C-H Stretchlng Modes and the Structure of n-Alkyl Chains. 1. Long

infrared spectra of lipid bilayers in going through the main phase transition to the liquid crystal. ... which has been used as diagnostic of chain ar...
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J. Phys. Chem.

1982, 86,5145-5150

5145

C-H Stretchlng Modes and the Structure of n-Alkyl Chains. 1. Long, Disordered Chains R. 0. Snyder,' H. L. Strauss,' Department of Chemistry. University of California, Berkeley, California 94720

and C. A. Elllger Western Regional Research Center, U.S. Department of Agrlcuiture, Berkeley, California 94710 (Received: March 12, 1982; In Final Form: August 18, 1982)

The C-H stretching modes of the conformationallydisordered polymethylene chain are analyzed. Fermi resonance interaction involving HCH bending overtones is dominant in determining the shape of the b a n C-H stretching spectrum. In addition, however, we have found that the C-H stretching frequencies are significantlyaffected by the conformation of adjoining C-C bonds. The estimated magnitude of this dependence is consistent with the shifts to higher frequenciesthat are observed upon melting n-alkanes and that have been reported for the infrared spectra of lipid bilayers in going through the main phase transition to the liquid crystal. However, similarities between the C-H stretching spectra of the ordered and disordered chains indicate that the small frequency dispersion observed for these modes in the crystal is, in part, maintained in the disordered chain, a conclusion that is supported by a simplified vibrational analysis. The effects of Fermi resonance interaction on the spectra of the disordered and ordered chain are also similar. The band near 2930 cm-' observed in hydrocarbon chain systems containing methyl groups is a composite of two overlapping bands, one from the polymethylene chain (2922 cm-l) and one from the methyl group (2938 cm-'). The 2922-cm-' component is affected by chain conformation while the 2938cm-' component is affected by the solvent. The distinction between the behavior of these bands is important since the peak-intensity ratio 12930/12850 is commonly used to interpret the structure of hydrocarbon assemblies.

Introduction Alkane chains are ubiquitous in biological and polymer systems and in many cases determine the arrangement of complex molecules in biological and polymer assemblies. The intimate correlation of the chains with structure has led to the use of infrared and Raman spectra as diagnostic of the chain conformation and thus of the structure of the molecular assembly.'V2 The C-H stretching region is of particular interest. It is usually the strongest feature in the infrared and Raman spectra. It can be easily modified for purposes of structural analysis by deuterium substitution which, for example, may involve partially deuterated molecules or mixtures of completely deuterated and undeuterated molecules. Until recently, the C-H region was seldom used for diagnostic purposes since the C-H frequencies do not change in any obvious fashion with changes of structure. However, the nature of the correlation between these bands and chain structure is now beginning to emerge. Part of the spectra/structure correlation is the direct result of changes of the stretching fundamentals with structure and conformation. Part is an indirect result such as, for example, the perturbation of stretches by Fermi resonance interaction with the methylene bends, which makes the C-H stretching region sensitive to conformation in a complicated but informative ~ a y . ~Spectral ? ~ changes in this region have also been used to indicate lateral chain-chain interaction in addition to conformational disorder.2 The effect of lateral interaction on the spectra can be separated into two components: an effect from direct intermolecular (1)D.F. H.Wallach, S. P. Verma, and J. Fookson, Biochin. Biophys. Acta, 559, 153 (1979),and references therein. (2)B.P. Gaber and W. L. Peticolas, Biochim.Biophys. Acta, 465,260 (1977). (3)R. G. Snyder, S. L. Hsu, and S. Krimm, Spectrochim. Acta, Part A, 34,395 (1978). (4) R. G. Snyder and J. R. Scherer, J. Chem. Phys., 71,3221 (1979).

