- The essence of the phenomenon
First of all, it should be noted that although the word “nuclear” is present in the name of this phenomenon, NMR has nothing to do with nuclear physics and has nothing to do with radioactivity. If we talk about a strict description, then one cannot do without the laws of quantum mechanics. According to these laws, the interaction energy of a magnetic core with an external magnetic field can take only a few discrete values. If magnetic nuclei are irradiated with an alternating magnetic field, the frequency of which corresponds to the difference between these discrete energy levels, expressed in frequency units, then the magnetic nuclei begin to move from one level to another, while absorbing the energy of the alternating field. This is the phenomenon of magnetic resonance. This explanation is formally correct, but not very clear. There is another explanation, without quantum mechanics. The magnetic core can be thought of as an electrically charged ball rotating around its axis (although, strictly speaking, this is not the case). According to the laws of electrodynamics, the rotation of a charge leads to the appearance of a magnetic field, i.e., the magnetic moment of the nucleus, which is directed along the axis of rotation. If this magnetic moment is placed in a constant external field, then the vector of this moment begins to precess, i.e., rotate around the direction of the external field. In the same way, the spinning wheel axis precesses (rotates) around the vertical, if it is unwound not strictly vertically, but at a certain angle. In this case, the role of the magnetic field is played by the gravitational force.
The precession frequency is determined both by the properties of the nucleus and by the strength of the magnetic field: the stronger the field, the higher the frequency. Then, if, in addition to a constant external magnetic field, an alternating magnetic field acts on the nucleus, then the nucleus begins to interact with this field - it, as it were, swings the nucleus more strongly, the precession amplitude increases, and the nucleus absorbs the energy of the alternating field. However, this will occur only under the condition of resonance, i.e., the coincidence of the precession frequency and the frequency of the external alternating field. It looks like a classic example from high school physics - soldiers marching across a bridge. If the step frequency coincides with the natural frequency of the bridge, then the bridge sways more and more. Experimentally, this phenomenon manifests itself in the dependence of the absorption of an alternating field on its frequency. At the moment of resonance, the absorption increases sharply, and the simplest magnetic resonance spectrum looks like this:
- Fourier spectroscopy
The first NMR spectrometers worked exactly as described above - the sample was placed in a constant magnetic field, and RF radiation was continuously applied to it. Then either the frequency of the alternating field or the intensity of the constant magnetic field changed smoothly. The energy absorption of the alternating field was recorded by a radio frequency bridge, the signal from which was output to a recorder or an oscilloscope. But this method of signal registration has not been used for a long time. In modern NMR spectrometers, the spectrum is recorded using pulses. The magnetic moments of the nuclei are excited by a short powerful pulse, after which a signal is recorded, which is induced in the RF coil by freely precessing magnetic moments. This signal gradually decreases to zero as the magnetic moments return to equilibrium (this process is called magnetic relaxation). The NMR spectrum is obtained from this signal using a Fourier transform. This is a standard mathematical procedure that allows you to decompose any signal into frequency harmonics and thus obtain the frequency spectrum of this signal. This method of recording the spectrum allows you to significantly reduce the noise level and conduct experiments much faster.
One excitation pulse to record the spectrum is the simplest NMR experiment. However, there can be many such pulses, of different durations, amplitudes, with different delays between them, etc., in the experiment, depending on what kind of manipulations the researcher needs to perform with the system of nuclear magnetic moments. However, almost all of these pulse sequences end in the same thing - recording a free precession signal followed by a Fourier transform.
- Magnetic interactions in matter
In itself, magnetic resonance would remain nothing more than an interesting physical phenomenon, if it were not for the magnetic interactions of nuclei with each other and with the electron shell of the molecule. These interactions affect the resonance parameters, and with their help, NMR can be used to obtain a variety of information about the properties of molecules - their orientation, spatial structure (conformation), intermolecular interactions, chemical exchange, rotational and translational dynamics. Thanks to this, NMR has become a very powerful tool for studying substances at the molecular level, which is widely used not only in physics, but mainly in chemistry and molecular biology. An example of one of these interactions is the so-called chemical shift. Its essence is as follows: the electron shell of the molecule responds to an external magnetic field and tries to shield it - partial shielding of the magnetic field occurs in all diamagnetic substances. This means that the magnetic field in the molecule will differ from the external magnetic field by a very small amount, which is called the chemical shift. However, the properties of the electron shell in different parts of the molecule are different, and the chemical shift is also different. Accordingly, the resonance conditions for nuclei in different parts of the molecule will also differ. This makes it possible to distinguish chemically nonequivalent nuclei in the spectrum. For example, if we take the spectrum of hydrogen nuclei (protons) of pure water, then there will be only one line in it, since both protons in the H 2 O molecule are exactly the same. But for methyl alcohol CH 3 OH there will already be two lines in the spectrum (if other magnetic interactions are neglected), since there are two types of protons - protons of the methyl group CH 3 and a proton associated with an oxygen atom. As the molecules become more complex, the number of lines will increase, and if we take such a large and complex molecule as a protein, then in this case the spectrum will look something like this:
- Magnetic cores
NMR can be observed on different nuclei, but it must be said that not all nuclei have a magnetic moment. It often happens that some isotopes have a magnetic moment, while other isotopes of the same nucleus do not. In total, there are more than a hundred isotopes of various chemical elements that have magnetic nuclei, but no more than 1520 magnetic nuclei are usually used in research, everything else is exotic. Each nucleus has its own characteristic ratio of the magnetic field and the precession frequency, called the gyromagnetic ratio. For all nuclei these ratios are known. Using them, one can choose the frequency at which, for a given magnetic field, a signal from the nuclei needed by the researcher will be observed.
The most important nuclei for NMR are protons. They are most abundant in nature, and they have a very high sensitivity. For chemistry and biology, the nuclei of carbon, nitrogen and oxygen are very important, but scientists were not very lucky with them: the most common isotopes of carbon and oxygen, 12 C and 16 O, do not have a magnetic moment, the natural nitrogen isotope 14 N has a moment, but it for a number of reasons it is very inconvenient for experiments. There are 13 C, 15 N and 17 O isotopes that are suitable for NMR experiments, but their natural abundance is very low and the sensitivity is very low compared to protons. Therefore, special isotopically enriched samples are often prepared for NMR studies, in which the natural isotope of one or another nucleus is replaced by the one needed for experiments. In most cases, this procedure is very difficult and expensive, but sometimes it is the only way to get the necessary information.
- Electron paramagnetic and quadrupole resonance
Speaking of NMR, one cannot fail to mention two other related physical phenomena - electron paramagnetic resonance (EPR) and nuclear quadrupole resonance (NQR). EPR is essentially similar to NMR, the difference lies in the fact that the resonance is observed on the magnetic moments not of atomic nuclei, but of the electron shell of the atom. EPR can be observed only in those molecules or chemical groups whose electron shell contains the so-called unpaired electron, then the shell has a non-zero magnetic moment. Such substances are called paramagnets. EPR, like NMR, is also used to study various structural and dynamic properties of substances at the molecular level, but its scope is much narrower. This is mainly due to the fact that most molecules, especially in living nature, do not contain unpaired electrons. In some cases, it is possible to use a so-called paramagnetic probe, i.e. a chemical group with an unpaired electron that binds to the molecule under study. But this approach has obvious drawbacks that limit the possibilities of this method. In addition, in EPR there is no such high spectral resolution (ie, the ability to distinguish one line from another in the spectrum) as in NMR.
