J. Phys. Chem. 1985,89, 3395-3398
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Ultraviolet Resonance Raman Studies of N-Methylacetamide L. C. Mayne, L. D. Ziegler,+and B. Hudson* Department of Chemistry, University of Oregon, Eugene, Oregon 97403 (Received: October 17, 1984; In Final Form: March 18, 1985)
Resonance Raman spectra of the simple peptide model compound N-methylacetamide have been obtained with 2 18- and 200-nm laser radiation. A large enhancement of the amide I1 vibration is observed relative to that of Raman spectra obtained with visible radiation. Replacement of the amide hydrogen by deuterium results in a spectrum with most of its intensity in the amide XI’ mode. Excitation of this deuterated species with 200-nm radiation results in intensity in the overtones of this,modes, a feature characteristic of resonance enhanced spectra. Isotopic substitution of the amide carbon and nitrogen by 13Cand I5N results in a spectral shift to lower frequency by nearly the amount expected for a normal mode consisting primarily of the motion of the amide C and N atoms. These results, taken together, demonstrate that the geometry change of N-methylacetamide upon electronic excitation to the m*state is dominated by a change in the C-N bond length. Studies of mixtures of the deuterio and protio forms show that a significant normal mode rotation occurs on isotopic substitution such that the amide 11’ of the deuterio form becomes approximately equally distributed between the amide I1 and I11 vibrations of the protio form. The amide I and I’ vibrations are very diffuse in aqueous solutions at the high dilutions used. These bands become sharp in acetonitrile. This behavior is interpreted in terms of a range of frequencies for this vibration due to a distribution of hydrogen-bonded species.
Introduction Advances in laser technology have made possible the generation of coherent radiation with wavelengths extending into the vacuum-ultraviolet region. We have used such radiation to obtain resonance Raman spectra with excitation wavelengths as short as 184 nm. For excitation in the vicinity of strongly allowed transitions, the resonance effect in Raman spectra preferentially enhances the intensity of vibrational normal modes that lie along the molecular geometry change which accompanies the excitation. Thus, in addition to a large increase in overall signal intensity, there is often a preferential enhancement of certain normal modes that may not even be observable with excitation far from resonance. This preferential enhancement gives information concerning the change in the geometry of the excited state relative to the ground state and may also result in a greatly simplified spectrum. For biological studies, the use of far-UV radiation has several advantages over conventional near-UV resonance Raman techniques.’ New chromophores are made available for resonance Raman studies. This includes the peptide bond, the aromatic amino acids, several other amino acid side chains including histidine, and the nucleic acid bases.2 This excitation is often resonant with states higher than the lowest energy excited state. This is the case for the fluorescent aromatic components of most biopolymers. Since these aromatic groups emit from their lowest excited states, the broad featurless fluorescence is at much longer wavelength than the Raman signal and does not intgrfere with collection of the resolved Raman spectrum. As part of a study of pepytides and proteins using this technique we have chosen N-methylacetamide [NMA, (CH3)CONH(CH3)] for preliminary study. N M A has been of considerable interest as a simple peptide analogue and has been the subject of many studies of both theoretical and experimental nature.’y3-” Results for the parent compound and three isotopic forms (N-D; amide 13C,I5N, N-H; and amide 13C, 15N, N-D) are presented. The primary objective here is the presentation of the new information that ultraviolet resonam(; Raman scattering provides concerning the excited-state geometry and normal modes of this simple peptide. Experimental Section N-Methylacetamide was purchased from the Aldrich Chemical Co. Deuterated N M A was prepared by exchange in D,O. IsN, I3C labeled N M A was a gift from Terry Oas of the University Present address: Department of Chemistry, Northeastern University, Boston, MA 02115.