TABLE I : Observed Frequencies of Methylene C-H Stretching Bands of t h e Polymethylene Chain in t h e Crystalline and Liquid States mode (cryst)

d+(n)

frequency, c m - ' activity

crystalline

liquid

IR

( 2 8 5 0 a (s) 2 8 5 0 b (S)

( 2 8 5 3 ' (s) 2856d (s)

Crystalline PE. Liquid n-alkanes.

a

Crystalline n-alkanes.

Liquid PE

vibrational coupling and an effect from the twisting-rotation mobility of individual chain^.^ The importance of understanding in detail the function of conformation and intermolecular interaction in determining C-H spectra has led to the present analysis of the spectra of disordered alkane chains. We first discuss in some detail the normal coordinates and the force constants associated with the C-H stretching vibrations of these chains. We then present C-H stretching spectra of polyethylene and other alkanes and discuss assignments. Finally, we discuss the intensity ratio 12850/12930, a quantity which has been used as diagnostic of chain arrangements. We conclude that this ratio is sensitive both to chain conformation and to solvent, though not necessarily to solvent polarity.

Normal Modes and Force Constants of the Disordered Chain We designate the modes of the disordered chain using the notation developed for the totally ordered (all-trans) ~~

( 5 ) R. G. Snyder, J. R. Scherer, and B. P. Gaber, Biochim. Biophys. Acta, 601,47 (1980).

0022-3654/82/2086-5 145$01.25/0 0 1982 American Chemical Society

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chain: d+ and d- are the symmetric and antisymmetric C-H stretches of the methylene group respectively, r+ and r- are the symmetric and asymmetric (degenerate) methyl C-H stretches, and 6 is the HCH methylene bend (scissors). In an ordered infinite chain, methylene modes exist for all values of the phase angle #. For these modes to appear in the spectra, the value of # must be 0 or a. Thus d*(O) and 6(0) are the Raman-active fundamentals, and the infrared-active fundamentah6 It is d*(a) and &(a) apparent that an estimate of the dispersion can be obtained by comparing the Raman (4 = 0) and infrared (# = a) frequencies for the ordered chain. Table I lists the frequencies for both an ordered system (crystalline polyethylene) and a disordered system (liquid polyethylene). The bands for the liquid are close to their crystalline counterparts. Therefore, we can immediately conclude that the C-H stretching modes as well as the HCH bending modes of the methylene group exhibit similar dispersion in the liquid and in the crystal. As we shall see, conformational disorder has only a small effect on the kinetic and potential energy terms associated with these modes and therefore has only a small effect on their overall frequencies. A first step in analyzing these small changes is to distinguish conformational effects that are intrinsic to the C-H modes from effects due to Fermi resonance interaction with the methylene bending-mode overtones. That the intrinsic dependence is relatively small is suggested by the temperature dependence of the Raman spectrum of liquid n-butane. Thus, we have examined this spectrum from -140 to 0 OC and find that over this temperature range the C-H stretching bands are not readily assigned to trans or gauche conformation^.^ Another indication of the magnitude of the C-H frequency dependence on conformation comes from examining the isolated C-H stretches of cyclohexane-dll or of the CHDz group in an n-alkane. The spectrum of cyclohexane shows axial and equatorial C-H stretching bands separated by 31 cm-' with the equatorial C-H bond having the higher frequency.s The C-H infrared spectrum of the CHD2group at low temperature shows a separation of 14 cm-' between the stretching frequencies of the in-plane and out-of-plane C-H bondsSDIn this case, the in-plane C-H bond has the higher frequency, a conclusion that follows from the assignments of Holland and Nielsen,lo who carried out IR polarization measurements on single crystals of the n-alkanes. In both cases detailed normal coordinate calculations show that less than 1 cm-' of the observed frequency separation can be attributed to changes in the kinetic energy. Let us designate k , and k , as the C-H stretching force constant incrementa that are appropriate to a H-C1 bond that is trans or gauche to the C2-Cj bond in the sequence HC,C2Cj. We take these increments to be additive for more than one Cj bonded to C2. Then for a CHDz group at the end of an n-alkane chain, the force constant difference between an in-plane (i) and out-of-plane (0)C-H bond is AKi, = k ,

- k,

(1)

(6) R. G. Snyder in "Methods of Experimental Physics", Val. 16, Part A, L. Marton and C. Marton, Eds., Academic Press, New York, 1980, p

Snyder et ai.

