home From timber Application of epr. Application of EPR spectroscopy Resonant absorption of electromagnetic radiation

Application of epr. Application of EPR spectroscopy Resonant absorption of electromagnetic radiation

EPR is observed in solids (crystalline, polycrystalline and powdery), as well as liquid and gaseous. The most important condition for observing ESR is the absence of electrical conductivity and macroscopic magnetization in the sample.

Under favorable conditions, the minimum number of spins that can be detected in the sample under study is 1010. The mass of the sample can range from several micrograms to 500 milligrams. During an EPR study, the sample is not destroyed and can be used in the future for other experiments.

Electron paramagnetic resonance

The phenomenon of electron paramagnetic resonance (EPR) is the resonant absorption of electromagnetic radiation in the radio frequency range by substances placed in a constant magnetic field, and is caused by quantum transitions between energy sublevels associated with the presence of a magnetic moment in electronic systems. EPR is also called electron spin resonance (ESR), magnetic spin resonance (MSR) and, among specialists working with magnetically ordered systems, ferromagnetic resonance (FMR).

The EPR phenomenon can be observed in:

atoms and molecules that have an odd number of electrons in their orbitals - H, N, NO 2, etc.;
chemical elements in different charge states, in which not all electrons in the outer orbitals participate in the formation of a chemical bond - first of all, these are d- and f-elements;
free radicals – methyl radical, nitroxyl radicals, etc.;
electronic and hole defects stabilized in the matrix of substances - O - , O 2 - , CO 2 - , CO 2 3 - , CO 3 - , CO 3 3 - and many others;
molecules with an even number of electrons, the paramagnetism of which is due to quantum phenomena of the distribution of electrons in molecular orbitals - O 2;
superparamagnetic nanoparticles formed during dissolution or in alloys with a collective magnetic moment that behave like an electron gas.

Structure and properties of EPR spectra

The behavior of magnetic moments in a magnetic field depends on various interactions of unpaired electrons, both among themselves and with their immediate environment. The most important of them are spin-spin and spin-orbit interactions, interactions between unpaired electrons and the nuclei on which they are localized (hyperfine interactions), interactions with the electrostatic potential created by ions in the immediate environment at the location of unpaired electrons, and others. Most of the listed interactions lead to a natural splitting of lines. In the general case, the EPR spectrum of a paramagnetic center is multicomponent. An idea of the hierarchy of basic splittings can be obtained from the following diagram (definitions of the notation used are given below):

The main characteristics of the EPR spectrum of a paramagnetic center (PC) are:

the number of lines in the EPR spectrum of a particular PC and their relative intensities.

Fine structure (FS). The number of TC lines is determined by the spin value S of the PC and the local symmetry of the electrostatic field of the immediate environment, and the relative integral intensities are determined by the quantum number mS (the magnitude of the projection of the spin onto the direction of the magnetic field). In crystals, the distance between the TC lines depends on the magnitude of the crystal field potential and its symmetry.

Ultrafine structure (HFS). HFS lines from a particular isotope have approximately the same integral intensity and are practically equidistant. If the PC core has several isotopes, then each isotope produces its own set of HFS lines. Their number is determined by the spin I of the isotope nucleus, around which the unpaired electron is localized. The relative intensities of the HFS lines from different PC isotopes are proportional to the natural abundance of these isotopes in the sample, and the distance between the HFS lines depends on the magnetic moment of the nucleus of a particular isotope, the hyperfine interaction constant, and the degree of delocalization of unpaired electrons on this nucleus.

Super ultrafine structure (USHS). The number of CCTS lines depends on the number nl of equivalent ligands with which the unpaired spin density interacts and the value of the nuclear spin In of their isotopes. A characteristic feature of such lines is also the distribution of their integral intensities, which in the case of I l = 1/2 obeys the law of binomial distribution with an exponent n l. The distance between the SCHS lines depends on the magnitude of the magnetic moment of the nuclei, the hyperfine interaction constant and the degree of localization of unpaired electrons on these nuclei.

spectroscopic characteristics of the line.
A special feature of EPR spectra is the form in which they are recorded. For many reasons, the EPR spectrum is recorded not in the form of absorption lines, but as a derivative of these lines. Therefore, in EPR spectroscopy, a slightly different terminology, different from the generally accepted one, is adopted to designate line parameters.

EPR absorption line and its first derivative: 1 – Gaussian shape; 2 – Lorentzian form.

The true line is a δ-function, but taking into account relaxation processes it has a Lorentz form.

Line – reflects the probability of the process of resonant absorption of electromagnetic radiation by the PC and is determined by the processes in which spins participate.

The shape of the line reflects the law of probability distribution of resonant transitions. Since, to a first approximation, deviations from resonant conditions are random, the shape of lines in magnetically diluted matrices has a Gaussian shape. The presence of additional exchange spin-spin interactions leads to a Lorentzian line shape. In general, the shape of a line is described by a mixed law.

The line width – ΔВ max – corresponds to the distance across the field between the extrema on the curved line.

Line amplitude – I max – corresponds on the signal amplitude scale to the distance between extrema on the curved line.

Intensity – I 0 – probability value at the MAX point on the absorption curve, calculated by integration along the contour of the recording line;

Integrated intensity - the area under the absorption curve, is proportional to the number of paramagnetic centers in the sample and is calculated by double integration of the recording line, first along the contour, then over the field.

