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EPR method using microwave radiation. Application of the method of electron paramagnetic resonance in the study of oils and dispersed organic matter




Magnetic resonance is based on the resonant (selective) absorption of radio frequency radiation by atomic particles placed in a constant magnetic field. Most elementary particles, like tops, rotate around their own axis. If a particle has an electric charge, then when it rotates, a magnetic field arises, i.e. it behaves like a tiny magnet. When this magnet interacts with an external magnetic field, phenomena occur that make it possible to obtain information about nuclei, atoms or molecules, which include this elementary particle. The magnetic resonance method is a universal research tool used in such diverse fields of science as biology, chemistry, geology and physics. There are two main types of magnetic resonances: electron paramagnetic resonance and nuclear magnetic resonance.

Electron paramagnetic resonance(EPR) was discovered by Evgeny Konstantinovich Zavoisky at Kazan University in 1944. He noticed that a single crystal placed in a constant magnetic field (4 mT) absorbs microwave radiation of a certain frequency (about 133 MHz).

The essence of this effect is as follows. Electrons in substances behave like microscopic magnets. If you place a substance in a constant external magnetic field and act on it with a radio frequency field, then in different substances they will reorient in different ways and the energy absorption will be selective. The return of electrons to their original orientation is accompanied by a radio frequency signal that carries information about the properties of the electrons and their environment.

Zeeman splitting corresponds to the radio frequency range. The line width of the split state spectrum is determined by the interaction of electron spins with their orbital momenta. This determines the time of relaxation oscillations of atoms as a result of their interaction with surrounding atoms. Therefore, EPR can serve as a tool for studying the structure of the internal structure of crystals and molecules, the mechanism of the kinetics of chemical reactions, and other problems.

Rice. 5.5 Precession of the magnetic moment (M) of a paramagnet in a constant magnetic field.

Rice. 5.5 illustrates the phenomenon of electron precession in a magnetic field. Under the action of the rotational moment created by the field, the magnetic moment performs circular rotations along the generatrix of the cone with the Larmor frequency. When an alternating magnetic field is applied, the intensity vector makes a circular motion with the Larmor frequency in a plane perpendicular to the vector. In this case, the precession angle changes, leading to the overturning of the magnetic moment (M). An increase in the precession angle is accompanied by the absorption of the energy of the electromagnetic field, a decrease in the angle is accompanied by radiation with a frequency .

In practice, it is more convenient to use the moment of onset of a sharp absorption of the energy of an external field at a constant frequency and a variable magnetic field induction. The stronger the interaction between atoms and molecules, the wider the EPR spectrum. This makes it possible to judge the mobility of molecules, the viscosity of the medium (>).

Rice. 5.6 Dependence of the absorption capacity of the energy of an external field by a substance on the value of its viscosity.

, , (5.4)

gyromagnetic ratio.

For example, when the frequency of electromagnetic influence should be within .

This method, which is one of the types of spectroscopy, is used in the study of the crystal structure of elements, the chemistry of living cells, chemical bonds in substances, etc.

On fig. 5.6 shows the block diagram of the EPR spectrometer. The principle of its operation is based on measuring the degree of resonant absorption by a substance of electromagnetic radiation passing through it with a changing strength of an external magnetic field.

Rice. 5.7 Scheme of the EPR spectrometer (a) and the distribution of magnetic and electric field lines in the resonator. 1 - microwave radiation generator, 2 - waveguide, 3 - resonator, 4 - magnet, 5 - microwave radiation detector, 6 - EPR signal amplifier, 7 - recording devices (computer or oscilloscope).

The discovery of EPR served as the basis for the development of a number of other methods for studying the structure of substances, such as acoustic paramagnetic resonance, ferro- and antiferromagnetic resonance, and nuclear magnetic resonance. At the appearance acoustic paramagnetic resonance transitions between sublevels are initiated by the imposition of high-frequency sound vibrations; the result is resonant sound absorption.

