J . Phys. Chem. 1987, 91, 3436-3442
3436
Raman-Actlve Bands Sensitive to Motlon and Conformation at the Chain Termini and Backbones of Alkanes and Lipids Kenneth G. Brown,* Department of Chemical Sciences, Old Dominion University, Norfolk, Virginia 23508
Ellen Bicknell-Brown,* and Meriem Ladjadj Department of Chemistry, Wayne State University, Detroit. Michigan 48202 (Receiued: December 26, 1985: In Final Form: February 9, 1987)
The Raman spectra of long-chain alkanes with methyl groups at both ends are compared with those having a substituted group at one end of the chain. A Raman-active band that appears between 890 and 900 cm-', and was previously assigned to CH3 rocking coupled with stretching of the terminal C-C bond and to acyl C1-C2 stretching in long-chain esters, was examined in detail in order to determine the accuracy of the assignments and the relative contributions of each band (if both assignments are correct) to the total Raman intensity observed in spectra of long-chain esters at about 895 cm-'. The assignment of this observed band assumes significance in studies of synthetic and biological polymers because a band at 895 cm-' in glycerides has been shown to be sensitive to molecular state, in particular local motional freedom and conformational randomness. The intensity of the 895-cm-l Raman band is shown to consist virtually entirely of chain end C-C stretching. Further, it is shown that, for long-chain esters, terminal acyl C-C stretching and terminal methyl end C-C stretching are superimposed. However, the acyl end C-C stretch band contributes much more to the total intensity than does the methyl end C-C stretch band. The spectra of the liquid alkanes contain broadened bands at about 875 and 895 cm-'. The 875-cm-' band has been assigned previously to C2CIstretching for disordered chain ends. Comparison of Raman spectra of crystalline dipalmitoylphosphatidylcholine (DPPC) and crystalline DPPC labeled with I3Cat the CI atom of chain 2 confirms the assignment of the band to acyl terminus CC stretching. Further, the 895-cm-' Raman band is assigned to the stretching mode for trans C2-C1 rotamers, while the 875-cm-I band is assigned to the stretching mode for gauche C2-C, rotamers. Therefore, the relative intensities of the 895- and 876-cm-' Raman bands may be used to determine the relative populations of trans and gauche rotamers at chain ends. The intensities of CH stretch bands and skeletal C-C stretch bands are compared for the liquid alkanes and melted DPPC. With the assumption that the 2885-cm-' CH2 antisymmetric stretch peak height for liquids depends on trans skeletal CC population while the peak height of the "all-trans" skeletal C-C stretch mode at 1130 cm-' depends on the population of all-trans segments, the interpretation of the spectra leads to the conclusion that gauche C-C rotamers are more randomly distributed along the liquid alkane chains than along the acyl chains of melted DPPC.
Introduction
Bicknell-Brown, Brown, and Person have reported that the Raman spectral region between 850 and 900 cm-' can be used to obtain structural information at the ester linkage in glycerides and phospholipids.2 In particular, a band at 891 cm-I in dipalmitoylphosphatidylcholine (DPPC) is observed to vary markedly in intensity and slightly in frequency as the molecule undergoes the gel-liquid crystal phase transition. This band was assigned to the terminal acyl C,-C2 stretching mode of the ester linkage end of the lipid acyl chains. This interpretation is based upon studies involving hydrogen-bonded fatty acids3 and small ester^.^ The C-C stretch mode due to the terminal C-C(=O) is assumed to be uncoupled from the C-C stretch modes of the hydrocarbon chain skeleton, which occur in the 1050-1 150 cm-' region. The acyl C2-C1stretch is expected to have a strong Raman intensity, based on Raman spectra of small esters and deuteriated
ester^.^ However, a normal-coordinate analysis of alkanes by Snyder and Schachtschneider assigns the 895-cm-' vibration in alkanes to a vibrational mode consisting of CH3rocking coupled with C-C stretching of the terminal CH2CH3group.' Therefore, the 895cm-' Raman band in long-chain esters is reinvestigated to determine whether this band is due to C-C stretching in the terminal acyl group or due to coupled CH3 rocking and C-C stretching at the terminal CH2CH3end or is a composite band with largely overlapping bands from each vibrational mode. (1) Snyder, R.G.; Schachtschneider, J. H. Spectrochim. Acta 1963, 19, 85-1 16. (2) Bicknell-Brown, E.; Brown, K. G.; Person, W. B. J . Raman Spectrosc. 1981, 11, 356-362. ( 3 ) Hagashi, S.;Umenura, J. J. Chem. Phys. 1970, 443, 2558-2562. (4) Oki, M.; Nakunishi, H. Bull. Chem. SOC.Jpn. 1970, 43, 2558-2562.
