Nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance spectroscopy (NMR) is a technique used mainly to elucidate molecular structures, although it can also be used for quantitative purposes and in kinetic and thermodynamic studies.

Some atomic nuclei subjected to an external magnetic field absorb electromagnetic radiation in the region of radio frequencies or radio frequencies. Since the exact frequency of this absorption depends on the environment of these nuclei, it can be used to determine the structure of the molecule in which these nuclei are found.

For the technique to be used, the nuclei must have a non-zero magnetic moment. This condition is not met by nuclei with mass number and even atomic number (such as 12C, ¹⁶O, ³²S). The most important nuclei in organic chemistry are: 1H, 13C, ³¹P, ¹⁹F and 15N. Other important nuclei: ⁷Li, ¹¹B, ²⁷Al, ²⁹Si, ⁷⁷Se, ¹¹⁷Sn, ¹⁹⁵Pt, ¹⁹⁹Hg, ²⁰³Tl, ²⁰⁵Tl, ²⁰⁷Pb

Nuclides with nuclear spin quantum number equal to 1/2 are preferred, since they lack an electric quadrupole moment that causes broadening of NMR signals. It is also better if the isotope is abundant in nature, since the intensity of the signal will depend on the concentration of those active nuclei. Therefore, one of the most useful in the elucidation of structures is ¹H, giving rise to proton nuclear magnetic resonance spectroscopy. 13C is also important in organic chemistry, although it is a scarcely abundant and insensitive nucleus.

The technique has been used in organic chemistry, inorganic chemistry, and biochemistry. The same technology has also ended up being extended to other fields, for example in medicine, where magnetic resonance imaging is obtained.

History

The first Nuclear Magnetic Resonance detection due to the formation of a difference in the energies of certain nuclei in the presence of a magnetic field was reported independently by the group of Felix Bloch of Stanford University (for liquid water) and the Edward Mills Purcell's group at Harvard University (for paraffin wax) in 1946. Purcell and Bloch shared the 1952 Nobel Prize in Physics for these discoveries. The chemical application of NMR was discovered in the early 1950s, when It was observed that the resonance frequency of a nucleus strongly depended on its chemical environment (chemical shift). Starting in the 1970s, the development of new techniques and larger magnetic fields (which increase both the sensitivity and the resolution of the signals) made it possible to study larger and larger molecules. The advent of multidimensional NMR and the use of ¹³C and ¹⁵N labeling marked the beginning of biological NMR.

Types of NMR spectroscopies

Continuous Wave (CW) NMR Spectroscopy

From its inception until the late 1960s, NMR spectroscopy used a technique known as continuous wave (CW) spectroscopy. The way to record an NMR spectrum in CW mode was to either hold the magnetic field constant and sweep the frequencies with an oscillating field, or, which was more often used, hold the magnetic field constant. oscillating field, and the intensity of the magnetic field was varied to find the transitions (peaks of the spectrum). In CW NMR the spectrum signals are recorded as resonance signals.

CW spectroscopy is limited by its low sensitivity, since each signal is recorded only once per scan and the nuclear magnetic resonance technique is already not very sensitive by itself; this means that the technique suffers from a low signal-to-noise ratio. Fortunately, in NMR it is possible to improve the signal-to-noise ratio by signal averaging. Signal averaging consists of repeating the acquisition of the experiment and adding the spectra obtained. In this way, the areas of the spectrum in which there are signals add constructively, while, for their part, the areas in which there is noise, due to its random nature, accumulates more slowly than the signal. Signal averaging increases the signal-to-noise ratio by a value that is the square root of the number of spectra that have been accumulated. This relationship is true for NMR spectra involving only one type of nuclei, for example, only ¹H, ¹³C, etc., also called homonuclear spectra.

Pulsed Fourier Transform NMR Spectroscopy

The Fourier Transform NMR (FT-NMR) technique is used in current spectrometers. One of the pioneers in this field is Richard R. Ernst, who developed it starting in 1966 and for which he was awarded the Nobel Prize in Chemistry in 1991.

FT-NMR allows you to drastically reduce the time required to acquire an accumulation (scan) of the full spectrum of NMR. Instead of performing a slow frequency sweep, one at a time, this technique simultaneously and instantly scans an entire range of frequencies. Two technical developments were essential to make the FT-NMR technique a reality: computers capable of carrying out the necessary mathematical operations to go from the time domain to the frequency domain, that is, to obtain the spectrum; and the knowledge about how to simultaneously excite a whole range of frequencies.

FT-NMR works with the sample (nuclear spins) subjected to a constant external magnetic field. The sample is irradiated with an electromagnetic pulse of very short duration in the region of radio frequencies. The shape that is usually used for this pulse is rectangular, that is, the intensity of the radiofrequency oscillates between a maximum and a minimum that is constant during the duration of the pulse. A pulse of short duration has some uncertainty in frequency (Heisenberg's uncertainty principle). The Fourier decomposition of a rectangular wave contains contributions from one of all frequencies. The pulse that is generated is therefore polychromatic and the shorter it is, it is capable of exciting a greater range of frequencies.

