Introduction to Chromatography, Mass Spectrometry, and Spectral Analysis

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Compilation and compilation of original series of materials of Haichuan Chemical Forum: (Haichuan ID) "Dalian Haixin Chemical" Editor: (Haichuan ID) "sea7000" beautification: (Ocean ID) “ghh” ————Ocean Chemical Forum———— Special reminder: The Haichuan Enterprise Platform is online, and the product promotion effect is very good. You are welcome to join and become a long-term VIP member of the Haichuan Enterprise Platform. www.hcbbs.com The table of contents of this book has a directory link under Adobe Reader. Section 1 Introduction to Chromatography, Mass Spectrometry and Spectral Analysis 1 Section 2 Chromatography Analysis 14 Section 3 Mass Spectrometry Analysis 59 Section 4 Organic Mass Spectrometry and Related Knowledge 92 Preface This book introduces the basic knowledge of chromatography, mass spectrometry and spectral analysis, and briefly introduces the methods and examples of chromatography analysis. For mass spectrometry analysis, it not only briefly introduces the relevant content of mass spectrometry analysis, but also briefly introduces organic mass spectrometry and related knowledge. I hope this book can bring some help to Sichuan friends in analytical positions. Section 1 Introduction to Chromatography, Mass Spectrometry and Spectral Analysis 1. Introduction to 3 Analysis Methods 1. Chromatographic analysis is a method of separation and analysis based on the differences in physical and chemical properties of the two phases of the mixture components in the system (such as adsorption, distribution differences, etc.). Russian MC Tsvet is internationally recognized as the founder of chromatography. When two phases in a chromatographic system move relative to each other, usually one of the phases is stationary and is called the stationary phase. ； The other phase is mobile and is called the mobile phase. During chromatographic analysis, the migration speed of substances depends on their relative interaction with the stationary phase and mobile phase. The attraction between the solute and the two phases is the intermolecular force, including dispersion force, induction effect, interfield effect, hydrogen bonding force and Lewis acid-base interaction. For ions, there is also the electrostatic attraction between ions. The solute that is strongly attracted to the stationary phase lags behind the solute that is strongly attracted to the mobile phase. As the movement is repeated and distributed multiple times, the components in the mixture are separated. The classification of chromatographic analysis methods is more complex. According to the difference between mobile phase and stationary phase, chromatography is divided into gas chromatography and liquid chromatography. ①The mobile phase of gas chromatography is gas, which can be divided into: Gas-solid chromatography, the mobile phase is gas and the stationary phase is solid ； In gas-liquid chromatography, the mobile phase is a gas and the stationary phase is a liquid coated on an inert solid. ②The mobile phase of liquid chromatography is liquid, and it can be divided into liquid-solid chromatography. The mobile phase is liquid and the stationary phase is solid. ； ②In liquid-liquid chromatography, both the mobile phase and the stationary phase are liquids. According to the adsorbent and its use form, it can be divided into column chromatography, paper chromatography and thin layer chromatography. According to the adsorption force, it can be divided into adsorption chromatography, ion exchange chromatography, distribution chromatography and gel permeation chromatography. According to the method of terminating the chromatographic operation, it can be divided into development chromatography and elution chromatography. According to the injection method, it can be divided into zonal chromatography, head-on chromatography and displacement chromatography. The components separated by chromatography are compared with known standard samples for qualitative analysis. Modern chromatography-mass spectrometry or chromatography-spectroscopy instruments are equipped with rich spectral libraries and microprocessors. The components flowing out of the chromatographic column are directly sent to the mass spectrometer and spectrometer for qualitative identification and quantitative data processing. The development of intelligent chromatographic analysis is the main direction of development. The characteristics of chromatography are: ①High separation efficiency. It can separate substances with very similar properties and can separate complex mixtures containing hundreds of components. ②Separation speed is fast. A separation operation of complex substances can be completed in a few minutes to tens of minutes. ③High sensitivity. Can detect substances with content below 10-12 grams. ④Large-scale preparation of pure substances is possible. Chromatography is widely used in various fields such as chemical industry, petroleum, biochemistry, medicine and health, environmental protection, food inspection, forensic inspection, agriculture and other fields. Among various chromatographic methods, gas-liquid chromatography and liquid-solid chromatography are the most widely used. Gas chromatography is ideal for separating medium and small molecular compounds. Medium-sized molecules can be separated using liquid-liquid chromatography and liquid-solid chromatography. Ion exchange chromatography is generally used for substances with ionic groups. When the molecular size is larger, gel permeation chromatography is used for separation. The analysis speed of thin layer chromatography and paper chromatography is fast, convenient and low cost. Column chromatography has higher resolving power than thin layer chromatography and paper chromatography. 2. Mass Spectrometry (Mass Spectrometry, MS) is a method that uses electric and magnetic fields to separate moving ions (charged atoms, molecules or molecular fragments, including molecular ions, isotope ions, fragment ions, rearranged ions, multiply charged ions, metastable ions, negative ions and ions generated by ion-molecule interactions) according to their mass-to-charge ratios and then detect them. Measuring the accurate mass of the ion can determine the compound composition of the ion. This is because the exact mass of a nuclide is to several decimal places. No two nuclides have the same mass, and the mass of one nuclide is never exactly an integer multiple of the mass of another nuclide. Analyzing these ions can obtain information such as the molecular weight, chemical structure, fragmentation rules of the compound, and certain interrelationships between certain ions formed by the decomposition of single molecules. In 1898, W. Wien used electric and magnetic fields to deflect positive ion beams and found that when the charges were the same, ions with small masses were deflected more and ions with large masses were deflected less. In 1913, JJ Thomson and FW Aston used a magnetic deflectometer to confirm that neon has two isotopes, Ne and Ne. In 1919, Aston built a mass spectrometer that could resolve one hundredth of a mass unit, which was used to measure the relative abundance of isotopes and identify many isotopes. But until 1940, mass spectrometers were only used for gas analysis and determination of stable isotopes of chemical elements. Later, mass spectrometry was used to analyze complex hydrocarbon mixtures in petroleum fractions, and after it was confirmed that complex molecules could produce definite and repeatable mass spectra, mass spectrometry was used to determine the structure of organic compounds, opening up a new field of organic mass spectrometry. Each component in the sample is ionized to generate ions with different charge-to-mass ratios. Under the action of the accelerating electric field, an ion beam is formed and enters the mass analyzer. The electric field and magnetic field are used to cause opposite velocity dispersion - the slower ions in the ion beam are deflected more after passing through the electric field, and the faster ions are less deflected. ； In a magnetic field, ions undergo opposite deflections of their angular velocity vectors, that is, slower ions still deflect more, while faster ions deflect less. ； When the deflections of the two fields compensate each other, their orbits intersect at a point. At the same time, mass separation can also occur in the magnetic field, so that ions with the same mass-to-charge ratio but different speeds are focused on the same point, and ions with different mass-to-charge ratios are focused on different points. They are focused separately to obtain a mass spectrum, thereby determining their mass. Mass spectrometry can also perform effective qualitative analysis, but it is incapable of analyzing complex organic compounds. Moreover, quantitative analysis of organic compounds requires a series of separation and purification operations, which is very troublesome. Chromatography is an effective separation and analysis method for organic compounds. It is especially suitable for quantitative analysis of organic compounds, but qualitative analysis is more difficult. Therefore, the effective combination of the two will provide a tool for efficient qualitative and quantitative analysis of complex compounds. Mass spectrometry, especially its method combined with chromatography and computers, has been widely used in the fields of organic chemistry, biochemistry, drug metabolism, clinical, toxicology, pesticide determination, environmental protection, petrochemistry, geochemistry, food chemistry, phytochemistry, aerospace chemistry and national defense chemistry. The use of mass spectrometers for multi-ion detection can be used for qualitative analysis. For example, in pharmacological and biological research, the presence of drugs and metabolites can be determined based on the retention time and corresponding mass fragmentation of drugs and their metabolites on the gas chromatogram. ； It can also be used for quantitative analysis, using the stable isotope of the tested compound as an internal standard to obtain more accurate results. In inorganic and nuclear chemistry, many low-volatility substances can be determined by mass spectrometry using high-frequency spark sources. This ionization method requires a pure sample electrode. If the sample to be tested is in powder form, it can be mixed with nickel powder and pressed into an electrode. This method is particularly valuable for the analysis of high-purity substances in processes such as alloys, minerals, atomic energy and semiconductors, and it is possible to detect impurities with a content of one part per billion. Using the decay of long-lived radioactive isotopes to determine the age of an object is of great significance in archeology and geography. For example, if there is radioactive uranium and its decay product lead in a certain radioactive mineral, and the decay rates of uranium 238 and uranium 235 are known, then the relative abundance of the isotopes of uranium and lead produced due to decay can be measured by mass spectrometry, and the age of the generation of the axis mineral can be estimated. There are many types of mass spectrometers, and the application characteristics of different instruments are also different. Generally speaking, samples that can vaporize at around 300C can be analyzed by GC-MS first. Because GC-MS uses EI source, it can obtain a lot of mass spectrum information, which can be used for library search. The separation effect of capillary column is also good. If it cannot be vaporized at around 300C, LC-MS analysis is required. At this time, the molecular weight information is mainly obtained. If it is tandem mass spectrometry, some structural information can also be obtained. If it is a biological macromolecule, LC-MS and MALDI-TOF analysis are mainly used to obtain molecular weight information. For protein samples, the amino acid sequence can also be determined. The resolution of a mass spectrometer is an important technical indicator. A high-resolution mass spectrometer can provide compound composition formulas, which is very important for structure determination. Dual focusing mass spectrometers, Fourier transform mass spectrometers, and time-of-flight mass spectrometers with reflectors all have high-resolution capabilities. Mass spectrometry has certain requirements for samples. The sample for GC-MS analysis should be an organic solution. Organic substances in aqueous solutions generally cannot be measured. They must be extracted and separated into organic solutions, or headspace sampling technology must be used. Some compounds are too polar and are easily decomposed during heating, such as organic acid compounds. At this time, esterification can be performed to convert the acid into an ester and then undergo GC-MS analysis. The structure of the acid can be inferred from the analysis results. If the sample cannot be vaporized or esterified, it can only be analyzed by LC-MS. The sample for LC-MS analysis is preferably an aqueous solution or methanol solution, and the LC mobile phase should not contain non-volatile salts. For polar samples, the ESI source is generally used, and for non-polar samples, the APCI source