TABLE 11: C-H Stretching Interaction Force Constants between Adjacent Group Coordinates, d- and d' a

a

Interacting methylene pairs are connected by a gauche

( G ) or trans (T) C-C b o n d .

and for cyclohexane the equatorial (e), axial (a) difference is N e , *

2(kt - kg)

(2)

Thus, we find = 2AKi,0,close to what is observed. Now consider the d+ and d- modes of a methylene group. The off-diagonal force constant F d between the two C-H stretching coordinates of a methylene group is small relative to the diagonal stretching constant and so the effect of conformational changes on the interaction constant can probably be ignored. The C-C bonds adjacent to the methylene group form a pair whose conformationsmay be tt, tg (or gt, g't, tg'), gg (or g'g'), and gg' (or g'g). The diagonal stretching force constant differences associated with these conformations (ignoring gg') are

AK,, = Ktg - Kt, = ' / ( k t - k g ) AK,, = K,, - Kt, = k , - k ,

(3)

Note that in this notation the subscripts on the K's refer to the conformation of the carbon skeleton, which is the usual specification of conformation, while those on the k's refer to the orientation of a C-H bond relative to the carbon skeleton. The probabilities for the occurrence of the tg conformation (0.132) and for the gg conformation (0.045) were calculated for a polymethylene chain at 25 "C by using E = 500 cal/mol and E,,! = 3000 cal/mol." Assuming taat the observed C-H frequencies are conformational averages! we calculate that the d+ and d- modes shift upward about 6 cm-' on going from the ordered to the disordered chain. A shift of roughly this amount is observed in the case of the n-alkanes (Table I) and also in the infrared spectra of lipids in bilayers in going through the main phase transition to the liquid crystal.'* We complete this section with a discussion of the interaction force constanh between valence C-H stretching coordinates on adjacent methylene groups. From studies on small molecules, McKean et al.13 have shown that it is necessary to include such constants. We have introduced them into our earlier n-alkane force fieldI4 and adjusted their values so that the calculated C-H stretching frequencies agree with values from recent experiments. We will now discuss this and then consider the effect of disorder. There are two possible interaction constants between C-H stretching coordinates on adjacent methylene groups and we designate these as fdt and fdg where t and g refer to the relative orientation (trans or gauche) of the two C-H bonds across the C-C bond connecting the methylenes. The connecting C-C bond can itself be trans (T)or gauche (G) depending on the orientation of the C-C bonds connected to it and this defines the relative orientation of the methylene groups. The interaction constants between

73.

(7) R. G. Snyder, unpublished. (8) J. Caillcd, 0. Saw, and J X . Lavalley, Spectrochim. Acta, Part A , 36, 185 (1980); J. Wong, R. A. MacPhail, C. B. Moore, and H. L. Strauss, J . Phys. Chem., 86, 1478 (1982). (9) R. A. MacPhail, R. G. Snyder, and H. L. Strauss, J . Chem. Phys., 77, 1128 (1982). (10) R. F. Holland and J. R. Nielsen, J . Mol. Spectrosc., 8, 383 (1962).

(11) P. J. Flory, "Statistical Mechanics of Chain Molecules", Interscience, New York, 1969. (12) D. G. Cameron, H. L. Casal, and H. H. Mantsch, Biochemistry, 19, 3665 (1980).

(13) D. C. McKean, J. C. Lavalley, 0. Saur, H. G. M. Edwards, and V. Fawcett, Spectrochim. Acta, Part A, 33, 913 (1977). (14) R. G. Snyder, J . Chem. Phys., 47, 1316 (1967).

