Proton-coupled carbon-13 J spectra in the presence of strong coupling. I.

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Proton-CoupledCarbon-13J Spectra in the Presenceof Strong Coupling.I. Some new experiments have recently been proposed which enable proton-coupled carbon-l 3 spectra to be recorded with significantly enhanced resolution by removing the limitation imposed by static magnetic field inhomogeneity (I, 2). They are extensions of the spin echo refocusing method used to produce proton spectra in a form where chemical shifts have been eliminated and the resonances appear at frequencies determined only by spin-spin coupling, the so-called J spectra (3). Used in conjunction with double Fourier transformation (4,s) the new techniques (6) generate proton-coupled carbon-13 J spectra in a two-dimensional display in which spin multiplets arising from different carbon-l 3 sites are completely separated (7) from one another. The improved resolution a:nd the elimination of overlap make this method particularly suited to the investigation of long-range proton-carbon spin coupling. The interpretation of proton J spectra is simple and straightforward only in cases where the coupling can be treated by a first-order approximation; otherwise the spectra a.re much more complex (8) and the extraction of the coupling parameters is considerably more difficult (9).This raises the question of the form of carbon-13 Jspectrain situations where there is strong proton-proton coupling. Many conventional proton-coupled carb’on-13 spin multiplets under these conditions lack the usual mirror symmetry about the chemical shift frequency (IO). The purpose of the present investigation is to examine the effects of strong proton-proton coupling on carbon-l 3 J spectra and to demonstrate that one of the possible modes of operation (the “gated decouple? method) generates a J spectrum identical in form with the conventional spectrum. Carbon-13 J Spectroscopy

The underlying principle of J spectroscopy is Fourier transformation of the echo modulation which may be observed in a Carr-Purcell spin echo experiment (II, 12). h’ormally, spin echoes are not modulated by heteronuclear coupling, but suitable J modulation can be introduced by one of two possible modifications of the experiment. In the gated decoupler method, the carbon-l 3 resonances are decoupled from the protons during the defocusing interval 0 to z, but are allowed to evolve while coupled to the protons in the refocusing interval z to 2r, building up phase errors on the multiplet components at the time of the echo; the phase errors constitute a phase modulation when the echo is monitored as a function of tl = 2t. The “proton flip” method achieves a similar phase modulation through the application of a 180” pulse to the protons in synchronism with the 180” refocusing pulse applied to carbon-l 3. The modulation arises in a manner analogous to that in homonuclear J spectroscopy in which both coupled nuclei experience the effects of the 180” pulse (3). The two techniques are by no means equivalent. For weak proton-proton coupling the proton flip experiment modulates each component of the carbon multiplet at a freCopyright 0 1977 by Academic Press, Inc. Al I rights of reproduction in any form reserved. Printed in Great Britain

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quency equal to its separation from the carbon-l 3 chemical shift (+J Hz in a two-spin system) whereas the gated decoupler experiment introduces modulation at half this frequency. There is an even more fundamental difference when these techniques are applied to systems with strong proton-proton coupling. J spectra obtained by the gated decoupler method are isomorphous with carbon-l 3 multiplets recorded by conventiona NMR spectroscopy, faithfully reproducing the asymmetry, whereas the proton flip version produces a J spectrum containing more lines than the conventional multiplet, and possessing an apparent mirror symmetry. Nevertheless it should be possible to extract all the usual parameters from such a spectrum. Experimental

