J. Phys. Chem. 1994, 98, 12191-12197
12191
Contribution of the Intermolecular Coupling and Librotorsional Mobility in the Methylene Stretching Modes in the Infrared Spectra of Acyl Chains V. Ramana Kodati,? Rachida El-Jastimi, and Michel Lafleur. D fpartement de Chimie, Universitf de Montr&al, Montr&al, Qufbec H3C 3J7, Canada
Received: June 20, 1994; In Final Form: September 13, 1994@
Methylene stretching modes are often used to probe the order of polymethylene chains in condensed material including lipids and polymers. The shifts of the bands related to these vibrations are generally interpreted in terms of conformational order. In this paper, we show that considerable shifts of the methylene stretching bands (for both hydrogenated and deuterated species) can be induced without variation of the conformational order. First, the results indicate that isotopic dilution affects the frequencies of the C-H stretching bands while the C-D stretching bands are less sensitive. This effect is more pronounced in systems for which interchain interactions are important such as lipids in the ordered gel phase. The origin of these shifts is attributed to a change in Fermi resonance caused by the variation in intermolecular coupling. Second, librotorsional motions also affect the methylene stretching modes. Shifts of about 2.5 cm-' are observed for the C-H stretching modes when hexadecane trapped into urea clathrate is warmed from -150 to 25 "C. Using deuterated hexadecane, a comparable change is observed for the antisymmetric C-D stretching mode whereas the frequency of the symmetric mode is temperature insensitive. The coupling between the stretching modes and the librotorsional modes of the carbon skeleton is proposed to be at the origin of these shifts. Bandwidths of the methylene stretchings appear to be essentially sensitive to intramolecular factors. Finally, as a side aspect, it is found that the method for conformational analysis based on the CH2 wagging bands is not sensitive to intermolecular coupling, confirming the reliability of this method. In conclusion, a warning about the interpretation of shifts of the methylene stretching bands exclusively in terms of conformational order is stated.
Introduction For a better understanding of the structural behavior of condensed material including polymers and lipids, the description of conformational order in polymethylene chains is an important key. Infrared spectroscopy has been broadly used for the characterization of chain order since several vibrations of the methylene groups constitute intrinsic probes. The frequencies and widths of the methylene C-H stretching (VC-H) bands of lipid acyl chains as measured on the IR spectra are often used to probe chain order (for a recent review, see ref 1). One of the major applications of lipid IR spectroscopy has been to determine gel-to-liquid crystalline phase transitions. Briefly, lipids undergo a transition implying the introduction of gauche conformers in the acyl chains and the increase of diffusion constants of the lipid molecules. During these transitions, the frequencies of the VC-H bands shift upward by about 3-4 cm-1.2 Similar experiments can be carried out with deuterated lipids looking at the C-D stretching (VC-D) vibration^.^^^ Generally, band shifts are small but thoroughly consistent within the same phase. In order to link the spectral information to molecular description, empirical relationships have been proposed. Shifts in frequencies have been proposed to be related to conformational order or, in other words, to the population of gauche conformers in the acyl chains whereas bandwidths have been associated with dynamic aspect^.^^^ In order to get a deeper understanding of fundamental membrane properties, a detailed description of chain order is required. The major problem in a detailed analysis of frequency and bandwidth changes is that several factors such as intermolecular vibrational coupling,
* To whom correspondence should be addressed. Present address: Department of Chemistry, University of California, Riverside, CA 92521. Abstract published in Advance ACS Abstracts, November 1, 1994. @
0022-365419412098-12191$04.5010
trans-gauche isomerization, and twisting motion along of the long axis of the chain may influence the frequencies of the methylene stretching modes. It is therefore essential to know the possible contributions of the different phenomena to spectral changes. Recently, a linear relationship between the 2H NMR order parameters and the infrared frequency shifts of VC-H bands of acyl chains has been observed in the liquid crystalline state of lipid systems.6 This correlation has provided experimental support to the empirical correlation between the frequency of the VC-H bands and conformational order. In this paper, we relate our attempts at getting a better molecular interpretation of the shifts of CH2 vibrational bands in the FTIR spectra of phospholipid bilayers by considering two other possible contributions: the intermolecular coupling and the librotorsional motion. First, the isolation of n-alkanes in a matrix of their deuterated analogues constitutes a straightforward experiment to highlight the contribution of interchain coupling in the VC-H and VC-D regions. The coordinates of methylene stretching vibrations on adjacent chains can couple with one another if their frequencies match. In the presence of deuterated analogue, the isotopic effect shifts the VC-D modes to different frequencies, preventing the intermolecular coupling with the neighboring C-H groups. The deuteration, which has a drastic effect from the vibrational spectroscopy point of view, does not alter the chemical properties significantly. Electron diffraction and calorimetric studies on the solutions of n-paraffins and their deuterated analogues with identical chain lengths have shown that they form ideal mixtures and that the intermolecular dispersion forces for the two types of chains are ~ i m i l a r .It~ is then possible by isotopic dilution to restrict the intermolecular vibrational coupling without altering significantly the conformational order and the acyl chain environment. This experiment has been 0 1994 American Chemical Society
Kodati et al.