The position of the line – B 0 – corresponds to the intersection of the dI/dB derivative contour with the zero line (trend line).

position of EPR lines in the spectrum.
According to the expression ħν = gβB, which determines the conditions of resonant absorption for a PC with spin S = 1/2, the position of the electron paramagnetic resonance line can be characterized by the value of the g-factor (analogue of the Lande spectroscopic splitting factor). The value of the g-factor is defined as the ratio of the frequency ν at which the spectrum was measured to the value of magnetic induction B 0 at which the maximum effect was observed. It should be noted that for paramagnetic centers the g-factor characterizes the PC as a whole, i.e., not a separate line in the EPR spectrum, but the entire set of lines caused by the PC under study.

In EPR experiments, the energy of an electromagnetic quantum is fixed, that is, the frequency ν, and the magnetic field B can vary within wide limits. There are some rather narrow microwave frequency ranges in which spectrometers operate. Each range has its own designation:

Range (BAND)	Frequency ν, MHz (GHz)	Wavelength λ, mm	Magnetic induction B0, at which the EPR signal of a free electron with g = 2.0023, G (T) is observed

The most widely used spectrometers are X- and Q-bands. The magnetic field in such EPR spectrometers is created by resistive electromagnets. In spectrometers with higher quantum energy, the magnetic field is created on the basis of superconducting magnets. Currently, the EPR equipment at the RC MRMI is a multifunctional X-band spectrometer with a resistive magnet, which allows experiments to be carried out in magnetic fields with induction from -11000 G to 11000 G.

The basic mode is the CW mode or the mode of slow differential passage through resonant conditions. In this mode, all classical spectroscopic techniques are implemented. It is intended to obtain information about the physical nature of the paramagnetic center, its location in the matrix of the substance and its immediate atomic-molecular environment. PC studies in the CW mode make it possible, first of all, to obtain comprehensive information about the possible energy states of the object being studied. Information about the dynamic characteristics of spin systems can be obtained by observing EPR, for example, at different temperatures of the sample or when it is exposed to photons. For PCs in the triplet state, additional photoirradiation of the sample is mandatory.

Example

The figure shows the spectrum of bison tooth enamel (lat. Bison antiquus) from the collection selected in 2005 by the Siberian archaeological expedition of the Institute of Humanities of the Russian Academy of Sciences, which carried out rescue excavations at the Upper Paleolithic monument Berezovsky cut 2, located on the territory of the Berezovsky 1 coal mine.

Tooth enamel consists of almost pure hydroxyapatite Ca(1) 4 Ca(2) 6 (PO 4) 6 (OH) 2. The structure of hydroxyapatite also contains 3-4% carbonates.

Irradiation of crushed tooth enamel with gamma radiation leads to the appearance of a complex asymmetric ESR signal (AS) near the value g=2. This signal is studied in the problems of dosimetry, dating, medicine and as a source of information about the structure of apatite.

The main part of the radicals generated during irradiation of tooth enamel are carbonate anions, i.e. CO 2 - , CO 3 - , CO - and CO 3 3- .

The spectrum recorded a signal from axially symmetric paramagnetic CO 2 centers - with g ‖ = 1.9975 ± 0.0005 and g ┴ = 2.0032 ± 0.0005. The signal is radio-induced, i.e. PCs were formed under the influence of ionizing radiation (radiation).

The intensity of the CO 2 signal carries information about the dose of radiation received by the object during its existence. In particular, dosimetric methods for analyzing and monitoring radiation are based on studies of CO 2 - signals in the spectra of tooth enamel (GOST R 22.3.04-96). In this and many other cases, it is possible to date a mineral sample using the ESR method. The age range covered by the EPR dating method ranges from hundreds of years to 105 and even 106 years, which exceeds the capabilities of the radiocarbon method. The sample whose spectra are shown in the figure was dated by EPR and has an age of 18,000 ± 3,000 years.

To study the dynamic characteristics of centers, it is advisable to use pulse methods. In this case, the FT mode of operation of the EPR spectrometer is used. In such experiments, a sample in a certain energy state is subjected to strong pulsed electromagnetic radiation. The spin system is brought out of equilibrium, and the system's response to this influence is recorded. By choosing different sequences of pulses and varying their parameters (pulse duration, distance between pulses, amplitude, etc.), one can significantly expand the understanding of the dynamic characteristics of the PC (relaxation times T 1 and T 2, diffusion, etc.).

3. ESE (electron spin echo technique)

The ESE method can be used to obtain an electron-nuclear double resonance spectrum to save recording time or when special ENDOR equipment is not available.

Example:

Test sample: tooth enamel, consisting of hydroxyapatite Ca(1) 4 Ca(2) 6 (PO 4) 6 (OH) 2. The signal of CO 2 - radicals located in the structure of hydroxyapatite was studied.

Free induction decay (FID) is represented by a set of oscillations called modulation. Modulation carries information about the resonant frequencies of the nuclei surrounding the paramagnetic center. As a result of the Fourier transform of the time dependence of FID, a nuclear magnetic resonance spectrum was obtained. At a frequency of 14 MHz there is a 1H signal, therefore, the CO 2 groups under study interact with protons located in their environment.

4.ENDOR

The most common double resonance technique is the electron-nuclear double resonance method - ENDOR, which makes it possible to study the processes of interaction of an unpaired electron both with its own nucleus and with the nuclei of its immediate environment. In this case, the sensitivity of the NMR method can increase tens and even thousands of times compared to standard methods. The described techniques are implemented in both CW mode and FT mode.

Example

The figure shows the ENDOR spectrum of biological hydroxyapatite (tooth enamel). The method was used to obtain information about the environment of paramagnetic CO 2 - centers contained in enamel. Signals from the nuclear environment of the CO 2 center were recorded at frequencies of 14 MHz and 5.6 MHz. The signal at a frequency of 14 MHz refers to hydrogen nuclei, and the signal at a frequency of 5.6 MHz refers to phosphorus nuclei. Based on the structural features of biological apatite, we can conclude that the paramagnetic CO 2 - center under study is surrounded by OH - and PO 4 - anions.