0022-3654/87/2091-3436$01 .50/0
This band assignment is particularly significant because a Raman temperature study of di- and triglycerides revealed that a band appearing at 890 cm-' is sharp for well-ordered solids but broadens and shifts to lower frequency upon melting of the solid and remains broadened and frequency-shifted if subsequent cooling is rapid. Therefore, this band offers a probe for monitoring order at the chain termini. However, it is necessary to more accurately determine the band assignment, particularly whether it is a single band due to one chain end, or whether it is a composite of bands due to uncoupled modes arising from different groups and/or different chain ends. If for substituted alkanes this band is a composite of two bands due to different modes from both chain ends, then it is important to determine the effect of end group substituents on C2-C' stretch mode intensity and frequency, so that parameters of this band are properly interpreted. Experimental Section
Raman Studies of Alkanes. The Raman spectrometer used in the measurements of the alkanes is a Spex 1400 double monochromator equipped with an uncooled RCA 100 photomultiplier for detection, located at Old Dominion University. All spectra were recorded at a spectral band-pass of 5 cm-'. The recorded frequencies are reproducible to within 1 cm-'. A Coherent Radiation Model 52 Ar laser operating at 488 nm was used, with laser power at the sample kept below 150 mW for all samples. For measurements carried out below room temperature, the sample in the capillary was placed in a Harney-Miller cell, cooled by passing cooled nitrogen through the cell. A thermistor was placed within the capillary as close to the laser focus point as possible. The temperature remained constant to within 1 OC throughout a run. All chemicals were purchased and used without further purification. The hydrocarbons and 1-bromohexadecane (cetyl 0 1987 American Chemical Society
Raman Spectra of Alkanes and Lipids
The Journal of Physical Chemistry, Vol. 91, No. 12, 1987 3437
n
I
I
I
I
800 850 900 950
I
I
I
I
1250 1300 1350 lL00
FREO Icm-'l
Figure 2. The 895- and 1 3 0 0 - ~ m -Raman ~ regions for liquids: (a) dodecane, (b) tetradecane, (c) hexadecane, (d) cetyl bromide.
--
850
900
9501250 1300 1350
FREQ (cm-')
Figure 1. The 895- and 1300-cm-' Raman bands for solids: (a) methyl palmitate, (b) hexadecane, (c) octadecane, (d) cetyl bromide, (e) cetyl hydroxide, (0 palmitic acid.
bromide) were obtained from Sigma Chemical with a stated purity of 99%. The other chemicals were reagent grade. Methyl palmitate was obtained from MCB, palmitic acid from Mallinckrodt, and 1-hydroxyhexadecane (cetyl alcohol) from Fisher. Raman Studies of Dipalmitoylphosphatidylcholine.Synthetic DL-a-dipalmitoylphosphatidylcholine(99%) from Sigma and 2-palmitoyl- 1-I3C phosphatidylcholine (90 atom % I3C) from Avanti Polar were used for the crystallization of the lipids. Two milligrams of the lipid was dispersed in 1 mL of solvent, (benzene or (CHCl,/acetone 3/2 v/v)), a t ambient temperature and the mixture was stirred with a vortex mixer. Both samples of DPPC, and DPPC-sn-2-I3C, in solvents were placed in Neslab water bath and were recrystallized by decrementing the temperature of the bath fluid from 25 to 18 OC by 1 OC every 2 h. The temperature was monitored by the temperature sensor within the bath fluid and controlled by a Neslab DCR4 temperature bath controller interfaced to a Spex Datamate computer by a Neslab interface. The Raman measurements of DPP were recorded with computer-controlled SPEX 14018 double monochromator (located at Wayne State University) equipped with a spatial filter to reduce stray light. The 514.5-nm line of a CR 8 argon ion laser was used for sample excitation. Spectral band-pass was set at 5 cm-'. The power at the sample was 80 mW. Scan accumulation and data measurements were controlled by the Spex Datamate computer.
Results Chain End Motion. The 850-950-cm-' spectral region and the methylene twisting band (- 1295 cm-l) are shown in Figure 1 for the solids studied and in Figure 2 for the liquids. The band-center frequencies and peak heights are listed in Table I, along with the bandwidths for the spectra of the solids. The CH, twisting band at about 1300 cm-' is used in this study as a standard for comparing the various spectra, because within a given phase (liquid or solid) it is relatively insensitive to the extent of hydrocarbon chain order. The CH, twist band increases in frequency by about 5 cm-I and increases in width over a narrow temperature range associated with the solid-to-liquid phase transition. Any apparent decrease in peak height as a solid-liquid phase change occurs is also accompanied by an increase in bandwidth, resulting in a relatively constant total integrated intensity for the band. W e assume the total integrated intensity of the CH, twist mode is proportional to the number of methylene groups in the hydrocarbon chain. Thus, CH, twist intensity
TABLE I: Frequencies, Bandwidths, and Peak Heights (arbitrary units) for the Raman Bands at 895 and 1300 cm-I
liquids dodecane tetradecane hexadecane cetyl bromide solids hexadecane octadecane
cetyl bromide cetyl alcohol methyl palmitate palmitic acid crystal I palmitic acid, crystal
894 895 895 894
0.25 0.22 0.19 0.18
1308 1306 1308 1304
2.5 2.6 2.7 2.5
895 895 898
11 11 21
0.33 0.26 0.35
1302 1301 1302
12 12 21
4.6 4.1 4.8
896 891
19 15
0.21 0.67
1305 1303
22 14
3.0 9.3
898
12
0.3 1
1302
13
4.3
894
9
1296
8
5.3
I1