12192 J. Phys. Chem., Vol. 98, No. 47, 1994 already performed in Raman spectroscopy to achieve a more quantitative interpretation of the YC-H region.8 The results showed a significant influence of intermolecular coupling on the Raman bands. In this paper, the interchain coupling experiments have been conducted in infrared spectroscopy, using n-alkanes and phospholipids. Second, the rotational diffusion of a chain along its long axis may be accompanied by a torsion of the chain. This motion leads to a departure from the planar zigzag all-trans geometry and introduces a change in torsion angles between adjacent methylene groups. This motion, generally referred as librotorsion, has been shown to couple with the YC-H m o d e ~ . ~InJ ~ order to investigate the influence of this motion on the methylene stretching modes, the infrared spectra of alkanes trapped in urea clathrates have been examined. Urea clathrates of hydrocarbons have been largely studied by spectroscopic techniques since they allow to work in a unique environment: the hydrocarbon chains trapped in the long channels formed by urea are isolated from one another, and they are expected to be in all-tran! configuration, owing to the small diameter (around 5.25 A) of these channels. Changes in temperature modulate the librotorsional mobility of the chain without introducing conformational disorder since it has been demonstrated that the concentration of gauche conformers for these trapped chains remains below 3%.'33'4 Although there are a number of vibrational spectroscopic studies on the urea inclusion compounds of n-alkanes, no results have been published on the methylene stretching frequencies. In this paper, we present an infrared study on hexadecane (hydrogenated and perdeuterated) in urea clathrate in the methylene stretching region in order to characterize the effect of twisting rotational motion on the methylene stretching bands. A similar investigation using Raman spectroscopy has shown a significant contribution of twisting-rotation motion in parameters used by this technique to estimate acyl chain order.15 More recently, CH2 wagging vibrations have been used to characterize the conformational order of lipid acyl chains. Based on extensive theoretical and experimental work,16 the bands at 1368,1352, and 1344 cm-I have been assigned to kink (GTG), double gauche (GG), and end gauche (TG) conformations, respectively. Distinct bands associated with the different conformers can be observed since the IR time scale is shorter than the transtgauche isomerization rate. These bands have been used to estimate the conformational defects quantitatively since the absorptivity coefficients (relative to that of the CH3 umbrella band) have been determined by comparing the area of heptadecane bands with the calculated conformer concentrations obtained from Flory's rotational isomeric state m0de1.l~ This method has been already applied to several system^.'^-'^ Using the spectra collected for the present investigation, we thought it would be of interest to verify whether the conformational description of alkanes shows any dependence on the intermolecular coupling, modulated by isotopic dilution.
