The invention concerns a method of two-dimensional, heteronuclear correlation spectroscopy for the investigation of solid state samples, containing a first (.sup.1 H) and a second (.sup.13 C) nuclear species, in a nuclear magnetic resonance (NMR) spectrometer by means of an NMR pulse sequence, which pulse sequence comprises a preparation interval, an evolution interval, a mixing interval and a detection interval, wherein during the preparation interval the first nuclear species is excited by at least one preparation RF pulse in a first frequency band and is exposed to evolution RF pulses inside the first frequency band during the evolution interval and wherein during the detection interval the first nuclear species is exposed to at least one decoupling RF pulse inside the first frequency band while the free induction decay in the second frequency band is detected and wherein the pulse sequence (1.ltoreq.p.ltoreq.n) is repeated n times in a row with identical preparation interval, mixing interval and detection interval but with changed evolution interval and wherein the sample rotates with a rotation frequency greater than 1 kHz about an axis which is tilted by about 54.degree. with respect to the axis of a homogeneous magnetic field and wherein the at least one preparation RF pulse is broad-banded with a center frequency in the center of the NMR spectrum of the first nuclear species of the sample effecting a rotation of the nuclear magnetization of the first nuclear species about an axis perpendicular to the direction of the magnetic field (X) with an angle of preferably 90.degree. and wherein the evolution RF pulses form a so-called FSLG sequence with two successive broadband evolution RF pulses, phase shifted with respect to each other by 180.degree. (Y, -Y), whose center frequencies are shifted in opposite directions with respect to the preparation RF pulse and which each effect a rotation of the nuclear magnetization of the first nuclear species by about 294.degree..
Such a method is known from EP-A 0 481 256 (U.S. Pat. No. 5,117,186).
Nuclear resonance (NMR) is a phenomenon occurring in relation to a selected group of atomic nuclei and that is based on the existence of magnetic nuclear moments of these atomic nuclei. If an atomic nucleus with nuclear spin is placed in a strong homogeneous and static magnetic field (so-called "Zeeman field") and is excited by means of a weak radio frequency (RF) magnetic field, the nuclear spin precesses with a natural resonance frequency, the Larmor frequency, which is characteristic for each nuclear species with nuclear spin and which depends on the magnetic field strength effective at the location of the nucleus. Typical atomic nuclei with magnetic moments are e.g. protons .sup.1 H, .sup.13 C, .sup.19 F and .sup.31 P. The resonance frequencies of the nuclei can be observed by observing the transverse magnetization occurring after a strong RF pulse. Usually, the measured signal is transformed into a frequency spectrum by Fourier transformation.
Although identical nuclei show the same frequency dependence on the magnetic field, differences of the immediate chemical surroundings of each nucleus can modify the magnetic field, so that nuclei of the same sample do not see the same effective magnetic field. The differences of the local magnetic fields effect spectral shifts of the Larmor frequency between two such chemically not equivalent nuclei which are called "chemical shifts". These chemical shifts are of interest since they yield information about the number and position of the atoms inside a molecule and about the relative arrangement of neighboring molecules inside a compound.
Unfortunately, it is not always possible to give an interpretation of the frequency spectra caused by chemical shift since there are additional and possibly dominant interactions present.
This is particularly the case in NMR spectroscopy of solids. In NMR spectroscopy of liquids, the fast molecular movements have the tendency to isolate the nuclei and to separate the nuclear interactions, so that it is much easier to recognize different nuclei in a spectrum. In solid state NMR there are many interactions between the molecules masking the result. E.g., magnetic moments of neighboring nuclei interfere with each other leading to interactions called "dipole-dipole couplings". These couplings broaden the characteristic resonance lines and mask the "fine" resonance structure caused by chemical shift. A further problem occurring in relation to solids and which is not present in liquids, is that the orientation of molecules in solids are relatively fixed with respect to the applied Zeeman field. Therefore the chemical shifts are anisotropic so that a contribution to the resonance frequency depends on the spatial orientation of the molecules with respect to the magnetic field. Therefore, it is important to suppress some of these interactions in order to obtain meaningful results for the others. Usually this is achieved by exciting the system at selected frequencies with the consequence that undesired interactions cancel or at least that they are averaged to a reduced amplitude. For example, in solids the above-mentioned anisotropy of the chemical shift is usually largely reduced by orienting the solid sample relative to the applied magnetic field at the so-called "magic angle" (54.degree. 44') and by rotating it at this angle with a comparatively fast frequency. This averages the anisotropic field components to zero.