is used. 3. Spectral analysis The method of identifying a substance and determining its chemical composition and relative content based on its spectrum is called spectral analysis. Its advantages are sensitivity and speed. Historically, many new elements have been discovered through spectral analysis, such as rubidium, cesium, helium, etc. According to the analysis principle, spectrum analysis can be divided into two types: emission spectrum analysis and absorption spectrum analysis. ； According to the form of the measured component, it can be divided into atomic spectroscopic analysis and molecular spectroscopic analysis. If the measured components of spectral analysis are atoms, it is called atomic spectrum, and if the measured components are molecules, it is called molecular spectrum. Since each atom has its own characteristic spectral lines, the spectrum can be used to identify a substance and determine its chemical composition. This method is called spectroscopic analysis. When doing spectral analysis, either the emission spectrum or the absorption spectrum can be used. The advantage of this method is that it is very sensitive and rapid. When the content of a certain element in a substance reaches 10^-10 (10 to the power of negative 10) grams, its characteristic spectral lines can be found in the spectrum, so it can be detected. Spectral analysis has wide applications in science and technology. For example, when checking whether the semiconductor materials silicon and germanium meet high purity requirements, spectral analysis is used. Historically, spectral analysis has also helped discover many new elements. For example, rubidium and cesium were discovered by seeing previously unknown characteristic lines in the spectrum. Spectral analysis is also useful for studying the chemical composition of celestial bodies. In the early 19th century, while studying the solar spectrum, it was discovered that there were many dark lines in its continuous spectrum. At first, we didn't know how these dark lines were formed. Later, people learned about the cause of the absorption spectrum, and they realized that this is the absorption spectrum produced when the strong light emitted from the sun passes through the relatively low-temperature solar atmosphere. By carefully analyzing these dark lines and comparing them with the characteristic spectral lines of various atoms, people know that the solar atmosphere contains dozens of elements such as hydrogen, helium, nitrogen, carbon, oxygen, iron, magnesium, silicon, calcium, and sodium. Complex color light is a pattern arranged in sequence according to wavelength after being split by a dispersion system. For example, sunlight is split into a color spectrum that is continuously distributed in the order of red, orange, yellow, green, blue, indigo, and violet. A wealth of knowledge has been accumulated about the structure, generation mechanism, properties of spectrum and its application in scientific research and production practice, and constitutes a very important subject - spectroscopy. Spectroscopy has a wide range of applications. Each atom has its own unique spectrum, which is as different as people's "fingerprints". They form several spectral line systems according to certain rules. The properties of the atomic spectral line system are closely related to the atomic structure and are an important basis for studying the atomic structure. Spectral analysis can be carried out by applying the principles and experimental methods of spectroscopy. Each element has its own unique identification spectrum line. By comparing the bright line spectrum generated by a certain substance with the identification spectrum lines of known elements, you can know which elements these substances are composed of. Spectroscopy can not only qualitatively analyze the chemical composition of a substance, but also determine the content of elements. Spectral analysis methods have extremely high sensitivity and accuracy. In geological exploration, spectral analysis can be used to detect trace amounts of precious metals, rare elements or radioactive elements contained in ores. Spectral analysis is fast, * * Improved work efficiency. Spectral analysis can also be used to study the chemical composition of celestial bodies and standard prototypes of calibrated lengths. After the polychromatic light is split by a dispersion system (such as a prism and a grating), the pattern is arranged in order according to the wavelength (or frequency). For example, sunlight passes through a prism and forms a color spectrum that is continuously distributed in the order of red, orange, yellow, green, blue, indigo, and violet. Red to purple, corresponding to the wavelength range from 7,700 to 3,900 Angstroms, are the visible parts that can be felt by the human eye. Beyond the red end is infrared light with longer wavelengths, and beyond the purple end is ultraviolet light with shorter wavelengths. Neither can be detected by the naked eye, but can be recorded with instruments. Therefore, according to different wavelength regions, the spectrum can be divided into infrared spectrum, visible spectrum and ultraviolet spectrum. ； According to the nature of production, it can be divided into atomic spectrum and molecular spectrum. ； According to different generation methods, it can be divided into emission spectrum, absorption spectrum and scattering spectrum. ； According to the different apparent forms of the spectrum, it can be divided into line spectrum, band spectrum and continuous spectrum. Emission spectrum analysis is to calculate the content based on the intensity of the characteristic spectrum emitted by the measured atoms or molecules in the excited state. The absorption spectrum is based on the characteristic spectrum of the element to be measured, and its content is calculated by the intensity attenuated after the ground state atoms of the element to be measured in the sample vapor absorb the spectrum of the element to be measured. It conforms to the Lambert-Beer law: A= -lg I/I o= -lgT = KCL where I is the transmitted light intensity, I0 is the emitted light intensity, T is the transmittance, and L is the optical path of light through the atomizer. Since L is a constant value, A=KC. The physical principle is: Atoms of any element are composed of a nucleus and electrons moving around the nucleus. The electrons outside the nucleus are hierarchically distributed according to their energy to form different energy levels. Therefore, an atomic nucleus can have multiple energy level states. The energy level state with the lowest energy is called the ground state energy level (E0=0), the remaining energy levels are called excited state energy levels, and the excited state with the lowest energy is called the first excited state. Under normal circumstances, the atom is in the ground state, and the electrons outside the nucleus move in their lowest energy orbits. If a certain amount of external energy, such as light energy, is provided to the ground state atom, when the external light energy E is exactly equal to the energy level difference E between the ground state and a higher energy level in the ground state atom, the atom will absorb the light of this characteristic wavelength, and the outer electrons will transition from the ground state to the corresponding excited state. After the original energy-providing light is split, some characteristic spectral lines are missing from the spectral lines, thus producing an atomic absorption spectrum. After the electron jumps to a higher energy level, it is in an excited state, but the excited state electron is unstable. After about 10-8 seconds, the excited state electron will return to the ground state or other lower energy level, and the energy absorbed during the electron transition will be released in the form of light. This process is called atomic emission spectrum. It can be seen that the atomic absorption spectroscopy process absorbs radiation energy, while the atomic emission spectroscopy process releases radiation energy. The spectrum is divided into the following forms:: ①line spectrum. A spectrum consisting of narrow spectral lines. The light waves emitted by monatomic gases or metal vapors all have linear spectra, so linear spectra are also called atomic spectra. When an atomic energy transitions from a higher energy level to a lower energy level, it radiates light waves of a single wavelength. Strictly speaking, this kind of monochromatic light with a single wavelength does not exist. Due to the certain width of the energy level itself and the Doppler effect, the spectral lines radiated by atoms will always have a certain width (see spectral line broadening). ； That is, a variety of different wavelength components are still included in a narrow wavelength range. The distribution of atomic spectra according to wavelength reflects the internal structure of atoms. Each atom has its own special spectrum series. Through the study of atomic spectra, the internal structure of atoms can be understood, or the components contained in the sample can be analyzed qualitatively and quantitatively. ②Band spectrum. It consists of a series of spectral bands that are radiated by molecules, so it is also called molecular spectrum. When observed with a high-resolution spectrometer, each band is actually composed of many closely spaced spectral lines. Band spectra are radiated by molecules when they transition between vibrational and rotational energy levels, usually in the infrared or far-infrared region. The structure of molecules can be understood through the study of molecular spectra. ③Continuous spectrum. The spectrum includes all wavelengths, and the spectrum radiated by hot solids is a continuous spectrum. Synchrotron radiation sources (see electromagnetic radiation) can emit a continuous spectrum from microwaves to X-rays, and the Bremsstrahlung radiation part emitted by X-ray tubes is also a continuous spectrum. ④Absorption spectrum. When a light wave with a continuous spectrum passes through a material sample, the sample atoms or molecules in the ground state will absorb light of a specific wavelength and transition to an excited state, so corresponding dark lines or dark bands appear on the background of the continuous spectrum, which is called the absorption spectrum. Each atom or molecule has an identified absorption spectrum that reflects its energy level structure. Studying the characteristics and patterns of absorption spectra is an important means to understand the internal structure of atoms and molecules. The absorption spectrum was first discovered by JV Fraunhofer in the solar spectrum (called Fraunhofer lines), and based on this, certain elements contained in the sun were determined. Specific element spectrum: Red represents sulfur, blue represents oxygen, and green represents hydrogen. Introduction to the core of China Spectrum Network: Spectroscopy is a branch of optics, which mainly studies the generation of spectra of various substances and their interactions with substances. A spectrum is an orderly arrangement of electromagnetic radiation according to wavelength. Depending on different experimental conditions, each radiation wavelength has its own characteristic intensity. Through the study of spectra, people can obtain knowledge about the energy level structure, energy level lifetime, electron configuration, molecular geometry, chemical bond properties, reaction kinetics and other aspects of material structure of atoms and molecules. However, spectroscopy technology is not only a scientific tool, it also provides important qualitative and quantitative analysis methods in chemical analysis. 