Experimental Section Hexadecane and decane were purchased from Sigma Chemical Co. (St. Louis, MO) and Aldrich Chemical Co. (Milwaukee, WI), respectively. Hexadecane-d34 and decane-d22 with a minimum isotopic purity of 99% were obtained from MSD isotopes (MontrCal, QuCbec). 1,2-Dipalmitoyl-sn-glycero-3phosphocholine (DPPC) and 1,2-bis(perdeuteriopalmitoyl)-snglycero-3-phosphocholine (DPPC-d62) were purchased from Avanti Polar Lipids (Birmingham, AL). Urea was purchased from Mallinckrodt Inc., (Paris, KY). Urea clathrate containing hexadecane was prepared according to the procedure given in
ref 20. Hexadecane diluted in decahydronaphthalene was mixed with the urea dissolved in excess of methanol. The precipitate was washed with isopentane and dried under vacuum overnight. The samples for FTIR were prepared by thoroughly mixing the inclusion compound with potassium bromide and by pressing the mixture into small disks. In order to control that the pellet making process did not damage the inclusion compound, the Raman spectra of the compound before and after pellet making were compared. Both spectra showed the strong C-N stretching band at 1024 cm-' and the NCO skeletal bands at 532 and 61 1 cm-' characteristic of channel forming urea in hexagonal symmetry.21 The temperature of the sample was varied from low to high using a Graseby-Specac automatic temperature controller. For isotopic dilution studies, hexadecane and hexadecaned34 were mixed in different proportions such that the mole fraction of each varied from 0 to 1. In the case of lipid dispersions, stock solutions of DPPC and DPPC-d62 were prepared in benzene:methanol (955). Appropriate volumes were mixed to obtain the desired mole fraction. The lipid solution was lyophilized, and the solid lipids were hydrated with a 20 mM Hepes buffer containing 100 mM NaCl and 2 mM EDTA, pH = 7.4. The sample was pressed between two CaF2 windows and kept in a brass cell mount whose temperature was controlled with a homemade device.22 A Nicolet 5DXB FTIR spectrometer equipped with a DTGS detector or a BioRad FTS-25 spectrometer equipped with a medium band MCT detector was used to record the spectra with 2 cm-' resolution. A minimum of 200 scans were coadded to get the final spectrum. The centers of gravity and the widths of the bands were determined using algorithms proposed by Cameron et aLZ3 For the clathrate samples, a fourth-order polynome fitting the edge of the N-H stretching band of the urea was subtracted prior to the determination of frequencies and bandwidths of the V C - H bands. For lipid dispersions, a similar procedure was used to correct for the contribution of YO-H water band. The water association band near 2150 cm-' was eliminated by subtracting the buffer spectrum recorded at the same temperature, before the analysis of the VC-D region. The Raman spectra were recorded with a computerized Spex Model 1401 double monochromator equipped with a side-on photomultiplier tube (R928, Hamamatsu, Bridgewater, NJ), and the sample was excited with the 514.5 nm line of a Coherent Innova 90 argon ion laser.
Results Isotopic Dilution. The effect of intermolecular vibrational coupling on the methylene stretching vibrations has been monitored by the isotopic dilution of hexadecane and decane in their respective deuterated analogues. Figure 1 shows the spectra of mixtures of hexadecane and hexadecane-d34 in the liquid state at 25 "C in the C-H and C-D stretching mode regions. The intense bands at 2854 and 2926 cm-I are assigned to symmetric and antisymmetric VC-H methylene vibration^.^^ The corresponding bands in YC-D region are found at 2095 and 2198 ~ m - l . The ~ terminal methyl symmetric and asymmetric stretching vibrations are observed at 2873 and 2959 cm-' in h e ~ a d e c a n and e ~ ~at 2071 and 2215 cm-I in he~adecane-d34.~ The intensity of the band at 2890 cm-I appearing as a shoulder of the antisymmetric YC-H methylene band increases with increasing proportion of hexadecane-ds4. It is assigned to the VC-H mode of a H-C-D group,24which is an impurity of the deuterated alkane. For each composition, the centers of gravity and widths of symmetric and antisymmetric stretching modes of CH:! and CD2 regions have been determined and then plotted
J. Phys. Chem., Vol. 98, No. 47, 1994 12193
Methylene Stretching Modes in Acyl Chains
C-H stretching region
C-D stretching region
2926
1
2198
2854
I
2095
I
2300 Wovenumbers (cm-1)
2230
21'60
2071
2090
2020
Wavenumbers (cm-1)
Figure 1. C-H and C-D stretching regions of mixtures of hexadecane and deuterated hexadecane at different mole fractions. (The mole fraction (MF) is relative to hexadecane for the C-H stretching region and relative to deuterated hexadecane for the C-D stretching region.) The spectra are recorded at 25 OC.
