JP2009541761A - Measurement of homonuclear J-coupling in ultra-low magnetic field by high resolution NMR spectroscopy - Google Patents

Abstract

The present invention relates to a method and apparatus for examining molecules by NMR spectroscopy. An object of the present invention is to comprehensively characterize a sample with high resolution. The purpose is to measure homonuclear and heteronuclear J-couplings in a small magnetic field and to use the measured homonuclear and heteronuclear J-couplings for sample characterization. Solved by a method and related apparatus for inspecting a sample using the method.
[Selection] Figure 1

Description

  The present invention relates to a method and apparatus for examining molecules by NMR spectroscopy.

Nuclear Magnetic Resonance Spectroscopy (NMR) and Magnetic Resonance Tomography (MRT) are highly successful non-destructive testing methods for imaging of materials or biological systems as well as elucidation of molecular structures. . Over the past 20 years, the great achievements of NMR and MRT have been reflected in three Nobel Prize winners (Richard Ernst 1991, Kurt Wuethrich 2002, Paul Lauterbur and Peter Mansfield, 2003). In particular, the elucidation of the structure of macromolecules and biomolecules by high-resolution or multidimensional NMR spectroscopy has advanced greatly. Basically, five interactions can be observed in NMR. It consists of:
1. Nuclear Zeeman interaction,
2. Quadrupole interaction of the electric nucleus,
3. Dipole-dipole interaction,
4. Chemical shift and 5. J-coupling.

  The last three interactions play an important role in elucidating the molecular structure. For example, the polarization transfer between different nuclei was pioneered through a dipolar cross relaxation mechanism (Nuclear Overhauser-effect spectroscopy, NOESY) for elucidating the structure of macromolecules. Publication “Ernst, RR, Bodenhausen, G. & Wokaun, A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions.” (Clarendon Press, Oxford, UK, 1987) ) ", The 2D COSY method employs not only chemical shifts but also J-coupling to measure the network of J-coupled nuclear spins and the resulting molecular structure. .

The chemical shift is caused by the magnetic field B ₀ generating a circular current through the molecular electrons, thereby generating an additional magnetic field of magnitude ΔB = σB ₀ at all positions of the observed nuclear spin. It is. Such an additional magnetic field is proportional to the magnetic field B _0, and the Larmor frequency of the nuclear spin observed with the amount of γσB ₀ :
ω ₀ = γB ₀
Fluctuate. Proton chemical species different in the molecule _{(e.g., CH 2, CH 3, OH} ) with respect to the chemical shifts in the range of 0 to 10 ppm, i.e., the Larmor frequency of protons species observed is only Larmor frequency It is only changed by a factor of 10 ^{−6 to} 10 ⁻⁵ multiplied by.

When B ₀ approaches 0, the chemical shift disappears. Thus, for small magnetic fields B ₀ , for example the geomagnetic field (5 · 10 ⁻⁵ T), the difference between the frequencies of two different proton species is only a few mHz. Such a slight difference cannot be observed in principle because the line width of the ¹ H-NMR spectral line exceeds 0.03 Hz. Therefore, the larger and more uniform the magnetic field B ₀ , the better the difference in chemical shift can be measured. This fact, and the signal-to-noise ratio that becomes larger as the magnetic field increases, is the reason for the trend towards larger and more expensive superconducting magnets.

  One exception to the rule that chemical shifts cannot be measured with a small magnetic field, ie, high-resolution NMR spectroscopy of Xe-atoms hyperpolarized in the earth's magnetic field, is described in the literature “High-resolution xenon nuclear magnetism in the earth's magnetic field. Resonance spectroscopy [Appelt, S., Haesing, FW, Kuehn, H., Perlo, J. & Bluemich, B. Mobile High Resolution Xenon Nuclear Magnetic Resonance Spectroscopy in the Earth's Magnetic Field. Phys. Rev. Lett. 94, 197602 (2005)] ”. The reason for the above exception is that the difference in chemical shift for Xe atoms is 100 times larger than that of protons and at the same time the line width of the Xe NMR line is very narrow (several mHz).

In the case of most nuclei, the chemical shifts cannot be measured at small magnetic fields, especially below 10 ⁻⁴ T, but for the last 50 years, in the following referred to as low-field NMR. Steady improvements have been made in the measurement of nuclear spin resonance in small magnetic fields. The first ¹ NMR spectrum in the geomagnetic field by Packard and Varian [References are Kaplan, J. Minutes of the Stanford Meeting December 28, 29 and 30,1953. Phys. Rev. 93, 939-954 (1954); page 941: Packard, M. & Varian, R. Beginning with Free Nuclear Induction in the Earth's Magnetic Field, development is the first heteronuclear ¹ H- ³¹ P and ¹ H- ¹⁴ NJ-cup Measurement of the ring [References are the applications of spectroscopy to the pre-magnetization method in Benoit, H., Hennequin, J. & Ottavi, H. (Champs faibles) Chimie Analytique 44,471-477 (1962); Bene, GJ, Nuclear Magnetism Of Liquid Systems In The Earth Field Range. Phys. Rep. 58,213-267 (1980)], water Of self-diffusion [Reference: Stepisnik, J., Kos, M., Planinsic, G. & Erzen, V. Strong Nonuniform Magnetic Field for Self-Diffusion Measurement by NMR in the Earth's Magnetic Field. J.Magn. Reson. Series A 107,167- 172 (1994)], measurement of water self-diffusion in Antarctic ice pores [reference is suitable for spin-echo measurements of self-diffusion pulse gradients under Antarctic conditions in Challaghan, PT, Eccles, CD & Seymour, JD An earth's field nuclear magnetic resonance apparatus suitable for pulsed gradient spin echo measurements of self-diffusion under Antarctic conditions.) Rev. Sci. Instrum. 68, 4263-4270 (1997)] [References are Planinsic, G., Stepisnik, J. & Kos, M. Relaxation-Time Measurement and Imaging in the Earth's Magnetic Field.] J. Magn. Reson. Series A110,170-174 (1994)], Earth magnetism Magnetic resonance tomography of the field [reference is Shushakov, A. Groundwater NMR in conductive water. Geophysics 61,998-1006 (1996)], and finally from micro Tesla to nano Tesla SQUID-tracking NMR in Range [McDermott, R. et al., Liquid-State NMR and Scalar Couplings in Microtesla Magnetic Fields. Science 295, 2247-2249 (2002); Trabesinger , AH et al., SQUID-Detected Liquid State NMR in Microtesla Fields. J. Phys. Chem. A108, 957-963 (2004); Burghoff, M., Hartwig, S ., Trahms, L. & Bernarding, J., Nuclear magnetic resonance in the nanotesla range. Appl. Phys. Lett. 87,054103 (2005); Greenberg, YS Conducted quantum interference device Rev.Mod.Phys.70,175-222 (1998); Clarke, J., SQUID sensors: Principles, configurations and applications (in Fundamentals, Fabrication) and Weinstock, H., Ed. (Kluwer Academic, Dordrrecht, Nederlands, 1996), 1-62.].

Although small chemical shifts are not measurable, in the case of most nuclei, for small magnetic fields, eg, less than 10 ^-4 T, molecules can be developed by developing a J-coupling mechanism. Information on the structure of the can be obtained. J-coupling between two nuclear spins (also known as spin-spin coupling) [Reference: On the nuclear magnetic moments of several stable isotopes (Proctor, WG & .Yu, FC On the Nuclear Magnetic Moments of Several Stable Isotopes.Phys.Rev.81,20-30 (1951 ))] , the nuclear spin I _a second being transported through the electrons in the chemical bonds between the I _a and I _B due to indirect mutual communication between nuclear spin I _B. The mathematical form of the J-coupling interaction energy is as follows:
H = 2π ・ J ・ I _A・ I _B
The J-coupling energy (generally 0.1-200 Hz for protons) depends on the relative orientation of the two nuclear spins (parallel or antiparallel to the B ₀ field), and This means that J-coupling is independent of the external magnetic field B ₀ , which is actually very important.

J-coupling between I _a and I _B may also occur across several chemical bonds. As a rule of thumb, the longer the bond distance (the more bonds are located between I _A and I _B ), the weaker the J-coupling constant or J-coupling energy. The independence of J-coupling from the magnetic field B ₀ means that the J-coupling constant can be measured with very high accuracy even with any small magnetic field.