The application of a polychromatic pulse in a narrow region of the radio frequency (MHz) band affects those nuclear spins that resonate in that region. A polychromatic pulse with a frequency width of a few kHz can simultaneously excite only the nuclear spins of the same type of atomic nucleus within a molecule, for example, all hydrogen nuclei (¹H). Before the pulse, the net polarization vector of each of the nuclear spins is in equilibrium, aligned in the direction of the magnetic field. During the time that the pulse is applied, the pulse introduces a second magnetic field in a direction perpendicular to the main field of the magnet and the polarization vector precesses. After the pulse ceases, the polarization vector of all affected spins can form a certain angle with the axis of the main magnetic field. At this moment, the spins, behaving like small polarized magnets, begin to precess with their characteristic frequency around the external magnetic field, inducing a small oscillating RF current in a receiving coil located in the vicinity of the sample. As the nuclei gradually return to their initial equilibrium situation aligned with the main magnetic field, the detected signal decreases in intensity until it becomes zero. This signal decay is known as Free Induction Decay (FID) and gives rise to the NMR spectrum.

The signal detected by FID (Free Induction Decay) is an oscillating signal that contains all the spectra signals and decays to zero.

The FID is a wave that contains all the signals in the spectrum in a way that is dependent on time. This wave can be converted into a spectrum of signals based on its frequency. For this, a mathematical function known as the Fourier transform is used. The result is what is known as an NMR spectrum (frequency spectrum).

Multidimensional NMR

The ability to excite the sample with one or more radio frequency (RF) pulses, each applied with a particular power, duration, frequency, shape, and phase, and introduced at specific moments in time during the NMR experiment, generally before the system has returned to equilibrium by relaxation, makes it possible to design a whole range of pulse sequences from which a wide variety of molecular information can be extracted.

A pulse sequence is a time distribution of one or several of the following elements: i) a certain number of RF pulses affecting one or more types of nuclei, ii) waiting times in which you do nothing but wait for the system to evolve in a certain way. These waiting times can be fixed or can be increased if their duration increases as the experiment is repeated. iii) magnetic field gradients and iv) a final stage in which the FID is acquired.

In a multidimensional NMR experiment the pulse sequence must consist of at least two pulses and these must be separated by an incremental waiting period. The pulse sequence is repeated a number of times with an FID being acquired each time. The phase of some of the pulses can be altered in each repetition as well as the duration of one or more variable waiting times can be increased. If the pulse sequence has an increasing waiting time, the experiment will have two dimensions, if it has two it will be three dimensions, if it has three the experiment will be four dimensions. Although in theory there is no limit to the number of dimensions in an experiment, experimentally there are limitations imposed by the consequent loss of signal due to relaxation that the detection of the different dimensions entails. Recording times of multidimensional NMR experiments can be drastically shortened with rapid NMR techniques developed in the present decade.

Multidimensional experiments can be classified into two main types:

Homonuclear correlation experiments: They are those in which all the dimensions correspond to the same nucleus. Examples: COZY (COrrelation SpectroscopY), TOCSY (TOtal Correlation SpectroscopY), NOESY (Nuclear Overhauser Effect SpectroscopY).

Heteronuclear correlation experiments: In these experiments, spectra whose dimensions belong to different nuclei are obtained. Examples: HMQC (Heteronuclear Multiple Quantum Correlation), HSQC (Heteronuclear Simple Quantum Correlation), HMBC (Heteronuclear Multiple Bond Correlation), HOESY (Heteronuclear Overhauser Effect SpectroscopY).

Roughly speaking, the interactions that can be detected by NMR can be classified into two types:

1. Interactions through links are based on the scale coupling
2. Interactions through space are based on dipolar coupling. In the case of dissolution samples, the dipolar coupling is manifested as a nuclear Overhauser effect that allows determining the distance between the atoms.

Richard Ernst in 1991 and Kurt Wüthrich in 2002 were awarded the Nobel Prize in Chemistry for their contributions to the development of 2-dimensional and multidimensional Fourier transform NMR. The advances achieved by them and by other groups of researchers have expanded NMR to biochemistry, and in particular to the determination of the structure in solution of biopolymers such as proteins or even small nucleic acids.

Solids

NMR in solution is complementary to X-ray crystallography since the first allows studying the three-dimensional structure of molecules in the liquid phase or dissolved in a liquid crystal, while X-ray crystallography, as its name suggests, studies the molecules in solid phase.

NMR can also be used to study samples in the solid state. Although in its current state it is far from being able to provide the three-dimensional structure of a biomolecule in good detail.

