Electron paramagnetic resonance (EPR) is composed of unpaired electronsmagnetic momentPrimordialmagnetic resonanceThe technology can be used to detect the unpaired electrons contained in atoms or molecules of substances qualitatively and quantitatively, and explore the structural characteristics of their surrounding environment.yesFree radicalIn terms of trackmagnetic momentIt hardly works, and most of the total magnetic moment (more than 99%) is contributed by electronsspinTherefore, EPR is also called "electronspinResonance "(ESR).
Chinese name
Electron paramagnetic resonance
Foreign name
electron paramagnetic resonance，EPR
Fundamentals
The motion of the electron produces torque
Detection object
Unpaired electron (or single electron) substance
Role
Explore the structural characteristics of its surrounding environment
Electron paramagnetic resonance (EPR) was first developed by E. K. Zavois, a physicist of the former Soviet Union, based on the research results of MnCl2, CuCl2, etcParamagnetismSalt found.Physicists initially used this technology to study the electronic structure, crystal structuredipole momentAnd molecular structure.Later, chemists clarified the chemical bond and electron density distribution in complex organic compounds and many problems related to the reaction mechanism based on the results of electron paramagnetic resonance measurements.In 1954, B. Comona and others in the United States first introduced electron paramagnetic resonance technology into the field of biology. They observed the existence of free radicals in some plant and animal materials.Since the 1960s, due to the continuous improvement of instruments and technological innovation, EPR technology has been widely used in many fields such as physics, semiconductor, organic chemistry, complex chemistry, radiation chemistry, chemical industry, marine chemistry, catalysts, biology, biochemistry, medicine, environmental science, geological prospecting, etc.[1]
Fundamentals
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An electron is a basic particle with a certain mass and negative charge, which can carry out two kinds of motions;One is to move in the orbit around the atomic nucleus, and the other is to spin the axis through its center.As the motion of electrons generates torque, current and magnetic moment are generated in the motion.In the external constant magnetic field H, the effect of the electronic magnetic moment is like a small magnetic rod or magnetic needleSpin quantum number1/2, so there are only two orientations of electrons in the external magnetic field: one is parallel to H, corresponding to the low energy level, and the energy is - 1/2g β H;One is inversely parallel to H, which corresponds to the high energy level. The energy is+1/2g β H, and the energy difference between the two levels is g β H.If the direction perpendicular to H and the electromagnetic wave with frequency of v can just meet the condition that hv=g β H, the low-energy electrons will absorb the electromagnetic wave energytransitionTo the high energy level, this is called electron paramagnetic resonance.In the above basic conditions for generating EPR, h isPlanck constant, g is the spectral splitting factor (g factor or g value for short), β is the natural unit of the electronic magnetic moment, calledBohr magneton。By substituting the g value of free electron=2.00232, β=9.2710 × 10-21 ergs/Gauss, h=6.62620 × 10-27 ergs · s into the above equation, the relationship between electromagnetic wave frequency and resonant magnetic field can be obtained: (Gauss)=2.8025 (MHz)[1]
Detection object
① A substance in which unpaired electrons (or single electrons) appear in molecular orbitals.For example, free radicals (molecules with one single electron), double and multi radicals (molecules with two or more single electrons), triplet molecules (molecules with two or more single electrons in the molecular orbital, but they are very close to each other and have strong magnetic interactions with each other, different from double radicals), etc.
② Materials with single electrons in the atomic orbit, such as alkali metal atoms, transition metal ions (including iron group, palladium group, platinum group ions, which in turn have unfilled 3d, 4d, 5d shells), rare earth metal ions (with unfilled 4f shells), etc.[1]
Spectrograph
Most of the instruments work in the microwave region, usually using a fixed microwave frequency v, while changing the magnetic field intensity H to achieve resonance conditions.But in fact, if V is too low, the size of the waveguide used should be increased, becoming cumbersome, inconvenient to process, and expensive;But v cannot be too high, otherwise H must be increased accordingly. At this time, the wire turns in the electromagnet should be increased, the wire should be thickened, and the magnet should be increased, which also makes processing difficult.[1]
The electron paramagnetic resonance spectrometer consists of four parts: ① microwave generation and conduction system; ②Resonant cavity system; ③Electromagnet system; ④Modulation and detection system.[1]
Main characteristics
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Electron paramagnetic resonance spectrometer
Since high-frequency field modulation is usually used to improve instrument sensitivity, what is recorded on the recorder is not the microwave absorption curve itself (the magnetic field intensity H is plotted by the absorption coefficient X ''), but its first differential curve to H.The two extreme values of the latter correspond to the two points with the maximum slope on the absorption curve, and the intersection point with the baseline corresponds to the vertex of the absorption curve.