C-H Stretching Modes and n-Alkyl Chains

pairs at local symmetry coordinates (d- or d+) are given in Table I1 for the T or G conformation of the connecting bond. The interaction constants may be evaluated from the zone-center frequencies derived from observed spectra. For the d- modes, the frequencies found in Table I for crystalline polyethylene may be used, but for the d+ modes the observed frequencies must be corrected for the effeds of Fermi resonance. An estimate of 2878 cm-' for the unperturbed frequency of the d+(O)fundamental is found in ref 4. We have extended this analysis to the d+(r) fundamental and estimate a value of 2865 cm-l for the unperturbed frequency of this mode. Altogether these four frequencies lead to values of -0.022 and 0.021 mdyn/A for the interaction constants fdt and fdB and may be compared with values -0.029 and 0.016 mdyn/A derived from an analysis of CHD2CHD2by McKean et al.13and with similar values from ab initio calculation^.^^ (When the CH interaction force constants were added to the valence force field reported in ref 14, the intramethylene constants Kd and Fdassumed values of 4.534 and 0.068 mdyn/A. All other force constants remained unchanged.) For the disordered chain, an "effective" value of the interaction constant is a weighted average over the conformationsgas in the case for the order-disorder frequency shifts discussed above: feffactive

= 2pTTfT

+ 4PTG(fT + f G ) + dPGGfG

where expressions for fT and fG in terms of ft and f are given in Table 11. In going from the ordered all-trans c!mn to the disordered chain in the liquid, the values of the interaction constants for the d- and d+ modes change from -0.086 and -0.001 to -0.071 and 0.006 mdyn/A. These changes correspond to calculated frequency shifta of about 3 and 1cm-' for the d- and d+ fundamentals, respectively. The shifts are upward for d-(O) and d+(?r)and downward for d-(r) and d+(O).

Raman Spectra of the Disordered Chain As a model, we consider the Raman spectra of polyethylene both as an oriented solid and as a liquid. Measurements on the polymer in these states have a great advantage in that the isotropic and anisotropic components of the Raman spectrum can be obtained separately. Separation of the components yields simpler spectra that can be much more easily interpreted. Isotropic Spectrum. Figure 1shows the cc spectrum of a highly oriented uniaxial sample of extruded polyethylenels and the isotropic spectrum of the liquid. In the case of the extrudate, the c axis of the crystal is parallel to the chain axis. The C-H stretching region of polyethylene is complex and we first consider the assignment of the bands. The intense low-frequency component at 2850 cm-l that appears as a prominent well-defined feature in the Raman spectra of all hydrocarbon chain systems has long been identified with the d+ fundamental. In addition, spectra of both crystalline and liquid polyethylene show two less intense bands near 2900 and 2925 cm-' which are also associated with the d+ fundamental and which are the result of Fermi resonance. These bands have not previously been well characterized because of the presence of the nearby intense d- band at 2890 cm-' and, in spectra of alkyl chains, of the methyl group r+ band at 2938 ~ m - l . ~ J 'These interfering bands are absent both in the (15) P. Pulay and W. Meyer, Mol. Phys., 27,473 (1974);C.E. Blom

and C. Altona, ibid., 31, 1377 (1976). (16)R. G. Snyder, S. J. Krause, and J. R. Scherer, J. Polym. Sci., Polym. Phys. Ed., 16, 1593 (1978).

The Journal of Physical Chemistry, Vol. 86, No. 26, 1982 5147

C r y s t . P E (25°C) cc pol.