4. Advantages and differences of chromatography, mass spectrometry, and spectral analysis in a nutshell: mass spectrometry: Qualitative and quantitative, can predict the composition of substances ； Chromatography: Quantitative, able to distinguish different substances in a sample ； spectrum: Qualitatively, determine the main groups in the sample and determine the substance category. The difference between spectroscopy and chromatography: (1) The analysis speed is fast. Atomic emission spectroscopy is used for analysis in front of steel-making furnaces. It can provide analysis results of more than 20 elements simultaneously within 1 to 2 minutes. ； (2) Easy operation. Some samples can be directly subjected to spectral analysis without any chemical treatment. Using computer technology, sometimes just pressing the keyboard can automatically perform analysis, data processing and print out the analysis results. In terms of poison alarms and air pollution detection, molecular spectroscopy is used for remote sensing. There is no need to collect samples. Alarms or pollution levels can be detected within a few seconds. ； (3) No pure sample is required, only known spectra can be used to conduct qualitative spectral analysis. This is a very prominent advantage of spectral analysis ； (4) Multiple elements or compounds can be measured simultaneously, eliminating complicated separation operations. ； (5) It has good selectivity and can measure elements and compounds with similar chemical properties. For example, when measuring niobium, tantalum, zirconium, hafnium and mixed rare earth oxides, their spectral lines can be separated without interference, making them a powerful tool for analyzing these compounds. ； (6) High sensitivity can be used for trace analysis using spectroscopy. At present, the relative sensitivity can reach one part per million to one part per billion, and the absolute sensitivity can reach 10-8g~10-9g. ； (7) The samples are less damaged and can be used in fields such as antiquities and criminal investigation. With the adoption of new technologies (such as the application of plasma light sources), the linear range of quantitative analysis becomes wider, allowing elements with different high and low contents to be measured simultaneously. Micro area analysis can also be performed. Chromatography compared with spectroscopy and mass spectrometry: (1) Spectrum and mass spectrometry are used for qualitative identification of substances, while chromatography has poor qualitative function ； (2) The main feature of chromatography is that it is suitable for the separation and analysis of complex multi-component mixtures. ； (3) The price of chromatograph is much lower than that of molecular spectrometer and mass spectrometer, and it has a wide range of applications. ； (4) Chromatographic detectors are more sensitive than molecular spectrometry and less sensitive than mass spectrometry. Limitations of spectral quantification: Spectral quantitative analysis is based on relative comparison. There must be a set of standard samples as a benchmark, and the composition and structural state of the standard samples should be basically consistent with the sample being analyzed, which is often difficult. Advantages of Spectrum Analyzers: (1) The sampling method is flexible, which can save the loss caused by sampling for the detection and analysis of rare and precious metals. (2) The test rate is high, multi-channel instant multi-point acquisition can be set, and real-time output through the calculator. (3) Non-destructive testing can be done for some mechanical parts without damaging the sample, which facilitates non-destructive testing. (4) The analysis speed is fast, and it is more suitable for pre-furnace analysis or on-site analysis to achieve rapid detection. (5) The accuracy of analysis results is based on chemical analysis standards. Disadvantages of Spectrum Analyzers: (1) It is difficult to accurately detect non-metals and elements between metals and non-metals. (2) It is not an original method and cannot be used as an arbitration analysis method, and the test results cannot be used as * * Certification basis. (3) Due to the relative monopoly of each company's products, the purchase and maintenance costs are relatively high and the price/performance ratio is low. (4) A large number of representative samples are required for chemical analysis modeling, which is obviously impractical for small batch sample detection. (5) The model needs to be constantly updated. When the instrument changes or the standard sample changes, the model also needs to change. (6) The modeling cost is very high, and the testing cost is also relatively high. Of course, when a large number of samples are tested, the testing cost will decrease. (7) It is easily affected by external or internal factors such as optical system parameters, and curve nonlinearity often occurs, which has a greater impact on the accuracy of detection results. Mass spectrometry characteristics: The only method that can determine molecular weight, especially modern biological mass spectrometry, is suitable for determining the molecular weight of biological macromolecules (hundreds of thousands). ； It has extremely high sensitivity, with a detection limit of 10-14g. Mass spectrometry applications: The most important application of mass spectrometry is to separate isotopes and determine their atomic masses and relative abundances. The accuracy of measuring atomic mass exceeds that of chemical measurement methods. The accurate mass of more than 2/3 of atoms is determined by mass spectrometry. Mass spectrometry methods can also be used for organic chemical analysis, especially trace impurity analysis, to measure the molecular weight of molecules and provide reliable basis for determining the molecular formula and molecular structure of compounds. Because compounds have unique mass spectra like fingerprints, mass spectrometers are also widely used in industrial production. Chromatography, spectroscopy, and mass spectrometry all have their own advantages and disadvantages. In order to maximize the advantages of each analytical instrument, two or three instruments can be combined to analyze samples. The combined technology can overcome the shortcomings of the instruments when used alone. This is the future development trend of analytical instruments. Section 2 Chromatographic Analysis 1. Introduction to Chromatography The characteristics of chromatography that distinguish it from other analysis methods: Complete separation and determination in one injection, and perform qualitative and quantitative measurements of multiple components at the same time. 1. Methods to improve separation R: Ⅰ. Capacity factor k (mobile phase flow rate, column temperature) When k=0-5, it is most effective to use retention to increase R. Generally, it is required not to exceed 10, otherwise it will * * Extend analysis time. 1) The most important thing in LC is to change the composition of the mobile phase. 2) Adjust the column temperature. It is possible to change the flow order. Lowering the column temperature in GC can increase k. 3) In GC, k can be increased by reducing the carrier gas flow rate Ⅱ. Selectivity α (mobile phase and stationary phase composition, column temperature) 1) Change the mobile phase composition, type and pH 2) Adjust the column temperature 3) Change the stationary phase 4) Use chemical modifier Ⅲ. Column efficiency N (column mass, column length, temperature, mobile phase) H=A+B/u+Cu 1) Eddy diffusion term: Use packing with narrow particle size distribution, use small particle size (should be moderate), and use small pore size columns to reduce loose arrangement and dead volume 2) Molecular diffusion term: (It plays a small role in LC) Use heavy load gas to reduce column temperature and increase flow rate 3) Mass transfer resistance term: Reduce particle size, small mobile phase viscosity, low flow rate, thin, uniform, low viscosity stationary phase film, increased column temperature, light carrier gas 4) Appropriate increase in column length 5) Small extra-column volume IV. Influence of extra-column volume (especially on small inner diameter columns and HPLC) Extra-column effect: The part of the flow path between the sampling system and the detector other than the chromatographic column has an impact on column performance due to factors such as the injection method and post-column diffusion. Ⅴ. Programmed temperature rise and gradient elution Programmed temperature rise: The column temperature changes with time during the separation process (samples with simple composition are best analyzed at a constant temperature, so that the analysis cycle will be shorter, especially when using a packed column. The baseline of the chromatogram during constant temperature analysis is much more stable than during programmed temperature rise. For samples with complex composition, programmed temperature separation is often required, because under constant temperature conditions, if the column temperature is low, the low-boiling-point components will be separated well, while the elution time of the high-boiling-point components will be too long, resulting in peak broadening, or even retention in the chromatographic column, causing contamination. ； On the contrary, when the column temperature is too high, it is difficult to separate low-boiling components. ) gradient elution: The composition of the mobile phase changes with time during the separation process 2. New progress in chromatography theory: Poppe Plot theory clarifies the relationship between particle size and separation time, N and column pressure drop Ⅰ. Column length ↑, N ↑, but the peak capacity is not necessarily large Ⅱ. The smaller the packing particle size, the better the column pressure drop & the relationship between packing particle size, column length and analysis time: Small particle size → High N → High R → Large peak capacity Small particle size → Large column pressure drop → Short column length → Short analysis time 2) Select the particle size according to the requirements for N. Small particle size will shorten the analysis time at the same N * * Add 3. mobile phase: Carry the sample through phase 4 of the chromatographic column. Solid-phase microextraction (SPE): It is a liquid-solid separation. The substance to be measured is extracted from the liquid phase to the solid phase, and then eluted with a small amount of solvent. The principle of the solid-phase extraction column is the same as that of chromatographic separation. Not only is it fast, but it also saves solvent and avoids the concentration step. 5. Reasons why standards are needed for chromatographic quantification: On most chromatographic detectors, the peak areas or peak heights obtained by different compounds of the same concentration under the same analysis conditions and on the same detector are often unequal. Therefore, standard samples must be used for calibration to obtain accurate quantitative analysis results. 6. Determine the peak order PEG-20M (strongly polar): cyclohexane, n-octane, benzene ； Propylene glycol, ethanol, isopropyl alcohol (hydrogen bonding) OV-1 (non-polar): Propylene glycol (56.5), isopropyl alcohol (82), isooctane (99.2) CZE pH3: aniline, toluene, benzoic acid ； Aniline, ethyl benzoate, naphthoic acid C18: Thiourea, p-nitrochlorobenzene, toluene ； Nitrotoluene, chlorobenzene, xylene 7, the living soul of chromatography: Look for differences between substances, create differences when necessary, and use differences to achieve the purpose of separating substances. 2. GC Disadvantages: It is difficult to characterize unknown substances. Solution: Combined with other analysis methods (mass spectrometry, infrared and electrochemistry, etc.) 1. Two types of GC: Packed columns and capillary columns (classified by column) ； Gas-solid chromatography and gas-liquid chromatography (classified by stationary phase) ； Partition chromatography and adsorption chromatography (classified by separation mechanism). Ⅰ. Advantages and Disadvantages of Capillary Column 1) Advantages: It has higher separation efficiency than packed columns (no solid filler, small gas resistance, so longer columns, smaller column inner diameters and higher carrier gas flow rates can be used), eliminating eddy current diffusion and reducing band broadening caused by longitudinal diffusion. The use of a thinner fixed liquid film offsets the increase in mass transfer resistance caused by the increase in carrier gas flow rate to a certain extent. 2) Disadvantages: The column capacity is small, and it requires high injection technology and more precise control of the carrier gas flow rate. Excessive injection volume can easily cause column overload, which requires high detector sensitivity. 3) For the analysis of permanent gases, packed columns (including packed capillary columns) have stronger separation capabilities. Ⅱ.Gas-solid chromatography - adsorption mechanism: Solid stationary phases, such as porous alumina polymer beads, are mainly used for permanent gases and organic compounds with low M. Gas-liquid chromatography-distribution mechanism: Liquid stationary phase, on which more than 90% of analyzes are based. 2. Retention mechanism: Ⅰ. Strongly polar fixative (such as PEG-20), the less polar peak will appear first ； Those with similar polarity and low boiling point peak first. Ⅱ. For weakly polar stationary solutions (such as OV-1, OV101, SE-30), the peak with the lower boiling point will appear first. ； Those with similar boiling points are inseparable. 3. Injection method (issues that should be considered include:: Sample stability ； The influence of the injection port on peak broadening, etc.) Ⅰ. Split injection: The operation is simple, but there is shunt discrimination and the sample may decompose. Ⅱ.Splitless injection: The operation is more complicated, but the analysis sensitivity is high and it is often used for trace analysis. Ⅲ. Injection on cold column: The sample enters the chromatographic column directly in a liquid state, without any split discrimination problem. The analysis has high precision and good reproducibility. It is suitable for samples with a wide boiling point range or unstable, and is often used for trace analysis (it can also be used for on-column concentration). Ⅳ.Programmed temperature vaporization and sample injection: The combination of split/splitless injection and cold column injection provides greater adaptability and flexibility. Ⅴ. Large volume injection: Programmable temperature vaporization or cold column injection port, combined with solution venting function. Ⅵ.Headspace sampling: The content of these components in the original sample is measured through the gas components above the sample matrix, and only the volatile components in the gas phase are taken. * * Reduces the impact of sample matrix on analysis. Ⅶ. Fragmentation injection: Under strictly controlled high temperatures, samples that cannot be vaporized or partially cannot be vaporized are cracked into vaporizable small molecule compounds, and then analyzed by GC, which is suitable for polymer samples. For thermally unstable compounds, it is best to use cold column injection technology. For thermally stable compounds, split/splitless injection is used. 4. Reasons for septum purging function: The injection septum is generally made of silicone rubber material and inevitably contains some residual solvents and low molecular polymers. ； Due to the influence of high temperature in the gasification chamber, silicone rubber will degrade and produce siloxane → Ghost Peak 5. Split Discrimination: It means that under a certain split ratio, the actual split ratios of different sample components are different. Ⅰ.Cause: 1) Uneven gasification (main reason) 2) Different sample components have different diffusion rates in the carrier gas, and the diffusion rate is proportional to the temperature. Ⅱ. Elimination method: 1) Higher vaporization temperature 2) The initial temperature of the column should be high 3) A small split ratio is more advantageous 4) Use a suitable liner 5) Column installation: The inlet end of the column is beyond the split point and is in the center of the gasification chamber liner. 