differences between molecules that are simply the result of changes in chain length are approximately eliminated by normalizing the 1300-cm-l methylene twist band intensity by dividing by the number of methylene groups. Therefore, the 895-cm-I peak heights for the different hydrocarbons in the same physical state are compared by using the normalized peak height of the band in the 1300-cm-I region as an internal reference. For the liquids, the unnormalized ~ 8 9 ~ / ~ratio ~ ~ varies 0 0 from 0.18 to 0.25 (Table I). The relative range of values (0.07/0.18) is high, about 40% of the lowest value. After normalization for chain length the ratio varies from 2.5 to 2.7, and the relative range of values is now much more narrow, about 8% of the lowest value. This result validifies the normalization procedure. Though cetyl bromide contains only one methyl end, while dodecane, tetradecane, and hexadecane contain two methyl ends, the normalized 1895/11300 ratio is approximately the same for all four molecules. Therefore, the Raman intensity at 895 cm-l cannot contain a significantly intense band belonging primarily to the methyl rocking mode, because in that case the cetyl bromide 895-cm-I band intensity would be approximately half that of the three unsubstituted hydrocarbons. Therefore, the band is due to essentially pure C-C stretching a t the chain ends. Moreover, because the band-center frequency remains relatively constant as the substituents at the end groups change, there cannot be significant coupling in the potential energy distribution of methyl rocking or other end group vibrational modes with the terminal C-C stretch. Such coupling of vibrational modes should affect the normal-mode frequency. Therefore, even for substituted hydrocarbons, the 895-cm-' band is a composite of two relatively pure chain end C-C stretch mode bands, one for each chain end, having nearly identical band center frequencies.
3438 The Journal of Physical Chemistry, Vol. 91, No. 12, 1987 TABLE 11: Frequencies and Relative Peak Heights" of Raman Bands in the CH2 Bend Region
Brown et al. TABLE 111: Frequencies and Peak Height Ratios for the Raman Methylene CHI Symmetric and Antisymmetric Stretch Bands"
liquids
dodecane tetradecane hexadecane
1449 1447 1445
h(CH~),
1455 (8.7)
(IO) 1455 (8.7)
a
1427 (4.0) 1423 (2.8) 1422 (6.6) 1428 (6.2)
1454 (10) 1454 (IO) 1450 (10) 1433 (10) 1448 (10) 1445 ( I O )
1466 ( I O ) 1466 (9.1) 1466 (9.2) 1468 (7.2) 1465 (7.2) 1468 (7.2)
1420 (5.9)
1437 (10)
1455 (4.8)
cm-I
2858 2857 2857 2856
2897 2897 2897 2894
1.41 1.43 1.39 I .48
2858 2855 2855 2856 2847 2847
2885 2886 2885 2885 2889 2885
1.72 1.68 1.71 1.61 1.70 1.63
~2R85/~2R1"
1i q u i d s
dodecane tetradecane hexadecane
cetyl bromide solids
hexadecane octadecane cetyl bromide cetyl alcohol methyl palmitate palmitic acid, crystal I palmitic acid, crystal I1
ua(CH2),
cm-
cetyl bromide
1465 (5.5)
Frequencies in cm-l. Peak heights are given in parenthesis.
The unnormalized and normalized Z8gs/Zl,, ratios for the solids hexadecane, octadecane, and cetyl bromide are compared (Table I). The relative range of values narrows for the normalized ratios (20% of the lowest value) compared to that for the unnormalized ratios (35% of the lowest value). The normalization removes much of the variance in ratios among the solid-state molecules also. Because the 895-cm-I bandwidth is approximately equal to the 1300-cm-] bandwidth for each molecule (Table I), the peak height ratios provide good values for the integrated intensity ratios. When the normalization using the number of methylene groups is performed, the resultant ratios are from 3.0 to 5.3 for those molecules in the solid state, except methyl palmitate. The normalized ratio for methyl palmitate is 9.3, approximately double the ratio that exists for most other solid-state molecules. The Z895/Z1300 ratios for solid hexadecane and octadecane average about 4.4. This value amounts to 2.2 per terminal CH2CH3 group. Therefore, the value of 9.3 for methyl palmitate should consist of contributions of about 2.2 from the CH2CH3 terminus and about 7.1 from the acyl terminus. The results indicate that the Raman 895-cm-' band intensity is due almost entirely to terminal C-C stretching and that the Raman-active acyl terminus C-C stretching is over three times as intense as the Raman-active methyl terminus C-C stretching. Hydrocarbon Chain Packing. Those bands which are sensitive to chain packing were examined for the possibility of any effect of crystal type on the 895-cm-I band parameters of the solids. Therefore, the C H stretch (2800-3100 cm-I), skeletal C-C stretch (1 050-1 150 cm-I), and the CH, bending (1400-1 500 cm-l) regions were also studied. The types of chain packing observed for long-chain alkanes are conveniently divided into two spectroscopic categories: (1) those with one chain per unit cell, which include base-centered triclinic packing and base-centered hexagonal packing, and (2) those with two chains per unit cell, which include base-centered monoclinic and base-centered orthorhombic packing. The Raman CH2 bending region at 1400-1500 cm-' can be used to distinguish between these two categories. Those solids in states with triclinic or hexagonal packing in the solid state produce two strong bands (about 1455 and 1465 cm-I) in the CH2 bend region. Those solids in states with monoclinic or orthorhombic packing produce a third strong band at about 1420-1430 cm-1.536 The CH, bending mode Raman band frequencies and relative peak heights are given in Table 11. Though cetyl bromide (solid) packing is orthorhombic or monoclinic, producing a strong 1427-cm-' band, the packing of hexadecane and octadecane are quite different, producing no 1420-cm-I band. Yet all three alkanes have about the same normalized 1895/113oo ratio (4.8, 4.6, and 4.1, respectively). Therefore, the crystal lattice packing type is not responsible for the large difference in the 895-cm-' C,-C, stretch intensities observed for methyl palmitate and unsubstituted alkanes. (5) Snyder, R.G.J . Mol. Spectrosc. 1961, 7, 116. (6) Boerio, F. J.; Koenig, J . L. J . Chem. Phys. 1970,52, 3405-3431.