The use of the EPR method provided valuable data on the structure of glasses, crystals, and solutions; in chemistry, this method made it possible to establish the structure of a large number of compounds, to study chain reactions, and to elucidate the role of free radicals (molecules with free valency) in the appearance and course of chemical reactions. Careful study of radicals led to the solution of a number of problems in molecular and cellular biology.

The EPR method is a very powerful research tool; it is practically indispensable for studying changes in structures, including biological ones. The sensitivity of the EPR method is very high and amounts to paramagnetic molecules. The search for new substances for quantum generators is based on the use of EPR; The EPR phenomenon is used to generate super-powerful submillimeter waves.

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

Under favorable conditions, the minimum number of spins that can be fixed in the test sample is 1010. The mass of the sample can be, in this case, from a few micrograms to 500 milligrams. During the EPR study, the sample is not destroyed and can be used later for other experiments.

Electron paramagnetic resonance

The phenomenon of electron paramagnetic resonance (EPR) consists in the resonant absorption of electromagnetic radiation in the radio frequency range by substances placed in a constant magnetic field, and due to 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 on:

  • atoms and molecules that have an odd number of electrons in their orbitals - H, N, NO 2, etc.;
  • chemical elements in various charge states, in which not all electrons in 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 stabilizing 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;
  • nanoparticles-superparamagnets 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 with each other and with the nearest environment. The most important of them are spin-spin and spin-orbit interactions, interactions between unpaired electrons and nuclei on which they are localized (hyperfine interactions), interactions with the electrostatic potential created by ions of the nearest environment at the location of unpaired electrons, and others. Most of the listed interactions lead to a regular splitting of the lines. In the general case, the EPR spectrum of a paramagnetic center is multicomponent. An idea of ​​the hierarchy of the main splits 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 (TS). The number of TS lines is determined by the value of the PC spin S and the local symmetry of the electrostatic field of the nearest environment, and the relative integrated intensities are determined by the quantum number mS (the value of the spin projection onto the direction of the magnetic field). In crystals, the distance between the TS lines depends on the magnitude of the potential of the crystal field and its symmetry.

Hyperfine structure (HFS). The HFS lines from a particular isotope have approximately the same integrated intensity and are practically equidistant. If the core of the PC has several isotopes, then each isotope gives its own set of HFS lines. Their number is determined by the spin I of the isotope nucleus, near which the unpaired electron is localized. The relative intensities of the HFS lines from various 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.

Superhyperfine structure (SHFS). The number of SHFS lines depends on the number nl of equivalent ligands with which the unpaired spin density interacts and the magnitude 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 I l \u003d 1/2 obeys the binomial distribution law with the exponent n l. The distance between the SHFS 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 feature of the EPR spectra is the form of their recording. For many reasons, the EPR spectrum is written not in the form of absorption lines, but as a derivative of these lines. Therefore, in EPR spectroscopy, a somewhat different, different from the generally accepted, terminology is adopted for designating line parameters.

EPR absorption line and its first derivative: 1 – Gaussian form; 2 - Lorentz form.

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

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

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

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

The amplitude of the line - I max - corresponds on the scale of the signal amplitude to the distance between the extrema on the curved line.

Intensity - I 0 - the value of the probability at the MAX point on the absorption curve, is calculated by integrating 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 integrating the recording line, first along the contour, then along 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).

the position of the EPR lines in the spectrum.
According to the expression ħν = gβB, which determines the conditions for 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 (analogous to the spectroscopic Lande 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 the 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 single line in the EPR spectrum, but the entire set of lines due to 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 over a wide range. There are some rather narrow ranges of microwave frequencies in which spectrometers operate. Each range has its own designation:

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

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

The base 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 designed to obtain information about the physical nature of the paramagnetic center, its location in the matrix of matter, and its nearest atomic and molecular environment. Studies of the PC in the CW mode make it possible, first of all, to obtain comprehensive information about the possible energy states of the object under study. Information about the dynamic characteristics of spin systems can be obtained by observing the EPR, for example, at different sample temperatures 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 Metallurgical Materials of the Russian Academy of Sciences, which carried out rescue excavations at the Upper Paleolithic site 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 emergence of a complex asymmetric signal (AS) of the EPR near the value of 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 that arise during the irradiation of tooth enamel are carbonate anions, i.e. CO 2 - , CO 3 - , CO - and CO 3 3- .

