J. Phys. Chem. 1984, 88, 6333-6340
6333
Molecular Structure and Packing of 1,2-sn-Dipalmitoylglycerol from Single Crystal Raman Spectra and Some New Features of the Spectra Norman Albon* and J. Franfois Baret Dspartement de Physique des Liquides, Uniuersite de Prouence, 13331 Marseille Cedex 3, France (Received: April 25, 1984; In Final Form: June 29, 1984)
The 1,2-sn-dipalmitoylglycerolmolecule is the largest part of the most common natural phospholipids and thus of major interest to the biological and physical sciences. Excellent single crystals gave high-resolution X-ray diffraction data from which the chain subcell packing was readily determined. However, a full crystal structure solution proved difficult because of slow refinement of chain structures since several different models were possible. The Raman spectra gave clear indications of the molecular folding from carbonyl stretching and longitudinal acoustical bands. Further, single-crystal spectra showed striking variations with orientation and new features of the methyl group spectra which demonstrate explicitly the influence of molecular environment on Raman scattering. The alignment of the methyl groups was established from the carbon-hydrogen stretching modes. This gave the molecular alignment in the unit cell and an unique detailed model for final refinement. Similar methods will probably be valuable for single-crystalstructural studies on other lipids and to clarify the interpretation of the spectra of liquid crystals, bilayers, and biological membrane systems.
Introduction X-ray diffraction of single crystals has been of immense value for the physical and biological sciences. However, severe experimental problems are often encountered in the preparaton of suitable single crystals, solution of the structures, and refinement of the diffration data. All these problems are acute in the study of amphiphilic compounds having polar groups attached to hydrocarbon chains. Such compounds form a variety of two- and three-dimensional structures, of interest to the physicist, many of which have important biological functions. Since the structural information from diffraction studies in the absence of single crystals is limited, a study of single-crystal phases was undertaken. Excellent single crystals of phases of several amphiphilic compounds were grown’ and examined by X-ray diffraction and other method^.^-^ As phospholipid phase systems are very ~ o m p l e x , ~ 1,2-sn-dipalmitoylglycerol(DPG) was included in these studies; this molecule comprises the hydrophobic part of natural phospholipids together with carboxy ester and hydroxyl groups. Thus, it is both amphiphilic and of major biological significance. DPG provides a system in which the effects of time and temperature on phase structures can be studied in the absence of water.2 Large single crystals of highly purified DPG were obtained.’ X-ray diffraction data were collected from samples cleaved from larger DPG crystak6 Of 3976 independent reflections scanned, 3494 (87.9%) were observed, even though very strong subcell reflections are present; the crystallographic data are given in Table Ia. The subcell structure was solved with an R factor of 12.8% after refinement of the single carbon and two hydrogen atoms. In DPG crystals, there is an orthorhombic “perpendicular” subcell packing of space group, Pbnm, as detailed in Table Ib. Both subcell orientation and Patterson synthesis show that the axes of the chains are parallel to the a-c plane and tilted at 30.55’ to the c axis. Thus, b (main cell) and b, (subcell) are coincident. The presence of a pseudo-twofold axis along b obscures other details of the Patterson. Despite knowledge of the subcell, several alternative structures are possible. Each would require prolonged refinement before there were definite indications as to whether a particular model was correct. Similar difficulties have been encountered for many hydrocarbon chain structures and these were (1) N. Albon, J . Cryst. Growth, 35, 105 (1976). (2) A. F. Craievich, A. M. Levelut, M. Lambert, and N. Albon, J. Phys., 39, 377 (1978). (3) N. Albon, J . Chem. Phys., 78,4676 (1983); 79, 469 (1983). (4) N. Albon and J. M. Sturtevant, Proc. Natl. Acad. Sci. U.S.A.,75, 2258 (1978). ( 5 ) E. Mushayakarara, N. Albon, and I. W. Levin, Biochim. Biophys. Acta, 686, 153 (1982). (6) N. Albon, J. Mcalister, and M. Sundaralingam, Am. Crystallogr. Assoc. Abstr. 2, 5, 69 (1977).
TABLE I: Crystallographic Data for 1,2-sn-Dipalmitoylglycerol (DPG) (a) Main Cell space group p 21
molecular formula crystal size dm, g/cm 4 3 g/cm Z cell constants
C35H6805
0.44 mm 1.049 1.050 2
a, A b, A c, A
0.26 mm X 0.16 mm
5.498 (2) 7.549 (2) 43.39 (1) 93.62 (2)
@,deg space group cell constants” a,, A b,, A c,, 8, a=6=
X
(b) Subcell: (0,) Pbnm 4.924 7.549 2.562
= 900
a b and b, are coincident.
described in some detail for tricaprin by Jensen and Mabis.’ Information about molecular conformations is given by spectroscopic data even in the absence of single crystals; however, the high purity of crystalline samples4 is advantageo~s.~ Molecular orientations in a crystal can also be established, as shown by Mathieu.* These methods have not been extensively used for hydrocarbon structures because the low transmittance only permits infrared observations of very thin crystals while crystals of suitable dimensions (thickness) for Raman observations at all orientations were not available. Infrared spectra of DPG single crystals measured by the Fourier transform method (Nicolet) were generally of poor quality because of the very low transmittance. All the crystals examined were much too thick. However, the spectra clearly show the extremely weak lines in regions not overlapped by strong lines. Excellent spectra were obtained by the same method for DPG powder with KC1. Comparison of these spectra, shown in Figure 1, revealed that lines at 1289, 1180, 1069, and 959 cm-l which were very strong in the powder spectra were absent or weak in the single-crystal spectra; in the absence of orientation effects, these lines would be saturated in the latter. Other lines are not extinguished, (7) L. M. Jensen and A. J. Mabis, Acta Crystallogr., 21, 770 (1966). (8) L. Couture-Mathieu and J. P. Mathieu, Acta Crystallogr., 5, 571 ( 1952).