solids hexadecane, 10 "C octadecane cetyl bromide, 10 OC cetyl alcohol methyl palmitate palmitic acid
uSymbols: u,(CH,), antisymmetric CH, stretch; us(CH2),symmetric CH, stretch.
Two different samples of crystalline palmitic acid were also compared. Crystal I1 produce spectra which contain signs of a higher degree of crystallinity than those of crystal I. In crystal I1 spectra, the third CH2 bending mode at about 1460 cm-' is split into a doublet at 1455 and 1465 cm-I. This does not occur for crystal I. (See Table 11.) In addition, bandwidths are more narrow in the spectra of crystal 11. (See Table I.) The Raman-active antisymmetric C H 2 stretch band at 2882 cm-' is sharper, with a greater peak height, for crystal 11. The intensity of the 895-cm-' C2-C1 stretch band for crystal I1 is greater, so that the normalized 1895/11300 ratio for crystal I1 is 5.3 while that of crystal I is 4.3. Therefore, a greater degree of crystallinity does appear to enhance the C2-C1 stretch intensity of a substituted polar alkane, by about 23% in this case. However, the much greater intensity of the C,-C, stretch in solid methyl palmitate compared to the unsubstituted alkanes hexecane and octadecane must clearly be attributed to the nature of the end group substituent. Hydrocarbon Chain Conformation. The Raman CH, stretch region of solid alkanes contains two predominant sharp bands at about 2850 and 2885 cm-' assigned to a symmetric CH, stretch mode and an antisymmetric CH, stretch mode, respectively, on the basis of polarization measurements. The region between 2840 and 2900 cm-' may contain more than one band due to backbone symmetric CH, stretch and may indeed contain a broad background due to polarized symmetric stretching.l The dominant C H 2 symmetric stretch at about 2850 cm-I varies in band shape with chain packing.l Two contributions to the 2885-cm-I antisymmetric CH2 stretch peak height vary with chain packing: (1) the height of the underlying broad background attributed to the symmetric CH, stretch bands' and (2) the width of the 2885-cm-I band. Therefore, the peak height ratio 1 2 8 8 5 / 1 2 8 5 0 is sensitive to chain packing type. The 2885-cm-' antisymmetric CH, stretch band also varies with tightness of chain packing, even within one type crystal packing (Bicknell-Brown and Roach, unpublished results for dimyristoylph~sphatidylglycerol~). In Table I11 the frequencies of the two major methylene stretches for the hydrocarbons used in this study are shown, along with their respective peak heights and the intensity ratio of the CH, antisymmetric stretch (2880 cm-]) to the symmetric stretch (2850 cm-I). The hydrocarbons fall into two distinct groups. The solids have intensity ratios between 1.61 and 1.72, with sharp 2885-cm-' bands. The C H stretch regions for the solids are shown in Figure 3. The higher ratios of the solids indicate well-ordered, tightly packed structures. There is relatively little variance in the ratios, indicating that no significant difference in packing tightness occurs. The 12890/12850 ratios for the liquids lie within the range 1.39-1.48 (Table 111). The CH, symmetric stretch bands for the liquid states studied are relatively invariant from molecule to molecule. The 2890-cm-I Fermi resonance band shape for the liquids is now dependent on chain conformation, gauche-trans ( 7 ) Bicknell-Brown, E.; Roach, T., manuscript in preparation.
The Journal of Physical Chemistry, Vol. 91, No. 12, 1987 3439
Raman Spectra of Alkanes and Lipids
A
2800
3000
2900
FREO [cm-li
FREQ icm-’1
Figure 3. The Raman C H stretch region for solids: (a) hexadecane, (b) octadecane, (c) cetyl bromide, (d) cetyl hydroxide, (e) palmitic acid, (f) methyl palmitate.
Figure 5. The Raman chain C-C stretch region for solids: (a) hexadecane, (b) octadecane, (c) cetyl bromide, (d) cetyl hydroxide, (e) palmitic acid, (f) methyl palmitate.