5. ELDOR (currently not available in the DC)

ELDOR (ELectron DOuble Resonance, electronic double resonance) is a type of double resonance technique. This method studies the interaction between two electron spin systems, with the EPR spectrum from one electron system being recorded by excitation of the other. To observe a signal, the existence of a mechanism connecting the “observed” and “pumped” systems is necessary. Examples of such mechanisms are dipole interaction between spins and molecular motion.

From the ESR spectra, it is possible to determine the valence of a paramagnetic ion and the symmetry of its environment, which, in combination with X-ray structural analysis data, makes it possible to determine the position of the paramagnetic ion in the crystal lattice. The value of the energy levels of a paramagnetic ion allows one to compare EPR results with optical spectra data and calculate the magnetic susceptibility of paramagnetic materials.

The EPR method makes it possible to determine the nature and localization of lattice defects, such as color centers. In metals and semiconductors, EPR is also possible, associated with a change in the orientation of the spins of conduction electrons. The EPR method is widely used in chemistry and biology, where in the process of chemical reactions or under the influence of ionizing radiation, molecules with an unfilled chemical bond - free radicals - can be formed. Their g-factor is usually close to , and the EPR linewidth
small Because of these qualities, one of the most stable free radicals (), with g = 2.0036, is used as a standard in EPR measurements. In ER biology, enzymes, free radicals in biological systems and organometallic compounds are studied.

EPR in strong magnetic fields

The overwhelming majority of experimental studies of paramagnetic resonance have been carried out in magnetic fields whose strength is less than 20 ke. Meanwhile, the use of stronger static fields and alternating fields of higher frequencies would significantly expand the capabilities of the EPR method and increase the information it provides. In the near future, permanent magnetic fields up to 250 will become available ke and pulsed fields measured in tens of millions of oersteds. This means that Zeeman splittings in constant fields will reach approximately 25
, and a in pulsed fields – values are two orders of magnitude greater. Lowe used a spectrometer with a superconducting magnet to measure EPR in fields H0 65 ke. Prokhorov and his colleagues observed EPR signals at the wavelength =1,21mm.

Strong magnetic fields should be of great benefit for the radiation of rare-earth ions in crystals, the intervals between the Stark sublevels of which are on the order of 10-100
. The EPR effect in ordinary fields is often absent due to the fact that the main Stark level turns out to be a singlet, or because transitions between Zeeman sublevels of the main Kramers doublet are prohibited. The effect is, generally speaking, possible due to transitions between different Stark sublevels. Further, the crystal field in rare earth crystals is characterized by a large number of parameters, for determining which knowledge g- the tensor of the main Kramers doublet is not enough.

Strong magnetic fields can also be used to study iron group ions, in particular such as

which have splittings of order 10 100
.

When applied to exchange-coupled pairs, strong magnetic fields will allow, by observing the effect caused by transitions between levels with different values of the resulting spin S pairs with spectroscopic accuracy to measure the exchange interaction parameter J.

Paramagnetic resonance in strong magnetic fields will have a number of features. Magnetization saturation effects will occur at relatively high temperatures. At not very low temperatures, the polarization of ionic magnetic moments will be so great that, in addition to the external magnetic field, it will be necessary to introduce an internal field into the resonance conditions. A dependence of the resonance conditions on the shape of the sample will appear.

ELECTRONIC PARAMAGNETIC RESONANCE (EPR)- resonant absorption of electromagnetic waves by substances containing paramagnetic particles. Methods based on EPR have found wide application in laboratory practice. With their help, they study the kinetics of chemical and biochemical reactions (see Kinetics of biological processes, Chemical kinetics), the role of free radicals in the vital processes of the body under normal conditions and in pathology (see Free radicals), the mechanisms of the occurrence and course of photobiological processes (see Photobiology) etc.

The EPR phenomenon was discovered by the Soviet scientist B.K. Zavoisky in 1944. Electronic paramagnetic resonance is characteristic only of paramagnetic particles, that is, particles capable of being magnetized when a magnetic field is applied to them) with an uncompensated electronic magnetic moment, which, in turn, is due to the electron’s own mechanical moment - spin. Electrons are characterized by a special kind of internal motion, which can be compared to the rotation of a top around its axis. The angular momentum associated with it is called spin. Thanks to the spin, the electron has a permanent magnetic moment directed opposite to the spin. In most molecules, electrons are located in orbitals in such a way that their spins are directed oppositely, the magnetic moments are compensated, and the EPR signal from them cannot be observed. If the magnetic field of an electron is not compensated by the spin of another electron (that is, the molecule contains unpaired electrons), then an EPR signal is recorded. Particles with unpaired electrons are free radicals, ions of many metals (iron, copper, manganese, cobalt, nickel, etc.), a number of free atoms (hydrogen, nitrogen, alkali metals, etc.).

In the absence of an external magnetic field, the direction (orientation) of the magnetic moment of the electron in space can be any; the energy of such an electron does not depend on the orientation of its magnetic moment. In accordance with the laws of quantum mechanics, in an external magnetic field, the orientation of the magnetic moment of an electron cannot be arbitrary - it can be directed either in the direction of the magnetic field or opposite to it.