0022-3654/84/2088-6333$01,50/0 0 1984 American Chemical Society
6334 The Journpl of Physical Chemistry, Vol. 88, No. 25, 1984
Albon and Baret
a
i
g-i !
c
I I'1
)
201
I
I
14b0
ij do0
ll
Y
Y
1
1doo
L.lcm-'
do0
1
$00
Figure 1. Infrared spectra of 1,2-sn-dipalmitoylglycerol (DPG), Nicolet Fourier transform: (a) single crystal, radiation incident along slowest growth axis, 2, and nearly parallel to crystal axis, c (Figure 2, Table I, and Discussion); (b) powder in KCl. Strong lines in powder spectra absent for the crystal are indicated by dashed lines. The spectral lines observed for CIS,CI9,and CZlhydrocarbon chains are shown.
including that at 879 cm-I assigned to the methyl group. The infrared spectra therefore gave clear indications of molecular orientation, as demonstrated for thin layers in a recent report? but this technique presents problems in flexibility of observation. Other details of the infrared spectra will be discussed later. Raman spectra of highly-purified, powdered crystals of DPG had been recorded previously, but only briefly d i s c ~ s s e d .The ~ single-crystal spectra described here were obtained with rectangular samples prepared by cleaving larger crystals and mounted on a goniometer, as used for X-ray diffraction. Both the surface and interior of the crystals were of excellent optical quality and good spectra were obtained. In addition to the features of the hydrocarbon chains, there are lines at about 1700 cm-l assigned to the carboxy groups and a hydroxyl line at 3500 cm-l which is very weak (this line is strong in the infrared spectra). Assignment of the hydrocarbon spectra has not been without difficulty and controversy and was discussed by Snyder et al.1° (with references therein) and Spiker and Levin." In this paper, the main topics of interest are the molecular conformation and orientations in the crystal but the spectra were found to contain new features which made interpretation easier. Chain packing in DPG is very similar to that in monoclinic subcell in a monoclinic main C36H7,,12 with an orthorhombic (01) cell. A monoclinic subcell can equally well be used to describe
d
II
e
Figure 2. Mounting of crystals and identification of axes: (a) typical crystal, arrow shows fastest growth direction; (b) first mounting of crystal 1; (c) orientation of unit cell axes in b, from spectra; the angle between c and 2 is about 3 O ; (d) second mounting of crystal 1; (e) mounting of crystal 2.
the packing.12 However, the main cell used for DPG differs from that of the C36H74 hydrocarbon in that the angles between the axes are 90' or close to 90' (93.6'), as given in Table Ia. The main cell has only a twofold screw axis, along b, and translation symmetry. DPG molecules are asymmetric and the absolute configuration of the carbon atom sn2 of the glycerol group is known. Although many features are similar, there are significant differences between the spectra of various compounds having hydrocarbon chains in the molecules. This can be seen in the infrared spectra of DPG and the corresponding lecithin phase 2 (unpublished data). They are also visible in comparing our DPG spectra with those published in ref 10 and 13. For example, the DPG infrared spectra show many strong lines in the region from 1050 to 1400 cm-l. Evidently, these spectra are quite sensitive to molecular packing and further understanding of these phenomena would be valuable for the applications of spectroscopy to liquid crystal and bilayer systems. The results of this investigation show that this understanding benefits substantially by studies of single-crystal spectra. Only the crystalline DPG phase 2 will be discussed here.
(9) J. F. Rabolt, F. C. Burns, N. E.Schlotter, and J. D.Swalen, J. Chem. Phys., 78, 946 (1983). (IO) R. G . Snyder, S. L. Hsu, and S . Krimm, Spectrochim. Acta, Part A,
Experimental Section Two crystals were used, mounted as shown in Figure 2. The apparatus for making spectral measurements of single crystals was partly improvised, so that only small changes in angle were possible for each setting. A second orientation of crystal 1 was obtained by cleaving the sample close to the mount and remounting. Crystals were oriented visually, using the large planar cleaved faces, with minor adjustments monitored by a spectral line. X , Y,and Z coordinates, as shown in Figure 2, were initially
(1975). (12) H. M. M. Shearer and V.
(13) G. Zerbi, R. Magni, M. Gussoni, K. H. Moritz, A. Bigatto, and S . Dirlikoy, J . Chem. Phyb., 75, 3175 (1981).