Quantum mechanical calculations show that splitting in the NMR spectrum due to J-coupling can be observed only if the difference between the Larmor frequencies of the nuclear spins I _A and I _B is greater than the J-coupling to be observed. Show. The difference between the Larmor frequencies of I _A and I _B are present either by their different chemical shifts or different gyromagnetic ratio gamma _a and gamma _B,.

When the nuclei are different species with different gyromagnetic ratios γ _A and γ _B (eg, ¹ H and ¹⁹ F), J-coupling is called heteronuclear. If I _A and I _B are of the same type (e.g., two protons), coupling is called homonuclear. Therefore, heteronuclear J- couplings, to very small magnetic fields are possible (approximately 10 to ^-7 T) measurement, which is the difference between the Larmor frequencies of I _A and I _B are even 10 ^-7 T Even so, it is larger than the J-coupling.

Isonuclear J-coupling is another case. In the geomagnetic field, for example, the typical ethanol ¹ H-linewidth is about 100 mHz, while the chemical shift of the CH ₂ or CH ₃ group protons is a few mHz. Thus, all protons in ethanol are magnetically equivalent in the geomagnetic field. Homonuclear J-coupling cannot be measured. This fact is illustrated for ethanol molecules in FIG. Hereinafter, in the case of a high magnetic field of about 1.5 T, referred to as a high magnetic field, the chemical shift is much larger than the ¹ H-line width. Thus, the ¹ H-spectrum shows on the one hand three chemical shifts of the OH, CH ₂ and CH ₃ groups, and on the other hand the CH ₂ group (coupling with protons of the CH ₃ group:-> quartet). And the homonuclear J-coupling of the CH ₃ group (coupling with protons of the CH ₂ group:-> triplet). The proton of the OH group does not show homonuclear J-coupling with the other two groups, because the proton can move freely by hydrogen bridge bonds. In high magnetic fields, the combination of measured chemical shifts and homonuclear J-coupling allows an association with the structure of the ethanol molecule. This in turn forms the basis for high resolution high field NMR.

The B ₀ field becomes smaller and moves the lines closer together until the OH, CH ₂ and CH ₃ lines are merged with a very low magnetic field. Such decay is evidenced by the illustration in FIG. 1 for three magnetic fields (1.5T, 0.35T, and 5 · 10 ⁻⁵ T of the geomagnetic field) using the complete ethanol spectrum, A complete ethanol spectrum consists of a single line without any structural information.

In recent years, more accurate measurements of heteronuclear J-coupling have been successfully performed for external magnetic fields smaller than 10 ⁻⁴ T. Several years ago, McDermott et al. [Reference: McDermott, R. et al. “Liquid-State NMR and Scalar Couplings in Microtesla Magnetic Fields.” Science 295, 2247 -2249 (2002)] proved the measurement of heteronuclear ¹ H- ³¹ PJ-coupling constants using a SQUID instrument in the nT range.

^{The 1} H-line width is about 1 Hz, although it depends on the inhomogeneity of the B ₀ magnetic field in the above experiment. Recent validity of high-resolution ¹ H, ¹⁹ F and ⁷ LiNMR spectroscopy in the Earth magnetic field, S. Appelt, H. Kuehn, W. Haesing und B. Bluemich is in Nature Physics 2,105-109 (2006) ¹ This was proved by showing the H-line width to 0.03 Hz. The heteronuclear ¹ H- ¹⁹ F and ¹ H- ²⁹ SiJ-coupling constants of various molecules can also be measured with an accuracy of a few mHz. Such accuracy cannot be improved any further since the measured line width is already the intrinsic line width (given by the reciprocal of the transverse relaxation time T ₂ ). Most importantly, however, the accuracy of the measured J-coupling constant is 10 times or 100 times better when compared to the accuracy obtained with a superconducting high field magnet. Such extremely high resolution allows for the precise classification or identification of the chemical structure of macromolecules as well as small molecules. In the future, this will have a wide range of effects, for example considering the technical realization of low-field NMR devices for performing chemical analysis with high accuracy.

As an illustration of the high resolution frequency in the geomagnetic field, Fig. 2 shows the heteronuclear ¹ H- ²⁹ SiJ-coupling of tetramethylsilane (TMS) measured with a very homogeneous (~ 3ppb) 9.4T high field magnet. And the same J-coupling constant measured in the geomagnetic field is shown. The ¹ H- ²⁹ SiJ-coupling in the geomagnetic field is 6.617 + 1 mHz, about 100 times better than the J-coupling measured with a high-field magnet (J = 6.6 ± 0.1 Hz). (Source: Nature Physics 2, 105-109 (2006)).

FIG. 3 shows another illustration for geomagnetic field NMR using a nonafluorohexene molecule with 8 heteronuclear ¹ H- ¹⁹ FJ-couplings, all of which are identified in the spectrum. It is possible. This is an example demonstrating that a J-coupling network can be measured over many bond distances in a single measurement, thereby learning the molecular structure. The ¹⁹ F-spectrum of nonafluorohexene (NFH) shown in FIG. 3 is characterized by eight heteronuclear ¹ H- ¹⁹ FJ-couplings. ^{The 19} F-spectrum consists of a superposition of 4 triplets and 4 doublets. The upper curve is a simulation of this superposition and shows that the molecular structure of NFH can be observed with a single measurement. The number on the left side of the J-coupling constant indicates the number of chemical bonds observed between ¹ H and ¹⁹ F nuclei (source: Nature Physics 2,105-109 (2006)).

  However, according to the latest technology in the industry so far, molecular characterization by ultra-high resolution low-field NMR is only possible with the aid of heteronuclear J-coupling. Even if high frequency resolution is successfully achieved, the information obtained from the heteronuclear J-coupling constant alone does not include any information for the molecular groups occurring in the sample.

  An object of the present invention is to comprehensively characterize a sample with high resolution.

  The above objective is solved by a method for inspecting a sample through nuclear magnetic spectroscopy that measures homonuclear J-coupling in a small magnetic field and uses the measured isonuclear coupling for sample characterization. .

The person skilled in the art had the opinion that only a heteronuclear J-coupling of the sample can be measured in a small magnetic field (see, for example, Applied Physics Letters 87,054103 (2005), in the nano Tesla range. Nuclear magnetic resonance in the nanotesla range, page 1, second paragraph on the left. In practice, however, experts consider that all organic or biomolecules are mainly composed of carbon, hydrogen and oxygen, and nuclei such as ¹⁹ F or ²⁹ Si are relatively rare. I did not. Carbon consists of 99% ¹² C (nuclear spin I = 0) and 1% ¹³ C (nuclear spin I = 1/2). 1% of the ¹ H nuclei of all organic molecules couple with ¹³ C nuclei in a heteronuclear manner. Thus, ¹ H spectra in low magnetic fields are mainly not only single ¹ H-lines with no information about the structure (already suggested in the geomagnetic field of FIG. 1), but also heteronuclear ¹ H- ^It consists of a very small peripheral line (approximately 200 times smaller than the main line) resulting from ¹³ CJ-coupling. Contrary to the expert opinion, the current structure determination is based on the above ¹ H coupled in a heteronuclear manner with at least ¹³ C nuclei (1% occurrence) or other rare nuclei (eg ¹⁵ N). It is possible for the nucleus. This, in turn, leads to a measurable high resolution homonuclear J-coupled H-spectrum, which simultaneously contains information about heteronuclear coupling.

  Contrary to expectations, which are the mainstream among experts, isonuclear J-couplings generated in samples can in principle be measured in small magnetic fields.

  In order to be able to actually capture the spectrum of homonuclear J-coupling, in one embodiment, for example, the sample to be examined is pre-magnetized, preferably in a strong magnetic field of at least 1 Tesla. In one embodiment, particularly strong magnets, preferably 1 to 2 Tesla permanent magnets, are used for this purpose. When measurements are performed in a small magnetic field, the specialists used, in principle, relatively weak (electro) magnets for pre-magnetization, but with this, the size of 0.1 to 0.3 Tesla A magnetic field was generated. In fact, such a weak magnetic field used for pre-magnetization usually results in measurement results that are insufficient to perform the claimed method. A stronger magnet is used to perform the pre-magnetization, thereby deviating from the path normally used in the art.