against the mole fraction (Figure 2). For the alkane mixtures, the contribution of the deuterated analogue has been corrected in the UC-H region; it has been found to have only a small effect on the VC-H frequencies and widths (maximum of 0.09 cm-I). As can be seen from Figure 2A, the centers of gravity of symmetric and antisymmetric YC-H methylene bands increase with increasing dilution. The shifts are about +1.0 and f 1 . 9 cm-I for symmetric and antisymmetric VC-H bands, respectively, going from pure to dilute hexadecane (0.1 mole fraction). The centers of gravity of symmetric and antisymmetric VC-D bands show a much less pronounced variation upon the dilution of the perdeuterated alkanes in the perhydrogenated alkane matrix (Figure 2A'); for both bands, the increase is less than 0.4 cm-' going from pure to dilute hexadecane (0.1 mole fraction). The widths of the symmetric and antisymmetric YC-H (Figure 2B) and YC-D (Figure 2B') bands hardly change with dilution. The dependence of VC-H and VC-D on isotopic dilution has been characterized for a few systems: hexadecane, decane, and DPPC in the gel (20 "C) and in the liquid crystalline phase (60 "C) (Figure 3). Trends similar to those mentioned for hexadecane are observed. The isotopic dilution shifts both the symmetric and antisymmetric YC-H bands toward high frequencies while it has a very limited effect on the VC-D modes. The increase in frequency is more limited for decane: it is of about f0.7 and +1.5 cm-' for the symmetric and antisymmetric VC-H bands, respectively, going from pure to 0.1 mole fraction decane in decane-d2z. The most pronounced effect is observed for DPPC in the gel phase whose symmetric and antisymmetric YC-H bands are shifted by +1.0 and f 2 . 2 cm-l, respectively, going from pure DPPC to 25% DPPC in its deuterated analogue. Using the set of spectra obtained from the alkane mixtures, the CH2 wagging region (1300- 1400 cm-') has been analyzed in order to determine the conformational order in a quantitative manner using the method proposed in ref 17 and presented in the Introduction. The concentration of kink, double gauche, and end gauche conformers (Le., the number of conformers per
chain) was determined for the alkane mixtures. The conformer concentrations are plotted against the mole fraction of decane (Figure 4A) and hexadecane (Figure 4B) diluted in the deuterated analogue, in the liquid state, at 25 "C. As can be seen, the concentrations of the different conformers obtained by this method are not significantly different in the various isotopically diluted mixtures, reinforcing the reliability of the method. The conformational description of the alkane chains can be compared to that of tridecane obtained previ~usly'~-theirmeasurement has been done at 30 "C, but this should not affect the conformer proportion in an important manner. As expected, the total number of gauche conformers increases from decane to tridecane to hexadecane. This is due to the increase of the number of kinks (0.31 for decane to 0.95 for hexadecane) and double gauche conformers (0.24 for decane to 0.80 for hexadecane). This is in agreement with previous results indicating that kinks are found to be the most probable form of gauche defects in acyl chains of tridecane,17SDS micelles,17 and DLPC bilayers.ls The concentration of end gauche conformers does not appear to be chain length dependent for alkanes. Urea Clathrate. Urea clathrate has been used to study the modulation of rotational mobility and its effect on the methylene stretching bands. Figure 5A,B shows the effect of temperature on the centers of gravity and widths of the symmetric and antisymmetric VC-H methylene bands of hexadecane trapped into urea clathrate. The results obtained in the UC-D region with the deuterated hexadecane in clathrate are also shown (A' and B'). The symmetric and antisymmetric V C - H frequencies and bandwidths are all increasing monotonously with increase in temperature. The shifts of the VC-H bands observed from - 150 to 25 "C are of about f 2 . 8 and +2.5 cm-' for the symmetric and the antisymmetric mode, respectively. The bandwidths increase by about 4 cm-' over this temperature range. This broadening is in agreement with previous result^.^ In the YC-D region, the frequency of the antisymmetric stretching mode also increases with increased temperature (+1.9 cm-I). Interestingly,
Kodati et al.