of Oregon. It was prepared by condensing [I3C]acetic acid and [ I5N]methylamine hydrochloride with carbonyldiimidazole in the presence of trimethylamine. The product was purified by vacuum distillation. The Raman apparatus consisted of a Quanta-Ray pulsed Nd:YAG laser with KD*P or KDP crystals for the production of the harmonics a t 532, 355, 266, and 213 nm. The far-UV wavelengths used in this study were generated by selecting one harmonic and focusing it in a cell containing hydrogen at a few atmospheres pressure.12 The resulting stimulated Raman frequencies, both Stokes and anti-Stokes, for several quanta of the hydrogen vibration are of sufficient intensity to be used for Raman spectroscopy. A more detailed description of the laser apparatus is presented elsewhere.’J3 The beam of radiation with the selected excitation frequency is directed to the sample. All spectra presented here were obtained with 217.85- or 199.76-nm radiation. The scattered light is collected in a backscattering geometry, focused into a 1-m monochromator, and detected by a solar-bline photomultiplier tube. The output of the photomultiplier was sampled by a boxcar averager triggered by a pulse from the laser. A second boxcar channel was synchronized with a chopped mercury lamp to calibrate the collected spectra. The analog output of the boxcar amplifier was processed by an analog to digital converter input to a DEC P D P l l / 3 4 minicomputer. All samples.were aqueous or acetonitrile solutions at concentrations of 10 mM or less. In order to avoid problems associated with focused UV radiation on a cell window, the samples were irradiated in a liquid jet in air produced by a simple circulating
(1) Hudson, B.; Mayne, L. C. Merhods Enzymol., in press. (2) Ziegler, L.; Hudson, B.; Strommen, D. P.; Peticolas, W. L. Biopolymers 1984, 23, 2067. (3) Nagakura, S.Bull. Chem. SOC.Jpn. 1952, 25, 164. (4) Yan, J. F.; Momany, F. A.; Hoffmann, R.; Scheraga, H. A. J. Phys. Chem. 1970, 74,420. ( 5 ) Nitzsche, L. E.; Davidson, E. R. J . Am. Chem. SOC.1978, 100, 7201. (6) Hoesterey, B.; Neely, W. C.; Worley, S.D. Chem. Phys. Lerr. 1983, 94, 311. (7) Klotz, I. M.; Franzen, J. S. J . Am. Chem. SOC.1962, 84, 3462. (8) Kaya, K.; Nagakura, S. Theoret. Chim. Acra 1967, 7, 117. (9) Nielsen, E. B.; Schellman, J. A. J . Phys. Chem. 1967, 71, 2297. (10) Momii, R. K.; Urry, D. W. Macromolecules 1968, I , 372. (1 1) Kitano, M.; Fukuyama, T.; Kuchitsu, K. Bull. Chem. Soc. Jpn. 1973, 46. 384. (12) Paisner, J.; Hargrove, S.“Energy and Technology Review”; UCRL52000-79-3, 1979; p 1. (13) Ziegler, L. D.; Hudson, B. J . Chem. Phys. 1981, 74, 982.
0022-3654/85/2089-3395$01.50/0 0 1985 American Chemical Society
3396 The Journal of Physical Chemistry, Vol. 89, No. 15, 1985
Mayne et al.
L
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Figure 2. 217.85-nm excitation Raman spectrum of N-methylacetamide in 1:l HzO/DzO. Note that the intensity of the amide 11’ peak at 151 1 cm-’ is approximately equal to the sum of the intensities of the amide I1 and 111 bands at 1581 and 1322 cm-I.
1600
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Figure 1. (a) Resonance Raman spectra of N-methylacetamide in water
taken with 217.85-nm excitation. Both I2C,l4N(solid line) and 13C,’SN (dasbe4 line) are shown. The concentrationin each case was 10 mM or less. The major peaks are the amide I1 and 111 modes. (b) Same as a but in deuterium oxide. In this case the major peak is the amide 11’ mode. system. Additional details of the experimental procedure are given elsewhere.’
Results The in-plane normal modes of the XHN-COY atoms of the peptide bond are conventionally labeled as “N-H stretch” and amide I-VI1 in order of decreasing frequency. Amide I is primarily a C - 0 stretching motion. Amide I1 is primarily the C-N stretch with a considerable admixture of C-N-H in-plane bending. Amide I11 is the other combination of the C-N stretch and CN-H bending mode. The 218-nm excitation Raman spectra of N M A and its C-13, N-15 isotopic form in HzO are shown in Figure 1A. The major peaks for the normal isotopic form are the amide I1 and I11 bands at 1595 and 1320 cm-I. The strong amide I1 band is not observed with visible excitation but has been seen weakly with 257.3-nm e ~ c i t a t i o n . ’ ~In these resonance Raman spectra the amide I V band a t 870 cm-’ is seen only weakly and the amide I band a t 1680 cm-’ is not seen a t all. This contrasts sharply with spectra obtained with visible excitation where both of these bands are quite strong. The spectrum of deuterated N M A is shown in Figure 1B. A large effect on the relative intensities is observed upon deuterium substitution. Most of the intensity of the amide I1 and amide I11 bands is now in the amide 11’ band at 1520 an-’.Again, the amide I’ band is weak or very broad as discussed below. Figure 2 shows the spectrum of N M A in 1:1 HzO:DzO. This spectrum shows that the intensity of the amide 11’ band is approximately equal to the sum of the intensities of the amide I1 and amide I11 bands. None of the other bands show significant (14) Harada, I.; Sugawara, Y . ;Matsura, H.; Shimanouchi, T. J . Raman Spectrosc. 1975,4,91. Sugawara, Y.;Harada, I.; Matsura, H.; Shimanouchi, T. Biopolymers 1978, 17, 1405.