In order to achieve good frequency resolution, it is particularly important to pay attention to the particularly good homogeneity of the weak magnetic field acting on the sample during the measurement. In one embodiment, the volume of the sample is selected as the smaller one in order to be close to the purpose. In particular, from the viewpoint of the present invention, the volume of a sample of less than 2 cm ³ is small. In general, a person skilled in the art selects a sample having a volume of 500 cm ³ or more for measurement in a weak magnetic field. From this aspect as well, we are moving away from normal measurements in this technical field.

In one embodiment, the sample is suitably enriched with, for example, ¹³ C nuclei to ensure an improved method that can be performed. Thereafter, the measured results are used for chemical characterization. Thereby, the ability to measure with considerably high accuracy and thus to characterize a sample, mainly an organic sample, in the geomagnetic field with very high accuracy is successfully mastered. In general, however, the process of the invention can also be carried out without such enrichment. Characterization with the geomagnetic field simplifies characterization, since the geomagnetic field does not require any technical effort and, of course, can be used freely.

As shown in FIG. 4, for example, the sample consists of molecules having the skeleton X ^M -A-B-X ^K. A and B are groups of any molecule to which a magnetically equivalent spin X is bound. m = hereinafter referred to as X ^M, X type 1, 2, ..., M-th spin, which is bonded to the group A of the molecule. k = X-type 1, 2,..., K-th spin, hereinafter referred to as X ^k , which is bound to the group B of the molecule. X represents, for example, the proton to be measured or another nucleus. Such a molecule contains, for example, ¹³ C atoms with a naturally occurring rate of 1% in part A of the molecule.

For example, M = 3 and K = 2. Two ¹ H- ¹³ C heteronuclear coupling constants of X ^K with X ^M and ¹³ C with a ¹³ C is specified as J _HA and J _HBA, ¹ H or the like between the X ^M and X ^K The nuclear coupling constant is designated as _JHH . Since the bond distance between X ^M and A is shorter than between X ^K and A, J _HA > J _HBA > J _HH is fundamentally applied. Thereafter, the H-NMR spectrum is measured periodically in a low magnetic field, and the first spectrum is typically a large center due to all magnetically equivalent uncoupled spins X ^M and X ^K. Includes lines. In addition, the spectrum includes two symmetric multiplet structures whose intensity is about 1/1000 compared to the intensity of the center line. The two multiplet structures generally consist of two quartets and two triplets whose splitting pattern is due to homonuclear J-coupling between X ³ and X ² . Four lines of a quartet structure with an intensity ratio of generally 1: 3: 3: 1 and a distance in the frequency space of J _HH are eight possible X ³ groups of three protons. It belongs to the NMR signal of two protons in the X ² group that are coupled in a homonuclear manner to the arrangement scheme. Conversely, three lines of triplet structure with an intensity ratio of 1: 2: 1 and the same distance J _HH are typically four possible types of two protons of X ² groups in a homonuclear manner. belonging to NMR signals of the three protons of the X ³ group for coupling to the array system. The quartet (or triplet structure) typically split symmetrically into two identical subgroups near the centerline indicates that the heteronuclear ¹ H- ¹³ CJ-coupling J _HBA ( Or J _HA ). In such a case, the ¹³ C nuclear spin can be parallel or anti-parallel to the magnetic field B 2 _O , and the quartet structure is separated from the center line by ± J _HBA / 2 (± J _HA / 2). (Triplet structure) is moved. This measurable spectrum is shown in FIG.

The homonuclear J-coupling between X ^M and X ^K spins in a low magnetic field can be measured in any case as long as the following equation can be met.
｜ J _X ^m _−A −J _X ^k _−B−A ｜ ＞ ｜ J _X ^m _X ^k ｜
In this case, J _XmXk is the _homonuclear J-coupling constant between the nucleus X ^m (m = [1, ... M]) and the nucleus X ^k (k = [1, ... K]). Defined. In principle, in a natural sample, there is at least one heteronuclear that is unequal to X at a sufficient concentration in part A of the molecule, and the two heteronuclear J-coupling constants J _XA and J _XBA are The amount varies. If the above equation can be met, the magnetic equivalence between X ^m and X ^k nuclei is eliminated, but this _causes coupling J _XA ≠ J _{XBA to} cause unequal splitting of the X nuclear NMR spectral lines. This is because the transition frequencies of X ^m and X ^k that were originally equivalent are moved to different ranges.
The nucleus X ^m then has its transition frequency (given by ω _H + J _XM / 2) sufficiently separated from the transition frequency of the nucleus X ^k (given by ω _H + J _XK / 2). Independent of the spin orientation of the nucleus X ^k , a magnetic transition can be made. If the difference in frequency between the core X ^m and X ^k is not to be larger than the homonuclear J- coupling between nuclei X ^m and X ^k, which is not the case, the nucleus X ^k However, this is because it “passes quickly” at the time of magnetic transition of the nucleus X ^m , and fundamentally no more homonuclear J-coupling can be measured. In fact, actual samples fundamentally satisfy these necessary boundary conditions, and the method of the present invention allows various samples to be successfully characterized.

  In one embodiment, an organic molecule is used as the sample because the organic sample in most cases meets the requirements under which the method of the invention can be performed. The method of the present invention is mainly used when positioning or characterizing a sample of organic molecules.

To ensure further improved operational capabilities of the method of the invention, the sample is selected such that there are at least two different heteronuclear J-couplings in the molecule that satisfy the following formula:
｜ J _X ^m _−A −J _X ^k _−B−A | >> | J _X ^m _X ^k |
In principle, this is the case for organic samples. The method of the present invention is applied particularly when positioning the sample. Alternatively, the sample is suitably enriched prior to performing the method of the present invention.

From the standpoint of the present invention, a small external magnetic field B ₀ is present, particularly when it is smaller than 10 ⁻⁴ T. Preferably, the geomagnetic field is used as a small external magnetic field, because the geomagnetic field is basically particularly homogeneous. A homogeneous external magnetic field is particularly advantageous for obtaining excellent measurement results.

  In order to obtain a further improved characterization capability, the sample's heteronuclear J-coupling is additionally measured and used for characterization. This overall allows the sample to be fully characterized in a manner that is significantly improved over the prior art.

  In order to measure in a further improved way, in one embodiment the sample is pre-magnetized by hyperpolarization of the nuclear spins. This enhances the signal to noise ratio.

  Despite the absence of chemical shift information, the method of the present invention allows for detailed structure determination. Those skilled in the art thought that in order to determine the detailed molecular structure, not only heteronuclear J-coupling but also chemical shift information had to be known. Such an idea is not necessary in the method according to the invention. This is because homonuclear J-coupling has been observed, which, coupled with heteronuclear J-coupling, allows detailed molecular structure determination in a much improved manner.

  On the other hand, the spectrum can be read. Otherwise, the sample can be analyzed by comparing the known spectrum with that spectrum. If an already known spectrum is found in the sample, this means that the sample has a known spectrum of material.

