Electron paramagnetic resonance spectroscopy
Electron paramagnetic resonance (EPR) is a magnetic resonance technique that originates from the magnetic moments of unpaired electrons. It can be used to qualitatively and quantitatively detect unpaired electrons contained in a material atom or molecule and explore The structural characteristics of the surrounding environment. For free radicals, the orbital magnetic moment is almost ineffective, and most of the total magnetic moment (over 99%) contributes to electron spin, so the electron paramagnetic resonance is also called “electron spin resonance” (ESR).
Research history
Electron paramagnetic resonance was first discovered by former Soviet physicist E.K. Zavois based on paramagnetic salts such as MnCl2 and CuCl2 in 1944. Physicists originally used this technique to study the electronic structures, crystal structures, dipole moments, and molecular structures of certain complex atoms. Later, based on the results of electron paramagnetic resonance measurements, chemists elucidated the chemical bond and electron density distribution in complex organic compounds and many problems related to the reaction mechanism. In 1954, B. Cammanner and others of the United States introduced the electronic paramagnetic resonance technology into the field of biology. They observed the presence of free radicals in some plant and animal materials. Since the 1960s, due to continuous improvement of instruments and continuous innovation of technologies, the electronic paramagnetic resonance technology has been used in physics, semiconductors, organic chemistry, complex chemistry, radiation chemistry, chemical engineering, marine chemistry, catalysts, biology, and biology. It has been widely used in many fields such as chemistry, medicine, environmental science, and geological prospecting.
An electron is a basic particle with a certain quality and a negative charge. It can perform two kinds of movements: one is in orbit around the nucleus, and the other is spin in the axis through its center. As the movement of the electrons produces a moment, currents and magnetic moments are generated in motion. In an externally applied constant magnetic field H, the electronic magnetic moment acts like a tiny magnetic rod or needle. Since the number of electron spin quantum is 1/2, the electrons have only two orientations in the external magnetic field: one is parallel to H and corresponds to low energy. Level, the energy is -1/2gβH; an antiparallel with H, corresponding to high energy levels, energy is +1/2gβH, and the energy difference between two energy levels is gβH. If in the direction perpendicular to H, plus the electromagnetic wave of frequency v makes it possible to satisfy the condition of hv=gβH, the electrons of the low energy level absorb the energy of the electromagnetic wave and transit to the high energy level, which is called electron paramagnetic resonance. In the above basic conditions for generating an electron paramagnetic resonance, h is Planck's constant, g is a spectrum splitting factor (abbreviated as g factor or g value), and β is a natural unit of an electronic magnetic moment, called Bohr magnetite. The free electron g = 2.00232, β = 9.2710 × 10-21 ergs / Gaussian, h = 6.62620 × 10-27 erg • s, substituting the above formula, can get the relationship between electromagnetic frequency and resonant magnetic field: (Mega ) = 2.8025H (Gaussian)
Detection object
Can be divided into two categories:
1 Substances that do not pair with electrons (or single electrons) appear in the molecular orbitals. Examples are free radicals (containing a single electron molecule), diradicals and polyradicals (molecules containing two or more single electrons), and triplet molecules (having two single electrons in the molecular orbital, but they are very close together. Recently, there is a strong magnetic interaction with each other, unlike the double base).
2 Single electrons appear in atomic orbitals, such as alkali metal atoms, transition metal ions (including iron, palladium, platinum group ions, which in turn have underfilled 3d, 4d, 5d shells), rare earth metal ions (with an unfilled 4f shell) etc.
The vast majority of instruments work in the microwave region, usually using a fixed microwave frequency v, and changing the magnetic field strength H to reach resonance conditions. However, if v is too low, the size of the waveguide to be used must be increased, it becomes bulky, the processing is inconvenient, and the cost is high; while v cannot be too high, otherwise H must be increased accordingly. At this time, the number of turns in the electromagnet must be increased. Add more, bolder wires, magnets to increase, but also make processing difficult.
The electronic paramagnetic resonance spectrometer consists of four components: 1 microwave generation and conduction system; 2 resonator system; 3 electromagnet system; 4 modulation and detection system.
The main features
Since high-frequency modulation is usually used to increase the sensitivity of the instrument, what is recorded on the recorder is not the microwave absorption curve (which is plotted against the magnetic field intensity H by the absorption coefficient X''), but its first derivative curve for H. The two extremes of the latter correspond to the two points with the steepest slope on the absorption curve, and the intersections with the baseline correspond to the vertices of the absorption curve.