In accordance with the orientation of the magnetic moment of an electron, its energy in a magnetic field can also take only two values: the minimum E1 - when the magnetic moment is oriented “along the field” and the maximum E2 - when it is oriented “against the field” and the difference in the energies of these states (delta E ) is calculated by the formula: ΔE = gβH, where β is the Bohr magneton (unit of measurement of the magnetic moment of an electron), H is the magnetic field strength, g is a constant depending on the electronic structure of the paramagnetic particle. If a system of unpaired electrons in an external magnetic field is exposed to electromagnetic radiation, the quantum energy of which is equal to ΔE, then under the influence of radiation the electrons will begin to move from a state with lower energy to a state with higher energy, which will be accompanied by the absorption of radiation by the substance.

EPR is classified as a radiospectroscopy method, since radiation in the radio frequency range of electromagnetic waves is used to observe electron paramagnetic resonance.

EPR is recorded using special instruments - radio spectrometers. They include: an electromagnet, a source of radio frequency radiation, a radiation transmission line from the source to the sample (waveguide), a resonator in which the sample under study is located, systems for detecting, amplifying and recording the signal. The most common radio spectrometers use electromagnetic radiation with wavelengths of 3.2 cm or 8 mm.

The EPR signal is recorded as follows. The strength of the magnetic field created by an electromagnet varies linearly within certain limits. At voltage values corresponding to the resonance condition, the sample absorbs the energy of electromagnetic radiation. The absorption line (EPR signal) represents the dependence of the radiation power absorbed by the sample on the magnetic field strength. In existing radio spectrometers, the EPR signal is recorded in the form of the first derivative of the absorption line.

To describe and analyze EPR spectra, a number of parameters are used that characterize the intensity of the lines, their width, shape, and position in the magnetic field. The intensity of EPR lines, other things being equal, is proportional to the concentration of paramagnetic particles, which allows for quantitative analysis.

When considering the ESR phenomenon, it should be taken into account that the magnetic moment of an unpaired electron interacts not only with the magnetic field of an electromagnet, but also with magnetic fields created by the electron’s environment: other unpaired electrons, magnetic nuclei (see Nuclear magnetic resonance). The interaction of unpaired electrons with nuclei often leads to splitting of the EPR spectrum into a number of lines. Analysis of such spectra makes it possible to identify the nature of paramagnetic particles and assess the nature and degree of their interaction with each other.

The participation of paramagnetic particles in chemical reactions, molecular motion and other kinetic effects also affect the shape of the ESR spectrum. Therefore, EPR is used to detect, estimate the quantity and identify paramagnetic particles, study the kinetics of chemical and biochemical reactions and molecular dynamics.

Due to its versatility, EPR is widely used in various fields of science. The use of EPR in biology and medicine is due to the presence in cells, tissues and biol. liquids of paramagnetic centers of different nature. Using ESR, the presence of free radicals was detected in almost all animal and plant tissues. The source of free radicals are compounds such as flavins, coenzyme Q and other substances that act as electron carriers in energy metabolism reactions in plant and animal cells; paramagnetic centers found in isolated tissues belong mainly to the electron transport chains of mitochondria, microsomes, and chloroplasts (see Respiration). It was found that the content of free radicals in tissues correlates with their metabolic activity. Numerous studies have shown a change in the amount of free radicals under various pathological conditions, for example, during oncogenesis (see), the development of radiation damage (see), toxicosis (see Intoxication), which is explained by a violation of energy metabolism in pathology (see Bioenergetics).

Using ESR, paramagnetic ions (iron, copper, manganese, cobalt, etc.) are determined in the tissues of animals and plants, which are part of metalloproteins involved in electron transfer reactions along electron transport chains and enzymatic catalysis, as well as in oxygen-carrying pigments ( hemoglobin). Using EPR, it is possible to study the redox transformations of metal ions and the nature of the interaction of ions with their environment, which makes it possible to establish the fine structure of metal-containing complexes.

Pathological changes in tissues lead to changes in the ESR signals of metalloproteins, which is associated with the disintegration of paramagnetic metal complexes, changes in the environment of paramagnetic ions, and the transition of ions to other complexes. However, studying the nature of paramagnetic centers of tissues, especially free radicals, is associated with certain difficulties due to the difficulty of deciphering EPR spectra.

With the help of EPR, it was possible to study the mechanisms of enzymatic reactions (see Enzymes). In particular, it is possible to simultaneously study both the kinetics of the formation and consumption of free radicals during enzymatic reactions, and the kinetics of redox transformations of the metals that make up the enzymes, which makes it possible to establish the sequence of stages of the enzymatic reaction.

Application of EPR in the study of radiation injury in biol. objects allows one to obtain information about the nature of radicals formed in biopolymers, about the mechanisms and kinetics of radical reactions that develop in irradiated objects and lead to a biological effect. The EPR method can be used in emergency dosimetry, for example, in case of accidental exposure of people to estimate the radiation dose, using objects from the irradiation zone.

EPR occupies an important place in the study of photobiological processes occurring with the participation of free radicals (see Molecule, Free radicals, Photobiology, Photosensitization). With the help of EPR, the processes of formation of free radicals in proteins, nucleic acids and their components under the influence of ultraviolet radiation, and the role of these radicals in the photodestruction of biopolymers are studied in detail (see Light). The use of EPR has provided important information about the primary mechanisms of photosynthesis (see). It has been shown that the primary reaction of photosynthesis is the transfer of an electron from a light-excited chlorophyll molecule and the formation of a chlorophyll radical cation. The nature of the molecules that accept the electron donated by the excited chlorophyll molecule has also been identified.