In the solid state, the molecules are static and, as occurs with molecules in solution, there is no averaging of the NMR signal due to the effect of the thermal rotation of the molecule with respect to the direction of the magnetic field. The molecules of a solid are practically immobile, and each of them experiences a slightly different electronic environment, giving rise to a different signal. This variation of the electronic environment decreases the resolution of the signals and makes their interpretation difficult. Raymond Andrew was one of the pioneers in the development of high-resolution methods for solid-state nuclear magnetic resonance. He was the one who introduced the Magic Angle Spinning (MAS) technique of rotation, which allowed increasing the resolution of solid spectra by several orders of magnitude. In MAS, the interactions are averaged by rotating the sample at a rate of several kilohertz.

Alex Pines in collaboration with John Waugh also revolutionized NMR of solids by introducing the cross-polarization (CP) technique, which manages to increase the sensitivity of sparse nuclei thanks to the transfer of polarization from protons to nearby more insensitive nuclei, usually ¹³C, ¹⁵N or ²⁹Yes.

Halfway between NMR in solution and in solid phase, is the HR-MAS (High Resolution with Magic Angle Spinning) technique, whose fundamental application is the analysis of gels and semisolid materials. The foundation of HR-MAS is to rotate the sample, at the magic angle, at a much higher speed than in normal solids. The effect achieved is high-quality one- and two-dimensional spectra, close to NMR in solution. The main application of this technique is the analysis of biological and polymeric matrices, such as resins for solvated solid phase synthesis.

Sensitivity

Because the intensity of the NMR signal, and the sensitivity of the technique depends on the strength of the magnetic field, since the early days of NMR there has been great interest in the development of stronger magnets. Currently the most powerful commercial magnets are around 22.31 T, or 950 MHz resonant frequency of 1H. Advances in audio-visual and computer technology have also improved aspects of pulse generation and signal reception and information processing.

The sensitivity of the signals also depends on the presence of magnetically-susceptible nuclei to NMR and, therefore, on the natural abundance of such nuclei. In the case of biomolecules, the most abundant and magnetically susceptible nuclei are the isotopes of hydrogen ¹H and phosphorus ³¹P. In contrast, nuclei such as carbon and nitrogen have NMR-useful isotopes, ¹³C and ¹⁵N, respectively, but occur in low natural abundance. To overcome this difficulty, there is the possibility of enriching the sample molecules with these isotopes (eg substitution of ¹²C by ¹³C and/or ¹⁴N by ¹⁵N) to be able to study them by NMR with sufficient sensitivity. These are perfectly stable isotopes that do not produce more than a small variation in the molecular mass of the molecule, without affecting at all other structural or chemical properties of the sample.

Nuclear magnetic resonance instrumentation: the spectrometer

An NMR spectrometer consists of the following fundamental parts:

• A magnet that generates a stable magnetic field, which can be of a variable intensity, defining the resonance frequency of each nucleus. Usually each spectrometer is identified by the proton resonance frequency, so in a magnet of 7.046 Tesla, the cores of the proton ¹H resound at 300 MHz, and therefore would be a spectrometer of 300 MHz. For the time being, the world's largest magnetic field magnet has been installed by Bruker in the UNESCO of Science and Technology King Abdullah in Saudi Arabia, of 950 MHz (22.3 Tesla).
• One probe, which is within the magnet, in which the sample is inserted and consists of the coils responsible for broadcasting and receiving radiofrequency (RF). The number of coils and their arrangement determine the type and applications of each probe.
• One console in which the RF pulses are generated and the rest of the electronic part of the spectrometer is controlled.
• A computer that serves interface with the spectrometer and with which all the information obtained is analyzed.

Information obtained by NMR

The fundamental application of NMR spectroscopy is the structural determination, be it organic, organometallic or biological molecules. For this, it is necessary to carry out different types of experiments from which certain information is obtained.

For the structural elucidation of organic and organometallic molecules, the most commonly used experiments are the following:

Example of a spectrum ¹H of RMN.

• Single-dimensional spectrum ¹H: Gives information about the number and type of different hydrogens in the molecule. The position in the spectrum (chemical displacement) determines the chemical environment of the nucleus, and therefore gives information of functional groups to which they belong or are close. The shape of the signal gives information of the near protons scalarly coupled.

Example of an APT spectrum, a type of experiment ¹³C.

• Single-dimensional spectrum ¹³C: Like in ¹H chemical displacement gives information of functional groups. Depending on the type of experiment performed, information on the number of hydrogens attached to each carbon can be obtained.

Example of a COSY spectrum.

• Homonuclear two-dimensional spectra: COSY and TOCSY experiments provide information on the relationships between the protons of the molecule, by scalar or dipolar coupling (NOESY)
• Heteronuclear two-dimensional spectra: HMQC and HSQC experiments indicate which hydrogens are attached to which carbons. The HMBC experiment allows to determine proton-carbon relationships at greater distance (2 or 3 links)
• Experiments with other nuclei: If the molecule has other active nuclei in MRI it is possible to measure it through one-dimensional or two-dimensional experiments (by indirect detection)