The spectrum shows a signal from axially symmetric paramagnetic centers of CO 2 - with g ‖ = 1.9975 ± 0.0005 and g ┴ = 2.0032 ± 0.0005. The signal is radioinduced, i.e., the PCs were formed under the action 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 the analysis and control of radiation (GOST R 22.3.04-96) are based on studies of CO 2 - signals in the spectra of tooth enamel. In this and many other cases, it is possible to date a mineral sample using the EPR method. The age range covered by the EPR dating method is 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 the EPR method and has an age of 18000 ± 3000 years.

To study the dynamic characteristics of centers, it is expedient to use impulse 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 taken out of equilibrium, and the reaction of the system to this impact 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 in the absence of special ENDOR equipment.

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 in the structure of hydroxyapatite was studied.

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

4. ENDOR

The most common method of double resonance is the method of double electron-nuclear resonance - ENDOR (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 nearest environment. In this case, the sensitivity of the NMR method can increase by tens and even thousands of times in relation to standard methods. The described techniques are implemented in both CW and FT modes.

Example

The figure shows the ENDOR spectrum of biological hydroxyapatite (tooth enamel). The method was used to obtain information about the environment contained in the enamel paramagnetic CO 2 - centers. Signals from the nuclear environment of the CO 2 center were registered - 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 investigated paramagnetic center CO 2 - is surrounded by anions OH - and PO 4 - .

5. ELDOR (currently not available in DC)

ELDOR (ELectron DOuble Resonance, electronic double resonance) is a variation of the double resonance technique. In this method, the interaction between two electron spin systems is studied, and the EPR spectrum from one electron system is recorded by excitation of the other. To observe a signal, the existence of a mechanism linking the "observed" and "pumped" systems is necessary. Examples of such mechanisms are the dipole interaction between spins, molecular motion.

The phenomenon of electron paramagnetic resonance

If a paramagnetic atom is placed in a magnetic field, then each of its energy levels will be split into the number of sublevels equal to $2J+1$(the number of possible $m_J)$. The interval between adjacent levels is equal to:

In the event that the atom in this state is placed in an electromagnetic wave having a frequency of $\omega $, which satisfies the condition:

then under the influence of the magnetic component of the wave, in accordance with the selection rule, transitions of the atom between neighboring sublevels, within one level, will occur. This phenomenon is called electron paramagnetic resonance (EPR). E.K. was the first to note him. Zavoisky in 1944. Since EPR is associated with resonance, transitions appear only at a certain frequency of the incident wave. This frequency can be easily estimated using expression (2):

With $g\approx 1$ and a typical magnetic field induction used in a laboratory, $B\approx 1\ T$, $\nu =(10)^(10)Hz$ is obtained. Which means that the frequencies are localized in the radio range (UHF).

When resonance occurs, energy is transferred from the field to the atom. In addition, when an atom passes from high Zeeman sublevels to lower sublevels, energy is transferred from the atom to the field. It should be noted that in the case of thermal equilibrium, the number of atoms with lower energy is greater than the number of atoms with higher energy. This means that transitions that increase the energy of atoms prevail over transitions to the side with lower energy. It turns out that the paramagnet absorbs the energy of the field in the radio range and at the same time increases its temperature.

Experiments with the phenomenon of electron paramagnetic resonance made it possible, using expression (2), to find one of the parameters: $g,B\ or\ (\omega )_(rez)$ from the rest of the quantities. Thus, by measuring $B$ and $(\omega )_(rez)$ with high accuracy in the state of resonance, one finds the Lande factor and the magnetic moment of the atom in the state with J.

In liquids and solids, atoms cannot be considered isolated. Their interaction cannot be neglected. It leads to the fact that the intervals between adjacent sublevels in Zeeman splitting are different, the EPR lines have a finite width.