EPR is also used to study the structure of biologically important macromolecules and biomembranes. For example, iron ions that are part of the heme in heme-containing proteins can be in a high-spin state (electrons in the outer orbits are not paired, the total spin is maximum) and low-spin (the outer electrons are completely or partially paired, the spin is minimal). Studies of the features of ESR signals of high-spin and low-spin states of iron ions in hemoglobin and its derivatives contributed to the understanding of the spatial structure of the hemoglobin molecule.

Significant advances in studying the structure of biomembranes and biopolymers were achieved after the advent of spin probe and label methods (see Biological membranes). Stable nitroxyl radicals are mainly used as spin labels and probes (see Free Radicals). The nitroxyl radical can be covalently bound to molecules (spin label) or retained in the system under study due to physical interactions (spin probe). The essence is that the shape of the EPR spectrum of nitroxyl radicals depends on the properties of the microenvironment: viscosity, the nature and molecular motion, local magnetic fields, etc. Spin marks covalently bound to various groups of biopolymers are an indicator of the state of the biopolymer structure. Using spin labels, the spatial structure of biopolymers, structural changes in proteins during denaturation, the formation of enzyme-substrate, antigen-antibody complexes, etc. are studied.

Using the spin probe method, packaging methods and mobility of lipids in biomembranes, lipid-protein interactions, structural transitions in membranes caused by the action of various substances, etc. are studied. Based on the study of spin labels and probes, methods for determining drugs in biol. liquids, and issues of directed transport of drugs, etc. are also being studied.

Thus, with the help of ESR, the wide distribution of electronic processes in the body is shown normally and in the event of any pathology. The creation of the theory and improvement of the technology of the EPR method formed the basis of quantum electronics as a branch of science and led to the creation of molecular generators and amplifiers of radio waves (masers) and light - lasers (see), which have found wide application in many areas of the national economy.

Blumenfeld L. A., Voevodsky V. V. and Semenov A. G. Application of electron paramagnetic resonance in chemistry, Novosibirsk, 1962, bibliogr.; Wertz J. and Bolton J. Theory and practical applications of the EPR method, trans. from English. M., 1975, bibliogr.; Ingram D. Electron paramagnetic resonance in biology, trans. from English. M., 1972; Kalmanson A.E. Application of the electron paramagnetic resonance method in biochemistry, in the book: Usp. biol. chem., ed. B. N. Stepanenko, vol. 5, p. 289, M., 1963; Kuznetsov A. N. Spin probe method. M., 1976; Lichtenstein G.I. Spin label method in molecular biology, M., 1974; Spin label method, ed. L. Berliner, trans. from English, M., 1979; Free radicals in biology, ed. W. Prior, trans. from English, vol. 1, p. 88, 178, M., 1979.

K. N. Timofeev.

The EPR method has gained great importance in chemistry, physics, biology, and medicine, since it allows one to determine the structures and concentrations of organic and inorganic free radicals. Free radicals can be created chemically, photochemically, or by exposure to high energy radiation.

The EPR spectrum is produced by free radicals, molecules with an odd number of electrons, triplet states of organic molecules, paramagnetic ions of transition metals and their complexes.

The EPR method began to be used in biological research in the 50s of the 20th century. Due to its fairly high sensitivity and ability to determine the nature of paramagnetic particles, this method has found wide application for studying a number of biological processes.

In addition to free radical signals, a number of metal signals (Fe, Cu, Mn, Ni, Co) are observed in tissues. These metals are part of metalloproteins, which take part in a number of enzymatic processes. Iron-containing proteins (cytochromes, ferredoxins) are components of electron transport chains in mitochondria and chloroplasts.

A number of enzymatic systems have been studied using the EPR method, and free radical products of substrates have been discovered. In a number of cases, it turned out to be possible to observe the redox transformations of metal ions included in the active center of the enzyme.

EPR spectroscopy is widely used in studies of photosynthesis: the mechanism of the primary stages of charge separation in reaction centers and the further transfer of electrons along the electron transport chain are studied.

In addition to studying the mechanisms of reactions occurring with the participation of paramagnetic particles, the EPR method is widely used to study the structural and dynamic properties of macromolecules and biomembranes.

Recently, “paramagnetic probe”, “spin label” and “spin trap” methods are often used to study biological and polymer systems. All of them are based on the use of stable nitric acid radicals of various structures, or more precisely on the analysis of changes in the linewidth of EPR spectra caused by the rotational and translational diffusion of these radicals.

The main idea of the spin label and probe method is to attach a free radical to a particular functional group of a protein and study the characteristics of its EPR signals. The most convenient in this regard are nitroxyl radicals containing a free radical group:

where R 1 and R 2 are different chemical groups.

Spin Mark Method consists in the fact that a stable radical is attached to a non-paramagnetic molecule by a covalent or some other bond so that the free valence is unaffected. The nature of the movement is clearly manifested in the shape of the spectrum and serves as an important source of information about the original molecule.

If a molecule is inserted into a protein molecule and held there by electrostatic forces or hydrophobic interactions, then such a molecule is called spin probe. The method is based on the study of the rotational and translational mobility of a radical probe in aqueous or organic media or in a polymer matrix. The mobility of a radical depends on the mobility of the molecules of the environment, so the radical is a kind of molecular sensor of structural and dynamic information about the local environment.

The shape of the EPR signal produced by a spin label or probe depends on the microenvironment of the nitroxide radical and, first of all, on the rotational mobility of the group it belongs to.

The main disadvantage of spin labels and probes is that although these molecules are small, when they are included in the lipid bilayer, they somewhat change its properties.

The basis of the method "spin traps" lies the reaction of a non-paramagnetic molecule (trap) specially introduced into the system under study with a short-lived radical, which results in the formation of a stable radical. The kinetic behavior of the resulting stable radical and its structure provide information about the kinetics and mechanism of processes in the system under study.