6. Programmed heating: The temperature changes with time during the separation process 7. Make-up gas: When the FID is used as a detector, the gas directly enters the detector from the chromatographic column outlet. Since the carrier gas flow rate in the capillary column is too low, helium is added as a makeup gas in order to meet the flame sensitivity requirements for the ratio of hydrogen, air, and nitrogen. Packed columns do not require makeup gas, while capillary columns mostly use makeup gas. The function is: Ⅰ.Adjust gas proportion to improve detection sensitivity. Ⅱ. Maintain flame stability. Ⅲ. The capillary column carrier gas flow rate is low. After the sample flows out of the chromatographic column, the spectrum band may be broadened due to volume expansion. The makeup gas plays a role of wrapping and accelerating, which can eliminate this extra-column effect. 8. GC carrier gas: H2: M is small, the mass transfer resistance term has little influence, the thermal conductivity is large, and the sensitivity is high when using a thermal conductivity detector (TCD) N2: Moderate M, easy to adjust flow rate, low cost He: M is small, and the mass transfer resistance term has a small impact on Ar: M is large and the effect of molecular diffusion terms is small. Dry air cannot be used as carrier gas because air has many components and impure gases will increase the noise of the detector, reduce the detection sensitivity, narrow the linear range, and may also affect the performance of the chromatographic column. 9. Detector 1) Flame ionization detector (FID): Mass type, quasi-universal type, high sensitivity to CH compounds 2) Thermal conductivity detector (TCD): Concentration type, universal type, suitable for the analysis of various inorganic gases and organic substances, mostly used for the analysis of permanent gases. 3) Electron capture detector (ECD): Concentration type, selective type, suitable for analyzing organic compounds containing electronegative elements or groups, mostly used for analyzing halogen-containing compounds. 4) Nitrogen and phosphorus detector (NPD): Mass type, selective type, suitable for the analysis of nitrogen- and phosphorus-containing compounds. 5) Flame photometric detector (FPD): Concentration type, selective type, suitable for the analysis of sulfur-, phosphorus- and nitrogen-containing compounds. 10. Two-dimensional chromatography: Using a suitable modulator to connect the two chromatographic columns, the components separated by the first-stage chromatography can be transferred to the second-stage chromatography for continued separation. center cut: Only the components that are not separated in the first-level chromatography are sent to the second-level chromatography for separation, and the separation capacity can be expressed as n+m. Comprehensive two-dimensional chromatography: All components of the first-stage chromatography are sent to the second-stage chromatography, and the separation mechanisms of the two columns are often different. The separation capacity can be expressed as n﹡m. 11. Advantages of backflush: High efficiency ； Extend column life ； Reduce detector contamination ； Reduce operating costs ； Reduce instrument maintenance frequency ； The background interference is small and the data is reliable. 12.GC-MS: Suitable for analyzing small molecules, volatile, thermally stable, and vaporizable substances. 1) GC requirements for MS: Molecular structure identification capability, fast response capability without loss of resolution, high sensitivity, online coupling 2) Interface: Jet separator: Separate and remove most of the carrier gas to concentrate the sample components ； At the same time, it reaches the vacuum level of mass spectrometry. Opening shunt type: When the chromatographic flow rate is large, the transmission capacity is low and is not suitable for packed column conditions. Direct capillary connection: No dead volume, no selectivity, limited by column capacity. 3) Selection of GC carrier gas Ⅰ. Chemically inert and does not interfere with mass spectra ； Ⅱ.Do not use N2: (Ionization energy 15.6eV, close to that of general organic matter), which interferes with the total ion flow ； The molecular ion peak signal is strong and is close to the starting mass of the usual MS scan. ； Ⅲ.Use He: Ionization energy 24.6eV, little interference to TIC ； The molecular ion peak does not affect the low mass region IV. High purity 4) Chromatographic column and septum background: The main components of the stationary phase and septum residue in the chromatographic column are siloxane polymers. Their decomposition at high temperatures is usually called "loss". This loss enters the ion source and is the main source of background in the GC-MS mass spectrum. Elimination method: Choose a septum that is resistant to high temperatures and has low loss. Change the septum frequently to prevent septum debris from entering the liner. 3. HPLC 1. Classification by adsorption mechanism: Ⅰ. Adsorption chromatography (liquid-solid chromatography): For the separation of weakly and moderately polar compounds, as well as homologues. Ⅱ.Distribution chromatography: It is further divided into liquid-liquid distribution chromatography and bonded phase distribution chromatography 1) Normal phase HPLC: From weak to medium polarity, the stationary phase polarity of the isomer is greater than the mobile phase polarity, and the smaller polarity peak appears first. 2) Reversed phase HPLC: Strongly polar, homologous and benzene series, it can also be used for ionized acidic or basic compounds (ion pair HPLC). Amines and water-insoluble compounds are difficult to analyze. Stationary phase polarity -OH, -NH2>-C=O>-NO2>-O->-X>aromatics>-CH3; C number ↓, degree of unsaturation ↑, branching ↑, then the polarity is ↑. Ⅲ.Ion exchange chromatography (IEC): organic or inorganic ions. Separation is based on differences in the ability of charged groups to undergo reversible ion exchange. Ⅳ. Size exclusion chromatography (SEC): For polymers and biomacromolecules with M>2000. Large molecules peak first. Ⅴ. Affinity chromatography (AC): Biochemical analysis and drug screening 2. Gradient elution: During the separation process, the composition of the mobile phase is changed, and the content of the organic modifier is gradually increased in the later stage. The distribution is transferred to the mobile phase, increasing the flow rate of the compound, and the speed of the tail is faster than the center of the peak, so that the peak is focused. advantage: Improve peak shape and resolution, increase separation power ； Improve detection sensitivity ； Reduce analysis time ； Reduces the possibility of poor column performance due to strongly retained components Disadvantages: sometimes causing baseline drift ； Strong retention additives are not applicable ； Not suitable for ion pair chromatography ； Normal phase HPLC on bare silica gel column, some detectors are not applicable 3. Column end-capping (packing end-capping): Add small molecule silanization reagents to react with the remaining silicone hydroxyl groups or amino groups on the surface of the stationary phase silica gel matrix, making the surface more inert and adsorbing alkaline compounds. * * Reduces the secondary retention effect and improves the separation effect. Divided into monomer bonding and polymer bonding (better inertness) 4. Ion suppression technology: Modifying reagents are added to reversed-phase HPLC to control the acidity and alkalinity of the mobile phase to improve the chromatographic peak shape and enhance separation. When analyzing weak acids, add trifluoroacetic acid, and when analyzing weak bases, add triethylamine. 5. Methods to improve α: See Part 1 6, Detector 7, Ultraviolet Visible (UV-Vis) Detector: It is suitable for most substances and can perform gradient analysis. The disadvantage is that it requires no absorption of the mobile phase. Refractive Index Detector: Suitable for detecting carbohydrates, gradient elution is not possible, and is greatly affected by temperature, pressure, and flow rate. ； Sensitivity is low and detection limit is high. Conductivity Detector (ECD): Selective detectors are widely used in ion chromatography. Not suitable for gradient elution. Fluorescence Detector (FLD): High sensitivity and high selectivity. Suitable for detection of aromatic hydrocarbons, steroids, amino acids, vitamins, enzymes, and proteins, and can perform gradient elution ； Little affected by changes in temperature and flow rate, high sensitivity ； The disadvantage is that it can only be used for detection of fluorescent active substances. Evaporative light scattering detector (ELSD): General-purpose detector, the sensitivity is 1-2 orders of magnitude higher than the differential refractive index detector, mainly used in sugar detection ； The disadvantage is that the operation is complicated and the cost is high. 8. Basic requirements for HPLC mobile phase: 1) High purity 2) Immiscible with the stationary phase to avoid degradation and collapse of the stationary phase 3) Sufficient solubility for samples to improve peak shape and sensitivity 4) Low viscosity to reduce mass transfer resistance and improve column efficiency 5) Compatible with detectors to reduce background signals and baseline noise 6) Low toxicity and good safety 9. Application of HPLC 1) Samples with M > 2000 need to be separated by SEC (size exclusion chromatography), fat-soluble macromolecules use GPC (gel permeation chromatography), and water-soluble macromolecules use GFC (gel filtration chromatography). 2) For M trailing buffer, under the action of an external electric field, the sample zone moves at a constant speed after the movement is balanced. Buffer ionic strength > sample, the conductivity of the sample zone is low, and the local electric field strength is large. Under the action of the high electric field, the high mobility is in front and the low mobility is behind, causing the sample to separate. 3) Purpose: Online sample concentration (on-column concentration) 5. Injection mode I. Pressure difference injection: It is divided into positive pressure injection, negative pressure injection and siphon injection. Ⅱ. Electric injection: Electroosmotic flow is relied upon to bring the sample into the capillary tube. There is injection discrimination, that is, the composition of the sample entering the capillary is different from the composition of the original sample due to the different electroosmotic mobility of each component. Without calibration, the error is larger than the pressure difference injection. 6. Detector Ⅰ.UV 1) On-column detection, so the detection cell {that is, the capillary} is cylindrical and the incident surface is a curved surface ； It is different from the general square sample cell (the incident surface is flat). Therefore, the linear range is small and the noise is high. The solution is: Bubble pool and Z-type pool 2) Indirect UV detection method: When the analyte does not have ultraviolet absorption properties, adding a substance with ultraviolet absorption properties to the separation medium will cause strong background absorption. When the sample components flow through the detection window, a negative peak will appear, and a normal electrophoresis pattern can be obtained by polarity conversion. 