lsb0
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Figure 3. Resonance Raman spectrum of NMA in DzO excited with 199.76-nmradiation. A longer scan is given to show the first and second overtones of the amide 11’ mode. Both the IzC,I4N(solid line) and l3C,lSN(dashed line) species are shown. The two large bands at -2500 and -3500 are DzOand HOD, respectively. The sharp line at 4155 cm-’ is the next shifted line from the H2 cell.
changes in intensity. This demonstrates that the change in the normal coordinates upon deuterium substitution only involves the amide I and I1 bands of the nondeuterated form. The spectra of 15N, I3C labeled N M A in H 2 0and DzOshown in Figure la,b show significant isotopic shifts. The isotopic shift observed in D20for the amide 11’ band of the deuterated species is considerably larger than that observed for either of the amide I1 or I11 bands in the protonated species. Figure 3 shows the overtone region of the spectrum of the deuterated species obtained with 200-nm excitation. Peaks involving 2 and 3 quanta of the amide 11’ vibration are quite strong under these resonance conditions. Figure 4 shows the resonance Raman spectrum of N-methylacetamide and deuterio-NMA in deuterioacetonitrile solution. Here the amide I C=O stretch is seen as a sharp band with moderate intensity. Discussion The absorption spectrum of N-methylacetamide in aqueous solution has a peak extinction coefficient of 8800 L/(mol cm) at 186 nm. The extinction coefficient at 218 nm is about 400 L/(mol cm) while at 200 nm it is 4000 L/(mol cm). Except for the overtone enhancement, the major features of the resonance Raman spectra obtained with 200- and 218-nm radiation are very similar in terms of the modes having the greatest enhancement. From this, and the absorption spectrum of the peptide chromophore,
The Journal of Physical Chemistry, Vol. 89, No. 15, 1985 3397
Resonance Raman Studies of N-Methylacetamide I .o
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equilibrium geometry. The vector points to the excited-stateequilibrium geometry. In B the coordinates have rotated so that the geometry change vector now is approximately parallel to the amide 11’ coordinate.
0
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Figure 5. Representation of the effect of deuteration on the amide I1 and 111 normal coordinates. A represents a slice through normal mode space showing the amide 11, 111 plane. The origin lies at the ground-state
b
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Firmre 4. 212.85-11111 excitation resonance Raman spectra of Nmethylacetamide (a) and N-deuterated N-methylacetamide (b) in deuterioacetonitrile at 10 mM concentration. In this solvent we do see the amide I mode.
we conclude that the state contributing most of the resonance Raman intensity in these spectra is the strong T-X* electronic excitation rather than the weak n l r * transition at lower energy.15 Because the resonance Raman process reflects the molecular geometry change that occurs on excitation, the intensities of the Raman peaks can provide information about the geometry of the resonant excited state not available from infrared or off-resonance Raman spectra. Of particular interest in this case is the fact that the amide I1 and I11 bands are by far the largest features of the N M A spectra under resonance conditions. The resonance enhancement of these bands is consistent with the preresonance spectra (257.5 nm) of Harada et al.I4 Upon deuteration, this intensity shifts uniquely to the amide 11’ band. This behavior demonstrates that replacement of a deuterium by a hydrogen at the amide nitrogen converts the amide 11’ vibration, which must be nearly collinear with the geometry displacement vector associated with electronic excitation, into a linear combination of the amide I1 and I11 motions of the protio form. This behavior is most easily represented in terms of a rotation of normal coordinates within the space of these two modes. The geometry change vector representing the displacement of atomic positions due to electronic excitation can be used as a fixed direction in understanding this rotation (see Figure 5). In this model, the excitation vector lies roughly in the amide 11, amide I11 plane in normal-coordinate space. In the protio form both of the normal mode vectors have significant projections on the excitation displacement vector. Upon deuteration there is a rotation of the normal coordinates so that in the deuterated form the excitation vector lies approximately along the amide 11’ coordinate. The amide 11’ band of [15N,13C]N M A shows a 46-cm-I (3.0%) shift to lower frequency relative to the normal isotopic form. If the only nuclear motion involved in this mode is that of the C and N atoms of the amide bond, then the predicted isotopic shift is 3.6% or 55 cm-’. The magnitude of this shift indicates that the (15) Dudik, J. M.; Johnson, C. R.; Asher, S.A. J. Phys. Chem., in press.