34, 395 (1978). (11) R. C . Spiker and I. W. Levin, Biochim. Biophys. Acta, 388, 361
Vand, Acta Crystallogr., 9, 379 (1956).
The Journal of Physical Chemistry, Vol. 88, No. 25, 1984 6335
Molecular Structure of DPG
Figure 3. Raman spectra of DPG single crystal, experiment 1. Incident polarization is along b axis (Table 11). Comparison with Figure 7 (ORTH) of ref 13 shows significant differences.
TABLE II: Raman Data for DPC Single Crystals' polarn crystal incident expt planes no. axis no. 1 1 a b2 3 4 5 6 7 8 9 10 11 12 13 14
1 1 1
1 1 lb lb lb lb 2 2 2 2
exit axis
b-
a
a a
bb ba bb bc cc ca cc cb
C
a a b b a a
aa
C
ab aa ac
C
C
a a b b b b C C
C
b
b
"See also Figure 2. Notation follows Porta,'* thus experiment 3 = a(bb)c. bSecond mounting of crystal 1. X = b, Y = a, Z = c, from spectra.
used to describe the various orientations. The slowest growth axis, Z , is perpendicular to the crystal plates so that Z is virtually parallel to the longest unit cell axis, c. However, the spectra allowed the crystal b axis to be identified as X and the a axis as Y and the results are tabulated on the basis of the unit cell axes, a, b, and c. A 90° geometry was used to observe the spectra. Raman spectra were recorded with a Jobin-Yvon Ramanor H G 25 spectrometer having a double monochromator with holographic gratin s with a stated resolution of about 1 cm-'. Excitation at 5145 was provided by a Spectra Physics Model 164 argon ion laser (250 mW). Photomultiplier voltages were adjusted to obtain optimum spectra overall and must be considered when comparing line intensities in different spectra. For the present discussion, the variations were so marked that a detailed correction was unnecessary. The variation in relative intensities between spectra for different orientations was often large. Some variation (in overall intensities) between crystals for the same orientation is probable. Spectra were obtained for the geometries listed in Table 11. In experiments 1 and 2, without analyzer, the ranges 0-1800, 2700-3050, and 3450-3550 cm-' were scanned. These observations were repeated in experiments 3 and 4,with the analyzer plane parallel and perpendicular to the incident beam. For experiments 5-14, also with the analyzer parallel and perpendicular, only the ranges 1000-1800 and 2750-3100 cm-' were scanned. A vertical polarization of the incident beam was used for all the observations. Good spectra, with high signal-noise ratio, were obtained in which many well-defined peaks are visible, as shown in Figure 3 (experiment 1 of Table 11). In experiments 7-10 there was some variation in the base line, thought to be due to the proximity of the mount to the laser beam. This was not important for the present interpretation of the spectra and could probably be eliminated by a modified mounting method.
x
chain assignmt
C
C
C
TABLE III: Carbonyl Stretching Modes for DPG and Related Compounds"
infrared Raman
freq, cm-I (a) DPG 1733 VS 1711 VS 1733 m 1707 m
(sn)
1 2 + H bond 1 2 H bond
+
(b) DPPC.2H20 infrared Raman*
1742VS 1732VS 1716VS 1743 W 1730 W 1716 W
1 1 2 2 1 molecule A 1 2 2 molecule B
+ +
(c) 1-0-hexadecyl-2-palmitoylPCe Raman 1716 2 1- palmitoyl- 2- 0-hexadecy 1 PC Raman 1737 1 1-0-hexadecyl-2-acetoyl PC 1739 2d "Data in (c) from ref 14. bReference 5 . 'PC denotes phosphatidylcholine. d N o gauche bond adjacent to C=O.
Molecular Conformation of DPG Information given by the spectra about the molecular conformation will be discussed first. Most of this can be obtained without single crystals but the high-quality spectra from the latter are advantageous. Carbonyl and Hydroxyl Groups. The carbonyl stretching mode appears as a doublet in DPG spectra, as listed in Table 111. In the infrared spectra the carbonyl lines are very strong and the Raman lines are distinct for most orientations as, for example, shown in Figure 3. As discussed in ref 5 , these lines are assigned to the snl and sn2 chain carboxyl groups and the frequency shift between snl and sn2 to the folding of the sn2 chain and and also (in DPG crystals) to hydrogen bonding of the sn2 group. Further evidence to support this assignment has been presented by Levin et a1.14 The hydrogen bonding is shown by the additional shift, by the relative broadening of the lower frequency band, and, in the infrared spectra, by a strong band at 3502 cm-'. Longitudinal Chain Vibrations. In experiments 1-4, lines with small frequency shifts were observed, some of which can be identified as longitudinal chain ~ i b r a t i 0 n s . l ~The relevant frequencies and assignments are given in Table IV and correspond to a folding of the sn2 chain as depicted in Figure 4, and confirming the conclusions of the preceding paragraph. (14) I. W. Levin, E. Mushakayarara, and R. Bittman, personal communication. (15) R. F. Schaufele and T. Shimanouchi, J . Chem. Phys., 47, 3605 (1967).