12194 J. Phys. Chem., Vol. 98, No. 47, 1994 2667
3-
s
2664
.-
I
B
I
B
--
+$-'-I T
I
8
0.6
1.0
I
I 15
0.2
0.4
0.6
W f a c ( b n , C,,D,
Figure 2. Variation of the centers of gravity (A and A') and the widths measured at 0.75 height (B and B') of the methylene stretching modes as a function of isotopic dilution of hexadecane, at 25 "C. (A) and (B) represent the results in the C-H stretching region, and (A') and (B') represent the results in the C-D stretching region: (W) symmetric and (A)antisymmetric mode. The emor bars represent the standard deviation on three
samples. the symmetric VC-D does not change significantly over the entire range of temperature. Both bandwidths increase with temperature, as in the VC-H region.
Discussion
Our results show that the frequencies of the methylene VC-H modes of acyl chains are shifted toward higher values when the intermolecular vibrational coupling is restricted. This phenomenon has been observed for liquid decane and hexadecane, as well as for phospholipid bilayers in both gel and liquid crystalline phases. The shifts of the bands are attributed to a change in the interchain Fermi resonance between the VC-H fundamental (at about 2900 cm-') and binary combinations of CH2 bending modes (at about 1460 cm-') as described in ref 24. In the infrared spectra of extended polymethylene chains, Fermi resonance should involve essentially the symmetric stretching since its symmetry is favorable for an interaction with a combination of bending modes. The resonance gives rise to two broad secondary bands observed near 2922 and 2898 cm-I in the spectrum of crystalline n-paraffhZ4 These bands appear at frequencies close to the antisymmetricVC-H band (2920 cm-') which is, as a matter of fact, more affected than the symmetric stretching. It should be noted that, in the investigated systems, the acyl chains contain some gauche conformers which lower the symmetry and loosen up the symmetry requirements; a more complex coupling system may therefore take place. A perturbation of the Fermi resonance caused by the restriction of the interchain vibrational coupling is proposed to be at the origin of the frequency shift observed for the VC-H bands. Even though no model has been proposed to describe this effect quantita-
tively, it is important to state that changes in intermolecular coupling between acyl chains can lead to significant shifts of the VC-H bands. This phenomenon persists even in a disordered milieu with high mobility such as liquid decane. The magnitude of the observed shifts appears to be related to the strength of interchain coupling. The changes in frequencies associated with isotopic dilution are more pronounced for the gel phase lipids compared to the other disordered systems (fluid phase lipids and liquid alkanes). In the gel phase lipids, the strength of the interchain coupling is more important due to the high order of the acyl chains. (The important order is illustrated by the low frequencies of the VC-H modes.) This disruption of the coupling by the presence of deuterated species has consequently a more pronounced effect. It should be noted that phospholipids bear two chains, and in this case, it was not possible to isolate a single chain in a completely deuterated surrounding. Previous results obtained by Raman spectroscopy have also indicated that interchain coupling between liquid alkane molecules or lipid molecules affects the profile of the VC-H r e g i ~ n . * , ~ ~ . ~ ~ Intermolecular coupling does not affect the VC-H and VC-D vibrations to the same extent, the changes of VC-D methylene bands being much less pronounced. Less pronounced sensitivity of the VC-D region has been also reported by Raman spectroscopy; VC-D modes have been shown to be relatively insensitive to DPPC-d62 pretran~ition,~ and the changes of their relative peak intensities are limited during the gel-to-liquid crystalline phase transition of 2-[6,6-2H2]DMPC?6 The origin of these observationshas been proposed to be a different pattem of Fermi resonance for the VC-H and the VC-D regions, the role played by Fermi resonance in the VC-D region being restricted. The
J. Phys. Chem., Vol. 98, No. 47, I994 12195
Methylene Stretching Modes in Acyl Chains
A'
A 2828
1
+
v .......................................