In the following, the invention is further illustrated by the drawings and embodiments.
FIG. 5 shows the structure H ³ —C—B—H ² in the case of the molecule X ^M -A-B-X ^K , that is, the weak correlation limit (| ω _H −ω _C | >> | J _HC |). The structure of the spectrum which carried out the isonuclear coupling in the low magnetic field with respect to the molecule | numerator which has is shown. ¹³ C nuclei have an incidence of 1% in the A group. The above structure of the spectrum in the low magnetic field looks completely different from that in the high magnetic field. When X represents a proton ( ¹ H), FIG. 5 further shows M = 3, K = 2, with 1% of all molecules X ^M —A—B—X ^{K in} the A group. There are ¹³ C nuclei with I = 1/2 ( ¹³ C spontaneous rate = 1%). ^The two ¹ H- ¹³ C heteronuclear coupling constants of X ^{M with 13} C and X ^K with ¹³ C are designated as J _HA and J _HBA , and X ^M with J _HH ω _H (ω _C ). The ¹ H isonuclear coupling constant between X ¹ and X ^K refers to the Larmor frequency for ¹ H ( ¹³ C) nuclei. Since the bond distance between X ^M and A is shorter than between X ^K and A, the following formula applies:
｜ J _HC −J _HBC ｜ ＞ ｜ J _HH ｜

The ¹ H-NMR spectrum in the low magnetic field in the case of the weak correlation limit (ω _H −ω _C >> J _HA ) shown in FIG. 5, on the one hand, is all magnetically equivalent and coupled. It consists of a large centerline, resulting from two spin multiplet structures that result from no spins X ^M and X ^K and otherwise have a thousandth intensity compared to the intensity of the center line. The two multiplet structures consist of two quartets and two triplets, and these splitting patterns are due to the homonuclear J coupling between X ³ and X ² . Four lines of a quartet structure with an intensity ratio of 1: 3: 3: 1 and a distance in the frequency space of J _{HH represent} eight possible arrangements of three protons of X ³ groups. It belongs to the NMR signal of the two protons of the X ² group, which are coupled in a homonuclear fashion. Conversely, three lines of triplet structure with an intensity ratio of 1: 2: 1 and the same distance J _HH are four possible arrangements of the two protons of the X ² group in a homonuclear manner. Belongs to the NMR signal of the three protons of the X ³ group that couple to The splitting of the quartet structure (or triplet structure) into two identical subgroups symmetrically around the central line indicates that the heteronuclear ¹ H- ¹³ CJ-coupling J _HBA (or J _HA ). In the above case, the ¹³ C nuclear spin may be parallel or antiparallel to the magnetic field B ₀ , and the quartet structure (triplet structure) is ± J _HBA / 2 (± Move away by J _HA / 2).

FIG. 6 schematically shows all isonuclear ¹ H- ¹ HJ-couplings observable at low magnetic fields for the more complex case of n-butanol in the weak correlation limit case.

The n-butanol molecule consists of a linear array of four carbon atoms with four possible positions (1-4 in FIG. 6) relative to the ¹³ C nuclear spin. ^{If 13} C nuclear spins situated in the 1-position of the butanol, the cup for one of the two protons of the CH ₂ groups, a plurality of proton of CH ₂ groups, the 2 and 3 positions in the homonuclear method Ring, or a plurality of protons in the CH ₃ group are coupled to the 4-position. The triplet set with frequency distances ² J _HC , ³ J _HC and ⁴ J _HC arranged symmetrically near the main line is the result. The number on the upper left side next to the J-coupling constant indicates the number of chemical bonds between the observed nuclei. The individual triplets, in turn, reflect the proton of the CH ₂ group at position ^{1 and the} isonuclear ¹ H- ¹ HJ-coupling constant of the CH ₂ or CH ₃ group (position 2-4). Because of different bond distances, there are three different isonuclear coupling constants ³ J _H-HC > ⁴ J _HH > ⁵ J _HH . Multiple J-coupled lines allow easy structural identification of molecules.

^{If the 13} C spin is in the 2nd or 3rd position, then a similar coupling pattern composed of 3 triplet sets will result, but the J-coupling constant has been shown previously All things may be different. ^{When the 13} C spin is in position 4, it also results in a set of three quartets with different isonuclear and heteronuclear J-coupling constants. The J-coupling pattern as a whole is characteristic for n-butanol molecules. In this manner, without using chemical shift information, the measurement of heteronuclear and isonuclear networks in low magnetic fields can be used to acquire a fingerprint of the n-butanol molecular structure, or according to the present invention The fingerprint can be measured. In general, the method according to the invention can also be applied to complex molecules without the use of complex high-frequency electronic systems, superconducting magnets or sophisticated cooling techniques (SQUID).

If the weak correlation limit condition is not met, the spectrum becomes more complex. As in the embodiment of FIG. 5, if we are concerned about the case of a strongly coupled CH ₃ group (ω _H −ω _{C to} J _HC ), the quadruple group becomes an octet group. The set becomes a set of 9 lines.

Because the homonuclear J-coupling constant between X ^M and X ^K nuclei can be very small (usually less than 1 Hz), the spectrometer used must be operated at the highest frequency resolution possible. Don't be. Ideally, the spectrometer should exhibit negligible line broadening in the instrument, and the spectral line width is close to the intrinsic line width (at the smallest possible).

For the same reason, it must be noted that the low field NMR spectrometer used is very sensitive. The signal-to-noise ratio exceeds 1000 for the main line, so that a small number of X nuclei coupled in a heteronuclear fashion to rare nuclei (eg ¹³ C generated at 1%) You must ensure that it is observed. If the rare nuclei are, for example, ¹⁵ N (I = 1/2), the requirement on the sensitivity of the spectrometer is much higher, but this is because the natural incidence of ¹⁵ N nuclei is 0.36%. Because there is.

If the sensitivity of the NMR spectrometer used is insufficient, the sample to be examined can be enriched with isotopes, for example ¹³ C. Then, the intensity of all homonuclear ¹ H- ¹ H coupling line is increased to 2 orders of magnitude. This means that the requirements for the sensitivity of the spectrometer are not higher. However, enrichment is a very expensive method and is hardly feasible for mobile applications (eg, on-line analysis of petroleum molecules).

FIG. 7 shows an example of the distribution of TMS molecular fragments with respect to the structure of the tetramethylsilane (TMS) molecule determined according to the present invention and the general arrangement of X ^M —A—B—X ^K molecules. In the above ^{examples, X = 1 H, M =} 3, K = 9, A = 13 C, and B = ⁽¹² C) is ₃ -Si.

For about 1% of all TMS molecules, ¹² C nuclei with spin I = 0 are replaced with ¹³ C nuclei with spin I = 1/2. Thus, in the above case, the A group is simply a ¹³ C nucleus and the B group consists of three ¹² C atoms bonded to a central Si atom. Naturally, silicon consists of 95% ²⁸ Si with a nuclear spin I = 0 and 4.7% Si with a nuclear spin I = 1/2. The measured X nucleus is a proton, M is three magnetically equivalent protons bonded to the A group (= ¹³ C), and K is the nine magnetic bonds bonded to the B group. Are equivalent protons. Since 4.7% of the total Si atoms are ²⁹ Si atoms with nuclear spin I = 1/2, the heteronuclearity of J _H-Si = 6.617 Hz between 12 protons and ²⁹ Si Coupling splits the ¹ H spectrum into two lines. Such J-coupled doublets ( ²⁹ Si nuclei are parallel or antiparallel to the magnetic field B ₀ ) have already been shown in FIG.

By the way, in only one of the 100 TMS molecules, there is a ¹³ C nucleus in the A group (1% incidence), which means that two different heteronuclear ¹ H- ¹³ C couplings (1J _HA ~ 120Hz and 3J _HBA ~ 2Hz), negating the magnetic equivalence of 12 protons. The nine magnetically equivalent protons of the B group are K, the M is the three magnetically equivalent protons of the A group having an isonuclear coupling constant of ⁴ J _HH << 1 Hz, etc. Coupled in a nuclear manner. 3 protons M, all of the ¹ H downward three upward or (all three of the ^{1 H} to have the eight possible sequences method, or two one in upward downward or 1, The two quartet structures have a frequency distance of 3./h−b−a, as shown in FIG. 8. ~ 2 Hz. Was the is shown, according to ¹ H-NMR, the measurement of homonuclear ¹ H- ¹ H coupling of TMS in the Earth magnetic field. At the same time, heteronuclear ¹ H- ²⁹ Si (J _H-Si = 6.617 Hz), ¹ H- ¹³ CJ-coupling (where 3J _HBA = 2.139 Hz) and ⁴ J _HH = 0.209 Hz. homonuclear ¹ H- ¹ H coupling with the triplet set is observed with a high resolution. Thus, very small homonuclear J-couplings (0.209 Hz) can be measured with the highest precision, thereby capturing even the fine structural differences of molecules. Thus, the entire molecular structure is captured in a single spectrum.