Liq. PE (150°C)

I

I

3040

2960

2880

2800

2720

cm-1 Figure 1. Raman spectra of crystalline and liquid polyethylene (resolutlon 2 cm-'). These spectra show only methylene symmetric C-H stretching (d+) bands. Top: cc polarized spectrum of extruded polyethylene at 25 OC (the chain axes are aligned parallel to c ) . Bottom: the isotropic spectrum of liquid polyethylene at 150 "C.

isotropic spectrum of liquid polyethylene and in the polarized spectrum of the extrudate. The fact that Fermi resonance components of the r+ (2938 cm-l) and d+ (2920 cm-l) modes tend to overlap has led to much confusion in interpretation. Thus, in the Raman spectra of hydrocarbon chain systems there always appears a band near 2930 cm-l and this band has been variously assigned to the methyl r+,17to the methylene d+,18 and to the methylene d-.l9 The assignment to d- modes, however, seems untenable for the following reason. The argument advancedlg in favor of this assignment is that the observed frequency of the band corresponds to that of the infrared-allowed d-(?r) fundamental, thereby suggesting that a Raman band at the same frequency may be the result of a relaxing of selection rules through chain disorder. The problem with this interpretation is that, since d- represents a very localized antisymmetric vibration, its Raman band should be depolarized. Our measurements show clearly that the 2930-cm-' band is polarized. The problem of resolving the r+ and d+ contributions to the 2930-cm-' band will be addressed in the last part of this paper. Only d+ modes appear in the crystalline and liquid spectra in Figure 1and they appear not as one but as three bands. The frequencies of these three bands change little upon changing phase, and the principal differences between the spectra of the solid and liquid phases are in the relative intensities of the components. The C-H stretching modes and, to a lesser extent, the HCH bending modes have dispersions in the liquid that are comparable to those (17)R.C. Spiker and I. W. Levin, Biochim. Biophys. Acta, 388,361 (1975). (18)R.Faiman and K. Larsson, J. Raman Spectrosc., 4,387 (1976). (19)M.R.Bunow and I. W. Levin, Biochim.Biophys. Acta, 487,388 (1977).

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The Journal of Physical Chemistry, Vol. 86, No. 26, 1982

lkd

-IO -I0[

% Uncoupled

-200

50

IO0

0

50

IO0

Yo Uncoupled

Flgure 4. Calculated dependence of the intensity ratio I 2830/ I 2850 and of the frequencies of the bands near 2930 and 2850 cm- on coupling between LHCH bending coordinates.

Yon

cno

n + e r C c * r q 3: ne

,?$

2-t.

n t f r c c t nq 3 ;

'*ref C-H

tcn3

2nd

28

?ends

Flgure 2. Fermi resonance interaction scheme for an isolated trans polymethylene chain of finite length. At left: the unperturbed C-H stretching and LHCH bending fundamentals and the LHCH overtones. At right: mixed states resulting from interaction. Approximate frequencies (cm-') of transitions are indicated. The thicker the lines marking the excited levels. the m e intense are the associated Raman bands. The character of the vibrations is indicated: dl+ and 6, are modes nearest the zone center; 26, designate LHCH overtones. 14701

I460

-,

I

14501

E 2

I

I

uncoupled

\

1

I

I

-1

Af

broad band is an approximate map of the inverse of the density of states of the HCH bending modes. In crystalline polyethylene, the Fermi resonance interaction is also affected by the interchain coupling which causes lateral dispersion of the bending modes. The effect is to broaden the component bands and to make the C-H stretching region to some extent dependent on the chain pa~king.~ The spectrum of the liquid differs a bit from that of the crystal. The difference is unlikely to be associated with a change in the value of the Fermi resonance constant since this constant is associated with interaction between internal coordinates on the same methylene group and thus is probably relatively insensitive to conformation. Moreover, the nearly in-phase C-H stretching fundamental involved in the resonance is not expected to be especially sensitive to conformation since it lies near the zone center in the flat region of the dispersion curve where its frequency does not vary much with phase angle. In contrast, many methylene bending modes are involved in the resonance through their overtones, and these modes are distributed over the entire dispersion curve. Moreover, unlike the C-H stretches, these modes tend to undergo significant intermolecular coupling. Finally, we note that the frequency perturbations of the bending fundamentals are amplified, since it is their overtones which affect the C-H stretching region. Therefore, we conclude then that the observed differences between the liquid and crystalline spectra are largely due to differences in the bending modes. Changes in the bending modes in going from the ordered to the disordered chain may be modeled by decreasing the coupling between the individual LHCH bending coordinates. This leads to a decrease in the dispersion of frequencies as a function of phase angle and a consequent decrease in the frequency spread of the overtone bands. The calculated values of the frequency shifts and the intensity ratio of the two C-H bands (2850 and 2930 cm-') that flank the broad continuum are plotted in Figure 4 as a function of the degree of coupling. The behavior predicted from this calculation is in agreement with that observed in going to the liquid; namely, the frequency separation becomes smaller while the intensity ratio 12930/12850 increases. A reduction in the coupling among the bending modes of less than 25% can account for the observed changes. Anisotropic Spectrum. The C-H stretching region of the anisotropic spectrum of molten polyethylene is dominated by a single intense band near 2895 cm-l (Figure 5 ) . This band is the d- methylene antisymmetric C-H stretch. It is flanked by weaker bands near 2930 and 2850 cm-l which are the anisotropic components of d+. The d- band is broad and asymmetric. Its width increases with temperature and decreases with chain length. In n-alkane-urea clathrates, in which the chains are con-