The sample was a IO-mm tube of pyridine containing 10 % by volume of heavy water. The latter provided the deuterium signal for the internal lock, and also improved sensitivity by lengthening the molecular correlation time through hydrogen bond formation (23) thus decreasing the spin-lattice relaxation times for the carbon-13 resonances. Spectra were recorded at 20 MHz on a Varian CFT-20 spectrometer slightly modified to improve the stability of the pulse-timing circuits. The program modifications for double Fourier transformation have been described elsewhere (6). Although it is by no means the universal practice for high-resolution spin echo work, these experiments used a spinning sample and achieved a magnet inhomogeneity linewidth of approximately 0.3 Hz. The J spectra were derived from echoes excited by a single refocusing pulse, according to the “Method A” described by Carr and Purcell (12). The pulse widths were carefully calibrated and no significant artifacts were observed (6). The most convenient form of two-dimensional display is obtained by double Fourier transformation of half-echoes acquired under proton-decoupled conditions. The F2 frequency dimension then corresponds to differences in carbon-13 chemical shifts, while the J spectra from the various carbon sites are displayed in the Fl dimension (2). Because of the intrinsic asymmetry, it is important to display the upfield and downfield portions of each spin multiplet separately, which requires that the negative Fl quadrant be distinguished from the positive Fl quadrant. This is achieved by taking suitable linear combinations of the four components that result from double transformation (the sine transform of the cosine transform, etc.). In this display all the lines exhibit the complex “phase twist” lineshape described by Bodenhausen et al. (6), but on any one trace in the Fl dimension the phase may be readily adjusted to the pure absorption mode throughout, as required for the highest resolution. The Spectrum of Pyridine

In principle a carbon-l 3 multiplet will lack mirror symmetry about the chemical shift frequency if it constitutes the X resonance of an AB2X spin system, or any system of higher complexity (14). A good example of such asymmetry is provided by the C2 resonance of pyridine, which is an ABCDEX system or an ABB’CC’X system if the very small carbon-13 isotope shifts of the proton resonances are disregarded. The conventional slow passage spectrum of proton-coupled pyridine (10) shows this lack of mirror symmetry quite clearly. When the gated decoupler method is used, the form of the J spectrum is determined solely by the evolution of the nuclear magnetization under the influence of the spin-

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FIG. 1. A portion of the two-dimensional J spectrum of pyridine corresponding to the C2 site, obtained by the “gated decouple? caused by the low sampling rate has drawn the two halves of the spectrum together by 72 Hz.

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coupled Hamiltonian during the refocusing interval r to 22, and the spin multiplets retain the same form as in the conventional spectrum, even though the protons are strongly coupled. Figure 1 shows a portion of the phase-sensitive two-dimensional spectrum of pyridine encompassing the chemical shift of the C2 resonance. Earlier work with strictly symmetrical multiplets (6) adopted a sign convention where the Fl dimension ran from negative frequencies on the left to positive frequencies on the right. For the present purposes it is desirable to reverse the direction of this axis in order to display J spectra in the same sense as multiplets in a conventional NMR spectrum where the magnetic field increases from left to right. The C2 multiplet consists of two well-separated groups of lines split by the large direct proton-carbon coupling. The sampling frequency was chosen so as to alias the frequencies of these groups in the F, dimension, with the result that the large proton-carbon splitting ($J = 89.29 Hz) appears to be decreased by 72 Hz, twice the sampling frequency. In this display the frequency steps in the F2 dimension are so coarse that only a single trace carries an appreciable signal, which obscures the “phase twist” effect (6). Adjustment to the pure absorption mode is then straightforward. For purposes of comparison a conventional proton-coupled carbon-13 spectrum of the same pyridine sample was required. Although no overlap of multiplets occurs in the pyridine spectrum, the limited data capacity of the CFT-20 (SK) makes it very difficult to obtain satisfactory digitization across the region of interest without introducing problems of interference from aliased parts of the spectrum. This difficulty was circumvented by recording a partial spectrum from the C2 multiplet alone by selective excitation of the proton-decoupled spectrum followed by acquisition of the proton-coupled free induction signal (1.5). A further comparison was made by fitting a simulated theoretical spectrum to the conventional spectrum using a modified version of the iterative computer program LAOCOON III (16). The coupling constants reported by Hansen and Jakobsen (10) for pyridine-containing deuteroacetone were used as initial parameters, while the proton chemical shifts of the pyridine/D,O sample were derived from measurements at 6.34 T; the proton spectrum of pyridine is approximately first order at this field. Only the carbon-13-proton coupling constants and the proton 2, 6 and 3, 5 isotope shifts were allowed to vary in the iteration, which resulted in a root-mean-square error of 0.021 Hz over the 36 measured line positions. The simulated spectrum was given a Lorentzian lineshape with a full linewidth 0.36 Hz. The optimum fit was obtained with parameters similar to those of Hansen and Jakobsen (IO), including a 0. l-Hz isotope shift on the H3 proton. The carbon-proton coupling constants showed changes of up to 6 %, attributable to hydrogen bonding between the nitrogen and D,O. Figure 2a shows a single trace extracted from the two-dimensional spectrum obtained by the gated-decoupler technique, compared with the conventional spectrum (Fig. 2b) and the computer simulation (Fig. 2~). Since the gated decoupler method halves all protoncarbon splittings, this comparison entails a compensating change in the frequency scale. If this scale change and the linewidth difference are disregarded, it is clear that the three spectra have precisely the same form, with all the higher-order effects of spin coupling faithfully reproduced. This provides strong confirmation that the gated decoupler method is directly applicable to asymmetric carbon-13 multiplets. Part II of