6336
The Journal of Physical Chemistry, Vol. 88, No. 25, 1984
Albon and Baret
TABLE IV: Longitudinal Vibrations in DPG Spectra (Raman)
obsd freq, cm-'
expt no. 1 2 3 4
meanvalue
120 119 120 121.5 120
163.5 162.5 164 165 164
320 320 320 319 320
440 ? 437.5 437 438
weak spectra
Hydrocarbon Valuesu
n Cl, Cl, C*O
rn 1 3 1
165 437 a(bb)c
118 322
C,, 3 assignmt Reference 15.
20/l
sn3
14/1
I4j3
20/3
*Or
1413
B
800
600
SH,FT
400
c,.q
"',P1 200
I
0
Figure 5. Longitudinal acoustic modes in DPG Raman spectra, experiment 3, a(bb)c, 0-700 cm-I. The 14/1, 14/3,20/1, and 20/3 vibrations are indicated and listed in Table IV. These lines are weak because of crystal orientation but are clearly visible in all spectra in which this region was scanned. TABLE V: Methylene Rocking-Twisting Modes (Frequencies, cm-' ) in DPG Infrared SpectraaSc
(obsd) DPG '15 hydrocarbonb powder single crystal 722 720s satd satd 733 729s 778 853 934 1017
778m 848m 932w 1017 s
778 m 853 m 933 m
satd
c
L19
hydrocarbon 722 744 780 836 903
720s 740m 778m 828? 904m
969 1030
975 w 1022s 1039 m
satd 740 m 778 m 828 m 903 905 m 970 mw
overlaps C,,
C21
hydrocarbon
Q
Figure 4. Molecular folding of 1,2-sn-dipalmitoylglycerol(DPG); hydrogen atoms are omitted except for the hydroxyl group: carbon atoms, partially filled circles; oxygen atoms, open circles. The asymmetric carbon atom sn2 is indicated by the star.
In this conformation, the relatively rigid (planar) carboxyester group sn2 is at right angles to both chains and will not transmit the longitudinal vibrations. The vibrations of the longer snl chain are evidently transmitted along the snl carboxy-ester group which is aligned along the chain axis. The frequency dependence is on m f n - 1 if we use the hydrocarbon data.15 Several other lines are visible in the low-frequency region as seen in Figure 5. None correspond to any longtitudinal modes that could be related to any possible alternative structure. Since there are two chain lengths, the longtitudinal lines are weaker that those shown in ref 13 and 15. However, this is mainly due to the unfavorable molecular orientation in the experiments in which this region was scanned. Methylene Rocking-Twisting Modes. These appear in hydrocarbon infrared spectral6 as a series of lines in the region from
722 730 761 806 862 923 980 1031
satd satd
720s 729s 756m 811w
751 ? mw 812 mw
shoulder ?
absent
923 m 975 w 1022s 1039 m
923 mw 970 mw
Overlaps'15.19 overlaps C,,
overlaps C,,
satd
Shown in Figure 1. Reference 16. Also observed in 7201050-cm-' region are the following: 878 cm-', methyl rock, very strong in both spectra; 956 cm-', very strong in powder: very weak for single crystal. Other lines in single crystal spectra are 751 m, 893 m, 993 mw, and 1006 w.
720 to 1050 cm-'. All the lines, as for a C15hydrocarbon, are clearly visible in both DPG spectra as shown in Figure 1 and listed in Table V. This is a further confirmation of the proposed molecular folding. As previously mentioned, the other, s n l , chain is more heterogeneous; this is evident from Figure 4. The rocking-twisting modes for such a chain may be considerably perturbed as com(16) R. G. Snyder and J. M. Schachtschneider, Spectrochim. Actn, 19, 85 (1963).
The Journal of Physical Chemistry, Vol. 88, No. 25, 1984 6337
Molecular Structure of DPG TABLE VI: Assignments in the C-H Stretching Region for Methylene and Methyl Groups in Hydrocarbons" and DPG
(a) Hydrocarbons infrared
Raman obsd, obsd, mode species cm-' mode species cm-' description CHI d-(?r) B2" 2920 d-(O) BI. 2880 antisym . . 2850 d+(O) A,' 2850 sym B3u 2962 ra2964 asym in skeletal CH3 rr plane 2953 rb2952 asym out of Tu' skeletal plane 2935 rt 2935 sym r+ r+ 2873 rt 2871 sym (bl 1.2-sn-DPGb Raman obsd, mode species cm-I ra-
2980
ri
2972 2963
description asym in skeletal plane
3000
methyl on sn2 chain
asym out of skeletal plane raasym in skeletal plane methyl on snl chain rb2955 asym out of skeletal plane r+ 2934 sym r+ 2875 sym "References 10 and 11. bCH2modes as above (d-(?r), 2916 cm-'; d+(?r),2849 cm-'). CH3 modes: infrared, rb- 2955 cm-', rt 2873 cm-I. pared with hydrocarbon spectra. A study of the spectra shows a series of lines at frequencies corresponding to those of a C19and another series at frequencies close to those of a Czl hydrocarbon chain. These observations are shown in Figure 1 and listed in Table V. The only deviations are that the line expected at 862 cm-l for Czl is absent in the single-crystal spectra and, instead of the line at 1030 crn-', there are lines a t 1022 and 1039 crn-'. The more intense lines are clearly visible in the powder spectra ( l b ) and the weak lines in the single crystal (la). In the single-crystal spectra a strong line at 956 cm-' is extinguished, further clarifying the spectra in this region. It is expected that these lines and especially those close to 720 and 1020 cm-l will be split as discussed by Snyder.I7 However, the geometry for the diglyceride chains is more complex than those disc~ssed.~'Splitting probably accounts for some of the lines observed. All the spectral data strongly supports the molecular conformation shown in Figure 4 but to determine the structure the orientations of the molecule in the cell must also be established. Orientation of Molecule in Cell Molecular vibrations involve all atoms in a molecule and are complex for compounds such as DPG. However, it is well-known that many vibrations are virtually confined to specific pairs or groups of atoms with characteristic frequencies. This depends upon symmetry, relative mass and bond strength, and the bond directions relative to the vibrations. So, to interpret the spectra it is possible to look at specific parts of the molecule. The intensity of the scattered radiation depends on the polarizability tensor which is made up of contributions from all the bonds. In a crystal the mean orientations of the molecules are fixed and vibrations of particular value for establishing these orientations are those in which an end atom vibrates against the whole molecule. For this, hydrogen is especially useful because of the large mass differences. For symmetric stretching vibrations, the tensor for each bond is cylindrically symmetrical with the major axis along the bond. Single-crystal spectra therefore contain information about bond or group orientations from which the molecular orientations may sometimes be deduced. (17) R. G. Snyder, J . Chem. Phys., 71, 3229 (1979).