1440pe j ' 5 0 % coupled

14301

0

I

I

1

1

I

30

60

90

120

150

I

180

(deg)

Figure 3. Dispersion curve for LHCH bending modes of the Isolated trans polymethylene chain as estimated in ref 4. Scalddown curves for reduced coupling between methylene groups are indicated.

of the ordered chain in the crystal. For this reason the C-H stretching spectra of the liquid resemble those of the crystal. The complex structure of the d+ bands in both cases is the result of Fermi resonance between the de fundamental and the many overtones of the HCH bending modes. In preparation for discussing the spectrum of the liquid in more detail, we will review briefly the nature of the resonance interaction in the case of the ordered chain. The resonance scheme for an isolated all-trans polymethylene chain of finite length is summarized in Figure 2. Only the interaction between the most in-phase C-H stretching dl+ mode (Le., the mode that is closest to d+(O)of the infinite chain) and the overtones of the HCH bending fundamentals is of importance. The frequencies of the overtones can be obtained from the bending dispersion curves in Figure 3.4 The resonance interaction results in two intense isolated bands and between them a series of closely spaced intensity-enhanced overtone bands which form a broad band. These overtone bands are only slightly displaced from their unperturbed frequencies. Those that gain the most intensity, which is assumed to come primarily from mixing with the dl+ mode, are those that are associated with bending fundamentals situated on the steepest part of the dispersion curve. Consequently the contour of the

The Journal of Physical Chemistry, Vol. 86, No. 26, 1982 5149

C-H Stretching Modes and n-Alkyl Chains

cm -1

cm -1

cm -1

Flgure 8. Isotropic Raman C-H stretching spectrum of liquid nC,H, at 25 OC (resolution 2 cm-'): neat; in CCi, solution (mole ratio 1:20); in n-C16D3, solution (mole ratio 1:lO). 1

I

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2960

2800

2800

2720

cm-I

Figure 5. Anisotropic Raman C-H stretching spectrum of liquid polyethylene at 150 OC (resolution 2 cm-').