EPR

So, the phenomenon of electron paramagnetic resonance consists in the absorption of microwave radio emission by a paramagnet due to transitions between sublevels of the Zeeman splitting. In this case, the splitting of the energy levels is caused by the action of a constant magnetic field on the magnetic moments of the atoms of the substance. The magnetic moments of atoms in such a field are oriented along the field. Simultaneously with this, there is a splitting of the Zeeman energy levels and redistribution according to the given levels of atoms. The occupancy of sublevels by atoms turns out to be different.

In the state of thermodynamic equilibrium, the average number of atoms ($\left\langle N\right\rangle $) inhabiting a given sublevel can be calculated using the Boltzmann formula:

where $\triangle E_(mag)\sim mH$. Sublevels with a lower magnetic quantum number ($m$) have more atoms as lower potential energy states. This means that there is a predominant orientation of the magnetic moments of atoms along the magnetic field, which corresponds to the magnetized state of the paramagnet. When an alternating magnetic field is applied to a paramagnet with a frequency equal to (a multiple of) the frequency of the transition between the Zeeman splitting sublevels, resonant absorption of electromagnetic waves occurs. It is caused by an excess of the number of transitions, which are associated with an increase in the magnetic quantum number by one:

over number of transitions like:

So, due to the resonant absorption of the energy of an alternating magnetic field, the atoms will make transitions from the lower, more filled levels, to the upper levels. Absorption is proportional to the number of absorbing atoms per unit volume.

If the substance is composed of atoms with one valence electron in state s, having a total magnetic moment equal to the spin magnetic moment of the s-electron, then EPR is most effective.

Resonant absorption of electromagnetic waves by conduction electrons in metals is considered a special paramagnetic resonance. It is related to the spin of electrons and the spin paramagnetism of the electron gas in such a substance. In ferromagnets, ferromagnetic resonance is isolated, which is associated with the reorientation of electronic moments in domains or between them.

Radiospectroscopes are used to study electron paramagnetic resonance. In such devices, the frequency ($\omega $) remains unchanged. Change the induction of the magnetic field (B), which creates an electromagnet (Fig. 1).

Figure 1. Electron paramagnetic resonance (EPR). Author24 - online exchange of student papers

A small sample A is placed in a cavity resonator R, which is tuned to a wavelength of about 3 cm. Radio waves of this wavelength are generated by a generator G. These waves are fed through a waveguide V to the resonator. Some of the waves are absorbed by sample A, some of them enter detector D through the waveguide. During the experiment, a smooth change in the magnetic field induction (B) is carried out, which is created by an electromagnet. When the magnitude of the induction satisfies the resonance occurrence condition (2), the sample begins to absorb the wave intensively.

Remark 1

EPR is one of the simplest methods of radiospectroscopy.

Examples

Example 1

Exercise: What is the magnetic moment of the $Ni$ atom in the $(()^3F)_4$ state if resonant absorption of energy occurs under the influence of a constant field with magnetic induction $B_0$ and an alternating magnetic field with induction $B_0$ perpendicular to the constant field. The frequency of the variable field is $\nu$.

Solution:

As is known, in the state of resonance the equality is fulfilled:

\[\hbar \omega =h\nu =\delta E=(\mu )_bgB\left(1.1\right).\]

From formula (1.1) we find the Lande factor:

For a given state ($(()^3F)_4$) we have: $L=3$, $S=1$, $J=4$. The magnetic moment is given by the expression:

\[\mu =(\mu )_bg\sqrt(J(J+1))=\frac(h\nu )(B_0,\ )\sqrt(20).\]

Answer: $\mu =\frac(h\nu )(B_0,\ )\sqrt(20).$

Example 2

Exercise: What useful information can be obtained from the study of electron paramagnetic resonance?

Solution:

Having empirically obtained the resonance from the resonance conditions, one of the quantities can be found: the Lande factor ($g$), the magnetic field induction under the conditions of resonant absorption of energy by an atom (B), the resonant frequency ($(\omega )_(rez)$). Moreover, B and $(\omega )_(rez)$ can be measured with high accuracy. Consequently, the EPR makes it possible to obtain the value of $g\$ with high accuracy and, consequently, the magnetic moment of the atom for a state with quantum number $J$. The value of the quantum number S is determined from the multiplicity of the spectra. If $g,\ J,\ S$ are known, it is easy to calculate $L$. It turns out that all the quantum numbers of the atom and the spin orbital and total magnetic moments of the atom become known.