SHIFT
2900
2800
c m-1
Figure 6. Spectra of C-H stretching region for DPG crystal observed in experiment 9, b(cc)n orientation, showing strong methyl group modes at 2963 and 2980 cm-l due to the asymmetrical modes of the snl and sn2 chains, skeletal plane (Table VI). Compare with Figure 3 for usual spectra. Other lines at 2875 and 2935 cm-' assigned to the methyl group are clearly visible.
In both infrared and Raman spectra, when hydrocarbon chains are present in the molecule, the lines assigned to carbon-hydrogen vibrations are by far the most intense, as shown in Figure 3. Assignments for the carbon-hydrogen stretching region of hydrocarbon spectra are given in Table VIa.lo,ll The number of methyl groups is often small compared with methylene so that the characteristic methyl frequencies appear as weak lines in the vicinity of the intense methylene modes. All the lines reported for hydrocarbons were found in DPG spectra with the methyl lines well resolved for the CH3/CHzratio in this compound as seen in Figure 3. I t was assumed that DPG spectra in the carbonhydrogen stretching region would be very similar to, if not identical with, to hydrocarbon spectra. This seemed to be confirmed on first examination of the DPG spectra. A close and detailed inspection revealed significant differences between these compounds which were unexpected. Hydrocarbon spectra frequently show a doublet assigned to the asymmetric methyl vibrations (in and out of the skeletal plane) at frequencies of 2952 and 2964 cm-', with the in-plane mode at 2964 cm-' more intense. These can be seen in Figure 2; (b), (c), and (d) or ref 10 and the DPG spectra shown in Figure 3 of this paper looks quite similar. But Raman spectra of DPG single crystals show striking variations in line intensities for different orientations and this is clearly visible in the carbon-hydrogen stretching region. The general appearance of the spectra changes completely. In some spectra, lines due to methyl vibrations are as strong or stronger than those assigned to the methylene group, as seen in Figure 6, for polarization b(cc)a. This shows two strong lines, of similar polarization characteristics, at 2963 and 2980 cm-' and led to a closer inspection of all the spectra. From this inspection, the conclusion was reached that the doublet assigned to the methyl asymmetric vibrations (in and out of the skeletal plane) in hydrocarbon spectra is present in the DPG spectra. There is also an almost identical doublet, shifted to higher frequencies by about 17 crn-', which is not present in hydrocarbon spectra. The lines can be assigned to the methyl groups attached to the snl and sn2 chains respectively, as listed in Table VIb. They appear as a result of the different environment of the two groups which will be discussed later. (As noted by a reviewer, the snl assignment is that commonly accepted for hydrocarbons and the sn2 assignment is new and dependent on the observations reported here.) As the lines in each doublet are only rarely resolved in a single spectra, a further discussion of the observations will be given. Analysis of the DPG methyl group spectra indicates that the
Albon and Baret
6338 The Journal of Physical Chemistry, Vol. 88, No. 25, 1984
c
z W
?