3
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.
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. - . - . . . - I 20 40 Bo 80 100 Hydragonatedspecies, mol%
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X - X . .... -.x...........x........ , , # .......... K 4. ,.+..-.. ....+...........+.........,+.........
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2
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x x x
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t
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a
m
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1 9 1 20 40 Bo Bo 100 Deuteriated species, mol%
Figure 3. Variation of the center of gravity for the symmetric (A and B) and the antisymmetric (A' and B') methylene stretching modes as a function of isotopic dilution for ( x ) decane at 25 "C, (+) hexadecane at 25 "C, (A)DPPC at 60 "C, and).( DPPC at 20 "C. 1.2
I
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...........
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........ ........... .......' . . . A ....... . . . . . . . . . . . . .
"-0
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1.0
0.0
0.2
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Figure 4. Conformer concentration as a function of isotopic dilution for decane (A) and hexadecane (B), at 25 "C:).( gauche, (A)kinks. The error bars represents the standard deviation on three samples.
independence of the methylene VC-D mode frequencies on isotopic dilution is therefore in good agreement with the facts that Fermi resonance is proposed to be at the origin of the shift observed in the VC-H region on isotopic dilution and that this phenomenon plays a limited role in the VC-D region. The second phenomenon that has been investigated is the coupling between librotorsional motion and the methylene stretching vibrations. In urea clathrates, hexadecane remains all-trans, but changes in the torsional angles result in small twists that lead to methylene group bisectors slightly shifted one relative to the other. The changes in the VC-H region observed
1.0 end gauche, (0)double
as a function of temperature are essentially related to the thermal excitation of torsional modes.g During the temperature variation of hexadecane trapped in urea clathrates, a change of about f 2 cm-' is observed for the symmetric and antisymmetric VC-H and the antisymmetric VC-D modes, going from -150 to 25 "C. This intramolecular coupling (intermolecular coupling is excluded since the hexadecane molecules in the clathrate are isolated one from the other) influences the VC-H and VC-D vibrations in a similar fashion, except the frequency of the symmetric VC-D vibration which is relatively independent of temperature. These shifts are probably related to intramolecular
Kodati et al.
12196 J. Phys. Chem., Vol. 98, No. 47, 1994
1
A'
Temperatun ( O C )
8
6
14
1
3 E
6 w
# + . . . . . . . I
-150
-100
-50
0
50
T e m p e " ( OC )
Figure 5. Variation of the center of gravity (A and A') and the width measured at 0.85 height (B and B') of the methylene stretching modes as a function of temperature for hexadecane trapped in urea clathrate. (A) and (B) represent the results in the C-H stretching region, and (A') and (B') represent the results in the C-D stretching region: (m) symmetric and (A)antisymmetric mode.