It is most difficult to explain the nature of NQR "on the fingers". Some nuclei have a so-called electric quadrupole moment. This moment characterizes the deviation of the distribution of the electric charge of the nucleus from spherical symmetry. The interaction of this moment with the gradient of the electric field created by the crystalline structure of the substance leads to the splitting of the energy levels of the nucleus. In this case, resonance can be observed at a frequency corresponding to transitions between these levels. Unlike NMR and EPR, NQR does not require an external magnetic field, since level splitting occurs without it. NQR is also used to study substances, but its scope is even narrower than that of EPR.
- Advantages and disadvantages of NMR
NMR is the most powerful and informative method for studying molecules. Strictly speaking, this is not one method, but a large number of different types of experiments, i.e., pulse sequences. Although they are all based on the NMR phenomenon, but each of these experiments is designed to obtain some specific specific information. The number of these experiments is measured by many tens, if not hundreds. Theoretically, NMR can, if not everything, then almost everything that all other experimental methods for studying the structure and dynamics of molecules can, although in practice this is, of course, far from always feasible. One of the main advantages of NMR is that, on the one hand, its natural probes, i.e., magnetic nuclei, are distributed throughout the molecule, and, on the other hand, it makes it possible to distinguish these nuclei from each other and obtain spatially selective data on properties of the molecule. Almost all other methods provide information either averaged over the entire molecule, or only about one of its parts.
There are two main disadvantages of NMR. First, this is a low sensitivity compared to most other experimental methods (optical spectroscopy, fluorescence, EPR, etc.). This leads to the fact that in order to average the noise, the signal must be accumulated for a long time. In some cases, the NMR experiment can be carried out for even several weeks. Secondly, it is its high cost. NMR spectrometers are among the most expensive scientific instruments, costing at least hundreds of thousands of dollars, with the most expensive spectrometers costing several million. Not all laboratories, especially in Russia, can afford to have such scientific equipment.
- Magnets for NMR spectrometers
One of the most important and expensive parts of a spectrometer is the magnet, which creates a constant magnetic field. The stronger the field, the higher the sensitivity and spectral resolution, so scientists and engineers are constantly trying to get the highest possible fields. The magnetic field is created by an electric current in the solenoid - the stronger the current, the greater the field. However, it is impossible to increase the current indefinitely; at a very high current, the solenoid wire will simply begin to melt. Therefore, superconducting magnets, i.e., magnets in which the solenoid wire is in the superconducting state, have been used for a very long time for high-field NMR spectrometers. In this case, the electrical resistance of the wire is zero, and no energy is released at any current value. The superconducting state can only be obtained at very low temperatures, just a few degrees Kelvin - this is the temperature of liquid helium. (High-temperature superconductivity is still only a matter of purely fundamental research.) It is with the maintenance of such a low temperature that all the technical difficulties in the design and production of magnets are connected, which cause their high cost. The superconducting magnet is built on the principle of a thermos matryoshka. The solenoid is in the center, in the vacuum chamber. It is surrounded by a shell containing liquid helium. This shell is surrounded by a shell of liquid nitrogen through a vacuum layer. The temperature of liquid nitrogen is minus 196 degrees Celsius, nitrogen is needed so that helium evaporates as slowly as possible. Finally, the nitrogen shell is isolated from room temperature by an outer vacuum layer. Such a system is able to maintain the desired temperature of the superconducting magnet for a very long time, although this requires regular pouring of liquid nitrogen and helium into the magnet. The advantage of such magnets, in addition to the ability to obtain high magnetic fields, is also that they do not consume energy: after the start of the magnet, the current runs through the superconducting wires with virtually no loss for many years.
- Tomography
In conventional NMR spectrometers, they try to make the magnetic field as uniform as possible, this is necessary to improve the spectral resolution. But if the magnetic field inside the sample, on the contrary, is made very inhomogeneous, this opens up fundamentally new possibilities for using NMR. The inhomogeneity of the field is created by the so-called gradient coils, which are paired with the main magnet. In this case, the magnitude of the magnetic field in different parts of the sample will be different, which means that the NMR signal can be observed not from the entire sample, as in a conventional spectrometer, but only from its narrow layer, for which resonance conditions are met, i.e., the desired ratio of magnetic field and frequency. By changing the magnitude of the magnetic field (or, which is essentially the same thing, the frequency of observing the signal), you can change the layer that will give the signal. Thus, it is possible to "scan" the sample throughout its volume and "see" its internal three-dimensional structure without destroying the sample in any mechanical way. To date, a large number of techniques have been developed that make it possible to measure various NMR parameters (spectral characteristics, magnetic relaxation times, self-diffusion rate, and some others) with spatial resolution inside a sample. The most interesting and important, from a practical point of view, the use of NMR tomography was found in medicine. In this case, the "sample" being examined is the human body. NMR imaging is one of the most effective and safe (but also expensive) diagnostic tools in various fields of medicine, from oncology to obstetrics. It is curious to note that doctors do not use the word "nuclear" in the name of this method, because some patients associate it with nuclear reactions and the atomic bomb.
- Discovery history
The year of the discovery of NMR is considered to be 1945, when the Americans Felix Bloch from Stanford and independently Edward Parcell and Robert Pound from Harvard first observed the NMR signal on protons. By that time, much was already known about the nature of nuclear magnetism, the NMR effect itself was theoretically predicted, and several attempts were made to observe it experimentally. It is important to note that a year earlier in the Soviet Union, in Kazan, the EPR phenomenon was discovered by Evgeny Zavoisky. It is now well known that Zavoisky also observed the NMR signal, this was before the war, in 1941. However, he had a poor quality magnet with poor field uniformity at his disposal, the results were poorly reproducible and therefore remained unpublished. In fairness, it should be noted that Zavoisky was not the only one who observed NMR before its "official" discovery. In particular, the American physicist Isidore Rabi (Nobel Prize winner in 1944 for the study of the magnetic properties of nuclei in atomic and molecular beams) also observed NMR in the late 1930s, but considered this to be an instrumental artifact. One way or another, but our country remains a priority in the experimental detection of magnetic resonance. Although Zavoisky himself soon after the war began to deal with other problems, his discovery for the development of science in Kazan played a huge role. Kazan is still one of the world's leading research centers for EPR spectroscopy.