The objects of research in chemistry using EPR spectroscopy are: 1) free radicals in intermediate products of organic reactions; 2) reaction kinetics; 3) chemistry of surface phenomena; 4) destruction resulting from irradiation; 5) polymerization caused by free radicals; 6) free radicals frozen at low temperatures; 7) metals of variable valence and their complexes.

The EPR method provides a valuable contribution to the study of the kinetics and mechanisms of chemical reactions. First, measuring the linewidth in EPR spectra can be used to determine the rate constants of processes involving paramagnetic particles, the characteristic lifetime of which lies in the range of 10 -5 -10 -10 s. Secondly, the EPR method makes it possible to detect paramagnetic particles with high sensitivity under different conditions, which provides valuable information about reaction mechanisms. Thirdly, an EPR spectrometer can be used as an analytical instrument for detecting the concentration of reacting paramagnetic molecules during reactions. The number of paramagnetic centers in a sample is proportional to the area under the absorption spectrum.

The EPR method is widely used to study rapid processes associated with changes in the molecular structure of radicals. These processes include inhibited rotation and conformational transitions.

For short-lived radicals, the sensitivity of the method can be increased by using a flow system or continuous irradiation. ESR spectra of unstable radicals can be obtained by recording them in glasses, frozen noble gas matrices, or crystals.

Interview Questions

1. Theoretical foundations of the method.

2. Analytical parameters of the EPR spectrum.

3. EPR spectrometers.

4. Application of EPR.

Test tasks

1. Resonance condition in the EPR method:

a) n= gH 0 (1-s) / 2p; b) δ = (ΔН/Н 0); c)hn=gβH 0; d) δ = (Δν/ν 0)/(ΔН/Н 0).

2. What happens at the moment of resonance in the EPR method:

a) absorption of radiation quanta occurs, spin reorientation does not occur;

b) absorption of radiation quanta and reorientation of spins occurs, i.e. transition from a lower energy state to a higher one and vice versa. The number of transitions from bottom to top is greater than the number of transitions from top to bottom.

c) absorption of radiation quanta and reorientation of spins occurs, i.e. transition from a lower energy state to a higher one and vice versa. The number of transitions from top to bottom is greater than the number of transitions from bottom to top.

3. EPR spectra parameters:

a) g-factor, absorption band width, absorption line intensity;

b) total number of signals, signal intensity, chemical shift, signal multiplicity;

c) g-factor, absorption band width, absorption line intensity, HFS of EPR spectra.

MASS SPECROMETRY

This method is fundamentally different from spectroscopic methods. Mass spectrometry methods are based on the ionization of a substance, the separation of ions, according to the ratio ( m/z), and recording the mass of the resulting fragments.

The theoretical and experimental foundations of mass spectrometry were laid by D.D. Thomson, who for the first time in 1912 created a device for obtaining the mass spectrum of positive ions. However, his instrument had low resolution. His student F. Aston in 1918 significantly increased the resolution and discovered isotopes of elements for the first time using his instrument. Almost simultaneously with F. Aston in Chicago, A. Dempster designed the first mass spectrometer, in which a transverse magnetic field served as an analyzer, and ion currents were measured by electrical methods. Its circuit is also used in modern devices.

Ionization of molecules must be carried out under conditions under which the resulting ion, regardless of the ionization method, does not undergo any collisions with other molecules or ions. This is necessary to establish the relationship between the properties of the ion and the molecule.

Ionization methods

Ionization can be carried out using various methods.

1. Electron impact ionization (EI) method.

This is the most common method for producing ions due to the simplicity and availability of ion sources and their high efficiency. Let us assume that a flow of electrons passes through the vapors of the substance, the energy of which can be gradually increased. If this energy reaches a certain level, then when an electron collides with a molecule, an electron can be “knocked out” from it with the formation of a molecular ion:

polyatomic molecule molecular ion (radical cation)

The lowest energy of bombarding electrons at which an ion can be formed from a given molecule is called ionization energy of a substance. Ionization energy is a measure of the strength with which a molecule holds the least bound electron. For organic molecules, the ionization energy is 9 ÷ 12 eV.

If the electron energy significantly exceeds the ionization energy, then the resulting molecular ion receives excess energy, which may be sufficient to break the bonds in it. The molecular ion disintegrates into particles of smaller mass (fragments). This process is called fragmentation . In the practice of mass spectrometry, electrons with an energy of 30÷100 eV are used, which ensures fragmentation of the molecular ion.

Molecular ions- these are ions whose masses are equal to the mass of the ionized molecule. Unfortunately, there are no direct methods for determining the structure of ions. Therefore, the assumption of the identity of the structure of the molecular ion (M +) and the neutral molecule (M) is often used. The likelihood of forming a molecular ion is greater for simple, small molecules. As the number of atoms in a molecule increases, the probability of fragmentation of a molecular ion increases.

There are two main types of molecular ion fragmentation known: dissociation and rearrangement.

Dissociation- decay of a molecular ion while maintaining the sequence of bonds. As a result of the process, a cation and a radical are formed:

Dissociation of hydrocarbons results in fragments with odd m/z ratios.

Regrouping is accompanied by a change in the sequence of bonds, resulting in the formation of a new radical cation of smaller mass and a neutral stable molecule (H 2 O, CO, CO 2, etc.):

Rearrangement of hydrocarbons and oxygen-containing compounds leads to a fragment with an even m/z ratio. Measuring the mass of the resulting fragments and their relative quantity provides valuable information about the structure of organic compounds.

