enhanced inthe bs-axis compared with the as-axis. Assuming that themolecular 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 theresults of the d(001) spacing (4.02 nm). The � value of20.8� obtained from the XRD method satisfactorilyagrees with the results obtained from the ATR method.Consequently, it was con®rmed that the acyl chains in

�01 of PPM are stacked in a unidirectional mannertoward the as-axis, while they are inclined alternatelyalong the successive layer toward the bs-axis. However, wecould not judge the two possibilities of the stacking modeswhich were proposed by previous researchers, either thechain 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-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

for confectionery science and technology. The primaryconcerns are, as recently reviewed (Sato, 1996), preciseinformation of polymorphic structures, kinetics ofpolymorphic crystallization, crystal morphology andnetwork, etc., all must be elucidated in relation to poly-morphic forms of cocoa butter (Wille & Lutton, 1966).The present FT±IR studies highlighted molecular-levelinformation of conformation and chain-chain interactionsin SOS, which are tightly related to structural stabilizationof 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 thecomplex phase transitions of SOS were assessed by theFT±IR spectra in the progression bands, polymethylenerocking and scissoring bands, ole®nic and glycerol groupbands. The basic properties of the conformational statesof stearoyl and oleoyl groups in the ®ve polymorphs ofSOS, shown in Fig. 8, are illustrated as follows. In � andsub-� forms, stearoyl chains mostly take all-trans con-formation, yet they contain conformational disorder to acertain extent. The oleoyl group is conformationally dis-ordered. There is no clear-cut conformational di�erencebetween the two stearoyl chains at sn-1 and 3 position. Inthe and �0 forms, one of stearoyl chains changes into abent conformation and the other stearoyl chain takes theall-trans form. The oleoyl group is still in a disorderedstate. Finally, in the � form the oleoyl group takes anordered conformation, as con®rmed with a strong IRband of �CÿH out-of-plane deformation (�CÿH) at688 cmÿ1 (Yano et al., 1993). This also indicated thatboth the methyl-side (the chain segment between an ole-®nic group and a CH3 group) and glycerol-side oleoylchains (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 usingduetrated SOS samples. Infrared CH2 scissoring andCH2 rocking regions are good indicators of lateralpacking, i.e. subcell packing (Abrahamsson, Dahlen,Lofgren & Pascher, 1978). However, the bands of oleoylgroups overlap with those of stearoyl groups for theusual hydrogenated specimens. To overcome this, par-tial deuteration has been attempted. Deuterated acyl

Fig. 8. Molecular models of polymorphic transitions in SOS.


groups show the CD2 scissoring �(CD2) and CD2 rock-ing r(CD2) bands around 1090 and 525 cmÿ1, instead ofthe �(CH2) and r(CH2) bands around 1460 and 720cmÿ1, respectively. Here presented are the spectralchanges of the �(CH2) and �(CD2) regions (Figs. 9 and10). Here, the symbols of �(CH2)o and �(CD2)s, meanthe �(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, andthe splitting width increased as temperature decreased.At 87 K the two components were observed at 1474 and1463 cmÿ1. In the � form of �-SOS, a single band wasobserved at 1467 cmÿ1 for �(CH2)o and at 1089 cmÿ1

for �(CD2)s (Fig. 10), corresponding to the hexagonalsubcell. On the transformation from � to sub-�, the�(CD2)s band split into two components at 1093 and1084 cmÿ1 while no marked changes occurred in the�(CH2)o band. The splitting of the �(CD2)s band isattributed to the inter-chain vibrational couplingbetween 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) bandhas been observed in many systems of long-chain mole-cules where hydrocarbon chains form a perpendiculartype subcell to a certain extent. We inferred that theorientational changes of stearoyl chains containingsomewhat conformational disorders from the hexagonalto 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 moietiesmake their own lea¯et (Sato et al., 1989). The �(CH2)band had two components (Fig. 9) for h-SOS; a strongband at 1472 cmÿ1 and the other weak band at 1467cmÿ1. Since the former band disappeared in the polar-ized spectra of d-SOS, we assigned this band to thestearoyl scissoring mode and the latter band to theoleoyl 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 thatthe stearoyl groups form a parallel packing and theoleoyl 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 splitinto two components at 1091 and 1088 cmÿ1 (Fig. 10).Although the polarization directions of the �(CD2)sbands were not clear due to the overlapping of othermodes, they showed the following characteristics: thebands at 1092 and 1086 cmÿ1 became maximum at the 0and 90� polarization directions, respectively. Thesespectral data indicate the O? subcell of the stearoyllea¯ets. This splitting width of the �(CD2)s bands (6.5

cmÿ1) was smaller than that of the typical O? subcell(7.8 cmÿ1) of deuterated n-alkanes, suggesting that thepacking of the stearoyl chains are not so tight as that ofn-alkanes (Snyder, Goh, Srivatsavoy, Strauss & Dorset,1992). The glycerol backbone probably hinders stearoyl

Fig. 9. FT±IR �(CH2) bands of hydrogenated-SOS. No-polarized

spectra were taken for the sub-� and � forms. For the and �0 forms,

polarized spectra were taken by the well-oriented specimens. For the �form, spectra were taken by the single crystal.