- Nobel Prizes in Magnetic Resonance
In the first half of the 20th century, several Nobel Prizes were awarded to scientists without whose work the discovery of NMR could not have taken place. Among them are Peter Szeeman, Otto Stern, Isidor Rabi, Wolfgang Pauli. But there were four Nobel Prizes directly related to NMR. In 1952, Felix Bloch and Edward Purcell received the prize for the discovery of NMR. This is the only "NMR" Nobel Prize in physics. In 1991, the Swiss Richard Ernst, who worked at the famous ETH Zurich, won the Chemistry Prize. He was awarded it for the development of multidimensional NMR spectroscopy methods, which made it possible to radically increase the information content of NMR experiments. In 2002, the prize winner, also in chemistry, was Kurt Wüthrich, who worked with Ernst in neighboring buildings at the same Technical School. He received the award for developing methods for determining the three-dimensional structure of proteins in solution. Prior to this, the only method that allowed determining the spatial conformation of large biomacromolecules was only X-ray diffraction analysis. Finally, in 2003, the American Paul Lauterbur and the Englishman Peter Mansfield received the Medical Prize for the invention of NMR imaging. The Soviet discoverer of the EPR E.K. Zavoisky, alas, did not receive the Nobel Prize.
allyl cleavage- addiction spin-spin interaction constants between protons in allyl systems ( 4 J ) which largely depends on the torsion angle between the planes formed by the atoms HC 2 C 3 and C 1 C 2 C 3 .
Annulens- cyclic conjugate systems.
atropic molecules- Molecules of compounds that do not give ring current.
Valence angle (θ) is the angle between two bonds on one carbon atom.
vicinal interaction - interaction between nuclei that are separated by three bonds.
Off-resonance decoupling(off resonance decoupling) - allows you to distinguish between the signals of CH 3 , CH 2 , CH groups and the Quaternary carbon atom. To observe the off-resonance decoupling, a frequency is used that is close to the chemical shift, but does not correspond to the resonant frequency of the signal. Such suppression leads to a reduction in the number of interactions, to such an extent that only direct J(C,H) interactions.
geminal interaction - interaction between nuclei that are separated by two bonds.
Heteronuclear correlation spectroscopy (HETCOR)- in these experiments, chemical shifts of 1 H spectra are placed on one axis, while 13 C chemical shifts are placed on the other axis. HETCOR - heteronuclear variant COZY, which uses indirect heteronuclear spin-spin interactions between 1 H and 13 C.
HMQC - HETeronuclearMultiQuantumcorrelation- registration 1 H with decoupling from 13 C.
HSQC - HETeronuclear MultyQuantum Correlation- HMQC variant
COLOC - CORrelation Long (very long)
HMBC (HETeronuclear MultiplBond Correlation)- a variant of the HMQC experiment for detecting long-range heteronuclear spin-spin interactions. HMBC gives a higher signal to noise ratio than the HMQC experiment.
Gyromagnetic ratio (γ ) - one of the characteristics of the magnetic properties of the nucleus.
Homoallylic interaction- interaction through 5 bonds in the allyl system.
further interaction - interaction between nuclei that are separated by more than 3 bonds (usually after 4-5 bonds).
Sensor- a device that provides the transmission of pulses to the sample and the registration of resonance signals. Sensors are broadband and selectively tuned. They are installed in the active area of the magnet.
Dihedral (torsion) angle- the angle that is formed by two planes between the considered bonds.
2DJ-spectra. Two-dimensional J-spectroscopy is characterized by the presence of one frequency coordinate associated with SSCC and the second coordinate associated with chemical shifts. The contour representation of two-dimensional J-spectra in two mutually perpendicular coordinates has received the greatest distribution.
2D NMR Spectroscopy - experiments using pulse sequences, which makes it possible to obtain the NMR spectrum in such a representation, in which the information is separated by two frequency coordinates and enriched with information about the interdependence of the NMR parameters. The result is a square spectrum with two orthogonal axes and a signal that has a maximum in the frequency representation at the point with coordinates (, ), i.e., on the diagonal.
delta scale (δ -scale) - a scale in which the chemical shift of TMS protons is taken as a zero value.
Diamagnetic shift- shift of the resonant signal to the region of a weak field (large values δ ).
Diatropic molecules- canceled from 4 n+2 π-electrons, which, in accordance with Hückel's rule, have an aromatic character.
doublet - the signal of two interacting nuclei, which is represented in the 1H NMR spectrum by two lines of the same intensity.
Isochronous nuclei- nuclei having the same chemical shift value. Often they are chemically equivalent, that is, they have the same chemical environment.
Integral signal intensity(area under the curve) - measured by the integrator and shown as steps, the height of which is proportional to the area and shows relative number protons.
Pulse spectroscopy - the method of excitation of magnetic nuclei is with the help of short and powerful (hundreds of kilowatts) high-frequency pulses. An impulse with a carrier frequency ν o and a duration t p creates an excitation band in the frequency range +1/t p . If the pulse length is calculated in several microseconds, and ν o approximately corresponds to the center of the resonance frequency region for a given type of nuclei, then the band will cover the entire frequency range, ensuring simultaneous excitation of all nuclei. As a result, an exponentially decaying sinusoid (SIS) is recorded. It contains information both about the frequency, i.e., in fact, about the chemical shift, and about the shape of the line. The more familiar form for us - the spectrum in frequency representation - is obtained from the SIS using a mathematical procedure called the Fourier transform.
Pulse NMR- a method of excitation of magnetic nuclei using short and powerful (hundreds of kilowatts) high-frequency pulses. During the pulse, all nuclei simultaneously are excited, and then, after the impulse stops, the nuclei return (relax) to their original ground state. The loss of energy by relaxing nuclei leads to the appearance of a signal, which is the sum of signals from all nuclei described by a large number of damped sinusoidal curves in the time scale, each of which corresponds to a certain resonant frequency.
Spin-spin coupling constant (SSCC)- quantitative characteristics of the interaction of different nuclei.
Correlation spectroscopy (COSY) - experiment with two 90 o pulses. In this kind of two-dimensional spectroscopy, the chemical shifts of spin-bound magnetic nuclei are correlated. Two-dimensional COZY spectroscopy, under certain conditions, helps to reveal the presence of very small constants, usually invisible in one-dimensional spectra.
COSY- experiments in which the pulse duration is varied. This reduces the size of diagonal peaks that make it difficult to identify nearby cross peaks (COSY45, COSY60).
DQF-COSY - double quantized filter - suppresses singlets on the diagonal and noise corresponding to them.
COSYLR (long range)- COZY experiment, which allows to determine long-range interactions.
TOCSY - TotalcorrelationSpectroscopy- shooting mode, which allows to obtain cross-peaks between all spins of the system in a signal-saturated spectrum by transferring magnetization along the bonds in the structural fragment under study. Most often used to study biomolecules.
Larmor frequency is the frequency of precession in NMR.
magnetically equivalent called such nuclei that have the same resonant frequency and a common characteristic value of the spin-spin interaction constant for all with the nuclei of any neighboring group.
Multiquantum coherences- states of superposition, when two or more interacting spins ½ reorient simultaneously.
Multidimensional NMR- registration of NMR spectra with more than one frequency scale.
Multiplet - signal of one group, manifested in the form of several lines.
Indirect spin interaction - interaction between nuclei, which is transmitted within a molecule through a system of bonds and is not averaged during rapid molecular motion.
Paramagnetic particles - particles containing an unpaired electron, which has a very large magnetic moment.
Paramagnetic shift- shift of the resonant signal to the region of a strong field (large values δ ).
paratropic molecules - are annulled with the number of π-electrons equal to 4 n.
Direct spin-spin interaction constant - a constant that characterizes the interaction between nuclei that are separated by one bond.
Direct spin-spin interaction- interaction between the nuclei, which is transmitted through space.