In a similar fashion, it is possible to reduce with known techniques spin-spin interactions by irradiating the nuclei with RF pulses at or close to the Larmor frequencies. By carefully selecting various polarizations and phases of the RF pulses, the magnetization of interfering nuclear spin systems in neighboring groups can be changed. Thereby spin-spin interactions can effectively be averaged out so that their contribution to the final measuring result is strongly reduced. Since for each nuclear species the Larmor frequency is different, an applied RF field will have a much greater effect on those spins with a Larmor frequency close to the applied frequency than on those spins with a significantly different Larmor frequency. In this way, applied RF fields can be used to influence one nuclear species whereas others remain unaffected.
Because of the special problems of solid state NMR, one usually applies a two-dimensional spectroscopy technique in the time domain in order to improve resolution. With this technique it becomes possible to investigate the interaction or "correlation" between two different nuclear species inside a solid--the interaction between protons and .sup.13 C nuclei is usually of great interest in many organic solids. The basic technique of two-dimensional heteronuclear correlation in relation to solids is well-known and described in many articles, as e.g. in "Heteronuclear Correlation Spectroscopy" by P.
Caravatti, G. Bodenhausen and R. R. Ernst, Chemical Physics Letters Vol. 89, No. 5, pp. 363-367 (July 1982) and in "Heteronuclear Correlation Spectroscopy in Rotating Solids" by P. Caravatti, L. Braunschweiler and R. R. Ernst, Chemical Physics Letters Vol. 100, No. 4, pp. 305-3107 (September 1983). We explicitly refer to the contents of these articles.
As described in the above-mentioned articles, the two-dimensional heteronuclear correlation technique comprises an "experiment" in the time domain generally consisting of four different sequential time intervals. The first interval is called "preparation interval". During this time, one of the two nuclear species under investigation is transferred into an excited, coherent nonequilibrium state, which changes or "evolves" during the following time intervals. The preparation interval can consist of irradiating of a single RF pulse or alternatively of a sequence of RF pulses. Usually, the preparation interval has a fixed length of time.
A second time interval is called "evolution interval" during which the excited nuclear spins "evolve" under the influence of the applied magnetic field, of the neighbor spins, possibly of irradiated periodic RF pulse sequences and of the sample rotation. Evolution of the excited nuclei during this interval makes it possible to determine these frequencies. A series of "experiments" or "scans" is executed, whereby the evolution time of the evolution interval is incremented systematically.
The evolution interval is followed by a "mixing interval". During the mixing interval, one or more RF pulses may be irradiated effecting the coherence or polarization transfer from the excited nucleus to the other investigated nuclear species. The coherence or polarization transfer, triggered by the mixing process, is characteristic of the investigated nuclear system.
Finally, the mixing interval is followed by a "detection interval" where the resonance frequencies of the second nuclear species are measured. Generally, during this time further pulses or continuous RF energy is irradiated in order to suppress a further interaction between both nuclear species (decoupling).
After Fourier transformation, the result of the multiple experiment is a two-dimensional spectral profile, called heteronuclear correlation spectrum (also: 2D HETCOR). One axis of the plot depicts the detected frequencies of the second nuclear species. The other axis represents the frequencies of the first nuclear species, derived via the repeated scans with incremented evolution times. Since the measured frequencies of the second nuclear species depend on the energy transfer from the originally excited first nuclear species and since on the other hand the state of the first nuclear species depends on the evolution time, the second plot axis effectively represents the chemical shifts due to the different first nuclear species in a specific molecule and on their spatial arrangement with respect to the second nuclear species. The measured peaks of the plots correspond to correlations between selected nuclei of first and second nuclear species within a given molecule. An advantage of the heteronuclear correlation is that it spreads the proton resonances over the by far greater chemical shift range of .sup.13 C. For this reason, this technique can yield well resolved information about the proton chemical shift of a sample, although it is impossible to resolve these chemical proton shifts with other one-dimensional spectroscopy techniques.