The potential of the proposed method for structure description is even higher when X = ¹³ C is used as the nucleus to be measured. When compared with the proton spectrum, the spectral resolution is more than 10 times better because the T ₂ relaxation time for ¹³ C nuclei can be very long (T ₂ -3 seconds to 3 minutes). ). However, due to the very small signal-to-noise ratio, strong pre-magnetization, such as PHIP (Para Hydrogen Induced Polarization Transfer), SEOP (Spin Exchange Optical Pumping), or SPINOE (Spin Polarized Induced Nuclear Overhauser Effect) method should be adopted [Reference: Nuclear-spin by Bowers, CR & Weitekamp, DP chemical reaction and nuclear magnetic resonance Transformation of symmetrization order to nuclear-spin magnetization by chemical reaction and nuclear magnetic resonance. Phys. Rev. Lett. 57, 2645-2648 (1986); Goldman, M., Johannesson, H. , Axelsson, O. & Karlsson, M. Polarization of ¹³ C through order transfer from parahydrogen: a novel contrast agent for MRI (Hyperpolarization of ¹³ C through order transfer from parahydrogen: A new contrast agent FOR MRI.). Magn.Reson.Imag.23,153-157 (2005); Happer, W. Optical Pumping.Rev.Mod.Phys.44,169-249 (1972) ;. Appelt, S. et al. ³ He and ¹²⁹ Xe spin - theory of exchange optical pumping (theory of spin-Exchange optical pumping of 3 He and 129 Xe), Phys.Rev.A58,1412-1439 (1998); Bouchiat , MA, Carver, TR & Varnum , nuclear polarization in ³ He gas to be induced in the CM optical pumping and dipole exchange ^{(nuclear polarization in He 3 gas induced} by optical pumping and dipolar exchange) .Phys.Rev.Lett.5,373- 375 (1960); Colegrove, FD , Schearer, LD & Walters, the polarization of the ³ He gas by GK optical pumping ^{(polarization of He 3 gas by optical} pumping) .Phys.Rev.132,2561-2572 (1963)].

To date, only the one-dimensional case of NMR spectroscopy for magnetic fields below 10 ⁻⁴ T is dealt with. In NMR spectra with various multiplet sets, it is possible to unambiguously estimate the number of numerous measurement nuclei (eg ¹ H, ¹³ C,...) Bound to the A or B group. . Even though all heteronuclear coupling constants can be determined from the spectrum, in all cases it is not possible to estimate which nucleus the measured nucleus will couple to. Such a problem is solved by incorporating a second dimension in the frequency space into the spectrum. In a manner similar to two-dimensional NMR [Reference: Ernst, RR, Bodenhausen, G. & Wokaun, A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions. Clarendon Press, Oxford, UK, 1987)], for example, during the measurement of a spectrum, it is possible to radiate low intensity radio frequencies continuously. The emitted frequency must be far enough away from the measuring Larmor frequency so as not to interfere with the measuring signal of the measuring nucleus. If the radio frequency resonates with an unknown nucleus that is heteronuclearly coupled to the nucleus being measured, the heteronuclear J-coupling on average causes the corresponding multiplet set to disappear from the spectrum. Since the radiation frequency is known, it is possible to determine what type the nucleus is. When repeating a one-dimensional experiment on a measuring nucleus at a number of different radio frequencies, all heteronuclear and homonuclear J-couplings of the measuring nucleus are scattered in the first-dimensional frequency space and are heteronuclear A two-dimensional NMR spectrum is acquired, in which all types of nuclei (except protons) coupled to the nuclei to be measured in the system are scattered in the second dimensional frequency space.

In the case of a TMS molecule in the geomagnetic field (4.698-10 ⁻⁵ T) with a corresponding Larmor frequency of protons at 2000 Hz (γ _H = 4.257 kHz / G), then the ¹³ Larmor frequency is At 503 Hz (γ _13c = 1.0708 kHz / G), the Larmor frequency of ²⁹ Si is 397.7 Hz (γ _29Si = 0.8465 kHz / G). For example, if radiated exactly at 503 Hz with a magnetic field strength B ₁ , the ¹³ C magnetization oscillates at the Rabi frequency V _R = γ _{13 C} B ₁ and averages heteronuclear ¹ H- ¹³ CJ-coupling. To do.
In the one-dimensional ¹ HTMS spectrum, the quartet set shown in FIG. 8 disappears. This is because the appearance condition of the quartet set is the presence of heteronuclear ¹ H- ¹³ C coupling. This is because. Similarly, heteronuclear ¹ H- ²⁹ SiJ-coupling averages when using 397.7 Hz radiation. As a result, a temporary fall of the multiplet structure appears accurately at the location where the heteronuclei in the proton 2D spectrum resonate. This is illustrated in FIG. 9, which shows a ¹ H2D spectrum simulated in the geomagnetic field for TMS molecules. More excellent molecular structure mapping is possible compared to 1D spectra. This is because, apart from all J-coupling constants, all nuclear types, including their associated J-couplings, are known from the 2D spectrum.

In one embodiment, the proposed 2D low field NMR method can be further improved if a larger magnetic field in the mT range is selected instead of the geomagnetic field. At 1 mT (= 10 ⁻³ T), the Larmor frequency of ¹³ C nuclei is about 10 kHz. ^Since the range of ¹³ C chemical shift is about 100 ppm, the difference in ¹³ C resonance frequency due to various chemical environments is about 1 Hz. However, if the magnetic field B ₀ = ¹ mT is very homogeneous (ΔB ₀ / B ₀ to 10 ⁻⁶ ) and temporarily stable, ¹³ C emitted in the ¹ H2D spectrum (˜42 kHz Larmor frequency) By changing the radio frequency (eg, every 10 mHz), it is possible to distinguish between various ¹³ C chemical shifts. In the 2D spectrum, this allows the assignment of all measured ¹³ C chemical shifts with an associated J-multiplet set, allowing final resolution of the molecular structure.

  Using the methods described in this application, for example, the structure of mobile petroleum molecules can be elucidated. Isonuclear and heteronuclear J-coupling, for example, isolates heptane, octane, benzol and other petroleum components on-site during oil exploration (already in or near the oil well), so that most Oil quality can be graded in an accurate manner.

  For example, chemical reactions in which technical changes occur under difficult conditions (eg, large-scale reaction chambers) can be characterized by (mobile) NMR according to the present invention while the reaction proceeds. This is important, for example, for on-line characterization of polymerization reactions during plastic manufacturing.

  Quality control of liquid food products (e.g., alcoholic beverages, ethanol, aromatics aqueous components, etc.) is another area of application of the method of the present invention.

  Finally, mobile NMR devices that map the structure of small to medium sized proteins or biomolecules are of great interest in the pharmaceutical industry. The method proposed in the present invention is substantially more flexible compared to the performance of structural analysis using a superconducting magnet, and allows the use of an inexpensive mobile NMR apparatus.

  In the following, an apparatus for carrying out the inventive method of mobile high resolution NMR will be described.

The central unit of the device shown in FIG. 10 consists of two Helmholtz coils (4) that generate as homogeneous a magnetic field (5) as possible (B ₀ = 0.2 to 200 G). In such a case, the homogeneity ΔB / B ₀ must be at least 1 ppm. In order to achieve sufficient temporary stability of the B ₀ field, the current stabilizer (6) must have a temporary stability of 1 ppm or more for over 1 hour. If necessary, the homogeneity must be obtained using an additional shim coil. Screening (10) is performed to avoid changes or distortions of the magnetic field due to the environment at the measurement location.

  The desired polarization of the sample (1) to be measured may be pre-magnetized by a Halbach magnet (2) or other hyperpolarization technique (11) followed by sample transfer (5) to the measuring coil (7). (SEOP, PHIP, SEOP + SPINOE). In the case of a sample having a short longitudinal relaxation time (T1 <1 second), the transfer from the Halbach magnet (2) to the measuring coil (7) can be performed within about 100 ms using compressed air.

  The sample is excited by a DC or AC pulse transmitter coil (8) generated by an NMR electronic system (9). The “free induction decay” (FID) is then measured with the receive coil (7) and the NMR electronics system (9), recorded by the data acquisition system, and then evaluated.

  The arrangement shown in FIG. 10 enables the movable performance of ultra-high resolution NMR spectroscopy. A highly sensitive NMR electronic system with thousands of signal-to-noise ratios for uncoupled protons with double magnet screening using a screening factor of at least 5000 is a special feature.