coupling between the stretching modes and the librotorsional mode of the carbon skeleton as proposed to interpret similar changes observed in the Raman spectra of alkanes trapped in urea lathr rate.^.'^ Similar to our results, the shifts observed in the Raman spectra were different for the various modes: for hydrogenated alkanes in urea clathrate, the symmetric stretching was not significantly affected by temperature while the antisymmetric mode was shifted upward by about 4 cm-' going from -180 to 25 "C. Temperature variation of the deuterated analogue in perhydrotriphenylene crystals did not lead to frequency shift of the YC-D modes. The coupling between the stretching modes and librotorsional mobility is proposed to be modulated by the symmetry of the mode (to rationalize the difference between the symmetric and antisymmetric modes) and the amplitude of the motions (to rationalize the difference between the hydrogenated and the deuterated species). lo We conclude therefore that frequency shifts of the methylene stretching modes can be induced by coupling between the methylene stretching and librotorsional modes, without the introduction of gauche conformer in the polymethylene chain. Libration rotation motion has a direct influence on the width of the infrared bands: the spectra of hexadecane (hydrogenated and deuterated) trapped in urea clathrate show a broadening of the methylene stretching bands as a function of increasing temperature. These results are in agreement with previous infrared and Raman s t u d i e ~ . ~This ~ ' ~ broadening has been explained by the modulation of the angles between the components of the dipole moment and the molecule-fixed frame as well as by the modulation of the vibration frequency via anharmonic coupling terms with libration t o r ~ i o n .On ~ the other hand, the isotopic dilution experiments have shown that bandwidths are not affected by isotopic dilution. This is
explained by the fact that isotopic dilution affects only vibrational energy resonance transfer due to transition dipoletransition dipole coupling, and this contribution to the relaxation is generally small in l i q ~ i d s . ~ Bandwidths ~,~~ appear therefore to be essentially sensitive to intramolecular factors including tradgauche isomerization, frequency distribution along the chain, and librotorsional mobility. It is however difficult to interpret this spectral feature in molecular terms because of these multiple contributions. Conformationalchanges are indeed reflected on the frequency and the width of the methylene stretching modes. When the torsional angle of the adjacent methylene groups results in gauche conformations, theoretical calculations on small molecules have shown that the interaction force constants between the adjacent methylene groups are a f f e ~ t e d , and ~~,~ these ~ changes affect the frequency of the methylene stretching modes. The new results presented here reveal that frequency and width associated to the methylene stretching bands not only are sensitive to conformational order but can also be affected by interchain vibrational coupling and librotorsional motions. In lipid systems, for example, the frequency shifts that are observed during the gel-to-liquid crystalline phase transition can find their origin in these three phenomena. At this point, their respective contribution is not evaluated; it is rather difficult to distinguish between them since they usually vary in a concerted manner. During the phase transition, several gauche conformers are introduced along the acyl chains, affecting the conformational order and intermolecular coupling. The rotational diffusion is also increased during the bilayer melting and might be accompanied by a change in the librotorsional motion. However, it should be noted that the magnitude of the frequency shifts caused by the variation of the librotorsional mobility of the
Methylene Stretching Modes in Acyl Chains alkanes into clathrates is very small (about 0.015 cm-'/'C); therefore, this source of frequency shift is likely insignificant in biological systems. Within a given fluid bilayer, the changes observed as a function of temperature should be mainly related to trandgauche isomerization. This is likely at the origin of the linear relationship observed between the 2H Nh4R order parameters and the VC-H frequency for several phospholipid systems.6 However, the present results provide some explanations for small irregularities which have not been discussed in the previous paper. A consistent dip has been observed in the variation of VC-H frequency versus cholesterol content (Figure 2 of ref 6 ) when cholesterol was about 30 mol %. This increase of frequency observed at 45 mol % cholesterol can be associated with the disruption of interchain vibrational coupling caused by the presence of cholesterol in the phospholipid bilayer. Because the interchain coupling should be more important at low temperatures and because its disruption causes a larger change for the antisymmetric mode, this explanation is consistent with the observation that the dip was more pronounced at low temperatures and for the antisymmetric stretching mode. A similar argument can be made to rationalize the small deviations of the high cholesterol content samples in Figure 5 of ref 6. It should be noted that these variations are small and confirm the limited role played by contributions other than trandgauche isomerization in the fluid phase. It constitutes also a warning about the interpretation of small frequency changes exclusively in term of conformational ordercaution which is specially serious when extra components that possibly break the intermolecular coupling are added. It should be noted that the VC-D bands are rather insensitive to intermolecular coupling (Figure 2 ) . If one assumes that the variations in librotorsional motions are negligible in biological systems, our results indicate that the frequencies of these bands constitute spectral probes essentially sensitive to lipid chain conformational order. Finally, we should comment briefly the results obtained on the effect of vibrational coupling on the CH2 wagging region. On the basis of previous study, it is likely that the deuteration does not cause significant changes in the structure of the alkane molecules. The concentrations of various conformers should then remain constant when an alkane is diluted in its deuterated analogue. It is found that the isotopic dilution does not influence the conformational description based on the CH2 wagging bands analysis; this method offers therefore one of the most straightforward and reliable IR methods for the characterizationof chain order.