amide 11’ mode has a very large component of C-N stretch. The smaller 13C, 14N isotopic shift observed for the protio form is consistent with the distribution of the C-N motion into two normal modes. Since we already know that the geometry change on excitation lies largely along the amide 11’ coordinate, we conclude that this geometry change is largely a change in C-N bond length. The deuterium isotope effect on the form of the amide I1 and I11 normal modes discussed above is consistent with the conclusions of previous workers.Ib2l A simplified description of this effect is that in the proto form the C-N-H bending motion has a frequency similar to that for the C-N stretch and these two motions mix strongly to form the normal modes. Deuterium substitution lowers the frequency of the C-N-D bending motion considerably so that it no longer mixes significantly with the C-N stretch. The appearance of strong overtones of a totally symmetric vibration is typical of conditions of resonance with an electronic transition when the excitation frequency is near the higher vibrational bands of the progression of this mode in the upper state. The fact that the 13C,I5Nlabeled species shows relatively greater overtone intensities (Figure 3) compared to the fundamental than does the 12C, I3Nspecies probably reflects the simple fact that amide 11’ has a lower frequency in the upper state for the heavier isotopic species. Because of this, a given excitation frequency is further into the manifold of vibrational levels for the I3C, I5N isotopic species than for the 12C, 14Nspecies so that the resonant vibronic states have larger Franck-Condon factors with the higher levels of the ground state. The information contained in these resonance Raman spectra provides a valuable test of calculation methods which might be used to predict the geometry of the excited state of the peptide group. An examination of previous theoretical treatment^^^,*^ indicates that most calculational methods predict much larger changes in the bond order of the C=O bond than the C-N bond. This is contradictory to our observations. The overtone and isotopic shift data also provide valuable information useful in refining potential energy surfaces and normal mode determinations for the ground state. Another interesting aspect of these resonance Raman spectra of N-methylacetamide is the lack of a discernible peak near 1650-1700 cm-’ for the amide I C=O stretch. Only a broad peak appears in this region in the aqueous spectra of Figure 1. On the basis of these spectra alone it is possible that the absence of this band is due to the fact that this mode has little intensity rather (16)
Sugawara,Y.;Hirakawa, A. Y.; Tsuboi, M. J . Mol. Spectrosc. 1984,
108, 206. (17) Miyazawa, T.; Shimanouchi, T.; Mizushima, S . J . Chem. Phys. 1958, 29, 611. (18) Miyazawa, T. In “Polyamino Acids Polypeptides and Proteins”; Stahman, M. A,, Ed.; University of Wisconsin Press: Madison, WI, 1962; p 201. (19) Jakes, J.; Schneider, B. Collect. Czech. Chem. Commun. 1968, 33, 643. . . (20) Jakes, J.; Krimm, S. Spectrochim. Acta 1971, 27A, 19. (21) Rey-Lafon, M.; Forel, M. T. Spectrochim. Acta 1973, 29A, 471. (22) Robb, M. A.; Csizmadia, I . G. Theoret. Chim Acta 1968, 10, 269. (23) Del Bene, J.; Jaffe’, H. H.; Ellis, R. L.; Kuehnlenz, G. QCPE 1974, 174.
3398
J. Phys. Chem. 1985, 89, 3398-3405
than being very broad. In order to investigate this point we have obtained the resonance Raman spectrum of N-methylacetamide in both its deuterio and protio forms in deuterioacetonitrile (Figure 4). The main point is that in this solvent the amide I and I' vibrations are clearly seen with moderate intensity and are quite sharp. We conclude from this that the lack of observation of this band in the aqueous environments is due to a very large line width probably because of a vibrationally heterogeneous distribution of hydrogen-bonding environments not present in acetonitrile.
Acknowledgment. This work was supported by N S F Grant PCM83-08529 and NIH Grant GM32323. L.C.M. was supported by an N I H predoctorial training grant, GM07759. We thank Terry Oas for the gift of the [13C,'5N]N-methylacetamide. A preliminary presentation of this work was made at the 28th Annual meeting of the Biophysical Society, San Antonio, TX (Biophys. J. 1984, 45, 322a.). Registry No. NMA, 79-16-3; N M A N-deuterated, 3669-70-3.