I
I
1010
1050
1
I
I
1090
1130
1170
FREO~ c m - ’ ~ I
I
I
I
I
2800
2850
2900
2950
3000
FREQ( c m - ‘ ~
Figure 6. The chain C-C stretch region for (a) dodecane, (b) tetradecane, (c) hexadecane, (d) cetyl bromide.
Figure 4. The CH stretch region for liquids; (a) dodecane, (b) tetradecane, (c) hexadecane, (d) cetyl bromide.
rotamerization kinetics, as well as interchain interaction^.^^^ The 2890-cm-I peak height is strong, though broadened, for the liquid state compared to the same band observed for lipids in aqueous dispersion in the liquid crystalline state. The symmetric CH2 stretch at 2850 cm-’ and the broad distribution of polarized bands underlying the 2890-cm-l band do not differ significantly in height and shape for the liquid alkanes and “melted” phosphatidylcholines. The large I2gS0/12g50 ratio of about 1.4 for the liquid alkanes compared to about 1.0 for liquid crystalline dipalmitoylphosphatidylcholine is due to differences in intensity of the 2890-cm-I band. The range of values for Z2890/Z2850for the liquid alkanes is from 1.39 to 1.48, showing consistency within this group. The main chain C-C stretching region from 1050 to 1 150 cm-’ has been shown to be sensitive to the relative amounts of gauche and trans conformers present in the hydrocarbon chain^.^ The (8) Snyder, R. G.; Scherr, J. R. J . Chem. Phys. 1979, 71, 3221-3228. (9) Snyder, R. G.J . Chem. Phys. 1979, 71, 3229-3235.
1000
I
1100
FREO icm-‘)
I
I
I
I
I
I
I
2800 2900 3000 3100 .
Figure 7. The chain C-C stretch and C H stretch regions for anhydrous
DPPC.
presence of a strong central band at approximately 1083 cm-l is due to randomization (interruption of all-trans conformation) is the chain. A band at about 11 30 cm-I a n d a band at about 1070
3440 The Journal of Physical Chemistry, Vol. 91, No. 12, 1987
Brown et al.
TABLE I V Frequencies and Intensities (Peak Heights, arbitrary units) of the Main Bands in the Skeletal C-C Stretching Region (1050-1150 cm-') molecule liquids dodecane tetradecane hexadecane cetyl bromide solids hexadecane octadecane cetyl bromide cetyl alcohol palmitic acid
u, cm-I
11070
1071 1071 1066
49 45 51
1064 1066 1069 1070 1066
79 38 51 69 47
u,
cm-I
Iinss
u,
cm-I
Iiiin
Iin8JIiim
>IO 2.4 2.4 >10
1085 1085 1083 1085
56 59 52 64
1138 1135
-0 25 22 -0
1107 1 I04 1 IO7
0 0 24 19 17
1139 1 I39 1136 1137 1135
103 43 54 58 43
cm-I are assigned to all-trans segments in the hydrocarbon chain.Isi0 The ratio of the peak height of the band at 1135 cm-' to the band at approximately 1080 cm-l is used to obtain information about the relative amounts of all-trans segments and gauche conformers present in the ~ h a i n s , ~ and J I hence, the extent of chain order. Figures 5 and 6 show the skeletal C-C stretch regions for the solids and the liquids, respectively. In Table IV the frequencies, peak heights, and relative intensities of the bands in the main chain C-C stretch region are given. The middle band of the C-C stretch triplet is observed as a strong broad band at about 1085 cm-I in the liquids, indicating a high degree of gauche rotamers present. Both "all-trans segment" bands at 1070 and 1135 cm-' are visible at low intensity in tetradecane and hexadecane, the longer chain unsubstituted hydrocarbons. The peak height ratios Zlogs/Z1 135, thought to be a measure of the gauche/trans segment ratio, are consistent at 2.4 for tetradecane and hexadecane. Those for dodecane and cetyl bromide could not be measured accurately because the all-trans-segment bands were so weak. The ratio of the 1085-cm-' peak height to the 1300-cm-' twisting mode peak height falls within the range 0.63-0.67 for all four liquids. Because the relative intensity of the 1085-cm-' band, assigned to gauche C-C rotamers, is about equal for all the liquids while the alltrans-segment 1 130-cm-' band peak height varies considerably, we conclude that the gauche C-C rotamer content is about equal for all four liquid alkanes, but the distribution of gauche rotamers differs. The distribution must be more random in the dodecane and cetyl bromide liquids thereby decreasing the all-trans-segment population and decreasing the intensity at 1130 cm-I, thereby producing a much higher Iloss/Zl 130 ratio. We conclude that within the group of liquid alkanes or within the group of solid alkanes, the C H stretch vibrations, CH2 bending vibrations, and C-C stretching vibrations show that some differences in packing exist but that these differences do not produce large difference in intensity in the 890-cm-I stretch. I3C1-LabeledPhosphatidylcholine. The assignment of the C2-C, stretch is most definitively studied by a carbon-13 label in the C2-Cl group. For Raman studies of diacyl phospholipids, it is of particular interest to determine whether the C2-Cl stretch mode differs for the two acyl chains, which have been shown to have different configurations. For this study, dipalmitoylphosphatidylcholine (DPPC) labeled at the acyl linkage in the sn-2 chain only was used. The configuration of the DPPC molecule a n d the position of t h e 13C label a t C1 of chain 2 are shown in Figure 8. Both labeled DPPPC (sn-2-l3C1)and unlabeled DPPC were recrystallized by slowly dropping solvent temperature from 25 to 18 'C, as described in the Methods section. Examination of head-group Raman bands between 800 and 1000 cm-' for a number of crystalline DPPC samples prepared has revealed that crystal may be prepared with one of two possible head-group conformations or with equal population of the two head-group conformations. (Bicknell-Brown and Brown, manuscript in preparation). The head-group bands for the two crystal samples (labeled and unlabeled) to be compared in this study (IO) Tasumi, M.; Shimanouchi, T. J . Mol. Spectrosc. 1962, 9, 261. ( 1 1 ) Lippert, J. L.; Peticolas, W. L. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 1572-1 576.