Resonant signal - a spectral line corresponding to the absorption of energy during the transition between eigenstates, caused by a high-frequency generator.
Relaxation processes - loss of energy at the upper level and return to the lower energy level due to non-radiative processes.
FROM vip- a gradual change in the magnetic field, as a result of which resonance conditions are achieved.
First order spectra- spectra in which the difference in chemical shifts of individual groups of magnetically equivalent nuclei ν o much larger than the spin-spin coupling constant J .
Spin-lattice relaxation - the process of relaxation (energy loss), the mechanism of which is associated with interaction with local electromagnetic fields of the environment.
Spin-spin relaxation - the relaxation process is carried out as a result of the transfer of energy from one excited nucleus to another.
Spin-spin interaction of electrons- interaction resulting from the magnetic interaction of different nuclei, which can be transmitted through the electrons of chemical bonds of directly unbound nuclei.
spin system- this is a group of nuclei that interact with each other, but do not interact with nuclei that are not included in the spin system.
Chemical shift - shift of the signal of the studied nucleus with respect to the signal of the nuclei of the standard substance.
Chemically equivalent nuclei- nuclei that have the same resonant frequency and the same chemical environment.
Shimmy - in NMR spectroscopy, this is the name given to electromagnetic coils that create magnetic fields of low strength, which correct inhomogeneities in a strong magnetic field.
Broadband isolation(1N broadband decoupling) - the use of strong irradiation, which covers the entire region of proton chemical shifts, in order to completely remove all 13 C 1 H interactions.
Shielding - change in the position of the resonant signal under the influence of induced magnetic fields of other nuclei.
Van der Waals effect- an effect that occurs with a strong spatial interaction between a proton and a neighboring group and causes a decrease in the spherical symmetry of the electronic distribution and an increase in the paramagnetic contribution to the screening effect, which, in turn, leads to a signal shift to a weaker field.
Zeeman effect- splitting of energy levels in a magnetic field.
roof effect- an increase in the intensity of the central lines and a decrease in the intensity of remote lines in the multiplet.
Effect of magnetic anisotropy(the so-called anisotropy cone) - the result of exposure to secondary induced magnetic fields.
Nuclear quadrupole resonance (NQR) - observed for nuclei with spin quantum number I > 1/2 due to the non-spherical distribution of the nuclear charge. Such nuclei can interact with the gradients of external electric fields, especially with the gradients of the fields of the electron shells of the molecule in which the nucleus is located and have spin states characterizing different energies even in the absence of an applied external magnetic field.
nuclear magneton The value of the nuclear magneton is calculated by the formula:
Nuclear magnetic resonance(NMR) is a physical phenomenon used to study the properties of molecules when the nuclei of atoms are irradiated with radio waves in a magnetic field.
nuclear factor - the ratio of the nuclear charge to its mass.
NMR spectroscopy
Nuclear magnetic resonance spectroscopy, NMR spectroscopy- a spectroscopic method for studying chemical objects, using the phenomenon of nuclear magnetic resonance. The most important for chemistry and practical applications are proton magnetic resonance spectroscopy (PMR spectroscopy), as well as carbon-13 NMR spectroscopy ( 13 C NMR spectroscopy), fluorine-19 (infrared spectroscopy, NMR reveals information on the molecular structure of chemicals However, it provides more complete information than IS, allowing one to study dynamic processes in a sample - to determine the rate constants of chemical reactions, the value of energy barriers of intramolecular rotation.These features make NMR spectroscopy a convenient tool both in theoretical organic chemistry and for the analysis of biological objects.
Basic NMR technique
A sample of a substance for NMR is placed in a thin-walled glass tube (ampoule). When placed in a magnetic field, NMR active nuclei (such as 1 H or 13 C) absorb electromagnetic energy. The resonant frequency, absorption energy and intensity of the emitted signal are proportional to the strength of the magnetic field. So in a field of 21 Tesla, a proton resonates at a frequency of 900 MHz.
chemical shift
Depending on the local electronic environment, different protons in a molecule resonate at slightly different frequencies. Since both this frequency shift and the fundamental resonant frequency are directly proportional to the strength of the magnetic field, this shift is converted into a magnetic field-independent dimensionless quantity known as the chemical shift. Chemical shift is defined as a relative change relative to some reference samples. The frequency shift is extremely small compared to the main NMR frequency. A typical frequency shift is 100 Hz, while the base NMR frequency is on the order of 100 MHz. Thus the chemical shift is often expressed in parts per million (ppm). In order to detect such a small frequency difference, the applied magnetic field must be constant within the sample volume.
Since the chemical shift depends on the chemical structure of the substance, it is used to obtain structural information about the molecules in the sample. For example, the spectrum for ethanol (CH 3 CH 2 OH) gives 3 distinctive signals, that is, 3 chemical shifts: one for the CH 3 group, the second for the CH 2 group and the last for OH. A typical shift for a CH 3 group is about 1 ppm, for a CH 2 group attached to OH-4 ppm and OH about 2-3 ppm.
Due to molecular motion at room temperature, the signals of the 3 methyl protons average out during the NMR process, which lasts only a few milliseconds. These protons degenerate and form peaks at the same chemical shift. The software allows you to analyze the size of the peaks in order to understand how many protons contribute to these peaks.
Spin-spin interaction
The most useful information for determining the structure in a one-dimensional NMR spectrum is provided by the so-called spin-spin interaction between active NMR nuclei. This interaction results from transitions between different nuclear spin states in chemical molecules, resulting in splitting of NMR signals. This splitting can be simple or complex and, as a result, is either easy to interpret or can confuse the experimenter.
This binding provides detailed information about the bonds of atoms in a molecule.
Second order interaction (strong)
The simple spin-spin interaction assumes that the coupling constant is small compared to the difference in chemical shifts between the signals. If the shift difference decreases (or the coupling constant increases), the intensity of the sample multiplets becomes distorted, becoming more difficult to analyze (especially if the system contains more than 2 spins). However, in high-power NMR spectrometers, distortion is usually moderate, and this makes it easy to interpret the associated peaks.
The second order effects decrease with increasing frequency difference between the multiplets, so the high frequency NMR spectrum shows less distortion than the low frequency spectrum.
Application of NMR spectroscopy to the study of proteins
Most of the recent innovations in NMR spectroscopy are made in the so-called protein NMR spectroscopy, which is becoming a very important technique in modern biology and medicine. A common goal is to obtain a high resolution 3-dimensional structure of a protein, similar to images obtained in X-ray crystallography. Due to the presence of more atoms in a protein molecule compared to a simple organic compound, the underlying 1 D spectrum is overflowing with overlapping signals, making direct spectrum analysis impossible. Therefore, multidimensional techniques have been developed to solve this problem.
To improve the results of these experiments, the labeled atom method is used, using 13 C or 15 N. Thus, it becomes possible to obtain a 3D spectrum of a protein sample, which has become a breakthrough in modern pharmaceuticals. Recently, methods (having both advantages and disadvantages) of obtaining 4D spectra and spectra of higher dimensions, based on nonlinear sampling methods with subsequent restoration of the free induction decay signal using special mathematical techniques, have become widespread.
Literature
- Gunter X. Introduction to the course of NMR spectroscopy. - Per. from English. - M., 1984.