strained by the urea channels to the all-trans conformation, but at the same time have a greater freedom for rotation and twisting about their long axis than in neat crystalline alkanes, the d- band has a temperature behavior similar to that of the l i q ~ i d .Thus, ~ the width is likely due to the rotational/twisting motion of the chain rather than to conformational disorder. We note that the temperature dependence of the width of the d- band of deuterated lipids has been used to monitor phase transitions in model membrane systems.20 An analysis of the shape of the dband and its temperature behavior is in progress. Zntensity Ratios and Chain Conformation. Peak intensity ratios of pairs of Raman C-H stretching bands have been used to characterize the structure and phase transitions of hydrocarbon chain systems such as lipid biomembranesl and polyethylene.21 The parameters most commonly used are the intensity ratios involving bands at 2850, 2890 cm-l and a band whose nominal frequency is 2930 cm-1.22-28The ratio Z2850/Z28w has been discussed in ref 3 and 5 and will not be considered here. Further progress in understanding the structural dependence of this ratio awaits a quantitative analysis of the aforementioned dependence of the shape of the 2890-cm-' band on temperature and chain length. The I2850/12930 ratio is, in a sense, a default parameter. It has been used to study biomembrane systems in which there is a high concentration of protein, since in this case the 12850/12890 ratio is difficult to measure because the 2890-cm-' band tends to be obscured by the 2930-cm-' band associated with the methyl group of the protein. Interpretation of changes in the ratio Z2m/Z2930 for lipid/protein biomembrane systems is made difficult by the (20) R. Mendelsohn and J. Maisano, Biochim. Biophys. Acta, 506,192 (1978). (21) S. L. Wunder, Macromolecules, 14, 1024 (1981). (22) S.-K. Hark and J. T. Ho, Biochim. B i ~ p h yActa, ~ . 601,54 (1980). (23) M. R. Bunow and I. W. Levin, Biochim. Biophys. Acta, 464,202 (1977). (24) S. P. Verma and D. F. H. Wallach, R o c . Natl. Acad. Sci. U.S.A. 73, 3558 (1976). (25) S. P. Verma and D. F. H. Wallach, Biochem. Biophys. Res. Commun., 74, 473 (1977). (26) E. Weideka", E. Bamberg, D. Brdiczka, G. Wildermuth, F. Macco, W. Lehmann, and R. Weber, Biochim. Biophys. Acta, 464,442 (1977). (27) S. P. Verma and D. F. H. Wallach, Biochim. Biophys. Acta, 436, 307 (1976). (28) S. P. Verma, R. Schmidt-Ullrich, W. S.Thompson, and D. F. H. Wallach, Cancer Res., 37, 3490 (1977).

fact that both methylene and methyl groups from the lipids and methyl groups from the proteins contribute to the intensity of the 2930-cm-' band. However, the ratio was employed in recent studies on the pH sensitivity of erythrocyte membranesz4and on the temperature sensitivity of ribonuclease/lecithin multibilayer systems.25 In the latter study, the 12860/12930 ratio was monitored through the phase transition and, from these measurements, it was concluded that, among other changes, the methyls of the protein side groups may undergo changes from a nonpolar (hydrocarbon) to a polar (aqueous) e n ~ i r o n m e n t . ~ ~ Our present results show that the Raman intensity ratio can change due to a variety of causes. In particular we note that changes in the Iz8,/IZg,, ratio from conformational change can be quite substantial as may be seen by comparing the Raman spectra of crystalline and liquid polyethylene. (A comparison of n-alkane spectra would lead to essentially the same result if the contribution from methyl bands were eliminated.) Thus, for the crystal (25 "C) the ratio 12@0/12930 is 3.1, and for the liquid (150 "C) it is 1.86 (Figure 1). The change is comparable to that observed for the erythrocyte2*and the ribonuclease/lecithin25systems. On the other hand, it is clear that the shape of the 2938-cm-' methyl band is indeed dependent on solvent. However, the dependency does not appear to be limited to polar solvents. Solvent effects involving polar solvents have been demonstrated in Raman studies in 1p r o p a n o i / ~ a t e and r ~ ~ 2-methyl-2-propanol/~ater~~ mixtures. In these cases, the methyl C-H stretching bands undergo changes in frequency and relative intensity as the polarity of the solvent is changed by varying the water concentration. Our own studies show that nonpolar solvents also affect the 2938-cm-' methyl band. Thus, we find that dilution by CCl, significantly affects the C-H stretching bands of n-Cl,H, by increasing the peak intensity of the 2930-cm-l band relative to that of the 2850-cm-' band (Figure 6). The value of the ratio 12850/12930 is 2.00 for neat n-C16H34 and 1.59 for a CCl, solution. Similar changes in the C-H stretching region of the Raman spectrum of n-C16HMhave been reported for dilution with the polar solvent CHC13.30 The solvents CCl, and CHC13 appear to have essentially the same effect on 12850/12930. An important question is whether the decrease in the intensity ratio Zm/ZBm observed upon dilution of n-Cl6H~ indicates that there is a decrease in the trans/gauche bond ratio since we know that this intensity ratio does, in fact, (29) K. Larsson and R. P. Rand, Biochim. Biophys. Acta, 326, 245 (1973). (30) S. Wunder and S. D. Merajver, J. Chem. Phys., 74,5341 (1981).