5
a-b plane
a
CY 3000
3000 2950
3000
SHIFT
2950
c m-1
3000
/c!&
2950
Chain axis
2950
Figure 7. Composite Raman spectra for C-H stretching region stretching showing that two doublets are present with lines at 2955 and 2963 cm-l (snl methyl) and 2972 and 2980 cm-l (sn2 methyl). See also Table VI and discussion in test. The numbers of the experiments are given on each spectra (Table 11). Asymmetric modes in the skeletal plane at 2963 and 2980 cm-I are indicated by longer line and the modes out of the plane by shorter line. The symmetrical mode at 2935 crn-l is indicated by heavy line. Other spectra confirm the pattern shown.
principal axes of the polarization ellipsoids are inclined to the crystal axes (see also Figure 8). In crystal spectra oriented favorably (cc) for the asymmetric modes, in the skeletal plane, the other modes are not evident as they are much weaker. When the orientations favor the out-of-plane modes (lower frequency) such as (aa) and (bb),the higher frequency modes are usually visible with a reduced intensity. These comments are illustrated by the composite spectra in Figure 7 and also Figure 6. When Figure 3 was reexamined, the two peaks were seen to be broader than in hydrocarbons and the upper peak extends to 2980 cm-'. This spectra is quite similar to the Raman spectra of powdered, crystalline, dipalmitoyllecithin dihydrate shown in ref 5 (Figure 4) where lines at 2961 and 2974 cm-I are visible. It is possible that in this phase the methyl spectra resemble that of DPG although not resolved in the powder spectra. Thus, the single-crystal spectra provide a dramatic confirmation of the proposed molecular folding and directly support previous assignments of the methylene and methyl groups in hydrocarbons for the carbon-hydrogen stretching region. Orientation ofMethy1 Groups. The two molecules in the cell are related by the twofold screw axis along b so that there are two related molecular orientations. Two alternatives exist for placing a molecule, folded as in Figure 4, into the subcell and main cell. These differ in the orientation of the methyl groups, all of which may either have the C-CH3 bond (i) aligned along the c axis or (ii) at an angle of about 70" to c, as shown in Figure 8. The spectra indicate that the C-CH3 bonds are inclined at about 70" to the c axis, as in (ii). This conclusion is evident from the persistence in the infrared spectra of the line at 879 cm-l for the single crystal where the polarization directions are all perpendicular to c. In single-crystal Raman spectra, the component of the methyl symmetrical carbon-hydrogen stretching vibration at 2934 cm-l is visible for all orientations of the incident polarization as expected for (ii) whereas for (i) this line should be significantly weaker for (cc) polarization, The line at 2875 cm-I is not visible in all spectra owing to the proximity of the very strong methylene mode at 2880 cm-'. Asymmetrical carbon-hydrogen stretching modes, out of the skeletal plane, at 2955 and 2972 cm-' (methyl) are clearly visible in spectra with (bb) and (aa) orientations and not visible for (cc), as expected for both models (i) and (ii). Finally, the asymmetrical carbon-hydrogen (methyl) stretching vibrations in the skeletal plane at 2963 and 2980 cm-' are much stronger for (cc) orientations, as expected for (i) whereas for (ii) the inverse behavior should occur. This conclusively indicates the
Figure 8. The two orientations of the methyl groups consistent with the subcell and molecular folding. Single-crystal Raman spectra show that (ii) is found in the crystal. c is the main cell axis, c, the chain and subcell axis, and the skeletal plane of the carbon atoms is indicated.
orientations of the methyl groups and thus of the whole molecule. As the configuration of carbon atom sn2 and the molecular folding are known, a precise model for the DPG crystal structure results. The large variations in the intensities of the methylene stretching modes at 2850 and 2880 cm-I indicate the chain orientation as known from the X-ray data. To briefly summarize the observations, in the 0, subcell, the carbon-hydrogen bonds are all in planes parallel to a,-b, and nearly along a, or b, as shown in Figure 3 of ref 19. This is for orthorhombic C36H74in which the subcell and main cell axes are parallel. On tilting the subcell about b,, as in the main cell of DPG,bonds along b, remain in the a-b plane and bonds along a, are tilted by about 30" to this plane. The skeletal carbon-carbon bonds are either nearly perpendicular to the a-b crystal main cell plane or tilted at about 23O to this plane. All spectra with polarization directions in the ab plane show strong lines at 2880 (antisymmetric) and 2850 cm-' (symmetric) for the methylene carbon-hydrogen stretching modes. As expected, (bb) orientations are stronger than (aa) because of the tilt direction. The lines at 2880 and 2850 cm-' are much weaker for the (cc) orientations although still lower intensities should be seen for the orientation (cscs), not measured. The antisymmetric and symmetric vibrations behave in a similar manner because of the chain and subcell symmetry. Chain orientations are clearly shown by the lines at 1130 and 1065 cm-I attributed to the carbon-carbon skeletal stretching modes, which appear in the spectra shown in Figure 9. These identify the 1 130-cm-' mode as the asymmetric carbon-carbon stretch along the chain axis and enable the a and b axes of the crystal to be assigned from the spectra. The line at 1065 cm-' is shown to be the symmetrical carbon-carbon stretch, in the skeletal plane and perpendicular to the chain axis. These are as given in previous which, however, are reversed in ref 13.