Let's consider the device of a mass spectrometer (Fig. 1). The mass spectrometer must contain components to perform the following functions: 1) ionization of the sample, 2) acceleration of ions by an electric field, 3) distribution of ions according to the m/z ratio, 4) detection of ions using a corresponding electrical signal.

Fig.1. Mass spectrometer device

1 - source of electrons; 2 - ionization chamber; 3 - accelerating plates (negative potential); 4 - magnet; 5 - slot;

6 - ion collector (ion detector)

To obtain a mass spectrum, vapors of substances are introduced into the ionization chamber in small quantities using a special injection system. (2) , where a deep vacuum is maintained (pressure 10 -6 mm Hg). Molecules of a substance are bombarded by a stream of electrons emitted by a hot cathode (1). The resulting ions are pushed out of the ionization chamber by a small potential difference (3). The resulting stream of ions is accelerated, focused by a strong electric field and caught in a magnetic field (4).

As a result of the bombardment of substance molecules by electrons, particles with a positive or negative charge, as well as neutral particles, are formed. When a stream of particles passes through a magnetic field, neutral particles do not change direction, but positive and negative particles are deflected in different directions. The amount of deflection of ions is proportional to their charge and inversely proportional to their mass.

Each individual ion, characterized by a specific m/z value, moves along its own trajectory at a given magnetic field strength. The mass scanning interval can be changed by varying either the magnetic field strength or the electric field potential.

In conventional mass spectrometry, it is customary to register only particles that have a positive charge, because When molecules are bombarded with electrons, there are usually more positively charged ions than negatively charged ones. If negatively charged ions also need to be studied, the sign of the acceleration potential (accelerator plates) should be changed.

If a recording device is installed at the exit of ions from a magnetic field, then particles with different m/z values will give separate signals. The intensity of the signals will be proportional to the number of particles with a given m/z value. The intensity of the signals is defined as their height, expressed in mm. The height of the peak with the maximum intensity is taken as 100% (base peak), the intensity of the remaining peaks is recalculated proportionally and expressed as a percentage.

As the m/z ratio increases, the difference in the deflection by the magnetic field of particles that differ by one atomic mass unit decreases. In this regard, an important characteristic of mass spectrometers is their resolution (R) , which determines the maximum mass of ions that differ by one atomic mass unit (for which the instrument separates the peaks by at least 90%):

where M is the maximum mass for which the peak overlap is less than 10%; ΔM is one atomic mass unit.

Standard devices have R ≈ 5000/1, and for devices with double focusing of ion flow R ≈ 10000/1 and even more. Such devices are capable of detecting differences in the molecular mass of ions up to 0.0001. A dual focusing mass spectrometer can easily separate peaks from ions with the same nominal molecular weights but different elemental compositions. For example, it can distinguish between N2 (28.0061), CO (27.9949), and C2H4 (28.0313).

Establishing an empirical formula from mass spectral data is not a simple task, but it can be solved using a suitable algorithm. To obtain a mass spectrum, a negligible amount of the substance is required - about 1 μg.

2. Chemical ionization (CI).

In this method, the sample is diluted with a large excess of “reagent gas” before irradiation with an electron beam. The probability of primary ionizing collisions between electrons and sample molecules is then so small that primary ions are formed almost exclusively from reactant molecules. Gases with low molecular weight are usually used as reagents, for example, CH 4, iso-C 4 H 10, NH 3 and inert gases (Ar, He). Secondary ions are formed by the transfer of a hydrogen atom or electron.

If methane is the reagent gas, then the reactions proceed in the following sequence:

CH 4 + ē → CH 4 + + 2ē

CH 4 + + ē → CH 3 + +H + + 2ē

CH 4 + + CH 4 → CH 5 + + CH 3

CH 3 + + CH 4 → C 2 H 5 + +H2

R-CH 3 + CH 5 + → R-CH 4 + + CH 4

where R-CH 3 is the molecule of the substance under study.

Research has shown that CH 5 particles + and C2H5 + together make up about 90% of the ions present in this system. Mass spectra obtained after chemical ionization are much simpler, contain fewer peaks, and are therefore often easier to interpret.

The electron paramagnetic resonance method is the main method for studying paramagnetic particles. Paramagnetic particles of important biological significance include two main types: free radicals and complexes of metals of variable valence (such as Fe, Cu, Co, Ni, Mn).

The method of electron paramagnetic resonance was discovered in 1944 by E.K. Zavoisky while studying the interaction of electromagnetic radiation in the microwave range with metal salts.

The EPR method is based on the absorption of electromagnetic radiation in the radio range by unpaired electrons located in a magnetic field.

The EPR method allows us to study the properties of paramagnetic centers by recording the absorption spectra of electromagnetic radiation by these particles. Knowing the characteristics of the spectra, one can judge the properties of paramagnetic particles.

The main characteristics of the spectra include amplitude, linewidth, g-factor and hyperfine structure of the spectra.