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Snyder et al.

CCI,

Soln

(I:IO)

crfl-' Flgure 7. Isotropic Raman C-H stretching spectrum of liquid perdeuterated n-hexadecane (>99% D) at 25 OC. The intense band is from CHD methylenes (resolution 2 cm-').

decrease with increased conformational disorder in the case of polyethylene (see above). It has been argued on the basis of the polyethylene results that there is a decrease in conformational order in liquid mC16H34 when it is diluted with CHClPm However, as we have recently reported in a preliminary comm~nication,~~ the change observed in the intensity ratio upon dilution is due to the effect of the solvent on the methyl contribution to the 2930-cm-l band. The experimental evidence in favor of this interpretation will now be summarized. The trans/gauche bond ratio for mC16H34 should be little affected in going from the neat liquid to solution in n-C16D3,. We have found that the effect on the C-H spectrum resulting from dilution with the deuterated n-alkane is essentially identical with what is observed for dilution with CC14 (and also probably CHCl,). The spectra of the two solutions are virtually the same except for the presence of a weak band near 2900 cm-' for the n-C1& solution as may be seen in Figure 6. The 2900-cm-l band is from C-H stretching of the residual hydrogen in the deuterated solvent. (The spectrum of the solvent in this region is shown in Figure 7.) The value of I2g,0/12930 is 1.59 for n-C&34 in C C 4 and 1.64 in nC16D34. The closeness of these values indicates that the changes in the spectrum observed upon diluting the sample result from decreased intermolecular interaction between vibrations of like molecules. The sensitivity of the r+ band of the alkyl chain to intermolecular interaction has also been noted by Hill and L e ~ i n . ~ ~ In direct support of this conclusion, we have found that the 12850/12930 ratio undergoes little change upon dilution if the 2938-cm-' methyl band is absent. This is indicated in Figure 8, which shows the isotropic spectra of n-CD,(31) R. G. Snyder, J. Chen. Phys., 76, 3342 (1982). (32) I. R. Hill and I. W. Levin, J. Chem. Phys., 70, 842 (1979).

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i

V I \

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4

3040

E'960

2880

2800

2 20

cm-i Flgure 8. Isotropic Raman C-H stretching spectrum of liquid n CD3(CH2),,CD3 at 42 OC (resolution 2 cm-'): neat and in CCI, solution (mole ratio l : l O ) ,

(CH2)20CD3a t 42 "C both as a neat liquid and in CC14 solution. Both spectra are quite similar to that of molten polyethylene with the "2930-cm-'" band appearing at 2922 cm-l rather than at 2938 cm-l which is the case for n-C&, (Figure 1). In going from the pure liquid to the solution, the ratio 128&)/12930 changes from 1.95 to 2.05 in sharp contrast to the situation for methyl containing n-C16H3, where much larger change (from 2.00 to 1.59) is observed (Figure 6). Thus, it appears that all or nearly all the change in the intensity ratio upon dilution can be attributed to changes in the methyl r+ band at 2938 cm-'.

Acknowledgment. We are indebted to Dr. James R. Scherer of the Western Regional Research Center, U.S. Department of Agriculture, for the use of his Raman facility and to Dr. Scherer and Ms. Victoria Shannon for help in measuring the spectra. This work was supported by a research grant from the National Institutes of Health (GM-27690-01).