Discussion An examination of the chain packing in the unit cell shows that if the chain axes pass through and 3/4aat b = 0 or 'I2,the T. C . Damen, S. P. S. Porto, and A. Tell, P h p . Rev., 142,570 (1960). P. W. Teare, Acta Crystallogr., 12, 294 (1959). R. G. Snyder, J . Mol. Spectrosc., 7, 116 (1961). M. J. Gall, P. J. Hendra, C . J. Peacock, M. E. A. Cudby, and H. A. Willis, Spectrochirn. Acta, 28, 1485 (1972). (18) (19) (20) (21)
Molecular Structure of DPG
The Journal of Physical Chemistry, Vol. 88, No. 25, 1984 6339
i
!n
P
i
jl SHIFT
I
cm-’
w
5
1
L U U V U L 9
I
I
1600
1400
I
1200
load
Figure 9. Raman spectra of DPG single crystals for different orientations showing the 1000-1500-cm-’ regions. Note large variations in line intensities. The lines at 1130 and 1065 cm-’ are briefly discussed in the text.
snl and sn2 chains are interdigitated and there is a pseudo-twofold axis along b. If the midpoint of the terminal carbon-carbon bond is on a, methyl carbon atoms on the same axis but in different molecules will be about 1 . 5 ~apart. ~ This distance is 3.84 8, compared with 3.91 8, found between the closest methyl groups in monoclinic C36H74.I’ Thus, the subcell may be virtually continuous across the planes of methyl atom contacts because these distances happen to be nearly identical. In most hydrocarbon structures, the subcells are appreciably displaced between the two zones in a bilayer crystal or across the methyl to methyl planes as in orthorhombic c36fI74.” When the molecule is placed as described in the preceding paragraph, the hydroxyl groups are near to ‘ I z aand 0, ‘l2b. This ensures interdigitation of the these groups and allows hydrogen bonding to the sn2 carboxy group, as indicated by the spectra. The model proposed for the structure is shown in Figure 10. This model will be used together with the X-ray diffraction data to confirm and refine the structure. In this process, modification of details of the model may be expected. Earlier, it was deduced from the geometry of the subcell and the molecular folding that both methyl groups have similar orientations. This was unexpectedly confirmed by the spectra because of the difference between methyl specta when attached to snl and sn2 chains. Thus Raman spectra may be used to determine the orientations of methyl groups in compounds with identical fatty acids in crystals and perhaps also in ordered layer systems. However, intensities of the methyl lines are low compared with the adjacent methylene bands except for the most favorable orientations. Further experiments may indicate whether the difference between s n l and sn2 methyl groups occurs in monolayers; for closely packed phases, the molecular folding will be similar to that in crystals. Enuironment of the Methyl Groups in DPG. In directions along the chain axes, the environment of both methyl groups is identical for atoms close enough to be significant. For directions perpen-
Figure 10. Model proposed for DPG crystal structure from X-ray and spectral data: (a) Projected on to the a-c plane and (b) projected on the b-c plane. Orthorhombic and monoclinic subcells are shown; b and b, are parallel and a, monoclinic is virtually parallel to a. Only a general position is given for carboxy ester group sn2. The structure is expected to be modified in detail on refinement, using the X-ray data. dicular to the chain axes, the closest hydrogen to hydrogen contacts of the s n l methyl group are with methyl groups and of the sn2 methyl group with methylene groups. Assignment of the bands can be made since the snl methyl group has an environment similar to that in hydrocarbon structures and similar frequencies. The sn2 group has a different environment with greater intermolecular forces and tighter packing giving higher frequency modes. It is observed that the snl asymmetric mode in the skeletal plane is more intense. These observations provide a good example of the details of the effects of crystal packing on Raman spectra which are also known to occur for the carbon-hydrogen methylene lines but not yet related to details of the structures. In all chain packings, the methyl group hydrogen atom which is in the skeletal plane continues the line of the carbon-carbon bonds. It is in the space occupied by a methylene group elsewhere in the structure and larger amplitude of vibrations are to be expected. For the asymmetric vibration in the skeletal plane, this atom has an amplitude which is about twice that of the two hydrogen atoms not in the skeletal plane. Since the atom in the skeletal plane is further from its neighbors while the other atoms are packed in a similar manner to methylene groups (see Figure lo), the asymmetric vibration (inplane) satisfies the intermolecular forces better than other possible modes. The spectral details correlate with the single-crystal X-ray diffraction structure of d l -dilaurylethanolamine reported by Shipley et al.,” in which very large temperature factors were observed for the last two carbon atoms of chain snl (Figure 2 of ref 22). Similar large temperature factors have previously been observed in the crystal structures of h y d r ~ a r b o n sand ’ ~ glycerides? The observations described link these effects with the vibrational modes of the methyl groups and especially to the asymmetric vibration in the skeletal plane. The appearance of modes due to 19 and 21 groups in the rocking-twisting spectra (infrared) could either be interpreted as due to the entire chain (21) and the portion from the methyl group to carbon atom sn2 (19) or, more plausibly, to ascribe the (22) M. Elder, P. Hitchcock, R. Mason, and G . G. Shipley, Proc. R . SOC. London, 354, 157 (1977).