Retention Theory for Fleld-Flow Fractionation In Annular Channels Joe M. Davis and J. Calvin Ciddings* Department of Chemistry, University of Utah, Salt Lake City, Utah 84II2 (Received: January 7, 1985)
The characteristics of field-flow fractionation (FFF) are summarized, and the desirability of expanding the methodology from a flat ribbonlike geometry to an annular geometry is explained. Annular systems are treated subject to the general force law F = A/?, where F is the force on an entrained particle directed along radial coordinate r and A is a constant. The retention ratio R , which describes the relative migration rate, is formulated in terms of complicated integrals involving concentration and velocity profiles. Approximations, which are accurate under most practical conditions, are developed for these integrals. With these approximations, a number of general and limiting expressions for R are obtained for both inner and outer wall retention as a function of n and of the inner-to-outer wall radius ratio. Equations for relaxation time in the annular system are also derived.
Introduction Field-flow fractionation (FFF) is a versatile family of separation The methods related in an operational way to strength of F F F lies in its ability to separate and characterize complex systems of macromolecules. Separation in FFF is achieved in a thin unobstructed flow channel whose narrow dimensions (50-500 pm) promote rapid mass transport and consequently rapid fractionation and measurement. In FFF, a field or gradient is applied in a direction perpendicular to the flow axis, inducing the movement of sample particles toward one wall. A steady-state cloud of particles is formed rapidly a t the wall. The thickness of this cloud depends upon the strength of interaction of the field with the particles. The level of interaction differs for different kinds of macromolecules and particles, leading to the formation of clouds of different thicknesses for different species. When laminar flow is initiated in the channel, the various particle clouds are carried downstream by virtue of their entrainment in the flowing fluid. However, the particle clouds forced closest to the wall by the strongest interactions have their downstream motion relatively impeded because the velocity of the transporting fluid approaches zero at the wall in accordance with normal viscous (generally parabolic) flow. Thus, the different particle types end up with a differentially retarded downstream motion, which leads to separation. The process is illustrated in Figure 1. Because of the open and regular geometry of FFF channels and the simple parabolic flow pattern, the displacement velocity of each particle cloud can be predicted rather exactly in terms of the field-particle interaction forces. Therefore, the rate of migration and the level of separation can be calculated for known particles. More often, particle characteristics are unknown, obscured by the presence of a variety of other particles. In this case each particle type is isolated by the normal separative process of FFF and at the same time, through the observation of the downstream velocity of the particle cloud, the force exerted on the particle by a specified field can be calculated. This calculation provides many avenues for characterizing the different particles (1) J. C. Giddinns. Anal. Chem.. 53. 1170A (1981)
(2j J. C. Giddin&,'M. N. Myers,'and K.D. Chdwdl, Sep. Sci. Technol.,
16, 549 (1981).
of the mixture because particlefield interactions generally reflect some desired property (such as charge, mass, or diameter) of the particles. This approach has been shown to be useful for a great variety of biological, environmental, and industrial macromolecules and particle^.^^^ In the great majority of F F F experiments to date, the channel has consisted of a ribbonlike space between flat parallel plates as shown in Figure 1A. This open parallel-plate channel (OPPC) has a number of advantages, including the simplicity of constructing such units. In such a configuration the sample and carrier fluid streams are simple to introduce and withdraw by means of triangular end pieces. The considerable breadth (1-6 cm) of such channels provides a reasonable sample capacity. More importantly, the OPPC geometry (or something very close to it) can be made compatible with various applied forces. It is preferable that the field vectors intercept the channel walls at right angles, so that the field-induced displacement occurs in a direction perpendicular to the wall rather than along the wall. Furthermore, the field strength should be uniform across the wall area. Most fields and gradients can be conveniently arranged to satisfy these requirements in OPPC systems. At the same time, the channel can be oriented in a direction perpendicular to gravity in order to offset potential convective effects, which may originate in the imposed gradients (such as thermal gradients) or in the local variation of density caused by the formation of the particle clouds. Using OPPC systems, we have implemented FFF using thermal gradients (thermal FFF), electrical fields (electrical FFF), sedimentation forces (sedimentation FFF), and liquid cross flow (flow FFF).4 More recently, magnetic forces have been used.s Each of these fields, by causing migration and separation on the basis of different physicochemical parameters, displays its own unique separation spectrum and selectivity. Furthermore, each field leads to the characterization of particles by a unique set of properties. An alternative geometry for FFF consists of an annulus formed by a cylindrical tube or wire of radius rl (the inner cylinder) centered within a cylindrical cavity of radius r2 (the outer cylinder), (3) J. C. Giddinp, G. Karaiskakis, K. D.Caldwell, and. M. N. Myers, J. Colloid Inrerface Sci., 92,66 (1983). (4) J. C. Giddings, Sep. Sci. Technol., 19,831 (1984). (5) T. C. Schunk, J. Gorse, and M. F. Burke, Sep. Sci. Technol., 19,653 (1984).
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