3) Detect discrimination: Bands of different components migrate at different rates in capillary electrophoresis and pass through the detection window at different rates, resulting in unequal peak areas for components with equal absorption coefficients and the same concentration. Therefore, standard sample calibration is required. Ⅱ.LIF (laser-induced fluorescence) is the most sensitive among CE detectors, but not all samples are fluorescent and require derivatization. Ⅲ.Electrochemical detector: Post-column detection 7. Three interface types of CE-MS: Sheath liquid type, sheathless liquid type, liquid contact type Advantages of sheath liquid type: The spray is more stable, forms a current loop, and can change the composition of the buffer solution. Disadvantages of the sheath liquid type: Will dilute the substance to be measured and make electrical contact 8. How to improve sensitivity: Optimizing electrophoresis conditions and using high-sensitivity detectors for on-column concentration: LIF, MS 9. How to improve reproducibility: Each glue filling capillary coating technology (PFG, PVA) strictly controls the condition parameters to be consistent each time, such as temperature, etc. 5. Basic knowledge of thin layer chromatography 1. Introduction and development of chromatographic analysis Thin layer chromatography is an important branch of chromatographic analysis technology. Because this method has the advantages of simplicity, rapidity, high sensitivity, and good separation efficiency, it has been widely used in various fields of chemical analysis. At present, thin layer chromatography is mostly used in food hygiene chemical analysis, especially for the separation and analysis of trace organic poisons in food. The advantages of thin layer chromatography are even more obvious, so it has become an important technology in food hygiene chemical analysis. Chromatography is a method of separating the components of a mixture. This method uses the components in the mixture to adsorb or distribute between the two mutually incompatible groups to achieve the purpose of separation. At the beginning of this century, botanist Tsw-ett used a chrysanthemum root powder column and petroleum ether to separate plant leaf extracts. As a result, chromatographic bands of several plant pigments were formed on the chrysanthemum root powder column, thereby separating various pigments in plant leaves. This method of separation and analysis is called chromatography, and Zweite is considered the founder of chromatography. However, this method has not been taken seriously by people in a long period of time. It was not until 1931 that people put forward reports on the principles and practical value of chromatographic analysis in the application of chromatographic analysis methods to separate carotenoids, and then gradually improved this technical method and promoted the development of chromatographic analysis. The chromatographic analysis method established by Zwit is called liquid-solid adsorption chromatography. This method is to separate the solution of the dissolved components to be separated through a solid adsorbent column. In 1941, Wartin et al. replaced the solid adsorbent in liquid-solid adsorption chromatography with an inert support containing water (such as water-containing silica gel), dissolved the sample in a water-insoluble organic solvent, and passed it through an inert support column containing water. Due to the different solubility of the separated components in the organic solvent and water, the components were distributed and separated between the two phases. This gave birth to liquid-liquid distribution chromatography. After that, Consden used filter paper instead of water-containing silica gel as the inert support agent, which was called paper chromatography. Martin et al. further changed the mobile phase in the chromatographic separation system from liquid to gas, and successfully separated fatty acids on a diatomaceous earth support coated with silicon copper oil liquid, creating a new chromatographic analysis method. Because this method has the advantages of high separation efficiency and fast speed, it has developed rapidly and has become an important technology in current chromatographic analysis - gas chromatography. In the mid-1950s, Stahl developed a new chromatographic analysis technique - thin layer chromatography. Due to the different static phase substances used in chromatographic analysis, two other chromatographic analysis methods have been produced.: (1) Ion exchange chromatography. This method uses ion exchange resin as the stationary phase in chromatographic analysis, and uses the polar chemical bonds in the exchange resin to cause the separated substances to form a reversible reaction between the two phases. (2) Gel permeation chromatography. This method uses molecular sieves as the stationary phase to separate compounds of different molecular sizes. 2. Classification of chromatographic analysis Chromatographic analysis continues to develop, and various types of chromatographic techniques have emerged. In any chromatographic analysis, the separated components are separated between two phases. One phase is a stationary substance with a large surface area. It is called the "stationary phase".” ； The other phase is the material flowing through or along the surface of the stationary phase, which is called the "moving phase". The stationary phase can be a solid or a liquid (this liquid is attached to an inert support) ； The moving phase can be liquid or gas. According to the two possible states of each phase in chromatographic separation, chromatography can be divided into four basic types, namely gas-solid chromatography, gas-liquid chromatography, liquid-solid chromatography, and liquid-liquid chromatography, as shown in Figure 1-1. However, this classification method cannot express the nature of chromatographic separation. Figure 1-1 Chromatography classification diagram. Dynamic phase gas liquid stationary phase solid gas-solid chromatography adsorption column chromatography thin layer chromatography liquid gas-liquid chromatography distribution column chromatography thin layer chromatography paper chromatography. Therefore, some people have proposed a method of classifying according to the physical and chemical properties of the chromatographic separation process.: (1) Adsorption chromatography: The static phase is solid adsorbent, including gas-solid adsorption chromatography and liquid-solid adsorption chromatography. The purpose of separation is achieved by utilizing the different adsorption properties of the various components in the mixture by the solid adsorbent. (2) Distribution chromatography: The static phase is the liquid attached to the carrier, including gas-liquid distribution chromatography and liquid-liquid distribution chromatography. Separation is carried out by utilizing the different solubility of each component in the mixture in the two phases and their different distribution coefficients. (3) Ion exchange chromatography: The static phase is ion exchange resin. The polar chemical bonds in the exchange resin are used to cause the separated substances to form a reversible reaction between the two phases to achieve the purpose of separating the mixture. (4) Gel filtration chromatography: The stationary phase is molecular sieve. The difference in molecular size of each component in the mixture is used to pass through the static phase of the molecular sieve for separation. 3. Brief description of thin-layer chromatography As early as 1938, Izmailov and others considered an open micro-column chromatography method after their attempt to reduce the inner diameter of the column chromatography to 1 mm to establish a "micro-column chromatography" failed. This method separates alkaloids from medicinal plant extracts on glass plates coated with a thin layer of aluminum oxide. At that time, only a few drops of sample solution were used, so it was called "drop chromatography". Later, people used this method to successfully separate terpenes, alkenes and other plant volatile oils, and this method developed and was called "band chromatography" or "plate chromatography". Ismailov's method was developed by Kirehner in the early fifties, but now widespread use of remote thin-layer chromatography is due to the work of Starr. In the process of studying the composition of plant cells, he explored highly sensitive microseparation methods, studied previous microchromatographic techniques in detail, and standardized the instruments, adsorbents, and operating conditions in the chromatographic method proposed by Ismailov, further developing this method into a new chromatographic analysis technology. Starr called this method "thin layer chromatography" (TLC for short). Thin layer chromatography is actually an improvement of column chromatography. The thin layer plate can be considered as an open chromatography column. But in terms of technical operation, it is very similar to paper chromatography. According to the properties of the stationary phase substances used in thin layer chromatography, thin layer chromatography can be divided into the following categories:: (1) Adsorption thin layer chromatography (2) Distribution thin layer chromatography (3) Ion exchange thin layer chromatography (4) Gel filtration thin layer chromatography. Due to the continuous development of thin layer chromatography, this micro-separation technology has shown to have more application value than paper chromatography. Its characteristics are as follows: (1) The mixture unfolds and separates quickly. Generally, it takes about 15-60 minutes to develop once, while paper chromatography usually takes several hours to more than ten hours, so thin layer chromatography is more suitable for rapid identification. (2) The separation efficiency is better than paper chromatography. Because the spread distance is shorter, the spots are denser. (3) Less sample solution is required. Generally, it is 1 microliter to several tens of microliters. (4) Easy to operate. No special expensive and complicated instruments are required, making it easy to popularize. (5) High sensitivity. Compared with paper chromatography, its sensitivity is about 10-100 times higher. (6) Little affected by temperature changes. Because the development time is short, it is not as difficult to control the temperature as paper chromatography. (7) Strongly corrosive developer can be used. Because most of the static phase substances are inert inorganic compounds, highly corrosive color-developing reagents such as concentrated sulfuric acid, concentrated nitric acid, and sodium hydroxide can be used. This is beyond the reach of paper chromatography. (8) The separation capacity of thin layer chromatography is larger than that of paper chromatography, so it is better to use preparative chromatography for separation of trace substances than paper chromatography. (9) It can be used as a purification method in conjunction with gas chromatography, infrared spectrophotometry and other methods. Although thin layer chromatography has the above advantages, it constitutes a unique branch in the field of chromatography analysis. But things always come in twos, and TLC has its shortcomings. First of all, due to limited operating conditions, standardization is not easy to strictly control, so the reproducibility of Rf values ​​in thin layer chromatography is not ideal. ； Secondly, due to the fragility of thin-layer plates, chromatograms are not easy to preserve. ； There are still certain problems in the application of volatile substances and high molecular weight compounds. Therefore, this technical method must be evaluated comprehensively and correctly. Table 1-1 is a comparison of various chromatographic analysis technologies in various aspects for reference. Table 1-1 Comparative methods of various chromatographic analysis techniques Separation time Separation efficiency Content determination Separation conditions Volatile substances Non-volatile substances Polymer substances Paper chromatography Thin layer chromatography Gas chromatography Liquid-solid column chromatography Gel filtration chromatography Ion exchange chromatography Capillary electrophoresis Electrophoresis Long short short medium short long medium medium extra high high medium high high Medium Difference Very Good Good Good Very Bad Useless Useless Available Available Useless Available Available Useless Available Available Useless Available Used Available Useable Difference Difference Useless Difference Available Useless Available Useable Fourth, the principle of thin layer chromatography is an improvement of column chromatography, that is, the open column color is thin, and at the same time it is similar to the operating technology of paper chromatography. Therefore the general principles of chromatography also apply to thin layer chromatography. In chromatographic analysis, it is mainly the process in which the moving phase solvent drives the mixture components to flow through the static phase (i.e. chromatographic column, thin layer plate, filter paper, etc.). According to the nature of the static phase, the separated components are distributed between the dynamic phase and the static phase based on one or several of the following factors to achieve the purpose of separation. (1) Dissolve and distribute in the liquid held on the surface or inside the stationary phase. (2) Adsorption and distribution on the surface or pores of the static phase. (3) Form polar bonds with ionic components of the stationary phase. According to the action principle of static phase on the separated substances in chromatography, thin layer chromatography can be divided into four types. The principles are as follows:: (1) Distribution thin layer chromatography: That is, the separated substances are distributed and separated between the moving phase solvent and the liquid contained in the stationary phase. Its principle is the same as paper chromatography, and