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this work will analyze the proton-coupled carbon-l 3 Jspectrum of pyridine obtained by the proton flip technique. ACKNOWLEDGMENTS The authors gratefully acknowledge an equipment grant and research studentships (G.A.M. and D.L.T.) provided by the Science Research Council. Dr. I. D. Campbell very kindly obtained the 270MHz proton spectrum of pyridine. The double Fourier transformation program was written by G. Bodenhausen, R. Niedermeyer, and D.L.T.

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REFERENCES G. BODENHAUSEN,R. FREEMAN,AND D. L. TURNER, J. Chem. Phys. 65,839 (1976). G. BODENHAUSEN,R. FREEMAN,R. NIEDERMEYER,AND D. L. TURNER, J. Magn. Resonance 24,291 (1976). R. FREEMANAND H. D. W. HILL, J. Chem. Phys. 54,301 (1971). J. JEENER,Ampere International Summer School II, Basko Polje, Poland, 1971; Second European Experimental NMR Conference, Enschede, Holland, 1975. W. P. AUE, E. BARTHOLDI, ANLI R. R. ERNST,J. Chem. Phys. 64,2229 (1976). G. BODENHAUSEN,R. FREEMAN,R. NIEDERMEYER,AND D. L. TURNER, J. Magn. Resonance 26,133 (1977). L. MULLER, A. KUMAR, AND R. R. ERNST,J. Chem. Phys. 63,549O (1975). A. ABRAGAM, “The Principles of Nuclear Magnetism”, Oxford University Press, London/New York, 1961. R. L. VOLD AND R. R. SHOUP, J. Chem. Phys. 56,4787 (1972). M. HANSEN AND H. J. JAKOBSEN, J. Magn. Resonance lo,74 (1973). E. L. HAHN AND D. E. MAXWELL, Phys. Rev. 88,107O (1952). H. Y. CARR AND E. M. PURCELL, Phys. Rev. 94,630 (1954). I. D. CAMPBELL, R. FREEMAN, AND D. L. TURNER, J. Magn. Resonance 20,172 (1975). P. DIEHL AND J. A. POPLE, Mol. Phys. 3,557 (1961). G. B~DENHAUSEN, R. FREEMAN, AND G. A. MORRIS,J. Magn. Resonance 23,171(1976); R. FREEMAN, G. A. MORRIS, AND M. J. T. ROBINSON, J. Chem. Sac. Chem. Commun., 754 (1976). A. A. BOTHNER-BY AND S. M. CASTELLANO, in “Computer Programs for Chemistry” (D. F. DeTan Ed.), Vol. 1, Benjamin, New York, 1968. RAY FREEMAN GARETH A. MORRIS DAVID L. TURNER

Physical Chemistry Laboratory Oxford University Oxford, EngIand Received January 5,1977