The principle of measurement of homonuclear J-coupling in an ultra-low magnetic field will be explained once again. Consider a single molecule consisting of two chemical groups A and B with no nuclear spin and proton ¹ H bonded to each other (FIG. 11a, left). ^There are two different chemical shifts of transition frequencies ω _{H, A} and ω _{H, B} for ¹ H-A and B ¹ H, respectively, and a ^homonuclear J-coupling constant J ^hom . The coupling constant can easily be measured at the weak correlation limit | ω _{H, A} −ω _{H, B} | >> 2π | J ^hom | However, in the ultra-low magnetic field with the magnetic field strength B ₀ <10 ⁻⁴ T, the strong correlation limit applies | ω _{H, A} −ω _{H, B} | << 2π | J ^hom |.
Two limitations are illustrated by the mechanical analogologon from FIG. 1a, the two coupled transducers on the right. Each vibrator represents a | position | = HA, HB of one chemical group ¹ HA—AB- ¹ H molecule, and consists of a spring having a spring constant D _i and a mass m. . These two vibration frequencies are ω ₁ = (D ₁ / m) ^1/2 . In this model, a homonuclear J-coupling between two protons corresponds to a spring having a constant D ^hom between the two masses. In a high magnetic field, the two groups are not magnetically equivalent and ω _{H, A} −ω _{H, B} | >> (D ^hom / m) ^1/2 , so ω _{H, A} ≠ ω _{H, B} And the spring incorporates a beat of frequency (D ^hom / m) ^1/2 . The retention spectrum (FIG. 11b) shows the splitting of the ¹ H line transferred by two frequency chemical shifts and homonuclear J-coupling. The two groups are magnetically equivalent so that the two spring constants coincide (D _{H, A} = D _{H, B} ) in an ultra-low magnetic field. The two masses are expected to oscillate at the following identical displacement phases, and the spring constant D ^hom does not affect the motion. Thus, the spectrum consists of a single line at frequencies ω _H = ω _H , _A = ω _{H, B} (FIG. 11c). In this case, the information for the homonuclear J-coupling constant is not valid.

である場合、超低磁場において定数Ｊ^homを有する等核性カップリングを見ることができる。量子力学的計算により、上記条件が、等核性及び異核性Ｊ-カップリングをすべて含めて、一般的に、マルチスピンシステムに対して有効であることが示される。予測スペクトル（図１１ｅ）は、多様な異核性Ｊ-カップリング定数を有する、周波数ω_H周りの対称的にグループ化した、２つの組の二重項からなる。ここで、等核性及び異核性Ｊ-カップリング定数は、超低磁場において測定可能である。 In FIG. 11d, the spring model was extended with two additional springs of constant D ^het _{H, C} ≠ D ^het _{H, B, C} in parallel with the springs of D _{H, C} and D _{H, B.} This induces different vibration frequencies for the two mass bodies, even if D _{H, C} = D _{H, B} , and induces an observable beat by D ^hom . If one of the chemical groups is not in the other but has a heteronuclear spin, the magnetic equivalence between the two protons is high. For simplicity, if only one carbon ¹³ C atom is considered as group A, one obtains the molecular structure ¹ H- ¹³ C-B- ¹ H (FIG. 11d, left side). The two protons show two different heteronuclear J-coupling constants J ^het _{H, C} , J ^het _{H, B, C,} which cause the chemical shift difference in ultra-low magnetic fields to disappear accordingly. In order to correct the two proton frequencies. as a result,

If so, a ^homonuclear coupling with a constant J ^hom can be seen in an ultra-low magnetic field. Quantum mechanical calculations show that the above conditions are generally valid for multi-spin systems, including all homonuclear and heteronuclear J-couplings. The predicted spectrum (FIG. 11e) consists of two sets of doublets symmetrically grouped around the frequency ω _H with various heteronuclear J-coupling constants. Here, homonuclear and heteronuclear J-coupling constants can be measured in an ultra-low magnetic field.

From this, the following liquids investigated: tetramethylsilane (TMS) with ¹³ C and natural abundance of silicon ²⁹ Si, and 99% ¹³ C-enriched methanol are discussed. Sample preparation, setting of geomagnetic field NMR, pre-magnetization and measurement procedures are described in document [41].

および｜¹Ｊ_Ha,C−³Ｊ_Hb,C｜＞＞｜⁴Ｊ_Ha,Hb｜であるため、予測される^１Ｈ-ＴＭＳスペクトルは、１組の四重項及び１組の４０本の線からなる。実際、ＴＭＳスペクトルの近景（図１２）は、１：３：３：１の強度比を有する１組の四重項を示し、それは、カップリングされていない^１Ｈ-線の付近で対称的にグループ化されている。それぞれの四重項構造の２つの中心の間の周波数分離から、³Ｊ_Hb,C＝２.１３９Ｈｚが測定された。
２個の後続する四重項線の周波数の差は、⁴Ｊ_Ha,Hb＝０.２０９Ｈｚである。もっとも小さい四重項線の強度は、カップリングされていないプロトンの強度Ａ_０よりも、１，０００倍以上小さい。 Initial density under the influence of an N + 1 spin Hamilton matrix to simulate the NMR spectrum of a J-coupled N + 1-spin system (N proton spins and one ¹³ C spin) The change over time of the matrix was calculated numerically. A 2 ^{N + 1} × 2 ^{N + 1} dimensional J-coupled Hamiltonian matrix is formed by N proton spin operators and one ¹³ C spin operator constructed by direct product approximation [42 ]. This was obtained by calculating the free transverse decay (FID) of the entire transverse proton spin multiplied by the density matrix. After Fourier transforming the calculated FID, the spectrum was phase corrected. The abscissa relaxation time was introduced by well elucidating essential spectral features.
Isonuclear J-coupling was first observed in the geomagnetic ¹ H-NMR spectrum of natural abundance tetramethylsilane (TMS). TMS has 12 protons J-coupled to the silicon isotope ²⁹ Si (amount f = 0.047), or ¹³ C (amount f = 0.01). In FIG. 12, the TMS J-coupled ¹ H-NMR spectrum is dominated by ¹ H- ²⁹ Si coupling. It is a ¹ H- ²⁹ Si doublet J-coupled with normalized strengths A ₀ = 1 and ² J _{H, Si} = 6.607 Hz (the number on the upper left of the J-coupling constant is coupled) The isolated centerline at 2060.3 Hz.
^{The 1} H ^-29 Si doublet has no substructure, which means that all heteronuclear ¹ H ^-29 SiJ-coupling constants are completely identical and the relation | ω _H −ω _This is to comply with _Si | >> 2π | J _{H, Si} |. Therefore, the 12 protons are all magnetically equivalent to ²⁹ Si, and the homonuclear J-coupling constants ⁴ J _{H and H} cannot be observed. However, the situation is quite different for 1% of ¹³ CH ₃ —Si— ( ¹² CH ₃ ) ₃ molecules. 3 pieces of protons, heteronuclear J- coupling constant ¹ J _Ha, but coupling to ¹³ C with C = 118Hz [41], which, ³ J _Hb, Cup ¹³ C with C = 2.139Hz It differs from the coupling constant of the nine protons that are ringed. Such differences in heteronuclear ¹ H- ¹³ CJ-coupling constants indicate a magnetic equivalence between three protons of ¹³ CH ₃ groups and nine protons of three ¹² CH ₃ groups. Destroy.
In the geomagnetic field

And | ¹ J _{Ha, C} ⁻³ J _{Hb, C} | >> | ⁴ J _{Ha, Hb} |, the predicted ¹ H-TMS spectrum is one set of quartets and one set of 40 Consists of lines. In fact, the close view of the TMS spectrum (FIG. 12) shows a set of quartets with an intensity ratio of 1: 3: 3: 1, which is symmetrical around the uncoupled ¹ H-line. Grouped. From the frequency separation between the two centers of each quartet structure, ³ J _{Hb, C} = 2.139 Hz was measured.
The difference in frequency between two subsequent quartet lines is ⁴ J _{Ha, Hb} = 0.209 Hz. Intensity of the smallest quartet line, than the strength A ₀ of protons that are not coupled, small more than 1,000 times.