the purpose of separation is achieved based on the difference in distribution coefficients between solutes in the separated mixture. Solutes with small distribution coefficients dissolve more in the moving phase and move farther with the moving phase. ； A solute with a large partition coefficient moves a small distance, thus allowing different compounds to be separated. (2) Adsorption thin layer chromatography: In thin layer chromatography, adsorption is also a major form. When developing with a mobile phase solvent, different compounds continuously adsorb, desorb, re-adsorb, and re-desorb between the adsorbent and the developing agent. (3) Ion exchange thin layer chromatography: Ion exchange refers to the mutual exchange of ions that occurs at the interface between solid particles and liquid solvent. The principle of ion exchange thin layer chromatography is also based on the basic principles of ion exchange and is applied to thin layer chromatography operating technology. On a thin layer plate where the ion exchange resin is a static phase, an appropriate amount of the substance to be separated is dotted. When developed with a developing solvent, a series of elution exchange and occlusion exchange phenomena occur. Depending on the various ion selection effects, the mixture is separated in a certain order. Commonly used in thin layer chromatography is ion exchange cellulose. (4) Gel filtration thin layer chromatography: Gel filtration chromatography (also known as gel permeation chromatography) is a method that uses molecular sieves as stationary phase substances and combines thin layer chromatography operating techniques to separate and analyze the components of a mixture. Gel is a porous material. During the chromatographic separation process, molecules of different sizes separated by the gel penetrate into the pores of the network tissue in the gel to varying degrees. This penetration is reversible, and small molecules that enter the pores can be washed out with the elution solvent. ； Larger molecules that cannot enter the pores are quickly eluted out by the passive phase solvent in the spaces between the gel particles. The small molecules entering the pores are separated in the order of first increasing and then decreasing at the speed at which the passive phase solvent elutes out. As shown in Figure 2-1. In general, gel chromatography separates based on the size of the pores within the gel particles and the size of the molecules of the compounds being separated. 5. Stationary phase substances. The stationary phase substances in thin layer chromatography are mainly adsorbents, supports, ion exchange resins, molecular sieves, etc. These substances play an important role in the separation effect of thin layer chromatography. Most of the static phase substances commonly used in column chromatography can be used in thin layer chromatography. The difference is that the particles of static phase substances used in thin layer chromatography are finer than those used in column chromatography. Kirchner systematically studied some inorganic adsorbents using starch as a binder and found that thin-layer plates made of silica gel and alumina had the best separation effect and are still the most widely used adsorbents in thin-layer chromatography. With the continuous development of thin layer chromatography, many widely used organic stationary phase substances have appeared in recent years, such as polyamide, cellulose, ion-exchange cellulose, and polydextrose gel used in gel filtration chromatography. The following is a brief introduction to commonly used static phase substances.: (1) Inorganic stationary phase substances: (1) Silicone: Silica gel is formed by dehydration of silica acid. Silica gel is the most commonly used adsorbent in thin layer chromatography. The structure of silica gel is porous, with pores ranging from 20 to 150 Angstroms. Pore ​​size and surface area have a great impact on adsorption performance. Experiments have proven that silica gel is suitable for thin layer chromatography, with a particle size of 200-250 mesh and a pore size of 80-150 Angstroms. The particle size is too coarse and the separation effect is poor ； If it is too thin, the expansion speed will be slower. Silica gel is also very hygroscopic. When the relative humidity is 45-75%, it can absorb 7-20% of water. This property often causes activated silica gel to absorb water and reduce its activity. There are a large number of -OH groups on the surface of silica gel particles, which have a certain acidity (pH 4-5), so they are suitable for the separation of acidic or neutral substances. Relatively speaking, silica gel is more suitable for separating hydrophobic substances, but is less effective at separating hydrophilic substances. Silica gel has affinity for glass and is easy to adhere to glass, which is also one of its advantages. At present, commercial silica gel suitable for thin layer chromatography is available at home and abroad. Therefore, it is no longer necessary to make homemade silica gel adsorbent in the laboratory. Common commercial silicone has the following specifications:: 1) Silicone without adhesive: That is, pure silica gel powder suitable for thin layer chromatography. The product is called Silica gel H or Silica gel N. 2) Silicone with full adhesive agent: Silica gel with 15% gypsum added, the product is called Silica Gel. Silica gel with 15% starch added, the product is called Silica Gel S. 3) Silica gel containing fluorescent indicator: This type of silica gel powder contains inorganic fluorescent substances, such as cadmium sulfide, etc., which produce green fluorescence under short-wave ultraviolet irradiation. The products are called silica gel GF254, silica gel Guv254 or silica gel HF254. 4) High purity thin layer silica gel: The products are called silica gel G-HR and silica gel HF-HR. (2) Alumina: Aluminum oxide is a thin-layer adsorbent second only to silica gel in terms of application range and has strong adsorption power. The adsorption rate is related to the structure of alumina. Generally, the specific surface area is 100-200 m2/g. The reactivation temperature of alumina is around 200°C. Excessive temperature will reduce its specific surface area. If the specific surface area of ​​alumina is less than 6 m2/g, it cannot be used as an adsorbent. Therefore, alumina cannot be heated above 500°C. Commercial alumina is mostly alkaline (approximately pH9), but there are also neutral (approximately pH7) and acidic (approximately pH4) alumina. Alumina thin-layer plates are mainly used to separate alkaline and neutral weakly polar compounds. In the case of sufficient deactivation, strongly hydrophilic compounds such as sugars, amino acids, etc. can also be separated. Binder gypsum is often added to commercial thin-layer alumina, which can weaken the alkalinity of the alumina and even reach neutrality. Therefore, when using alkaline thin-layer boards, care should be taken. Commonly used aluminum oxides are: 1) Alumina without binder: That is, pure alumina powder suitable for thin layer chromatography, the product is called alumina H, or alumina N. 2) Alumina with binder: The commercial product of alumina with gypsum added is called alumina G. 3) Aluminum oxide containing fluorescent indicator: The product is called alumina GF254, and alumina HF254 is also called alumina Guv254. (3) Diatomaceous earth: Diatomaceous earth is a hydrate of crude silica, formed by the remains of diatoms accumulated on the seabed. Its adsorption force is extremely weak, but it has a large number of cavities, so it can be used as an inert support (carrier) in distribution thin layer chromatography to separate hydrophilic compounds. The particle size of diatomite for thin layers is within 60 nanometers. Commercial diatomite includes diatomite H without binder, diatomite G with gypsum binder, and diatomite GF254 containing fluorescent substances. (4) Other inorganic substances: In addition to the above three commonly used inorganic adsorbents, there are also some adsorbents, such as magnesium silicate, calcium silicate, zinc carbonate, glass powder, etc. These adsorbents are only used for some special purposes and are not as widely used as silica gel and alumina. (2) Inorganic stationary phase substances: (1) Polyamide powder: Polyamide is a polymer of lactam. There are many amide bonds in its molecule, which can form hydrogen bonds with compounds such as phenols and acids. The amine groups in the amide bonds can form hydrogen bonds with aromatic nitro groups and quinone groups in quinoid compounds, so it has a certain adsorption effect on these compounds. These compounds have different abilities to form hydrogen bonds with polyamide, so the separation effect can be achieved by using polyamide thin-layer plates. Polyamide is particularly suitable for the separation of phenolic compounds and is used in the analysis of pigments and preservatives in food. The adhesive ability of polyamide is worse than other adsorbents, so binders such as cellulose or starch need to be added. (2) Cellulose: Cellulose is cellobiose that contains a large number of β-1,4-glucoside chains. Because it contains a large number of hydroxyl groups, cellulose has good hydrophilicity and is therefore suitable for the separation of hydrophilic compounds. Cellulose thin layer plates belong to partition chromatography. The cellulose thin layer plate is similar to the filter paper in paper chromatography. The main difference is that the cellulose used in the thin layer is shorter than the cellulose filter paper. Generally, the length of cellulose is between 2 and 200 nanometers. Its specific surface area is approximately 1500 cm/g. Due to short fibers. Therefore, when unfolded on a cellulose thin-layer plate, the diffusion is very small and the spots formed are dense. The same separation effect as paper chromatography can be obtained in a short distance, so the development time is shorter than that of paper chromatography. However, compared with inorganic adsorbents, the development time is relatively long. In addition, there are also disadvantages that corrosive color developers cannot be used. There are three types of cellulose for thin layers: 1) Natural cellulose: Made of cotton, the product name is MN-300, MN-300VU254 cellulose with fluorescent substance), MN-300HR (high purity MN-300). 2) Crystalline cellulose: It is obtained by hydrolyzing natural cellulose with hydrochloric acid, and its trade name is Aviecl. 3) Acetylated cellulose: This acetylated cellulose can be used for reversed phase chromatography, and the commercial product is called MN-300AC. (3) Ion exchange cellulose: In ion exchange thin layer chromatography, commonly used static phase materials are ion exchange resins and ion exchange cellulose. The latter is the esterification and etherification of natural cellulose, so that the hydroxyl groups in the fiber interact with some acidic or alkaline groups to form cellulose with ion exchange properties. These substances are very effective in separating high molecular weight compounds. Commonly used ion exchange cellulose and ion exchange resins are shown in Table 3-1 and Table 3-2. Table 3-1 Commonly used ion exchange resins Resin type name Resin properties Ion exchange capacity Strong acidic cation exchange resin Amberlite IR-120 Dowex 50 Zeokarb 225 polystyrene ； -SO3-H+ polystyrene ； -SO3-H+ polystyrene ； -SO3-H+ H or Na H or Na H or Na 4.2 4.3 5.0 Weakly acidic cation exchange resin Amberlite IRC-50 polyacrylic acid ； Polymethacrylic acid H 10.0 Strongly basic cation exchange resin Amberlite IRA-400 Amberlite IRA-410 Dowex 1 Dowex 2 Quaternary amine CH2-N (CH3) 2CH2CH2OH Polystyrene ； -CH2-N (CH3) 3 -CH2-N (CH3) 2CH2CH2OH CI CI CI CI 3.3 3.1 3.3 3.3 Weakly basic anion exchange resin Amberlite IR-4B Amberlite IR-45 Dowex 3 Polyamine ； -NHR, -NR2 polystyrene ； -NHR, -NR2 polystyrene ； -NHR, -2NR OH OH OH 10.0 5.0 5.5 Table 3-2 Commonly used ion exchange cellulose powder resin types Exchange group Chemical formula Name Abbreviation Symbol Strongly acidic cation exchange resin -O-CH2-SO3- -O-CH2-CH2-SO3- Sulfomethylsulfoethyl SM SE Weakly acidic cation exchange resin -O-CH2-COO- Hydroxymethyl CM Strongly basic anion exchange resin-O-CH2-CH2-N (C2H5)3 Triethylaminoethyl TEAE Weakly basic anion exchange resin-O-CH2-CH2-NH (C2H5)2 -O-CH2-CH2-NH2 -N (CH2-CH2OH)3 Diethylaminoethylaminoethyltriethanolamine DEAE AE ECTEOLA (4) Hydrophilic gel: Polydextrose gel is a representative of this type of substance. It is a kind of concatenated polydextrose and is widely used in gel filtration thin layer chromatography. The trade name is Sephadex. According to the molecular weight of this gel, Sephadex is divided into 11 specifications, as shown in Table 3-3. Generally, G-10 to G-25 are not suitable for thin layer chromatography. ； It is suitable for thin layer chromatography above G-50. The particle size is finer than that used in column chromatography, ranging from 10 to 40 microns. In addition, Bio-gelP is also used in thin layer chromatography. (5) Other organic substances ； Organic compounds such as starch, sucrose, and mannitol have also been used in thin layer chromatography, but they have rarely been used. Table 3-4 General properties of commonly used static phase substances Name Acid-base adsorption Active separation principle Silica gel Alumina Diatomaceous earth Magnesium silicate Calcium hydroxide Calcium phosphate Iron hydroxide Activated carbon Cellulose Polyamide Ion exchange Cellulose Polyglucose Glucose Acid-alkaline Neutral - Alkaline Slightly alkaline - - Neutral Neutral Alkaline - Acidic - Strong Strong None Weak Weak Strong Strong Strong None None No adsorption ； Distribution Adsorption ； Distribution Distribution Adsorption Adsorption Adsorption Adsorption Distribution Ion Exchange Gel Filtration 6. Adhesives and other additives According to the characteristics of thin layer chromatography, stationary phase substances usually adhere to completely inert glass plates, and it is very difficult for a simple adsorbent to adhere to the glass plate. Therefore, it is often necessary to add appropriate adhesive agents to improve the mechanical strength of the thin layer and the adhesion ability on the glass plate. In addition, according to other purposes, such as spectrum display, separation, etc., some necessary substances, such as fluorescent agents, acid-base solutions, etc., are often added to the adsorbent. Some commonly used adhesive agents and additives are introduced below. (1) Binder (1) Forged gypsum: Calcined gypsum is the most commonly used inorganic binder in thin layer chromatography, with medium adhesion. It is made of calcium sulfate (CaSO4) containing two molecules of crystal water. 