Generally, in a typical two-dimensional heteronuclear correlation experiment, applied to an organic material, the correlation between hydrogen nuclei (protons) .sup.1 H and .sup.13 C nuclei are investigated inside the sample. In order to do this, an RF pulse is applied during the preparation interval, exciting the hydrogen protons. In theory, the proton spins would now perform a free precession motion during the evolution interval. During the mixing interval, the protons interact with the .sup.13 C nuclei via direct heteronuclear dipole-dipole coupling. Finally, during the detection interval the .sup.13 C frequencies are measured. One of the advantages of such an experiment is that the heteronuclear coupling between the protons and the .sup.13 C nuclei depends exclusively from the distance between the nuclei, independent of the chemical shift. Therefore, the correlation offers a possibility to investigate the stereochemistry of individual molecules as well as the relative arrangement of neighboring molecules.
The problem of this technique is that other couplings, as e.g. a "homonuclear" dipole-dipole coupling between protons and the "heteronuclear" dipole-dipole coupling between protons and carbon nuclei can mask the desired result if these interactions are allowed to be present during the evolution interval, since they have an influence on the measurement of chemical shifts in the proton spectrum. These last two interactions effect a peak broadening of the proton chemical shifts, leading to an overlap of different proton sites and, as a consequence, to a smearing out of the assignment to the individual sites. Therefore it is necessary to suppress these two very strong interactions during the evolution interval. For certain conditions, if an element more abundant than .sup.13 C is investigated, e.g. phosphorus or aluminum, it may be necessary to suppress the homonuclear interaction between these nuclei.
Generally, one has to apply carefully selected RF pulse sequences in order to ensure suppression of the homonuclear and heteronuclear interactions during the evolution interval, wherein the pulses are either irradiated to the protons, to the .sup.13 C nuclei or to both simultaneously. The object of these pulse sequences is to suppress the results of the unwanted interactions or to average them out. Many pulse sequences of this type are known in the corresponding prior art. For example, there are prior art pulse sequences suppressing relatively effectively the homonuclear interactions between protons. In addition, other pulse sequences are known to suppress heteronuclear interactions between protons and .sup.13 C nuclei. Experiments to simultaneously suppress both, homonuclear and heteronuclear interactions, simply combined the known RF pulse sequences. However, since the known pulse sequences were not designed in view of a combination, very long sequences of RF pulses resulted that were necessary to suppress both interaction types and the methods did not yield satisfying results. Therefore, the number of resolved, non-equivalent proton sites was considerably limited. This on its turn limited the number of compounds that could be successfully investigated.
The publication EP-A 0 481 256 cited at the beginning, describes an improved method suppressing heteronuclear interactions more effectively. The suggested pulse sequence is designed such that it can be used in combination with one of the previously known pulse sequences. In this way, homonuclear as well as heteronuclear interactions are suppressed. In addition, the suggested pulse sequence effectively suppresses homonuclear interactions. Therefore it can be used in relation to a multitude of nuclear species. In detail, during the preparation interval the first nuclear species is excited by a preparation pulse and is irradiated during the evolution interval for homonuclear decoupling between nuclei of the first species (generally protons) with a so-called BLEW-12 sequence (phases X Y -X -X -Y -X X Y X X -Y -X), whereas for decoupling between both nuclear species (generally .sup.13 H-.sup.13 C) and of the nuclei of the second species (generally .sup.13 C--.sup.13 C) the second nuclear species is irradiated with a pulse sequence of 12 90.degree. RF pulses with a predetermined phase sequence, the so-called BB-12 sequence (-X Y -X X Y -X -X Y X -X Y -X). Since in this way the homo- as well as the heteronuclear interactions are decoupled, the protons can freely evolve being only under the influence of their chemical shift, leading to an improved resolution. After the evolution interval, two separated pulses (.theta. and .phi. pulses) are irradiated onto the protons in order to--in view of the following detection--tilt the magnetization formed during the evolution interval into a plane perpendicular to the magnetic field. The .theta.-pulse is a 90.degree. pulse and the .phi. pulse has an angle of 63.degree. (with -Y phase). These two pulses are followed by the so-called WIM-24 ("Windowless Isotropic Mixing") sequence selectively transferring nuclear polarization from the protons to directly coupled carbon nuclei via direct heteronuclear dipole interaction. In addition, the WIM-24 sequence suppresses the proton and .sup.13 C chemical shifts as well as the proton-proton and .sup.13 C--.sup.13 C homonuclear couplings. However, it leaves unaffected the proton-.sup.3 C heteronuclear coupling. The WIM-24 sequence consists of a 24 pulse sequence irradiated onto the protons and a corresponding 24 pulse sequence simultaneously irradiated onto the .sup.13 C nuclei. The sequence is prior art and is described in detail in the article "Heteronuclear Correlation Spectroscopy in Rotating Solids" by P. Caravatti, L. Braunschweiler and R. R. Ernst in Chem. Phys. Letters 100, No. 4, pp 305-310 (1983).