0 0 0.44 0.33 0.40
Figure 8. Configuration of the DPPC molecule assumed from X-ray diffraction study of DLPE of ref 12: (*), the I3C label at C , of the sn-2-chain; (6+), the acyl C2-C, stretch modes.
s 00
850
900
( c m -1)
Figure 9. Raman spectra from 800 to 900 cm-' for (a) unlabeled crystalline DPPC and (b) crystalline D P P C - S ~ - ~ - ~ ~ C , .
showed that both samples contain a single head-group conformation is identical for the two samples. Therefore, differences in the Raman spectra in the 800-900-cm-' region for the two DPPC samples are due to the isotope effect only and not to differences in head-group geometry. The Raman spectra from 800 to 900 cm-I for crystalline DPPC and crystalline DPPC-sn-2-l3C1 are compared in Figure 9, a and b. In the spectrum of unlabeled DPPC (Figure 9a), two relatively strong bands are observed at 892 and 877 cm-l, and a weaker band is observed at 844 cm-I. The bandwidths of the 892- and 877-cm-' bands, given in Table V, are both 11.7 cm-'. The peak height of the 877-cm-l band is 1.5 times that of the 892-cm-I band. The
The Journal of Physical Chemistry, Vol. 91, No. 12, 1987
Raman Spectra of Alkanes and Lipids TABLE V Comparison of the 800-900-cm-' Raman Spectra of Crystalline DPPC and D P P C - S ~ - ~ - ' % ~ "
comwund unlabeled DPPC, cryst
labeled DPPC-sn-2I T I , cryst
assignment gauche C2-Cl st,
+ C,N+ def
trans C2-Cl st gauche C2-C, st (sn-2-chain) C,N
+
def trans C2-C1,st (sn-1 chain)
freq, bandwidth, re1 peak (em-,) cm-' height 876 11.7 15 11.7 14.3
-
876
17.1
891
14.3
-10 10
892
859
10
10
"Peak heights are relative to that of the trans C2-C, stretch (891 cm-I), which was set equal to 10. 892-cm-' band has been assigned to the acyl C2-C1 stretch for an ordered acyl interface2because this band decreases in relative peak height as the lipid melts. The 876-cm-' Raman band for phosphatidylcholines has been assigned to two frequency-coincident bands, the choline C4N+deformation band (because the 876-cm-' band is stronger for phosphatidylcholine than for phosphatidylethanolamine) and the acyl C2-C1stretch for the disordered acyl interface (because this band increases in relative peak height as di- and triglycerides and phospholipids melt. The Raman spectrum from 800 to 900 cm-' for the crystallized labeled DPPC-sn-2-l3C1is shown in Figure 9b. The peak frequencies and relative heights are presented in Table V. Upon comparing the spectrum of the labeled DPPC to that of the unlabeled DPPC, we observe that the 876-cm-' band is reduced considerably in relative intensity, while a new band appears at 859 cm-I. Clearly, the new band is due to a shift of the sn-2 acyl C2-CI stretch from 876 to 859 cm-I, brought about by the increased reduced mass of the I3C2Clgroup. The remaining band at 876 cm-' is assigned to the choline C4N+deformation. The bandwidths of the 891- and 859-cm-' bands in the labeled DPPC are both 14.3 cm-I, while the remaining band at 876 cm-' has bandwidth 17.1 cm-'. The similarity in width and height of the bands at 891 and 859 cm-' corroborates the assignment of both bands to acyl C2-C, stretches, the 891-cm-' band to the sn-1 chain acyl C2-Cl stretch, and the 859 cm-' band to the sn-2 chain acyl C2-CI stretch. Furthermore, the sn-2 and sn- 1 chain C2-C, stretch frequencies are totally distinct in both the solid and gel phases, which suggests that these bands can be very important in monitoring the acyl region of spectroscopically distinct chains. Therefore, we now assign the sn-2 chain acyl C2-C1 stretch in well-ordered phospholipids to the 876-cm-' Raman band and the sn-1 chain acyl C2-Cl stretch to the 891-cm-I Raman band. X-ray crystallographic studies of dilaurylphosphatidylethanolamine show that the rotameric configuration is trans about the sn-1 Cl-C2bond and gauche about the sn-2 CI-C2bond. The frequency difference between the C2-C1 stretches for the two chains may be due to their differences in C2-C1 dihedral angle. This conclusion is borne out by chain melting studies. As phospholipids undergo the Raman chain melting transition, the 892-cm-l peak height decreases as the 876-cm-l peak height increases (Bicknell-Brown and Brown, unpublished results). We now attribute this initial change in relative peak intensities at 892 and 876 cm-l to trans-gauche isomerization about the sn-1 acyl C2-Cl bond. The 876-cm-' band increases in bandwidth as the band increases in height. A bandwidth increase is to be expected with motional freedom at the acyl interface; that is, disorder at the interface. Therefore, we conclude that a sharp 876-cm-' band indicates the presence of a well-defined, ordered gauche rotamer at the acyl C2-C, bond, while a sharp 892-cm-' band indicates the presence of a well-defined, ordered trans rotamer at the acyl C,-C, bond. We also conclude that a broadened 876-cm-' band is due to C2-C, rotamers which have achieved rotational freedom, with a preferred gauche configuration, as in a melted acyl region. Further corroboration of this interpretation is obtained from the Raman