Electron paramagnetic resonance (EPR) is a phenomenon of resonant absorption of electromagnetic radiation by a paramagnetic substance placed in a constant magnetic field. It is caused by quantum transitions between magnetic sublevels of paramagnetic atoms and ions (Zeeman effect). The EPR spectra are observed mainly in the microwave frequency range.

The electron paramagnetic resonance method makes it possible to evaluate the effects that appear in the EPR spectra due to the presence of local magnetic fields. In turn, local magnetic fields reflect the pattern of magnetic interactions in the system under study. Thus, the EPR spectroscopy method makes it possible to study both the structure of paramagnetic particles and the interaction of paramagnetic particles with the environment.

The EPR spectrometer is designed to record spectra and measure the parameters of the spectra of samples of paramagnetic substances in a liquid, solid or powder phase. It is used in the implementation of existing and development of new methods for studying substances by the EPR method in various fields of science, technology and healthcare: for example, to study the functional characteristics of biological fluids from the spectra of spin probes introduced into them in medicine; to detect radicals and determine their concentration; in the study of intramolecular mobility in materials; in agriculture; in geology.

The basic device of the analyzer is a spectrometric unit - an electron paramagnetic resonance spectrometer (EPR spectrometer).

The analyzer provides the ability to study samples:

  • with temperature controllers - sample temperature control systems (including those in the temperature range from -188 to +50 ºС and at liquid nitrogen temperature);
  • in cuvettes, ampoules, capillaries and tubes using automatic sample change and dosing systems.

Operation features of the EPR spectrometer

A paramagnetic sample in a special cell (ampoule or capillary) is placed inside a working resonator located between the spectrometer electromagnet poles. Electromagnetic microwave radiation of constant frequency enters the resonator. The resonance condition is achieved by a linear change in the magnetic field strength. To increase the sensitivity and resolution of the analyzer, high-frequency modulation of the magnetic field is used.

When the magnetic field induction reaches a value characteristic of a given sample, resonant absorption of the energy of these oscillations occurs. The converted radiation then goes to the detector. After detection, the signal is processed and fed to the recording device. High-frequency modulation and phase-sensitive detection convert the EPR signal into the first derivative of the absorption curve, in the form of which the registration of the electron paramagnetic resonance spectra takes place. Under these conditions, the integrated EPR absorption line is also recorded. An example of the recorded resonance absorption spectrum is shown in the figure below.

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 high energy radiation.

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

The EPR method began to be used in biological research in the 1950s. Due to its rather high sensitivity and the possibility of determining the nature of paramagnetic particles, this method has found wide application in 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 the metalloproteins involved 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 by the EPR method, and free-radical products of substrates have been found. 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 an electron 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 also widely used to study the structural and dynamic properties of macromolecules and biomembranes.

Recently, the methods of "paramagnetic probe", "spin labels" and "spin traps" are often used to study biological and polymeric systems. All of them are based on the use of stable nitric acid radicals of various structures, or rather, on the analysis of changes in the linewidth of the EPR spectra caused by rotational and translational diffusion of these radicals.

The main idea of ​​the spin label and probe method is to attach a free radical to one or another 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 label 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 motion is clearly manifested in the form of the spectrum and serves as an important source of information about the original molecule.

If a molecule is integrated into a protein molecule and is 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 the probe radical in aqueous or organic media or in the polymer matrix. The mobility of the radical depends on the mobility of the molecules of the environment; therefore, 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 nitroxy radical and, first of all, on the rotational mobility of the group in which it is included.

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.

At the heart of the method "spin traps" is 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 due to free radicals; 6) free radicals frozen at low temperatures; 7) metals of variable valency and their complexes.