J. Phys. Chem., Vol. 98, No. 47, I994 12197
Acknowledgment. This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Fonds pour la Formation de Chercheurs et 1'Aide B la Recherche, and CAFIR-UniversitC de MontrCal. References and Notes (1) Mantsch, H.H.;McElhaney, R. N. Chem. Phys. Lipids 1991, 57, 213. (2) Umemura, J.; Cameron, D. G.; Mantsch, H.H. Biochim. Biophys. Acta 1980, 602, 32. (3) Gaber, B. P.; Yager, P.; Peticolas, W. L. Biophys. J. 1978,22, 191. (4) C a d , H. L.; Cameron, D. G.; Smith, I. C. P.; Mantsch, H. H. Biochemistry 1980, 19, 444. (5) Casal, H.L.; Cameron, D. G.; Jarrell, H. C.; Smith, I. C. P.; Mantsch, H.H. Chem. Phys. Lipids 1982, 30, 1726. (6) Kodati, V. R.; Lafleur, M. Biophys. J. 1993, 64, 163. (7) Dorset, D. L. Macromolecules 1991, 24, 6521. (8) Gaber, B. P.; Peticolas, W. L. Biochim. Biophys. Acta 1977, 465, 260. (9) Wood, K. A,; Snyder, R. G.; Strauss, H. L. J. Chem. Phys. 1989, 91, 5255. (10) Zerbi, G.; Roncone, P.; Longhi, G.; Wunder, S. L. J . Chem. Phys. 1988, 89, 166. (11) Snyder, R. G.; Aljibury, A. L.; Strauss, H.L.; Casal, H.L.; Gough, K. M.; Murphy, W. F. J. Chem. Phys. 1984, 81, 5352. (12) McKean, D. C. Chem. SOC.Rev. 1978, 7, 399. (13) Lee, K. J.; Mattice, W. L.; Snyder, R. G. J . Phys. Chem. 1992,96, 9138. (14) Casal, H.L. J. Phys. Chem. 1990, 94, 2232. (15) Snyder, R. G.; Scherer, J. R.; Gaber, B. P. Biochim. Biophys. Acta 1980, 601, 47. (16) Snyder, R. G. J. Chem. Phys. 1967, 47, 1316. (17) Holler, F.; Callis, J. B. J. Phys. Chem. 1989, 93, 2053. (18) Casal, H.L.; McElhaney, R. N. Biochemistry 1990, 29, 5423. (19) Senak, L.; Davies, M. A.; Mendelsohn, R. J . Phys. Chem. 1991, 95, 2565. (20) Zimmerschied, W. J.; Dinerstein, R. A.; Weitkamp, A. W.; Marschner, R. F. J. Am. Chem. Soc. 1949, 71, 2947. (21) Casal, H.L. Appl. Spectrosc. 1984, 38, 306. (22) PBzolet, M.; Boul6, B.; Bourque, D. Rev. Sci. Instrum. 1983, 54, 1364. (23) Cameron, D. G.; Kauppinen, J. K.; Moffatt, D. J.; Mantsch, H.H. Appl. Spectrosc. 1982, 36, 245. (24) Snyder, R. G.; Hsu,S. L.; Krimm, S. Spectrochim. Acta 1978,34A, 395. (25) Snyder, R. G.; Strauss, H. L.; Elliger, C. A. J . Phys. Chem. 1982, 86, 5145. (26) Bansil, R.; Day, J.; Meadows, M.; Rice, D.; Oldfield, E. Biochemistry 1980, 19, 1938. (27) Tokuhiro, T.; Rothschild, W. G. J. Chem. Phys. 1975, 62, 2150. (28) van Woerkom, P. C. M.; de Bleijser, J.; de Zwart, M.; Burges, P. M. J.; Leyte, J. C. Ber. Bunsen-Ges. Phys. Chem. 1974, 78, 1303. (29) McKean, D. C.; Lavaleey, J. C.; Saur, 0.;Edwards, G. G. M.; Fawcett, V. Spectrochim. Acta 1977, 33A, 913.