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spectra of dipalmitoylphosphatidylethanolamine in the solid (anhydrous) and gel (hydrated) phases which contain both bands at approximately equal intensity,*the 876-cm-l band of the gauche rotamer sn-2 C2-C1 stretch and the 892-cm-' band of the trans rotamer sn-1 C2-CI stretch. Gauche-Trans Isomerization at the C2-Cl Bond. The room temperature Raman spectra of alkanes and substituted alkanes in the liquid state shown in Figure 2 contain broadened bands at both 870 and 890 cm-'. We now assign the broadened 870-cm-l band to gauche chain end C-C rotamer states with motional freedom and the broadened 890-cm-' band to trans chain end C-C rotamers with motional freedom. The presence of both bands indicates that there is residual order in the liquid states, at least at the chain ends, because both the lower energy trans rotamer state and the higher energy gauche rotamer state are abundantly populated. The intensity ratio of the 870- and 890-cm-' bands should provide an estimate of the gaucheltrans population ratio and one means, along with the bandwidths, to monitor the degree of disorder in the liquid state. This principle has previously been applied to measurements of disorder and motional freedom in the acyl interface region of phospholipids,2 although the band assignments were not as clearly defined as in the present work.
Discussion Assignment of the 890- and 875-cm-' Bands. The normalcoordinate analysis of hydrocarbons by Snyder and Schachtschneider' led to the conclusion that the coordinate for the 890-cm-' mode consists of a methyl rocking mode, with contribution from the C-C stretching motion for the terminal C-C bond. The experimental studies described herein support the assignment of the Raman band at 890-895 cm-' virtually entirely to the chain end C-C stretching motion. The intensity ratio I895/ZI3m is significantly greater for methyl palmitate and slightly greater for palmitic acid crystal I1 than for any of the other hydrocarbons in this study. In addition the frequency of the terminal mode occurs at 891 cm-l which is lower frequency than for the other solids. The frequencies for the other substituted hydrocarbons are essentially equivalent and about that for the unsubstituted hydrocarbons. In summary, two significant conclusions are reached which are necessary to use for evaluating chain order and chain terminus order in substituted hydrocarbons. First, the 895-cm-' Raman intensity is due virtually entirely to the terminal C-C stretches. Second, for some substituents, such as the ester functional group, the contribution to the total intensity may differ for the two ends. Acyl terminal ends contribute 3 times the intensity to the Raman C2-CI stretch band as do methyl terminal ends. The 895-cm-' band broadens and shifts to lower intensity as motional freedom increases. Therefore, when monitoring this band in long esters (e.g., lipids), one must recognize that the large majority of the intensity is due to the acyl terminus C2-Cl stretch, with a much smaller contribution from the methyl terminus C-C stretch. The comparison of the sn-2-13C1DPPC spectrum with the DPPC spectrum for crystallized samples with a single identical head group population showed that the sn-2 chain acyl C2-C1 stretch for solid DPPC occurs at 876 cm-l while the sn-1 chain acyl C2-C, stretch occurs at 892 cm-'. The difference in C2-C, stretch frequencies for the two chains can be associated with conformational differences at the acyl linkages in the two chains, which are gauche at the C2-C,linkage for the sn-2 chain and trans at the C2-C1 linkage for the sn-1 chain.I2 Therefore, the results indicate the C2-C1 stretch bands may be very useful for monitoring chain end conformation and motional freedom. The 876-cm-' C2Cl stretch observed in the liquid alkane and phospholipid spectra is assigned to gauche C2-C1 rotamers. The 892-cm-I band, which we assign to trans C2-CI rotamers, is present in the Raman spectra of alkanes and lipids in both the (12) Hitchcock, P. B.; Mason, R.; Thomas, K. M.; G. Shipley Proc. Narl. Acad Sci. U.S.A. 1974, 71, 3036-3040. (13) Snyder, R. G.; Maroncelli, M.; Strauss, H. L. Am. Chem. SOC.1983, 105, 133.