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The essence of the NMR phenomenon can be illustrated as follows. If a nucleus with a magnetic moment is placed in a uniform field 0 directed along the z axis, then its energy (with respect to the energy in the absence of a field) is equal to -mzH0, where mz is the projection of the nuclear magnetic moment onto the direction of the field.
As already noted, the nucleus can be in 2I + 1 states. In the absence of an external field 0, all these states have the same energy. If we denote the largest measurable value of the magnetic moment component by m, then all measurable values of the magnetic moment component (in this case, mz) are expressed as mm, where m is the quantum number, which, as is known, can take the values
m=I,I–1,I–2,…,-(I+1),-I.
Since the distance between the energy levels corresponding to each of the 2I + 1 states is equal to mH0 / I, then the nucleus with spin I has discrete energy levels:
MH0,-(I–1)/ImH0,…(I–1)/ImH0,mH0.
The splitting of energy levels in a magnetic field can be called nuclear Zeeman splitting, since it is similar to the splitting of electronic levels in a magnetic field (the Zeeman effect). Zeeman splitting for a system with I = 1 (with three energy levels).
The NMR phenomenon consists in the resonant absorption of electromagnetic energy due to the magnetism of the nuclei. This implies the obvious name of the phenomenon: nuclear - we are talking about a system of nuclei, magnetic - we mean only their magnetic properties, resonance - the phenomenon itself is resonant in nature. Indeed, it follows from Bohr's frequency rules that the frequency n of the electromagnetic field causing transitions between adjacent levels is determined by the formula:
hν=μH0/I, or ν=μH0/hI.
Since the vectors of the angular momentum (angular momentum) and the magnetic momentum are parallel, it is often convenient to characterize the magnetic properties of nuclei by the value g defined by the relation
where γ is the gyromagnetic ratio having the dimension radian*oersted-1*second-1 (rad*Oe-1*s-1). With this in mind, we find
ν=γ0/2π. (3.2)
Thus, the frequency is proportional to the applied field.
If, as a typical example, we take the value of $\gamma$ for a proton equal to 2.6753*104 rad/(E*s) and H0 = 10000 Oe, then the resonant frequency
ν=42.577 (MHz)
Such a frequency can be generated by conventional radio techniques.
NMR spectroscopy is characterized by a number of features that distinguish it from other analytical methods. About half ($\sim$150) of the nuclei of known isotopes have magnetic moments, but only a minority of them are systematically used.
Prior to the advent of pulsed spectrometers, most studies were carried out using the phenomenon of NMR on hydrogen nuclei (protons) 1H (proton magnetic resonance - PMR) and fluorine 19F. These nuclei have ideal properties for NMR spectroscopy:
high natural content of the "magnetic" isotope (1H 99.98%, 19F 100%); for comparison, it can be mentioned that the natural content of the "magnetic" carbon isotope 13C is 1.1%; large magnetic moment; spin I = 1/2.
This determines, first of all, the high sensitivity of the method in detecting signals from the nuclei mentioned above. In addition, there is a theoretically rigorously substantiated rule according to which only nuclei with a spin equal to or greater than unity have an electric quadrupole moment. Consequently, 1H and 19F NMR experiments are not complicated by the interaction of the nuclear quadrupole moment of the nucleus with the electrical environment.
The introduction of pulsed NMR spectrometers into everyday practice has significantly expanded the experimental possibilities of this type of spectroscopy. In particular, the recording of 13C NMR spectra of solutions - the most important isotope for chemistry - is now actually a familiar procedure. The detection of signals from nuclei, the intensity of NMR signals of which is many times lower than the intensity of signals from 1H, including those in the solid phase, has also become a common phenomenon.
High-resolution NMR spectra usually consist of narrow, well-resolved lines (signals) corresponding to magnetic nuclei in various chemical environments. The intensities (areas) of the signals during the recording of the spectra are proportional to the number of magnetic nuclei in each group, which makes it possible to carry out a quantitative analysis using NMR spectra without preliminary calibration.
Another feature of NMR is the effect of exchange processes involving resonating nuclei on the position and width of resonant signals. Thus, NMR spectra can be used to study the nature of such processes. NMR lines in liquid spectra typically have a width of 0.1 - 1 Hz (high-resolution NMR), while the same nuclei examined in the solid phase will give rise to lines with a width of the order of 1 * 104 Hz (hence the concept of NMR wide lines ).
In high-resolution NMR spectroscopy, there are two main sources of information about the structure and dynamics of molecules:
chemical shift; spin-spin interaction constants.
Under real conditions, resonating nuclei whose NMR signals are detected are a constituent of atoms or molecules. When the substances under study are placed in a magnetic field (0), a diamagnetic moment of atoms (molecules) arises due to the orbital motion of electrons. This movement of electrons forms effective currents and, therefore, creates a secondary magnetic field proportional to the field 0 in accordance with Lenz's law and oppositely directed. This secondary field acts on the nucleus. Thus, the local field in the place where the resonating nucleus is, loc=0 (3.3)
where σ is a dimensionless constant called the screening constant and does not depend on 0, but strongly depends on the chemical (electronic) environment; it characterizes the decrease in lok compared to 0 .
The value of $\sigma$ varies from a value of the order of 10-5 for a proton to values of the order of 10-2 for heavy nuclei. Taking into account the expression for lok, we have: ν=γΗ0(1−σ)/2π (3.4)
The screening effect is to reduce the distance between the levels of nuclear magnetic energy, or, in other words, leads to the convergence of the Zeeman levels. In this case, the energy quanta that cause transitions between levels become smaller and, consequently, resonance sets in at lower frequencies (see expression (3.4)). If the experiment is carried out by changing the field 0 until resonance sets in, then the strength of the applied field should have a large value compared to the case when the core is not shielded.
Influence of electronic shielding on Zeeman levels of the nucleus: a - unshielded, b - shielded
In the vast majority of NMR spectrometers, spectra are recorded when the field changes from left to right, so the signals (peaks) of the most shielded nuclei should be in the right part of the spectrum.
The shift of the signal depending on the chemical environment, due to the difference in screening constants, is called the chemical shift.
For the first time, messages about the discovery of a chemical shift appeared in several publications in 1950-1951. Among them, it is necessary to single out the work of Arnold, who obtained the first spectrum with separate lines corresponding to chemically different positions of identical 1H nuclei in one molecule.
There are three types of protons in this molecule: three protons of the methyl group CH3-, two protons of the methylene group -CH2- and one proton of the hydroxyl group -OH. It can be seen that three separate signals correspond to three types of protons. Since the intensity of the signals is in the ratio 3: 2: 1, the decoding of the spectrum (assignment of signals) is not difficult.
Since chemical shifts cannot be measured on an absolute scale, that is, relative to a nucleus devoid of all its electrons, the signal of a reference compound is used as a conditional zero. Usually, chemical shift values for any nuclei are given as a dimensionless parameter δ defined as follows:
δ=(H−Het)/Het*106, (3.6)
where (H - Nat) is the difference between the chemical shifts for the test sample and the reference, Nat is the absolute position of the reference signal with the applied field (H0) .