The EPR method makes a valuable contribution to the study of the kinetics and mechanisms of chemical reactions. First, linewidth measurements in EPR spectra can be used to determine the rate constants of processes involving paramagnetic particles whose characteristic lifetime lies in the range 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 on the reaction mechanisms. Thirdly, the EPR spectrometer can be used as an analytical device for detecting the concentration of reacting paramagnetic molecules in the course of 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 fast processes associated with changes in the molecular structure of radicals. These processes include hindered rotation and conformational transitions.

For short-lived radicals, the sensitivity of the method can be increased by using a flow system or continuous irradiation. EPR spectra of unstable radicals can be obtained by fixing them in glasses, matrices of frozen noble gases, 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 \u003d (ΔH / H 0); c) hn \u003d gβH 0; d) δ = (Δν/ν 0)/(ΔН/Н 0).

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

a) radiation quanta are absorbed, spin reorientation does not occur;

b) radiation quanta are absorbed and spins are reoriented, i.e. transition from a lower energy state to an upper one and vice versa. The number of transitions from bottom to top is greater than the number of transitions from top to bottom.

c) radiation quanta are absorbed and spins are reoriented, i.e. transition from a lower energy state to an upper one and vice versa. The number of transitions from top to bottom is greater than the number of transitions from bottom to top.

3. Parameters of the EPR spectra:

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 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 registration of the mass of resulting fragments.

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

The ionization of molecules should be carried out under conditions under which the formed ion, regardless of the method of ionization, would 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 by various methods.

1. Electron impact ionization (EI) method.

This is the most common method for obtaining ions due to the simplicity and availability of ion sources and their high efficiency. Let us assume that a stream 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 the bombarding electrons at which the formation of an ion from a given molecule is called ionization energy of matter. The ionization energy is a measure of the strength with which a molecule holds the electron least bound to it. 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 bonds in it. The molecular ion decays into particles of smaller mass (fragments). Such a process is called fragmentation . In the practice of mass spectrometry, electrons with an energy of 30÷100 eV are used, which ensures the fragmentation of a 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 about the identity of the structure of the molecular ion (M +) and the neutral molecule (M) is often used. The probability of forming a molecular ion is greater for simple, small molecules. With an increase in the number of atoms in a molecule, the probability of fragmentation of a molecular ion increases.

There are two main types of fragmentation of a molecular ion - dissociation and rearrangement.

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

The dissociation of hydrocarbons leads to fragments with odd m/z ratios.

regrouping 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.):

The 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 amount provides valuable information on the structure of organic compounds.

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

Fig.1. Mass spectrometer device

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

6 - ion collector (ion detector)

To obtain a mass spectrum, vapors of substances are introduced into the ionization chamber in small amounts using a special puffing 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 ion flow is accelerated, focused by a strong electric field and enters a magnetic field. (4).

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

Each individual ion, characterized by a specific value of m/z, moves along its own trajectory for 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 it is necessary to study negatively charged ions, the sign of the acceleration potential should be changed (acceleration plates).

If a recording device is installed at the exit of ions from the magnetic field, then particles differing in m/z values ​​will give separate signals. The signal intensity 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.

With an increase in the m/z ratio, 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 the ion flux R ≈ 10000/1 and even more. Such devices are able to capture the difference in the molecular weight of ions up to 0.0001. A dual focusing mass spectrometer can easily separate the peaks of ions with the same nominal molecular weights but different elemental compositions. For example, it can distinguish between N 2 (28.0061), CO (27.9949), and C 2 H 4 (28.0313).

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

2. Chemical ionization (CI).

In this method, the sample is diluted with a large excess of "reagent gas" prior to 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. Low molecular weight gases such as CH 4 , ISO-C 4 H 10 , NH 3 and inert gases (Ar, He) are usually used as reactants. Secondary ions are formed as a result of the transfer of a hydrogen atom or an electron.

If methane is the reactant 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 + +CH3

CH 3 + + CH 4 → C 2 H 5 + +H2

R-CH 3 + CH 5 + → R-CH 4 + +CH4

where R-CH 3 is the molecule of the test substance.

Studies have shown that CH 5 particles + and C 2 H 5 + together they 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.