6340
J . Phys. Chem. 1984,88, 6340-6344
C19 series to the increased vibrations of the last two carbon atoms of the chain. There are many other details of interest in these spectra which warrant further studies. Conclusions The data obtained in these experiments may be used to clarify details of assignments of Raman spectra for hydrocarbon chains in glycerides and phospholipids. For the methylene carbon-hydrogen stretching mode the components observed nearly perpendicular to the bond directions are small; a more precise estimate of the spatial variation may be obtained by observations along and perpendicular to the chain axis directions, Le., a,, b,, and c, rather than the unit cell axes. Both infrared and Raman spectra give considerable information about molecular conformations and orientations and are of value for the study of lipid crystal structures. Infrared spectra are useful for very thin crystals such as are often obtained from less highly purified samples. In addition to the carbon-hydrogen and carbon-carbon modes of the methylene and methyl groups, carbon-oxygen bonds, especially in the carbonyl group, can be used to indicate conformations and orientations. Vibrational spectra have great potential value for investigations of the structures and dynamics of thin films and liquid crystal systems. Raman spectra are especially useful in the presence of water such as in monolayers, bilayers, and biological membranes. Interpretation of this data is extremely difficult without precise information such as is obtained from single crystals as in the present study. Measurements of compounds, such as DPG, with the Same configuration as naturally occurring lipids are particularly valuable. Further experiments will show whether the methyl spectra reported here can be observed in bilayers, and some orientation of the layers is probably essential. A recent report2j shows spectra
of the 2800-3000-~m-~region in which the intensity between 2960 and 2980 cm-’ displays a marked increase for the x ’ ( Z Z ) y geometry as expected from the present results. This is evidently the envelope of the two lines at 2963 and 2980 cm-’ reported here. Also, the spectra for x ’ ( 2 X j y show less intensity in this region shifted slightly to a lower frequency. The nature of the phase examined may vary considerably with the water content but is probably near to the boundary between phases 5 and 10, with tilted chains as described in ref 3. This has an hexagonal chain packing. In most biological membranes, the fatty acid attached to the sn2 carbon atom is longer so that the methyl spectra will more resemble that of hydrocarbons. In certain specialized systems, such as the lungs, relatively high concentrations of dipalmitoyllecithin are present and the spectra reported here may be of value in the investigation of these systems. These experiments show that DPG and probably all related phospholipids have spectra which differ in detail from hydrocarbon spectra. They show that in these diglycerides one of the methyl groups is more tightly packed than in hydrocarbons with a greater vibrational frequency of the asymmetric modes.
Acknowledgment. The infrared measurements given here were made by M. J. Pourcin, Department de Physique des Interactions Moleculaires, Centre Saint-JCr6me and extensive Raman spectra of single crystals were obtained by M. M. Guiliano, Centre Infrarouge et Raman Sptroscopie, Centre Saint-JErbme. We thank M. Pourcin and M. Guiliano for their invaluable cooperation. Thanks are also due to Dr. I. W. Levin for his introduction (N.A.) to Raman spectroscopy. Registry No. DPG, 30334-71-5. (23) W. L. Peticolas, M. Harrand, and R. Dupeyrat, J. Raman Spectrosc., 12, 130 (1982).
Enthalpy Change for the s-Trans to s-Cis Conformational Equilibrium in 2-Methyl-I ,3-butadiene (Isoprene), As Studied by High-Temperature Ultraviolet Absorption Spectroscopy’!* Philip W. Mui* and Ernest Grunwald Department of Chemistry, Brandeis University, Waltham, Massachusetts 02254 (Received: May 2, 1984, In Final Form: August 6, 1984)
Conformational equilibrium in 2-methyl-l,3-butadiene(isoprene) was studied by measuring UV apparent extinction coefficients at wavelengths (258-268 nm) where only the s-cis conformer absorbs. Temperature dependence of the extinction coefficients of the s-cis conformer was explicitly accounted for. In the temperature range of 418 to 592 K, the bandshape of the s-cis absorption band was found to be well represented by a Gaussian distribution with a temperature-independent mean and an increasing width, in agreement with theoretical predictions. The enthalpy difference (AH0,,9) between the two stable conformations of isoprene was determined to be 1.78 A 0.15 kcal/mol.
Introduction Conformational equilibria and barriers to internal rotation around single C-C bonds in alkyl-substituted derivatives of 1,3butadiene are of interest because they elucidate the role played by nonbonded interactions in the rotational isomerization process. However, in contrast to 1,3-butadiene, its alkyl-substituted analogues have received much less attention. Isoprene represents the simplest possible alkyl-substituted 1,3-butadiene. Evidence from m i c r o ~ a v e ,electron ~ , ~ diffracti~n,~ and IR studied shows that the most stable conformation of *Present address: Department of Chemistry, Cornell University, Ithaca, NY 14853.
0022-3654/84/2088-6340$01.50/0
isoprene is planar s-trans. Concerning the structure of the lessstable conformer, experimental evidence from two recent inde(1) This project was supported in part by BRSG Grant S07-RR07044, awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health. P.W.M. gratefully acknowledges the award of a Career Development Fellowship from Brandeis University. (2) Abstracted in part from the Ph.D. dissertation of P.W.M., Brandeis University, 1984. (3) D. R. Lide and M. Jen, J . Chem. Phys., 40, 252 (1964). (4) S . L. Hsu, M. K. Kemp, J. M. Pochan, R. C. Bensen, and W. H. Flygare, J. Chem. Phys., 50, 1482 (1969). ( 5 ) M. Traetteberg, G.Paulen, S. J. Cyvin, Yu.N. Panchenko, and V. I. Mochalov, J . Mol. Struct., 116, 141 (1984).
0 1984 American Chemical Society