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.


groups being packed tightly. As to the oleoyl lea¯et, the�(CH2)o mode became slightly sharper in �0 than in ,but its frequency was still 1468 cmÿ1, suggesting a lat-eral packing similar to the hexagonal subcell in �.In the � form of h-SOS, the single �(CH2) band

appeared at 1470 cmÿ1 with a clear polarization nearlyparallel to the b-axis (Fig. 9); maximum at � � 70� andminimum at � � 160�. The �(CH2)o and �(CD2)s bandsof d-SOS were also observed as single bands at 1471 and1092 cmÿ1 with a clear polarization (Fig. 10). The�(CH2)o bands appeared at a higher frequency than inthe �0 form. These results suggested that both the stear-oyl and oleoyl moieties were packed in T== subcell.The FT±IR analyses using the d-SOS samples also high-

lighted the molecular aspects of the reversible sub-� and �transition occurring in the bilayered structure, where theoleoyl and stearoyl chains are packed in the same lea¯ets(Yano et al., 1999). Although not represented here, it wasfound that the orientational ordering of the stearoyl chainswith decreasing temperature in the sub � form results in thereduction of the entropy factor, and thereby the enantio-tropic transformation occurs between � and sub �. Suchenantiotropic transitions observed in the higher entropyphases may be due to the characteristics common to thehighly disordered hydrocarbon chain assemblies such as therotator phases of n-alkanes (Doucet, Denicolo & Craie-vich, 1981; Sirota, King, Singer & Shao, 1993; Snyder,Maroncelli & Strauss, 1983; Ungar, 1983). From thebiological 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®nicbond. 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 thestearoyl and oleoyl moieties in the polymorphic formsof SOS are summarized in Table 2.In SOS, the � to transition is initiated by the stabi-

lization of the stearoyl moieties. Correspondingly, the �form having bilayer structure transforms to the form

having trilayer structure. The molecular conformationof the stearoyl chains in takes the same ordered con-formation as those of more stable �0 and � forms inwhich one chain extends to form the all-trans conforma-tion, whereas the other takes a bent conformation in theneighborhood of the ester bond. These stearoyl chainstake a parallel type subcell in and change to O? sub-cell in �0, but the oleoyl lea¯et maintains the hexagonalpacking in and �0. Although the subcell structure isthe same in � through �0, the molecular conformationsof the oleoyl chain are di�erent, judging from the spec-tral features of the oleoyl progression bands in the1350±1150 cmÿ1 region. In addition, the NMR studiesshowed that the methyl-side and the glycerol-side of theoleoyl chain in SOS are not conformationally equivalentin and �0 unlike in � (Arishima, Sugimoto, Kiwata,Mori & Sato, 1996). We infer that the gauche bonds inthe oleoyl chain are concentrated in the vicinity of themethyl-side chain in the and �0 phases and are rela-tively delocated in the � and sub � phases.The structural di�erence of the oleoyl chain between

and �0 is probably due to the di�erence in the con-centration of gauche bonds. Finally, the �0 to � transi-tion corresponds to the stabilization of the oleoyl lea¯etwhere the methyl-side and glycerol-side chains take anextended 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 andunsaturated acyl chain in fats and lipids having layeredstructures. This steric hindrance results in the occur-rence of various metastable phases and complicatedpolymorphic transformations. In the TAG polymorph-ism having the monotropic characteristics, the transi-tion from the least stable form through to the moststable one can be regarded as a process of the crystalgrowth in a solid state. The saturated acyl chains trans-form to a stable molecular conformation at the ®rststage of polymorphism and then the unsaturated moi-eties are stabilized successively with the changes of thesubcell structure. The interfacial energy of the stablepolymorph is larger than those of the least stable ormetastable forms. Namely, the crystallization rates arealways higher in the less stable forms and the formationof the stable molecular conformation from melt is steri-cally hindered.

5. Summary

In this paper, we focused on the usefulness of thevibrational spectroscopy for the structural study ofTAG crystals, especially for the metastable phases andtiny crystals which cannot be used for the X-ray struc-tural analysis. In the ®eld of the physicochemical studiesof fats, further studies will be needed to resolve the

Table 2

Chain-length structures and subcell packing of SOS polmorphs

Polymorphic

form

Chain-length

structure

Subcell

structure

Stearoyl-lea¯et Oleoyl-lea¯et

� Double 4.83 (nm) Hexagonal

Triple 7.05 //-type Hexagonal

�0 Triple 7.00 O? Hexagonal

��2�a Triple 6.75 T// T// or O0//

�1 Triple 6.60 T// T//

a The results from Yano et al., 1993.


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�erentpolymorphs.

Acknowledgements

The authors are indebted to Dr. F. Kaneko, OsakaUniversity, Dr. S. Ueno, Hiroshima University, Prof.D. M. Small, Boston University and Dr. D. R. Kodali,Cargill Research for their cooperative work and valuablediscussion. Thanks are also expressed to Prof. A. Mar-angoni, University of Guelph for inviting us to submit thispaper to the present Journal.

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