2H2O) is heated at 120°C for 4 hours to lose water. The calcined gypsum after losing water is calcium sulfate (CaSO4) containing half a molecule of crystalline calcium. 1/2H2O). In addition, if the temperature is too high during the heating process, it will further lose water. When it reaches above 200°C, it will completely lose water and become anhydrous calcium sulfate, and lose its coagulability. When calcined gypsum is combined with a small amount of water, it gradually hardens and expands. Adsorbents using gypsum as a binder include silica gel, alumina and diatomaceous earth. The gypsum content is between 5-15%. The products are called silica gel G, alumina G and diatomite G. The advantage of using gypsum as a binder is that it does not react chemically with the separated compounds and developer, and corrosive developers can be used. Its disadvantage is that the adhesion is not strong, the mechanical strength of the thin layer is poor, and it is easy to break. However, this weak adhesion is a favorable condition for the determination of elution content of thin layers and preparative thin layer chromatography. (2) Starch: The adhesive force is medium, but stronger than that of gypsum. It is easy to be interfered by corrosive color developers and color developers that react chemically with starch. The preparation method is also more troublesome than gypsum. (3) Other adhesives: Both polyvinyl alcohol and methylcellulose have adhesives that can be used as thin layers. The latter have strong adhesion, and the thin laminate produced has good mechanical strength, wear resistance, and is simple to prepare. But it is not easy to remove, which affects the elution content determination. (2) Additives: (1) Fluorescent substances: In thin layer chromatography, fluorescence quenching is a common method. To apply this method, some fluorescent substances need to be added to the adsorbent. Common ones include cadmium sulfide, zinc silicate, etc. Thin-layer plates containing fluorescent substances produce green fluorescence when irradiated by short-wave ultraviolet light (254nm), and the separated compounds produce green spots due to fluorescence quenching. Thin-layer plates with this fluorescent substance are commercially known as silica gel GF254 and alumina GF254. In addition, the thin-layer plate with Rhodamine 6G fluorescent substance added, under the irradiation of long-wave ultraviolet light (366nm), the bottom of the plate will fluoresce blue, and the product is called silica gel GF366. (2) Other adsorbents: According to the special needs of quilt separation materials, such as improving separation effect, improving reaction sensitivity, etc., appropriate adsorbents can be added. For example, adding boric acid to the adsorbent can improve the separation of carbohydrate isomers, because boric acid can form complexes with these isomers. ； Silver nitrate is added to the thin layer plate to help separate unsaturated compounds. Another example is using dilute acid or dilute alkali instead of distilled water to make plates. The former is called an acidic thin layer plate, which can separate acidic substances, such as phenols and organic acids. ； The latter is called an alkaline thin layer plate, which can separate organic bases and organic amines. 7. The choice of developing solvent in mobile phase solvent thin layer chromatography is very important for successful thin layer separation. Because among the three factors of adsorbent, separated substance and developing solvent, the properties of the first two are fixed under certain conditions, the properties of the developing solvent will have an important impact on the thin layer separation effect. When using silica gel, alumina and other inorganic adsorbents for adsorption thin layer chromatography separation, the principles of solvent system selection are similar to those of adsorption column chromatography. The most important of these is the polarity of the developing solvent. Generally speaking, medium-polar substances to be separated require medium-active adsorbents and medium-polarity developing solvents. ； Non-polar substances to be separated require highly active adsorbents and non-polar developing solvents. ； For polar substances to be separated, use low-activity adsorbents and highly polar developing solvents. The choice of development solvent should take into account the polarity of the substance to be separated, and this polarity is closely related to the structure of the compound. The polarity of various functional groups increases in the following order. -CH2-CH2-＜CH=CH-＜OCH3＜-COOR＜=C=O＜-CHO＜—SH＜-NH2＜-OH＜-COOH The polar order of various solvents is slightly different from each other. Table (3-5) is for reference. In order to select the best developing solvent, two or more mixed solution systems are usually used. This solvent consists of two parts: "base solvent" and "elution solvent". Basic solutions commonly use solvents with low polarity, such as n-hexane, petroleum ether, benzene, carbon tetrachloride, chloroform, etc. ； Elution solutions are mostly highly polar solutions, such as acetone, ethanol, methanol, ethyl acetate, etc. Table (3-6) shows several common binary solution systems. It can be seen that the polarity changes caused by increasing the elution solvent. Figure 3-4 shows the impact of a binary solvent system consisting of carbon tetrachloride as the base solvent and ethyl acetate as the elution solvent on the hRf value of the separation of four organophosphorus ester pesticides on thin layer plates when mixed in different proportions. When using cellulose, diatomaceous earth, etc. for partition thin-layer chromatography analysis, the solvent selection principles are the same as those for paper chromatography. It mainly depends on the solubility ratio of the separated compound in the developing solvent and the stationary phase, that is, the distribution coefficient. When polyamide is used as the adsorbent, the solvents are arranged in the following order according to their ability to form hydrogen bonds in the solvent.: Water＜ethanol＜methanol＜acrylic acid＜dilute alkali (ammonium hydroxide or sodium hydroxide)＜formamide＜dimethylformamide To choose the developing solvent in thin layer chromatography, the following two simple pre-test methods can be used: (1) Micro-thin layer method: Use an ordinary microscope to put the slide on, coat it with adsorbent, spot it on the bottom edge after activation, spread it 5 cm in a staining vat, take it out and blow dry before developing color. If the mixture can be separated into individual spots, a developing solvent can be used. (2) Micro-ring method: Spot the sample solution on the thin layer plate. You can count the points at the same time, with a distance of 2-3 cm between each point. Then use a capillary tube to absorb the development solvent to be tested respectively, and drop it into the origin of each sample sequentially, and develop color after drying. If a certain solvent forms the mixture to be separated into concentric rings with obvious separation, it is a more suitable developing solvent. Table 3-6 Elution capabilities of mixed solvents in polar order cyclohexane phenylcyclohexane: Ethyl acetate (95+5) benzene: Ethyl acetate (95+5) Chloroform cyclohexane: Ethyl acetate (85+15) Benzene: Ethyl acetate (85+15) chloroform: Bingtong (9+1) Benzene: Methanol (95+5) cyclohexane: Ethyl acetate (1+1) benzene: Ethanol (9+1) Chloroform: Bingtong (85+15) Benzene: Ethyl acetate (1+1) cyclohexane: Ethyl acetate (1+4) Ethyl acetate Ethyl acetate: Methanol (99+1) Benzene: Acetone (1+1) Acetone dimethylformamide dimethyl methyl sulfone 8, thin layer chromatography operation technology 1, preparation and properties of thin layer plates The preparation of thin layer plates is to coat the adsorbent on a plate of appropriate size to form a film of a certain thickness. Preparing thin-layer plates of certain specifications is an essential condition to ensure satisfactory separation results and good Rf value reproducibility. The specific preparation methods and related issues are discussed below.: (1) Carrier board: The wear plate should be stable to various developing solvents, chemical chromogenic reagents and temperatures, and should also have certain mechanical strength for repeated use. Among the commonly used wearing plates, glass is the best. Generally, glass only needs to have a flat and smooth surface, a thickness of 2-4 cm, and a tolerance of ±0.1-0.2 mm. There are no absolute regulations on the size of the wearing plate. The standard specifications recommended by Starr are 20×20 cm and 20×10 cm. As a general and simple rapid thin layer separation, a microscope slide can also be used as a plate. For preparative TLC, plates up to 20 × 100 cm are used. The wearing plate should be very clean to ensure good adhesion. Therefore, the glass plate should be washed with soapy water first, then soaked in dichromic acid washing solution, then cleaned, and finally rinsed with distilled water, dried and set aside. The disadvantage of glass panels is that they are difficult to store. Among the wearing plates made of other materials, the most commonly used ones are plastic plates. The operation of preparing thin-layer boards from plastic laminates is complicated, so most of them are sold as finished thin-layer boards. Its advantage is that it is easy to use, but its disadvantage is that it has poor corrosion resistance at high temperatures. Others such as stainless steel plates are also used. (2) Preparation of adsorbent paste: After selecting the appropriate wearing plate, an adsorbent homogenate of a certain viscosity should be prepared as needed for the preparation of thin layer plates. The preparation methods of various commonly used adsorbent pastes are as follows:: (1) Silicone 1) Add gypsum adhesive: Take 20 grams of silica gel G, place it in a glass mortar, add 40 ml of distilled water twice, add 30-35 ml for the first time, grind it thoroughly, and then add the remaining water. Grind quickly and carefully until a gel begins to form and plate immediately. The amount of water added to the adsorbent and the stirring time after adding water are very important and are the key to the success or failure of preparing thin layer plates. If too much water is added, the adsorbent will not gel easily. ； If it is too little, the gel will gel too quickly and cause difficulty in coating. There is no specific requirement for stirring time, but it is generally around 45-65 seconds. It is best when the adsorbent begins to appear in a gel state. At this time, the viscosity increases and the gloss is white. ； If this time is exceeded, the gel will solidify and become difficult to apply. At present, the amount of water added and stirring time of different brands and batch numbers of adsorbents are different, and the best conditions can be selected according to the specific situation. (The preparation methods of other inorganic adsorbents such as alumina G and diatomite G are similar to silica gel G) 2) Add starch binder: Generally, about 5% starch and twice the amount of water are added to the adsorbent, heated on a boiling water bath, stirred continuously until it reaches a certain consistency, and then laid. 3) Add hydroxymethylcellulose adhesive: Usually, take 55 grams of silica gel or 60-80 grams of alumina, add 100 ml of 1% hydroxymethylcellulose aqueous solution, mix it into a paste, and lay it out. (2) Cellulose powder 1) Natural cellulose powder: Prepare a 15% cellulose water suspension and mix it on an electromagnetic stirrer for 30-60 seconds. The cellulose powder can be paved without adding any adhesive. 2) Microcrystalline cellulose powder: Prepare a 15%-30% water suspension, mix thoroughly on an electromagnetic stirrer for 1 minute, and then plate it. Stirring for too long may result in gel formation and failure. 