The intensity of the multiplet line can be estimated. Define M as the number of all proton spins, N as the number of proton spins in one group, and MN as the number of proton spins in the other group. When MN spin is observed, the intensity of the minimum line of the N + 1 multiplet is given by A _{N + 1} = A ₀ × f × (1/2) × ((MN) / M) / 2 ^N It is done. For TMS with M = 12, N = 3, f = 0.01 and A ₀ = 1, the intensity of the minimum quartet line is A ₄ = 5 × 10 ⁻⁴ , which is It coincides with the minimum quartet line of the TMS spectrum.

The ¹ H-spectrum of the three protons of the ¹³ CH ₃ group is initially four lines due to a strong coupling such as | ω _H −ω _C | = 2π × 1542 Hz≈13 × 2π | ¹ J _{Ha, C} | Split into one set. The reason for the split into four lines will be explained in the next section. Each of the four lines is further split into multiples with ten lines by homonuclear J-coupling to the nine protons of the other three methyl groups. When N spin is observed, the intensity of the minimum line of the MN + 1 multiplet is A _{M−N + 1} = A ₀ × f × (½) × (N / M) / 2 ^MN . In the case of TMS, A ₁₀ = 2 × 10 ⁻⁶ . The intensity of the smallest of the 40 lines is given by A ₄₀ = (1/4) A ₁₀ ≈5 × 10 ⁻⁷ , which cannot be measured with the spectrometer of the present invention. In the high-field ¹ H-spectrum of the corresponding TMS with a line width of about 1 Hz, the ¹ H- ²⁹ SiJ-coupling constant can hardly be resolved, and the quartet set is not coupled. Hidden in the wide of protons. In high magnetic _{^{field, 1 J H, C = 1}} H- 13 CJ- -coupled doublet of 118Hz is observed [41], a pair of multiplets having a 10 mains each 0.209Hz interval The complete structure consisting of is not decomposed. Note that the geomagnetic TMS spectrum provides more structural information than the high field spectrum, even though chemical shift information cannot be extracted from the ultra low magnetic field ¹ H-spectrum.

である。それぞれ４本の線のある１組の多重項が観察される（図１３ａ、上側）。
非対称的なパターン及び可変的な線空間配置が、¹Ｊ_H,C＝１４０.６Ｈｚ⁴⁴の強くカップリングされた４-スピン¹³ＣＨ₃-ハミルトニアンの影響下に展開される横軸^１Ｈ-磁化の量子力学的計算と完全に一致する（図１３ａ、下方）。分裂は、純粋に低磁場における強い異核性^１Ｈ-¹³ＣＪ-カップリングに起因するものであり、この場合、対応する対角要素の差に比べて、ゼーマン（Ｚｅｅｍａｎ）固有原則では、¹³ＣＨ_３-ハミルトンマトリックスにおける非対角要素がもはや小さくないことに留意する。これは、エネルギー固有値の多様な変動、及び、２つのＪ-カップリングされた線の縮重の上昇の原因となる。それぞれのＪ-カップリングされた線に対する４本線の数は、下記のように説明することができる：¹³ＣＨ₃基の１つのプロトンを観察する場合、他の２つのプロトンスピンは、磁場に関して、３つの可能な配向の三重項状態及び１つの可能な配向の一重項状態のいずれかに分類することができる。これは、異なる周波数で合計１＋３＝４本の線をもたらす。プロトン化学シフト情報は、地球磁場スペクトルからは得られないが、¹³ＣＨ₃基の化学構造は、４本線の１組に分裂されることにより、同定することができる。同様の現象が、低磁場で強くカップリングされたＰＨ₂基に対する２本線の１組を観察したＢｅｎｏｉｔらによって最初に報告された［３９］。一般に、強くカップリングされたＹＸ_N基（Ｙ＝スピン１/２、Ｘ＝測定されたスピン１/２、Ｎ＝自然数）に対して、それぞれの多重項線の数Ｋが以下のように与えられる１組の多重項を測定するシミュレーションから発見した：

式中、Ｕは、奇数の集合を示す。言い換えれば、Ｎ＝１、２、３、４の強くカップリングされた¹³ＣＨ_N基は、１、２、４及び６本のＪ-カップリングされた線の１組として、それぞれ、超低磁場スペクトルにおいて同定することができる。 FIG. 13 shows the geomagnetic field ¹ H-spectrum of ¹³ C enriched methanol (f = 0.99) measured at two different temperatures. At T = 60 ° C., since OH-protons are rapidly exchanged between different molecules (residence time τ << 0.1 s), they perform any homonuclear or heteronuclear J-coupling. Absent. The uncoupled proton is visible in the ¹ H-spectrum by the center line at 2061.29 Hz on the top of FIG. 13a. Under the prevailing rapid exchange, the J-coupled geomagnetic spectrum is an example of a strongly coupled 4-spin system with 3 protons and 1 ¹³ C, in this case ,

It is. A set of multiplets, each with four lines, is observed (FIG. 13a, upper side).
Asymmetrical pattern and variable line space _arrangement, ¹ J H, _C = 140.6Hz strongly-coupled 4-spin ¹³ CH ₃ ⁴⁴ - horizontal axis ¹ H- magnetization is expanded under the influence of Hamiltonian Is completely consistent with the quantum mechanical calculation of (Fig. 13a, bottom). The splitting is purely due to strong heteronuclear ¹ H- ¹³ CJ-coupling in a low magnetic field, in which case the Zeeman eigenprinciple, compared to the corresponding diagonal element difference, is ¹³ Note that off-diagonal elements in the CH ₃ -Hamiltonian matrix are no longer small. This causes various fluctuations in the energy eigenvalue and an increase in the degeneracy of the two J-coupled lines. The number of four lines for each J-coupled line can be explained as follows: When observing one proton of the ¹³ CH ₃ group, the other two proton spins are There are three possible orientation triplet states and one possible orientation singlet state. This results in a total of 1 + 3 = 4 lines at different frequencies. Proton chemical shift information cannot be obtained from the geomagnetic spectrum, but the chemical structure of the ¹³ CH ₃ group can be identified by splitting it into a set of four lines. A similar phenomenon was first reported by Benoit et al. Who observed a set of two lines for a PH ₂ group strongly coupled in a low magnetic field [39]. In general, for a strongly coupled YX _N group (Y = spin 1/2, X = measured spin 1/2, N = natural number), the number K of each multiplet line is given by Found from a simulation that measures a set of multiplets:

In the formula, U represents an odd set. In other words, a strongly coupled ¹³ CH _N group with N = 1, 2, 3, 4 is a very low magnetic field as a set of 1, 2, 4, and 6 J-coupled lines, respectively. Can be identified in the spectrum.

At a temperature T = −80 ° C. (FIG. 13b, upper side), the residence time of the OH-protons is sufficient so that the homonuclear J-coupling between the OH— group and the ¹³ CH ₃ group does not fall on average. Long (τ> 1 s) [45, 46]. From _{here, | ω H -ω C | ≧} 2π | 1 J H, C | and _{^{| 1 J H, C - 2}} J H, C | ≧ | 3 J H, H | a is two conditions strongly correlated limit Below, a 5 spin system is observed for H—O— ¹³ CH ₃ . Isonuclear J-coupling begins to be observable, and each of the four strongly coupled ¹ H-lines in the line group of FIG. 13a is split into doublets, each of which is an 8-line external pair. (FIG. 13a, upper side). The frequency separation between the doublets is approximately the homonuclear coupling constant ³ J _{H, H} = 5.0 Hz ⁴⁴ . The observed outer set of each eight lines is in complete agreement with the quantum mechanical calculations shown below in FIG. 13b. Here, the J-coupling constants are assumed to be ¹ J _{H, C} = 140.6 Hz, ² J _{H, C} = -2.4 Hz and ³ J _{H, H} = 5.0 Hz [44, 47]. As shown at the bottom of FIG. 13b, the calculation also predicts a set of 6 transitions observable around the ¹ H-line that is not coupled at 2061.29 Hz. The number of six lines is formally similar to the strongly heteronuclear coupled ¹³ CH ₄ molecule, but one OH-proton is strongly isonuclear with respect to the other _three protons of the ¹³ CH ₃ group. is J- coupling _{^{(| 1 J H, C -}} 2 J H, C | ≧ | 3 J H, H |) is different. Note that a small peak at the frequency of the uncoupled ¹ H-line is visible in the experimental spectrum of FIG. 13b (but not in the simulated spectrum). This is due to the 1% uncoupled ¹ H-signal of methanol molecules containing ¹² C without nuclear spin.