From the viewpoint of the resonance condition hv=gβH, h and β are constants. After the microwave frequency is fixed, v is also a constant. The remaining g and H are inversely proportional to each other, so g is sufficient to indicate the position of the resonant magnetic field. The g value essentially reflects the characteristics of a local magnetic field within a material molecule, which is mainly derived from the orbital magnetic moment. The stronger the coupling of the spin motion and the orbital motion, the greater the value of the g value to the ge (the g value of the free electron), and thus the g value can provide the information of the molecular structure. For free radicals containing only C, H, N, and O, the g value is very close to ge, and its value is only a few thousandths.
When a single electron is localized to a sulfur atom, the g value is 2.02-2.06. The g value of most transition metal ions and their compounds is far away from ge, because the orbital magnetic moments in their atoms contribute a lot. For example in an Fe3+ complex, the g value is as high as 9.7.
The line width is usually expressed in terms of the distance between the two extreme values ​​on the first derivative curve (in Gaussian units), and the “peak-to-peak width” is referred to as ΔHpp. The line width can be used as a measure of the magnetic interaction between the electron spin and its environment. The theoretical line width should be infinitely small, but it is actually greatly widened for a variety of reasons.
Hyperfine structures, such as the presence of magnetic nuclei in the vicinity of a single electron, split the single resonant absorption line into many narrow spectral lines through the interaction of the spin magnetic moments of the two. They are called hyperfine structures of the spectrum. . Let n be the number of magnetic cores and I be its nuclear spin quantum number. The original single-peak spectrum will be split into (2nI+1) spectral lines, and the relative intensity obeys certain rules. The most common magnetic nuclei in chemistry and biology are 1H and 14N, and their I are 1/2 and 1 respectively. If n 1H atoms are present, then (n+1) spectral lines are obtained, and the relative intensity is subject to the binomial distribution coefficient in (1+x)n. If n 14N atoms are present, then (2n+1) spectral lines are obtained, and the relative intensity is subject to the trinomial partition coefficient in (1+x+X2)n. Hyperfine structures are of great value for the identification of free radicals.
The area under the absorption curve can be calculated by integrating the first derivative curve twice. Compared with a standard sample containing a known number of single electrons, the single electron content in the sample can be measured, that is, the spin concentration.
Biological applications
1. Study free radicals in biological tissues
Free radicals were detected in lyophilized animal tissues and plant tissues, while free radicals were highly contained in metabolically active tissues (such as green leaf, liver, kidney). Also in the ant, fruit fly, mouse tail. Free radicals were detected in humins, plant resins, and melanin of various animal and plant origins.
2. Study free radicals in enzymatic reactions
Directly confirms L. Michaelis’s hypothesis that there is a phased oxidation of biological substrates (see Biooxidation). It is known that semi-anthraquinone radicals are generated as intermediate products, and the free radical concentration varies with the electron transfer rate or the enzyme. Increased in activity. In some cases, ultrafine structures can be used to identify free radicals, and in turn provide information about the catalytic mechanism of the enzyme and probe the structure of the active site of the relevant enzyme.
3. Study photosynthetic primary reactions
It is shown that free radicals are generated by light in chloroplasts, live algae, and photosynthetic bacteria, all of which participate in the photosynthetic electron transport chain. This helps clarify the nature of the conversion of solar energy into chemical energy.
4. Study the original process of radiation
For qualitative and quantitative detection of free radicals produced by high-energy radiation of biological substances, information on the degree of radiation damage and the site of damage has been provided. Also from more in-depth studies, many very important results concerning the original mechanism of radiation effects, oxygen effects, energy transfer, spin transfer, radiation sensitivity of biological substances, radiation protection and radiation sensitization have been derived.
5. Study free radicals in the process of cancer
It has been observed that the content of free radicals in certain cancer tissues is higher than normal tissues. After feeding a variety of carcinogens to rats, a characteristic signal can be detected in the liver, which may have important value in the diagnosis of cancer. It also demonstrated the formation of free radicals in tissues by carcinogens.
Electron paramagnetic resonance
6. Studying Paramagnetic Metal Ions in Biological Tissues
Including transition metal ions, EPR signals of copper (II), manganese (II), or iron can be seen in some animal tissues, plant materials, and microorganisms. EPR technology has been used to verify that the activity of some paramagnetic metal-containing enzymes is directly related to the valence state of these metals. These metal ions may participate in the binding of substrates and enzymes, such as molybdenum and succinate deamination in xanthine oxidase. Iron in the enzyme, copper in plasma ceruloplasmin.

For single crystals of hemoglobin, myoglobin, and several derivatives thereof, the orientation of the heme plane to the external crystal axis measured by the EPR method is more accurate than that obtained by other methods, and provides information about the central iron atom of the molecule. The information of the chemical bonds, wells prove that the four heme planes within the hemoglobin molecule are not parallel to each other.
The discovery of many iron-sulfur proteins is mostly due to the results of electron paramagnetic resonance measurements. Electron paramagnetic resonance also makes a major contribution to the identification of its active sites and understanding of the relationship between structure and function.
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