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The Journal of Physical Chemistry, Vol. 91, No. 12, 1987
solid and liquid states. This result shows that the Raman spectra can be used to observe the instantaneous distribution of gauche and trans C2-Cl rotamers, and also shows that both rotamers exist with significant populations in the liquid state. Distribution of Gauche Rotamers in Hydrocarbons and Phospholipids in the "Melted" State. The backbone methylene C-C stretch region for the liquid alkanes and liquid substituted alkanes showed very little intensity or no measurable intensity remaining in the 1060- and 1130-cm-' bands, which have been assigned to all-trans segments of hydrocarbon chains.' It has been pointed O U ~ ' ~ that . ' ~ the decreases in intensity of the 1130-cm-l band with increase in gauche bond concentration is nonlinear. The interruption of an all-trans segment by a few gauche bonds may effect a large decrease in intensity of the C-C stretching band at 1130 cm-I. This interpretation is upheld by comparison of the skeletal C-C stretch region with the C2-Cl stretch region (870-890 cm-l) and the CH stretch region (2800-3100 cm-I) for the liquids. The C2-Cl stretch region for the liquids showed intensity in both the gauche rotamer band at 870 cm-l and the trans rotamer band at 890 cm-l. The C H stretch region showed relatively strong and broadened intensity in the Fermi-resonance-enhanced Ramanactive CH2 stretch at 2885 cm-I. The broadening of the 2885-cm-' band indicates motional freedom in the backbone. However, the height of the 2885-cm-' band, which is attributed to the population of trans rotamers in the chain and to lateral chain interaction, is considerably greater than that observed for liquid crystalline phospholipids. Therefore, the CH stretch region indicates that a significant population of trans bonds does exist for the liquid alkanes. The C-C stretch intensity at 1130 cm-' is greatly reduced in peak height and intensity, showing that its response to the introduction of a mixed gauche-bond/trans-bond population is less linear and more sudden than is that of the C2-Cl stretch bands and the C H stretch bands. The C H stretch region and backbone C-C stretch region for liquid hexadecane and anhydrous DPPC in the melted chain state can be compared by using Figures 4c, 6c, and 7. Though the height of the 2885-cm-' band is greater for the liquid alkane compared to the anisotropically melted DPPC, the backbone C-C stretch regions are quite similar. This apparent discrepancy might be explained by a difference in distribution of gauche and trans rotamers in the two systems. The acyl regio nof phospholipids is the most rigid portion of these molecules, while the methyl chain ends and the head group have the greatest degree of motion. Therefore, a model for the introduction of gauche rotamers into (14) Snyder, R. G.; Cameron, D. G.; Casal, H. L.; Compton, D. A. C.f Mantsch, H. H. Biochim. Biophys. Acta 1982, 684, 11 1-1 16. (15) Pink, D. A,; Green, T. J.; Chapman, D. Biochemistry 1980, 19, 349-356.
Brown et al. phospholipids would include the premise that statistically the gauche rotamer population would be more concentrated at the methyl ends of the chains, while the trans rotater population would be more concentrated at the acyl ends of the molecule. Thus, all-trans segments may exist at the acyl ends while gauche rotamers form a significant population at the methyl ends. The relative intensities of the "gauche" 1085-cm-I backbone C-C stretch and the all-trans 1130-cm-' backbone C-C stretch would therefore closely reflect the gauche/trans rotamer ratio for this case, because the gauche rotamers are concentrated at the methyl end of the acyl chains. Therefore, the decrease in intensity of the 2885-cm-' va(CH2)stretch for the phospholipid occurs with the decrease in all-trans CC stretch intensity at 1130 cm-l, because both intensities reflect the trans rotamer content for this model of the phospholipid chain conformation. It has been shown that the decrease in intensity of the 1 130-cm-] all-trans segment C-C stretch and rise of the "gauche" C-C stretch at 1100 cm-' parallels the decrease in the 2885-cm-' peak height with temperature in a melting study of anhydrous DPPC. If gauche rotamers are introduced at the chain methyl end and ascend the chain with increasing temperature, then there would be parallel rates of decrease in trans rotamers and all-trans segments. Then parallel temperature behavior of the chain C-C stretch bands and C H stretch bands would be observed even though the former depend on the population of all-trans segments while the latter depend on trans rotamer population. The above interpretation of the dependence of backbone stretch intensities on conformation leads to the conclusion that, for liquid alkanes, in contrast to phospholipids the gauche C-C rotamers must be more randomly distributed along the length of the backbone, more effectively disrupting all-trans segments. Therefore, for alkanes the all-trans segment band at 1130 cm-' would disappear more quickly as the number of gauche rotamers increases. The relative intensities of the trans terminal C C stretch and gauche terminal C C stretch are comparable for the liquid alkanes, reinforcing a model in which gauche rotamers are distributed over the length of these longer chain alkanes. Snyder and co-workers found using IR studies of selectively deuteriated methylene groups that gauche C C rotamers in alkanes are more concentrated a t the chain ends than in the chain center.I6 The results in the present communication show that this gauche conformer gradient along the chain is much greater for phospholipids in bilayers than for liquid alkanes.
Acknowledgment. This work was supported in part by N I H grant GM-27744. (E.B, M.L). (16) Maroncelli, M.; Straws, H. L.; Snyder, R. G. J . Phys. Chem. 1985,
89. 4390-4395.