In summary, the inventor has shown that valuable information regarding molecular structure can be acquired by NMR in ultra-low magnetic fields where no valid chemical shift information is available. There are two possible reasons for this: 1) The heteronuclear strong correlation limit (| ω _H −ω _C | ≧) that induces the characteristic heteronuclear splitting of ¹ H-resonance into a set of multiplets. By the action of 2π | ¹ J _{H, C} |), one chemical group (for example, ¹³ CH ₃ ) can be identified in an extremely low magnetic field. 2) If two or more different chemical groups constitute a molecule, and one of these contains a rare spin, eg, a ¹³ C nucleus, the homonuclear and heteronuclear coupling networks are multiplet sets. As observed. The underlying mechanism for multiplet generation is that the magnetic equivalence between protons of various chemical groups is increased by the presence of various heteronuclear J-coupling constants from protons of various chemical groups to rare spins. It is to be. At low magnetic fields, the spectral pattern can be used as a fingerprint of the molecular structure, even though the spectral complexity of large molecules with rare spins of natural abundance is high. As a result, not only the molecular structure, but also dynamic processes such as ¹ H-exchange between molecules can be studied with high accuracy in an ultra-low magnetic field.

FIG. 2 shows a J-coupled ¹ H-NMR mechanical analog. (A) A bead-spring model of two spins ¹ H-A-B- ¹ H (left side) interacting via a chemical group A and B through a ^homonuclear J-coupling with a coupling constant J ^hom Right). D _{H, A} , D _{H, B} and D ^hom represent the corresponding spring constants, and m is the mass of the two coupled mechanical oscillators. When D _{H, A} = D _{H, B} , D ^hom is not measurable. The corresponding high field spectrum (b) and ultra-low field spectrum (c) were sketched as retention spectra. (D) 3-spin system ¹ H- ¹³ C-B- ¹ H (left side) and corresponding mechanical analogologon (right side). J ^het _{H, C} and J ^het _{H, B, C} are two different heteronuclear J-coupling constants, corresponding to D ^het _{H, C} and D ^het _{H, B, C} in the bead-spring model To do. Note that D ^hom can be measured even if D _{H, C} = D _{H, Be} . (E) Corresponding retention spectra in very low magnetic fields showing all three homonuclear and heteronuclear J-couplings. It is the figure which showed the ¹ H-spectrum of the geomagnetic field by which J-coupled tetramethylsilane (TMS). Nine scans at T = 60 ° C. were averaged. The uncoupled proton line with transition frequency 2060.3 Hz (B ₀ = 4.8391 × 10 ⁻⁵ T) is normalized to one. ^The doublet of ² J _{H, Si} = 6.607 Hz corresponds to heteronuclear ¹ H ^-29 SiJ-coupling derived from TMS molecules containing ²⁹ Si but not ¹³ C. Heteronuclear J-coupling constant ³ An additional set of quartets separated by J _{Hb, C} = 2.139 Hz is derived from a TMS molecule containing ¹³ C but not ²⁹ Si. Coupling constant ⁴ appears with J _{Hb, Ha} = 0.209 Hz. The line width of each quartet line is about 0.05 Hz. FIG. 6 shows experimental and simulated simulated J-coupled geomagnetic field ¹ H-spectrum of 99% ¹³ C enriched methanol. All experimental spectra are the average of 9 scans. The width of all simulated lines is 0.16 Hz (T ₂ = 2s). (A) The ¹ H-spectrum (top) measured at T = 60 ° C. shows a set of four lines with irregular spacing. The width of the experimental J-coupled line is about 0.07 Hz. The simulation of the spectrum is shown at the bottom. (B) ¹ H spectrum measured at T = −80 ° C. (top). Homonuclear J-coupling between OH-group and ¹³ CH ₃ -group protons can be observed by slow intermolecular ¹ H-exchange. The line width of the J-coupled line is about 0.6 Hz. The simulated spectrum of the 5-spin HO- ¹³ CH ₃ is shown at the bottom.

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Claims (22)

  A method for inspecting a sample by nuclear magnetic spectroscopy, characterized in that homonuclear J-coupling is measured in a small magnetic field and the measured isonuclear coupling is used for characterization of the sample. . The method according to claim 1, wherein the volume of the sample is 0.1 to ⁴ cm ³ , preferably 1 to 2 cm ³ .   3. A method according to claim 1 or 2, wherein the sample is pre-magnetized with a magnetic field having a strength of at least 0.1 Tesla, preferably at least 1 Tesla.   The method according to any one of claims 1 to 3, wherein an organic molecule is used as a sample. The method according to any one of claims 1 to 4, wherein a molecule having the structure X ^M -A-B-X ^K is examined as a sample. The method according to any one of claims 1 to 5, wherein ¹ H- ¹ HJ-coupling of the sample is measured and used for characterization.   7. A method according to any one of the preceding claims, wherein the sample is provided or selected such that there are at least two different heteronuclear J-couplings in the sample. 8. A method according to any one of the preceding claims, characterized in that the magnetic field is weaker than ^10-3 Tesla, preferably weaker than ^10-4 Tesla, more preferably a geomagnetic field.   The method according to any one of claims 1 to 8, wherein the heteronuclear J-coupling of the sample is further measured and used for characterization.   10. A method according to any one of claims 1 to 9, wherein the sample is pre-magnetized, in particular by hyperpolarizing the nuclear spin of the sample. 11. A method according to any one of the preceding claims, wherein radio frequency radiation is provided continuously during measurement of the proton spectrum, and the emitted frequency is preferably significantly different from the ¹ H Larmor frequency. A method for inspecting a sample by nuclear magnetic spectroscopy, in which homonuclear and heteronuclear J-couplings are measured with a small magnetic field (<5 · 10 ⁻³ T), and the molecular structure in the sample is multiplexed in association. A method characterized in that it can be determined from a term structure. The method of claim 12, wherein the NMR spectrometer including the B ₀ field is much smaller than the measured intrinsic linewidth of nuclei, and wherein the resulting line width extension by the instrument.   14. Method according to claims 12 and 13, characterized in that the NMR spectrometer is very sensitive enough to measure the small intensity of the multiplet structure with a sufficient signal to noise ratio. The volume of the sample is 0.1~4cm ^3, preferably A process according to any one of claims 12 to 14 is 1 to 2 cm ^3.   16. A method according to any one of claims 12 to 15, wherein the sample is pre-magnetized with a magnetic field having an intensity of at least 0.1 Tesla.   17. A method according to any one of claims 12 to 16, wherein molecules having at least two different heteronuclear J-couplings are used. The method according to any one of claims 12 to 17, wherein a molecule having the structure X ^M -A-B-X ^K is used as a sample. 19. A method according to any one of claims 12 to 18, wherein the magnetic field is weaker than ^10-3 Tesla, preferably weaker than ^10-4 Tesla, more preferably a geomagnetic field.   20. The method according to any one of claims 12 to 19, wherein the nuclear spin to be measured is polarized by a hyperpolarization technique (spin-exchange optical pumping, PHIP, DNP). To determine the type of nucleus in which the J-coupling occurs, provide radio frequency radiation continuously while measuring the NMR spectrum of the X-type nucleus ( ¹ H, ¹³ C, ¹⁹ F, ...) The radiated frequency resonates with a coupled heteronuclear nucleus belonging to group A or B and is remote from the frequency of the X nucleus so as not to interfere with the measurement. 21. The method according to any one of 20. An apparatus for performing the method according to any one of claims 1 to 21, wherein means for polarizing the sample, a transmitter coil (8) for exciting the sample, and preferably for more than 1 hour. An apparatus using means for generating a homogeneous magnetic field having a homogeneity of 1 ppm or more over 1 cm ³ and a magnetic screening having a screening factor of at least 5000.

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