In real experimental conditions, it is possible to measure the frequency more accurately than the field, so $\delta$ is usually found from the expression:
δ=(ν−νet)/ν0*106, (3.7)
where (ν – νet) is the difference between the chemical shifts for the sample and the standard, expressed in units of frequency (Hz); NMR spectra are usually calibrated in these units.
You should use not ν0 - the working frequency of the spectrometer (it is usually fixed), but the frequency νet, that is, the absolute frequency at which the resonant signal of the standard is observed. However, the error introduced by such a replacement is very small, since ν0 and νet are almost equal (the difference is 10-5, that is, by the value of σ for a proton). Since different NMR spectrometers operate at different frequencies ν0 (and, consequently, at different fields H0), it is obvious that δ must be expressed in dimensionless units.
The unit of chemical shift is one millionth of the field strength or resonant frequency. Spin-spin interaction.
In 1951-1953, when recording the NMR spectra of a number of liquids, it was found that there are more lines in the spectra of some substances than follows from a simple estimate of the number of nonequivalent nuclei. One of the first examples is the resonance on fluorine in the POCl2F molecule. The 19F spectrum consists of two lines of equal intensity, although there is only one fluorine atom in the molecule. Molecules of other compounds gave symmetrical multiplet signals (triplets, quartets, etc.).
This interaction is due to the mechanism of indirect communication through the electronic environment. The nuclear spin tends to orient the spins of the electrons surrounding the given nucleus. Those, in turn, orient the spins of other electrons and through them - the spins of other nuclei. The energy of the spin-spin interaction is usually expressed in hertz (that is, the Planck constant is taken as a unit of energy, based on the fact that E=hν). It is clear that there is no need (unlike the chemical shift) to express it in relative units, since the discussed interaction, as noted above, does not depend on the strength of the external field. The magnitude of the interaction can be determined by measuring the distance between the components of the corresponding multiplet.
The simplest example of splitting due to spin-spin coupling that can be encountered is the resonance spectrum of a molecule containing two kinds of magnetic nuclei A and X. The nuclei A and X can be either different nuclei or nuclei of the same isotope (for example, 1H ) when the chemical shifts between their resonant signals are large.
Spin echo methods.
In experiments, when a high-frequency field 1 acts continuously on a sample in a uniform magnetic field 0, a stationary state is reached, in which two opposite tendencies are mutually compensated. On the one hand, under the action of a high-frequency field 1, the occupation numbers of the Zeeman levels tend to equalize, which leads to demagnetization of the system, and on the other hand, thermal motion prevents this and restores the Boltzmann distribution.
Completely different unsteady processes are observed when the high-frequency field 1 is turned on for a short time. The practical implementation of experiments of this kind is possible, since the characteristic time parameters of the electronic equipment are small compared to the decay time of the Larmor precession T2.
For the first time, the reaction of a system to pulses of a high-frequency field was observed by Khan in 1950, having discovered the phenomenon - spin echo. This discovery marked the beginning of the development of pulsed NMR techniques.
The action of field 1, rotating at a resonant frequency, is reduced to a deviation of the magnetization from the initial equilibrium direction parallel to field 0. If the field is turned on only for a short period of time, and then turned off again, then the angle of deviation of the magnetization vector depends on the pulse duration. After field 1 is turned on, the magnetization vector will precess around field 0 until its components perpendicular to field 0 disappear, either due to relaxation or due to other causes. The inductive signal that is observed after turning off the high-frequency field 1 is the damping of free precession, first considered by Bloch.
If the strength of field 1 is high and the duration of the pulse tw is so short that relaxation processes can be neglected during the pulse, then the action of field 1 will be reduced to a rotation of the magnetization vector by an angle g1tw (g1 is the angular velocity with which field 1 deviates the vector from the z axis ). If the values 1 and tw are chosen in such a way that g1tw=1/2p, (3.8) then the vector after rotation will be in the xy plane. Such pulses are called 900 turn pulses (or 900th pulses). Those pulses for which g1tw=p are called 1800 turn pulses (1800th pulses).
The action of the last pulses on the magnetization vector leads to a change in its initial direction to the opposite. The effect of 900-th pulses can be better understood by considering them in a coordinate system rotating with an angular velocity equal to the frequency of field 1. If the pulse duration is short, so that the final result depends little on the magnitude of the deviation of the field 1 frequency from the resonant value, then in such a system coordinates, the magnetization vector M immediately after the end of the pulse action will be directed along the v axis.
If the constant field 0 is completely uniform, then the behavior of the magnetization vector after the end of the pulse is determined only by relaxation processes. Therefore, the component of the magnetization vector located in the plane perpendicular to field 0 will rotate around this direction with the Larmor frequency, while its amplitude will tend to zero according to the law exp(-t/T2).
In the case when the inhomogeneity of the magnetic field H0 cannot be neglected, the attenuation occurs faster. This phenomenon can be visualized using a series of diagrams showing the position of the vector on the
magnetization in different parts of the sample at certain moments of the damping process. Let us assume that the sample is divided into several regions, and within each region the magnetic field is uniform, and the magnetization is characterized by its own vector i. The presence of a magnetic field inhomogeneity 0 will lead to the fact that instead of the precession of the resulting magnetization vector with a certain Larmor frequency w0, there will be a precession of a set of magnetization vectors with frequencies distributed according to a certain law.
Let us consider the motion of these vectors in a coordinate system rotating with an angular velocity equal to the average velocity of the Larmor precession corresponding to some average value of the field H0. Vectors i are called spin isochromats.
However, due to the fact that they have different precession rates, since are in regions of the pattern with different field values of 0, then some of them will rotate faster and some will rotate slower than the coordinate system. Therefore, in a coordinate system rotating with a certain average angular velocity, spin isochromates will scatter into a “fan”. Because the receiving coil of the induction system responds only to the vector sum of these moments, then signal attenuation is observed.
Hahn found that the impact on the system of the second pulse after a time interval τ after the first one leads to the appearance of an echo signal after an equal time interval 2τ. The echo signal is observed even when the free precession signal is completely attenuated within 2τ.
1. Initially, the system is in thermal equilibrium, and all magnetization vectors are parallel to the constant field 0.
2. Under the influence of a high-frequency field directed along the x΄ axis of the rotating coordinate system, the magnetization vectors during the first pulse deviate from the direction of the z axis to the direction of the y΄ axis.
3. After the end of the 900th pulse, all magnetization vectors are located in the equatorial plane in the direction of the y΄ axis (the vector product is a vector perpendicular in this case to the z΄x΄ plane). If the pulse duration tω is sufficiently small, then no relaxation or scattering of the magnetization vectors into a "fan", associated with the inhomogeneity of the field 0, will be observed.
4. Immediately after switching on the high-frequency field H1, the free precession decays, which leads to the scattering of spin isochromats into a "fan" located in the x΄y΄ plane.
5. After a time interval τ, an 1800-th pulse with a duration of 2tω acts on the system. As a result of this impulse, the entire system of vectors i rotates by 1800 around the x΄ axis.
6. At the end of the second pulse, each of the magnetization vectors in the rotating coordinate system continues to move in the same direction. However, now, after turning by 1800, this movement does not lead to scattering, but to the folding of the ″fan″ of vectors.