Finally, during the detection interval, a continuous wave (CW) signal of relatively high intensity is irradiated at the proton frequency in order to decouple in a known way the protons from the .sup.13 C nuclei and the .sup.13 C FID is measured.
During the entire experiment, the solid state sample is routinely rotated about the "magic angle" in order to reduce broadenings caused by the anisotropy of the chemical shift.
In the EP-A 0 481 256 (U.S. Pat. No. 5,117,186) mentioned at the beginning, it is also pointed out that instead of the WIM-24 sequence other prior art pulse sequences can be used, too, in order to effect selective cross polarization during the mixing interval and still to simultaneously suppress the homonuclear dipole interaction. The WIM-24 sequence is mentioned to be preferred but a phase and frequency switched Lee-Goldburg sequence (FSLG) in combination with a phase switched .sup.13 C sequence might effect a similarly effective selective cross polarization during the mixing interval. This mixing method is described in detail in the article "Frequency-Switched Pulse Sequences: Homonuclear Decoupling and Dilute Spin NMR in Solids" by A. Bielecki, A. C. Kolbert and M. H. Levitt in Chem. Phys. Letters 155, Nos. 4,5, pp. 341 (1989).
However, the method known from EP-A 0 481 256 (U.S. Pat. No. 5,117,186) has the disadvantage that the BLWE-12 sequence applied during the evolution interval has to be relatively long. Generally, it is limited to more than 36 .mu.s because of the otherwise endangered safety of the probehead against high voltage breakdown. This on the other hand limits the possible spin rates of the sample rotation about the magic angle since the rotation time has to be large compared to the time period of the BLEW-12 sequence. In practice, spin rates are limited to below 5 kHz by this, whereas present conventional probeheads already allow spin rates around 15 kHz. The article J. Magn. Res. A 120, p. 274-277 (1996) describes a method where even without additional narrowing of the proton spectrum indications of a resolution of the chemical shift can be achieved by a pulse sequence at high fields.
The article J. Magn. Res. A 121, p. 114-120 (1996) describes a method of NMR imaging where the line narrowing effect of the FSLG sequence is used to effect slice selection.
In the German patent DE 196 48 391 C1 (GB 2 319 848 A), in a dipolar HETCOR experiment the nuclear spins are decoupled during the evolution interval with respect to dipole coupling by an FSLG RF pulse sequence irradiated in the proton frequency band. Since this sequence--compared to the ones used so far--can be very short and there is no need to simultaneously irradiate radio frequency in the range of the S nuclei, this method is particularly suited to high rotational speeds of the sample and high magnetic fields where it effects a decisive resolution improvement.
All carbon-proton correlation experiments reported so far, are based on a magnetization transfer by dipolar couplings. Various schemes of polarization transfers have been suggested and were investigated in view of their sensitivity and distance selectivity, e.g. experiments from Hartmann-Hahn cross polarization to WIM (windowless isotropic mixing) multipulse sequences. Since all these experiments make use of interactions through space, one of the main problems remains to ensure the sufficient selectivity of the magnetization transfer in order to usefully interpret the spectrum. In other words, only magnetization from protons to directly bonded carbons shall be transferred but not to carbon nuclei that are further removed. Whereas correlation signals between not-bonded pairs may yield valuable information about molecule conformation, they nevertheless severely complicate analysis of a two-dimensional spectrum.
There is therefore a need for a method mentioned at the beginning enabling an improved selective magnetization transfer.