Application of spin tags

Spin labels are chemically stable paramagnetic molecules that are used as molecular probes to study the structure and molecular mobility of various physicochemical and biological systems. The essence of the spin label method is as follows. Paramagnetic molecules are introduced into the system under study as spin probes, which produce characteristic electron paramagnetic resonance (EPR) signals. The EPR signals of spin labels depend on their molecular mobility and the physicochemical properties of the immediate environment. Therefore, by observing the EPR signals of molecular probes, it is possible to study the structural characteristics of the system under study and the dynamics of the molecular processes occurring in it. The term “spin marks” comes from the English word “spin” (spindle, top), which refers to the intrinsic mechanical momentum of an electron. An electron, as is known from quantum mechanics, has a mechanical moment equal to the value "/2, and its own magnetic moment, where " is Planck's constant, e and m are the charge and mass of the electron, c is the speed of light. The paramagnetic properties of molecular probes are determined by the presence of an unpaired electron in them, which has spin and is the source of the ESR signal. Stable nitroxyl radicals are usually used as spin labels. All molecules of spin labels, despite the diversity of their chemical structure, as a rule, contain the same paramagnetic fragment - a chemically stable nitroxyl radical (>N-OJ). An unpaired electron is localized on this radical, serving as a source of the ESR signal. The specific choice of spin labels is determined by the research problem. For example, in order to monitor conformational rearrangements of proteins using spin labels, label molecules are usually “sewn” to certain regions of the protein. In this case, the spin label must contain a special reaction group that can form a covalent chemical bond with the amino acid residues of the protein molecule. To study the properties of artificial and biological membranes, fat-soluble spin labels are usually used that can be incorporated into the lipid layer of the membrane.

The EPR phenomenon can be observed in:

* atoms and molecules that have an odd number of electrons in their orbitals - H, N, NO2, etc.;
* chemical elements in different charge states, in which not all electrons in the outer orbitals participate in the formation of a chemical bond - first of all, these are d- and f-elements;
* free radicals - methyl radical, nitroxyl radicals, etc.;
* electronic and hole defects stabilized in the matrix of substances - O-, O2-, CO2-, CO23-, CO3-, CO33- and many others;
* molecules with an even number of electrons, the paramagnetism of which is due to quantum phenomena of the distribution of electrons in molecular orbitals - O2;
* superparamagnetic nanoparticles formed during dissolution or in alloys with a collective magnetic moment that behave like an electron gas.

Structure and properties of EPR spectra

The main characteristics of the EPR spectrum of a paramagnetic center (PC) are:

* number of lines in the EPR spectrum of a particular PC and their relative intensities.
* Fine structure (TS). The number of TC lines is determined by the spin value S of the PC and the local symmetry of the electrostatic field of the immediate environment, and the relative integral intensities are determined by the quantum number mS (the magnitude of the projection of the spin onto the direction of the magnetic field). In crystals, the distance between the TC lines depends on the magnitude of the crystal field potential and its symmetry.
* Ultrafine structure (HFS). HFS lines from a particular isotope have approximately the same integral intensity and are practically equidistant. If the PC core has several isotopes, then each isotope produces its own set of HFS lines. Their number is determined by the spin I of the isotope nucleus, around which the unpaired electron is localized. The relative intensities of the HFS lines from different PC isotopes are proportional to the natural abundance of these isotopes in the sample, and the distance between the HFS lines depends on the magnetic moment of the nucleus of a particular isotope, the hyperfine interaction constant, and the degree of delocalization of unpaired electrons on this nucleus.
* Super ultrafine structure (USHS). The number of CCTS lines depends on the number nl of equivalent ligands with which the unpaired spin density interacts and the value of the nuclear spin Il of their isotopes. A characteristic feature of such lines is also the distribution of their integral intensities, which in the case of Il = 1/2 obeys the law of binomial distribution with the exponent nl. The distance between the SCHS lines depends on the magnitude of the magnetic moment of the nuclei, the hyperfine interaction constant and the degree of localization of unpaired electrons on these nuclei.
* spectroscopic characteristics of the line.

A special feature of EPR spectra is the form in which they are recorded. For many reasons, the EPR spectrum is recorded not in the form of absorption lines, but as a derivative of these lines. Therefore, in EPR spectroscopy, a slightly different terminology, different from the generally accepted one, is adopted to designate line parameters.

EPR absorption line and its first derivative: 1- Gaussian shape; 2- Lorentzian form.

* The true line is a d-function, but taking into account relaxation processes it has a Lorentz form;
* Line - reflects the probability of the process of resonant absorption of electromagnetic radiation from the PC and is determined by the processes in which spins participate;
* Line shape - reflects the law of probability distribution of resonant transitions. Since, to a first approximation, deviations from resonant conditions are random, the shape of lines in magnetically diluted matrices has a Gaussian shape. The presence of additional exchange spin-spin interactions leads to a Lorentzian line shape. In general, the shape of a line is described by a mixed law;
* Line width - DVmax - corresponds to the distance across the field between the extrema on the curved line;
* Line amplitude - Imax - corresponds on the signal amplitude scale to the distance between extrema on the curved line;
* Intensity - I0 - the probability value at the MAX point on the absorption curve, calculated by integrating along the contour of the recording line;
* Integral intensity - the area under the absorption curve, is proportional to the number of paramagnetic centers in the sample and is calculated by double integration of the recording line, first along the contour, then along the field;
* The position of the line - B0 - corresponds to the intersection of the contour of the derivative dI/dB with the zero line (trend line);
* position of EPR lines in the spectrum.

According to the expression hн = gвB, which determines the conditions of resonant absorption for PCs with spin S = 1/2, the position of the electron paramagnetic resonance line can be characterized by the value of the g-factor (analogue of the Lande spectroscopic splitting factor). The value of the g-factor is defined as the ratio of the frequency n at which the spectrum was measured to the value of magnetic induction B0 at which the maximum effect was observed. It should be noted that for paramagnetic centers the g-factor characterizes the PC as a whole, i.e. not a single line in the EPR spectrum, but the entire set of lines caused by the PC under study.

In EPR experiments, the energy of an electromagnetic quantum is fixed, that is, the frequency n, and the magnetic field B can vary within wide limits. There are some rather narrow microwave frequency ranges in which spectrometers operate.

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Application of epr. Application of EPR spectroscopy Resonant absorption of electromagnetic radiation

EPR in strong magnetic fields