7. After a time interval of 2τ after the start of the first pulse, all magnetization vectors located in the x΄y΄ plane will have the same direction and will create a strong resulting magnetic moment in the negative direction of the y΄ axis. This leads to a pickup in the receiving coil of a signal called an echo.
8. After the appearance of the echo signal, the magnetization vectors again scatter into a "fan", and the usual damping of the free precession is observed. The decay of the echo signal (beginning from the time 2τ) coincides in form with the decay of the free induction signal after the first 900th pulse. Immediately after the 1800th pulse, no signal of free induction occurs.
The shape of the echo signal, as well as the shape of the free precession damping signal, depends on the time law, which obeys the spreading of the magnetization vector into a "fan". If the magnetic field is non-uniform, then coherence is lost quickly and the echo will be narrow; its width is of order (γΔΗ0)-1. Thus, the spin echo mechanism eliminates the usual undesirable effect of the inhomogeneity of a stationary magnetic field.
If the molecules remain for a long time in the same parts of the sample, then the amplitude of the echo signal is determined only by relaxation processes and, therefore, is proportional to exp(-2τ/T2). However, in liquids and gases, diffusion processes can not always be neglected. Therefore, due to the movement of molecules in an inhomogeneous magnetic field, the rate of dispersion into a "fan" of some magnetization vectors changes.
As a result, there is some additional loss of coherence. In this case, the amplitude of the echo signal turns out to be dependent on τ as follows:
exp[–2τ/T2 –k(2τ)3/3]. (3.9)
For echoes received for a sequence of 900 and 1800 pulses
k=1/4γ2GD , (3.10)
where D is the diffusion constant;
G is the average value of the magnetic field gradient (dH0/dt)av.
If the condition is met
12/γ2G2D<< T32, (3.11)
then the main role in the attenuation of spin echo signals will be played by diffusion processes rather than relaxation processes. Similar phenomena are also observed for any other pulses, and not only for a sequence of 900 and 1800 pulses. If a sequence of 900th pulses is applied, then after the second pulse there is a decay signal of free precession, which is not present when a sequence of 900th and 1800th pulses is applied. This is because after time τ, due to the action of the spin-lattice relaxation mechanism, the magnetic moment directed along the z axis is partially restored. This process can be characterized by the function:
f=1 – exp (–τ/T1). (3.12)
As a result, the impact of the second 900th pulse leads to a free precession decay signal, the amplitude of which is f times less than the amplitude of the first signal. In the case when the second pulse is the 1800th pulse, this restoring magnetic moment will be directed in the negative direction of the z axis and, therefore, its projection on the xy plane is zero.
Spin echo experiments can be carried out with a large number of pulses. There are general calculation methods. Suitable for any pulse train.
If the sample contains nuclei with different resonant frequencies and spin-spin interaction takes place between them, then the spin echo pattern becomes more complicated. In this case, the dependence of the attenuation of the amplitude of the spin echo signal on the interval between pulses τ does not obey the law (3.9), but also contains some terms oscillating in time. Now let us dwell on how it is possible to control the phase of the alternating voltage of the second pulse so that in the rotating coordinate system the field 1 is again directed along the +x΄ axis, as in the case of the first pulse. The fact is that in the so-called coherent equipment, a highly stable frequency generator produces a stationary alternating voltage, which enters the power amplifier through a key circuit.
The key circuitry passes the RF signal (field 1) and it is amplified only during the period of time when these circuits are opened by the strobe pulse. Thus, the powerful RF pulses at the output of the amplifier coincide in time with the gate pulses. The output voltage of the amplifier is applied to the sample coil, in which an RF field is created 1. If the generator frequency ω is finely tuned to resonance, i.e. ω=ω0, then the phase of this field is always the same in a coordinate system rotating with frequency ω0.
NMR spectrometers.
The NMR spectrometer must contain the following main elements:
1) a magnet that creates a magnetic field 0 polarizing the nuclear spin - system;
2) a transmitter that creates a probing field 1;
3) a sensor in which, under the influence of 0 and 1, an NMR signal appears in the sample;
4) a receiver that amplifies this signal;
5) recording system (recorder, magnetic recording, oscilloscope, etc.);
6) information processing devices (integrator, multichannel spectrum accumulator);
7) a system for stabilizing resonance conditions;
8) sample temperature control system;
9) a transmitter that creates field 2 for double resonances;
10) programming system for NMR registration: for a spin-spectrometer - a sweep of the field 0 or frequency n0 in a given interval with the required speed required by the number of spectrum realizations; for pulsed spectrometers – selection of the number, amplitude and duration of probing pulses, the tracking time of each point and the number of points of the interferrogram, the repetition time of the interferrogram, the number of cycles of accumulation of the interferrogram;
11) magnetic field correction systems. This schematic enumeration shows that a modern NMR spectrometer is a complex measuring system.
According to the purpose of NMR - spectrometers are divided into devices of high and low resolution. The boundary here is conditional, and more and more often the characteristics of high and low resolution NMR spectrometers are combined in one universal instrument. A typical low resolution instrument should have a magnet providing a relative resolution of about 10-6 h
To ensure high sensitivity, a modulation method of signal observation is used: field 0 (frequency n0) is modulated according to a sinusoidal law; the frequency nm and the amplitude Am are chosen from considerations of optimizing the sensitivity and the signal distortions introduced by such modulation. Since the spin-lattice relaxation time T1 in crystals can reach several hours, the low-resolution spectrometer must be able to detect NMR at extremely low levels of the radio frequency field 1 to avoid signal saturation. The sensitivity of the modulation method depends on the ratio Am/d, and this ratio for weak signals has to be chosen comparable to unity. But then there is a strong modulation broadening, which must be taken into account when processing signals. Difficulties increase even more if the NMR line has a wide and narrow components - with a single recording it is impossible to correctly convey the ratio of the intensities of these components.
Recently, pulsed methods for detecting broad NMR lines in solids have become increasingly popular, but here their own difficulties arise. In order to excite all transitions in a spin system in the same way, it is necessary to use very short pulses of duration t and £ 1 μs; this requires powerful RF sources. In addition, the time response of the spin system for broad lines (T2~10 μs) decays very quickly; in order to produce a sufficient number of readings in a few microseconds, an analog-to-digital converter with a speed of about 0.1 μs per channel is required.
Great difficulties arise due to the ringing of the circuit in the sensor and the overload of the receiver after a powerful pulse. The advantage of the pulse technology is that in one experiment all parameters of nuclear magnetism in the sample can be determined - moments, line shape and relaxation times. According to the Fourier theorem, large frequencies correspond to small times. Therefore, impulse methods are being developed to analyze phenomena that occur in a negligibly short time after the end of the impulse. They increase the accuracy of determining the highest moments of the NMR line up to n=14.
To implement pulse narrowing (high resolution in a solid state), the number of transmitter pulse channels must be at least four. Powerful pulses are formed in the amplification mode of oscillations created by an accurate master oscillator. The duration of its operation should be large enough to implement the required accuracy of frequency tuning and the phase of the radio-frequency filling of the pulses. In addition, the coherence of the spectrometer allows high frequency synchronous detection to improve sensitivity.