From the resonance condition hv=g β H, h and β are constants. After the microwave frequency is fixed, v is also constant. The remaining g is inversely proportional to H, so g is enough to indicate the position of the resonance magnetic field.The g value essentially reflects the characteristics of the local magnetic field in the molecule of a substance, which mainly comes from the orbital magnetic moment.The stronger the coupling effect between spin motion and orbital motion, the greater the increment of g value to ge (g value of free electron), so g value can provide information about molecular structure.For free radicals containing only C, H, N and O, the g value is very close to ge, and its increment is only a few thousandths.[1]
When the single electron is localized in the sulfur atom, the g value is 2.02-2.06.The g values of most transition metal ions and their compounds are far away from ge, because the orbital magnetic moment in their atoms contributes greatly.For example, in a Fe3+complex, the g value is as high as 9.7.
The line width is usually expressed by the distance between the two extreme values on the primary differential curve (in Gauss), which is called "peak to peak width" and recorded as Δ Hpp.The linewidth can be used as a detection of the interaction between the electron spin and the magnetic field generated by its environment. Theoretically, the linewidth should be infinitely small, but in fact, it has been greatly widened due to a variety of reasons.[1]
Hyperfine structureIt originates from the interaction of spin magnetic moment between magnetic nucleus and electron.Specifically, when the nucleon is a non-magnetic nucleus, a single resonance absorption spectrum line will be observed; when the nucleon is a magnetic nucleus, multiple absorption spectrum lines with narrower linewidth will be observed, which are called hyperfine structures of the spectrum.The energy of this interaction comes from two parts:
Is the quantum number of the electron orbit,
Is the electron spin quantum number,
Is the nucleon spin quantum number.
This energy can finally be expressed as[3]：
Here,
,
J is the total angular momentum of the electron, F is the total angular momentum of the whole atom (all electrons and nucleons) (to reducePlanck constantћAs a unit).
Is the fine structure spacing, and A is the hyperfine structure spacing, also known asHyperfine coupling constant。
Let n be the number of magnetic nuclei and I be its nucleusSpin quantum numberThe original single peak spectrum is split into (2nI+1) spectral lines, and the relative strength is subject to certain laws.The most common magnetic nuclei in chemistry and biology are 1H and 14N, and their I is 1/2 and 1 respectively.If n 1H atoms exist, (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 exist, (2n+1) spectral lines are obtained, and the relative intensity is subject to the distribution coefficient of the three terms in (1+x+X2) n.Hyperfine structureIt is valuable for the identification of free radicals.[1]
The area under the absorption curve can be calculated by twice integrating from the first differential curve, and compared with the standard sample containing a known number of single electrons, the content of single electrons in the sample can be measured, that is, the spin concentration.[1]
Spin labeling
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It was founded by H. M. McConnell of the United States in 1965, which means that a stable free radical (most commonly usedNitroxide radical）Combine to a single molecule or a specific part of a molecule in a more complex system, and obtain information about the marker environment from the electron paramagnetic resonance spectrum.In the process of spin labeling, attention should be paid to maintaining specificity as far as possible and reducing disturbance to biological and molecular characteristics of natural systems.[2]
Spin markers have four advantages: ① they are sensitive to the polarity of solvents, so the hydrophobicity or hydrophilicity of the surrounding environment of the markers can be explored; ②It is extremely sensitive to the molecular rotation rate, so it can measure the allowable activity level of the marker in the environment, especially the change of biomolecule conformation caused by some biochemical process; ③EPR spectrum is simple and easy to analyze. The three peak spectrum caused by 14N can provide many valuable information; ④There is no interference signal from the diamagnetic environment.[2]
Spin markers can be connected to the target through covalent bonds or non covalent gravitations contained in the interactions between enzymes and coenzymes, enzymes and substrates, antibodies and haptens, and membranes and steroids.Spin labelingIt has been widely used to study the conformation of biopolymers, the structure of the active site of enzymes, the structure of liposomes and biofilms, and in immunoassay.[2]