Along with synchronous detection, signal accumulation with the help of multichannel storage devices is very widely used. The stability of NMR - spectrometers provides a long-term unambiguous correspondence of each spectral interval Dn to the number of the storage memory channel.
High-resolution spectrometers are divided into stationary and pulsed spectrometers according to the method of finding the resonance conditions. In stationary spectrometers, the resonance is found by changing (sweeping) one of the parameters (n or 0) while fixing the other. In pulse spectrometers with a constant external field 0, the sample is irradiated with a short high-frequency pulse of duration t and frequency n, i.e. frequency spectrum, the main power of which is in the band n±1/t. In this band, all the corresponding NMR transitions are excited, giving a response - a signal of the decay of free induction. Fourier transform of this signal gives the usual NMR spectrum.
Spectrometers operating in stationary mode consist of the following main units:
A magnet that creates a very uniform field;
Signal sensor containing the test sample and the receiving coil;
A scanner that allows you to change within a small range the main magnetic field according to a certain law;
RF generator operating in the meter range;
RF receiver and amplifier;
Oscilloscope and recording potentiometer for observing and recording spectra.
Sufficiently fast rotation of the sample makes it possible to effectively get rid of the influence of magnetic field gradients 0. This circumstance, due to the continuous growth of the used values of 0, leads to the fact that the achieved relative resolution, measured as the ratio DH/0, where DH is the observed inhomogeneity of the magnetic field, is in interval 10-9 - 10-10. Lines measured in tenths and hundredths of a hertz, whose width is determined by the length of the relaxation time in the liquid (10–20 s), lead to a significant difficulty. Therefore, it may take several hours for a single realization of the spectrum. This imposes very high requirements on the system for stabilizing resonance conditions, which is usually carried out using NMR (for an additional sample - external stabilization or for one of the lines of the sample under study - internal stabilization). The most successful results are obtained with a combination of internal and external stabilization.
Nuclear magnetic resonance (NMR) spectroscopy is the most powerful tool for elucidating the structure of organic substances. In this type of spectroscopy, the sample under study is placed in a magnetic field and irradiated with radio frequency electromagnetic radiation.
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Rice. 11-13. Protons in a magnetic field: a - in the absence of a magnetic field; b - in an external magnetic field; c - in an external magnetic field after absorption of radio frequency radiation (spins occupy a higher energy level)
radiation. Hydrogen atoms in different parts of the molecule absorb radiation of different wavelengths (frequency). Under certain conditions, other atoms can also absorb radio frequency radiation, but we will limit ourselves to hydrogen spectroscopy as the most important and widespread form of NMR spectroscopy.
The nucleus of a hydrogen atom consists of one proton. This proton rotates around its axis and, like any rotating charged object, is a magnet. In the absence of an external magnetic field, the proton spins are randomly oriented, but in a magnetic field, only two spin orientations are possible (Fig. 11-13), which are called spin states. The spin states in which the magnetic moment (shown by the arrow) is oriented along the field have somewhat lower energy than the spin states in which the magnetic moment is oriented against the field. The energy difference between the two spin states corresponds to the energy of a photon of radio frequency radiation. When this radiation is exposed to the sample under study, protons pass from a lower energy level to a higher one, and energy is absorbed.
Hydrogen atoms in a molecule are in different chemical environments. Some are part of methyl groups, others are connected to oxygen atoms or a benzene ring, others are near double bonds, etc. This small difference in the electronic environment is enough to change the energy difference between spin states and, consequently, the frequency of the absorbed radiation.
The NMR spectrum arises as a result of the absorption of radio frequency radiation by a substance in a magnetic field. NMR spectroscopy makes it possible to distinguish between hydrogen atoms in a molecule that are in different chemical environments.
NMR spectra
When scanning the radiation frequency at certain frequencies, absorption of radiation by hydrogen atoms in the molecule is observed, the specific value of the absorption frequency depends on the environment of the atoms
Rice. 11-14. Typical NMR spectrum: a - spectrum; b - integral curve giving the area of the peaks
hydrogen. Knowing in which region of the spectrum the absorption peaks of certain types of hydrogen atoms are located, one can draw certain conclusions about the structure of the molecule. On fig. 11-14 shows a typical NMR spectrum of a substance in which there are three types of hydrogen atoms. The position of the signals on the chemical shift scale 5 is measured in parts per million (ppm) of the RF frequency. Usually all signals are located in the area in Fig. 11-14 the chemical shifts of the signals are 1.0, 3.5 and The right part of the spectrum is called the high-field region, and the left part is called the low-field region. In NMR spectra, the peaks are traditionally depicted as directed upwards, and not downwards, as in IR spectra.
Three types of spectral parameters are important for interpreting the spectrum and obtaining structural information from it:
1) signal position in -scale (characterizes the type of hydrogen atom);
2) signal area (characterizes the number of hydrogen atoms of a given type);
3) multiplicity (shape) of the signal (characterizes the number of closely spaced hydrogen atoms of other types).
Let's get acquainted with these parameters in more detail using the spectrum of chloroethane as an example (Fig. 11-15). First of all, let's pay attention to the position of signals in the spectrum, or, in other words, to the values of chemical shifts. Signal a (the protons of the group are at 1.0 ppm, which
Rice. 11-15. NMR spectrum of chloroethane
(see scan)
indicates that these hydrogen atoms are not located next to an electronegative atom, while the shift of the signal b (protons of the group ) is The most important chemical shifts are given in table. 11-2.
Then we analyze the area of the peaks, which is proportional to the number of hydrogen atoms of a given type. On fig. 11-15 relative areas are indicated by numbers in brackets. They are determined using an integral curve located above the spectrum. The area of the signal is proportional to the height of the "step" of the integral curve. In the spectrum under discussion, the signal area ratio is 2:3, which corresponds to the ratio of the number of methylene protons to the number of methyl protons.
Finally, consider the shape or structure of signals, which is usually called multipetality. The methyl group signal is a triplet (three peaks), while the methylene group signal consists of four peaks (quartet). The multiplicity gives information about how many hydrogen atoms are bonded to the neighboring carbon atom. The number of peaks in a multiplet is always one more than the number of hydrogen atoms of the neighboring carbon atom (Table 11-3).
Thus, if there is a singlet signal in the spectrum, this means that the substance molecule includes a group of hydrogen atoms, in the neighborhood of which there are no other hydrogen atoms. In the spectrum in Fig. 11-15 the megillic group signal is a triplet. This means that the adjacent carbon atom has two hydrogen atoms.
Similarly, the methylene group signal is a quartet because there are three hydrogen atoms in the neighborhood.
It is useful to learn how to predict the expected NMR spectrum from the structural formula of a substance. Having mastered this procedure, it is easy to move on to solving the inverse problem - establishing the structure of a substance from its NMR spectrum. Below you will see examples of spectra prediction based on structure. You will then be asked to interpret the spectra in order to establish the structure of the unknown substance.
Prediction of NMR spectra based on the structural formula
To predict the NMR spectra, do the following procedures.
1. Draw the complete structural formula of the substance.
2. Circle the equivalent hydrogen atoms. Determine the number of hydrogen atoms of each type.
3. Using the table. 11-2 (or your memory) determine the approximate values of the chemical shifts of the signals of hydrogen atoms of each type.
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