3) Acetylated cellulose powder: Depending on the degree of cellulose acetylation, weigh an appropriate amount, add 95% ethanol, stir thoroughly and then plate. (3) Polyamide powder: Take 12 grams of polyamide powder, add 50 ml of methanol, shake vigorously and then spread the plate. If 2 is added. 5 grams of cellulose powder and 40 ml of methanol are mixed on an electromagnetic stirrer to form a slurry, which can be used to make a strong thin layer board. (4) Ion exchanger 1) Ion exchange resin: Take 5 grams of cellulose MN300, add a small amount of water and stir for a few minutes, add 20-30 ml of distilled water, continue stirring, add 30 grams of Dowex50 resin, and finally add 25 ml of water, and lay the plate. 2) Ion exchange cellulose: Use distilled water to prepare 10-20% ion-exchange cellulose, and lay it according to the general adsorbent laying method. The adhesion effect is good. (3) Thin-layer plate preparation: Currently, the commonly used methods for preparing thin-layer boards include dipping, pouring, spraying and coating. The most commonly used method is coating. Each method is briefly described as follows:: (1) Dip method: Used for the preparation of small thin-layer plates, such as glass slide plates. The adsorbent paste used is best prepared with organic solvents, such as chloroform-acetone or chloroform-methanol mixture. Place two glass slides back to back, dip them in the adsorbent paste, take them out and place them on a horizontal plate to form a thin layer on the glass slides. (2) Pour method: Similar to the dipping method, it is easy to operate and does not require special equipment. Pour a certain amount of adsorbent paste evenly on the glass slide, and then place it on a horizontal plate to form a thin layer with a relatively uniform thickness. (3) Spray method: Using the gas pressure spray method, the adsorbent paste is evenly sprayed on the glass slide to form a thin layer. (4) Coating method: This method is the most commonly used thin-layer plate preparation method in the laboratory. The thin layer coater consists of a coating table and a coating instrument, and is sold commercially. You can also make your own. The basic structure is as shown in Figure 4-1. The material can be stainless steel or organic glass. Figure 4-2 shows a homemade simple applicator. This kind of applicator has a movable gate. When coating, the adsorbent flows out from the gap under the gate and is coated on the carrier plate. According to the different requirements of the thickness of the thin layer, the gate can be made into a variety of gap specifications for easy replacement. Generally, there are gates with specifications of 250, 275, 500, 750, 1000 and 2000 microns. There are many types of commercial applicators, and the most commonly used one is the Stahl type applicator. The biggest advantage of thin-layer plates prepared by coating method is that the thickness can be precisely controlled. The other three methods all have the disadvantage that it is not easy to control the thickness of the thin layer. (4) Activation and storage of thin-layer plates: The thin layer plate of adsorption thin layer chromatography should first have a certain adsorption activity in order to achieve good separation results. The activity of the thin-layer plate is related to the moisture content in the adsorbent. If the moisture content is high, the activity will be weakened. Therefore, in order to achieve a certain adsorption activity, the water in the thin layer should be removed by heating. This process is called activation of the thin layer plate, and its operation is: Place the coated thin-layer plate at room temperature for 15-20 minutes to allow the water to evaporate and the adsorbent to solidify. During this process, the thin-layer plate has the following phenomena:: (1) The surface of the thin layer plate begins to have a shiny luster. (2) The gloss disappears after a few minutes. (3) Finally, the thin layer solidifies, turns white, and about 50% of the water evaporates. The activity level of the thin layer plate can be measured with standard pigments. The method is as follows: (1) Activity measurement of silica gel thin layer plate: Weigh 40 mg each of the standard pigments butter yellow, Sudan red and indophenol blue, dissolve them in 100 ml of benzene, place this mixed solution on the activated TLC plate, and expand it to 10 cm with n-hexane or petroleum ether. All pigments should stay at the origin. ； When expanded to 10 cm with benzene, it should be separated into three clear spots. Their Rf values ​​should be respectively: Butter yellow 0.58, Sudan red 0.19, indophenol blue 0.08. And the rising speed of the unfolding solvent front should move 10 cm in 30 minutes. (2) Activity measurement of alumina plate: Weigh 30 mg of azobenzene, 20 mg each of p-methoxyazobenzene, Sudan yellow, Sudan red and p-aminoaphenyl, respectively, dissolve them in 50 ml of carbon tetrachloride, and place 10 μl of each spot on an activated alumina plate. Use carbon tetrachloride as the developing solvent and find out its activity level based on the Rf value of the standard pigment (table below). Alumina plate activity level Beckman classification dye name Rf value Activity level II Activity level III Activity level IV Activity level V Azobenzene p-methoxyazobenzene Sudan yellow Sudan red p-aminoazobenzene 0.59 0.16 0.01 0.00 0.00 0.74 0.49 0.25 0.10 0.03 0.85 0.69 0.57 0.33 0.08 0.95 0.89 0.78 0.56 0.19 After activation, the thin-layer plate should be used immediately. If storage is required under special circumstances, it can be stored in a sulfuric acid dryer immediately after activation to avoid absorbing moisture and certain gases in the air and affecting the activity. The storage time is about one week, not too long. (5) Preparation and properties of sintered thin-layer plates: At present, silica gel and alumina thin-layer boards are widely used, but these thin-layer boards still have shortcomings. First, it must be prepared temporarily, which is troublesome and can only be used once. The adsorbent consumption is large and not economical enough. ； Secondly, this kind of thin-layer board is very fragile and inconvenient in terms of use and storage. (6) Quality specifications of thin laminates: The quality of thin laminates made according to the above methods should meet the following requirements. (1) Appearance characteristics: The surface should be flat, smooth, without marks and bubbles, and should be observed evenly under light. (2) Mechanical strength: When the thin layer plate is immersed in the developing solvent and sprayed with the developer, the thin layer will not fall off and will not crack during the heating process. (3) Capillary performance: During the development process, the solvent front should be in a horizontal line and the rising speed should be appropriate. When a thin layer with a thickness of 250 microns is developed with benzene, it should take 30-40 minutes to rise 10 centimeters. (4) Thickness of thin layer: The thickness of the thin layer should be uniform and consistent with the experimental requirements. The thin layer plate used for analysis is generally 250-500 microns, while the thin layer plate used for preparation is 500-2000 microns. The thickness of the thin layer affects the speed of expansion of the dynamic phase solvent [5]. Using n-hexane-ethyl acetate (9+1) as the developing solvent, it was developed on silica gel plates of different thicknesses within a specified period of time. As a result, the unfolding distances of each plate were different, as shown in Figure 4-5. It can also be seen from the figure that within a thickness of 250 microns, the thinner the thin layer plate, the slower the expansion speed. 2. In spot thin layer chromatography, there is a certain relationship between spotting and the formation of spots. The origin of the sample solution required to be dropped should be as small as possible, and generally the diameter should not exceed 3 mm. For thin layer quantitative measurement, the area size of each origin should be basically the same. There are two methods for spotting: ； (1) Direct spotting method: First, use a hard pencil or dissecting needle on the TLC board to gently trace the starting line of the dots 3 cm or 2.5 cm from the bottom edge. And draw the arrival line of the developing solvent front on the upper end of the thin layer plate according to the specified spreading distance (10 cm or 12 cm). Then use a microsyringe or a hemoglobin pipette with a fine tip to add sample solution points directly to the starting line, with a distance of 1.5 cm between each point. When spotting, air flow can be used to slowly blow over the origin of the spot, so that the solvent can evaporate quickly and the area of ​​the origin will not be too large. Microsyringes are best suited for spotting thin layer chromatography. However, it is generally difficult for droplets to fall off with sharp needles, and it is easy to rub and break the thin layer, so it is not suitable. A simple improvement method can grind the needle flat, but if you are not careful, the pinhole will be blocked by adsorbent particles. If the sample solution is within 10 microliters, manual operation is sufficient. However, when the sample solution is larger than 10 microliters, manual spotting will inevitably cause errors and is also too tiring and cumbersome. Therefore, the syringe is usually fixed on a rack for operation. The distance between the needle tip and the thin layer plate and the speed of dripping are controlled constant, so the size of the origin can also be controlled, and several samples can be spotted at the same time, so it has many advantages. When spotting, care should be taken not to use the needle tip to poke small holes in the thin layer plate to avoid forming non-circular irregular spots. Because if there is a small hole at the origin, the solvent passing through the central axis of the origin during expansion will be slower than the solvent around the small hole. In this way, the color spots of compounds with higher Rf values ​​will be triangular, while the color spots of compounds with lower Rf values ​​will be crescent shaped. If the volume of the spotting solution is too large, the area of ​​the origin will also increase, which will affect the results. In this case, you can choose a developing solvent that can make the Rf value of all compounds to be separated equal to 1, and first develop it over a short distance. At this time, the compounds to be separated will form a thin line parallel to the bottom edge along the front of the developing solvent. This line can be used as the starting line of the sample, but the origin is not a dot, but a thin line. For preparative thin layer chromatography, the preparation volume needs to be increased as much as possible, and the spotting volume needs to be increased. Therefore, linear spotting is often used, and the spotting volume can reach hundreds of microliters. Thin layer spotting should be done quickly, generally no more than 10 minutes. Because the thin-layer plate is exposed for too long, it will absorb moisture in the air and change the activity, affecting the separation effect. Therefore, skilled skills are required when spotting. (2) Filter paper sample transfer method: With the direct spotting method, it is difficult to make the origin area consistent and control the diameter within 3 mm, and it is easy to damage the surface of the thin layer. Especially when measuring area quantification on a thin layer plate, these shortcomings will affect the measurement results. The filter paper sample transfer method can overcome the above shortcomings, and its operation method is as follows ； Use a belt with an inner diameter of 3 mm to punch the chromatographic filter paper into a disc. Use a fine specimen needle to pick up the filter paper disc. Slowly apply the sample solution on the disc in batches. After each spotting, wait until the solution is completely evaporated before dotting a second time, as shown in Figure 4-10. Add an external layer to the starting line of the thin layer plate, use a belt punch to lightly press the thin layer to form a 3 mm round hole, put a little paste starch in the hole, carefully put the dotted filter paper disc into the hole, and let it stick. 3. The development method of thin layer chromatography is similar to that of filter paper chromatography. The basic types can be divided into three types: upward expansion, downward expansion and horizontal expansion. The downward expansion method has few applications at present and will not be introduced in this section. (1) Upward expansion method: This method is the most commonly used method, and its deployment instruments and methods include the following:: (1) Unsaturated expansion tank: The commonly used one is a rectangular standard cylinder with a glass cover that can be tightly sealed. The size of the expansion slot is suitable for placing thin-layer boards and should not be too large. First, pour the developing solvent into the tank. The general unfolding height is 10-12 cm from the origin, and can be increased to 15 cm depending on the situation. Since this method is in an unsaturated development tank, the development solvent on the TLC plate continuously evaporates from the TLC plate, and the evaporation speed gradually increases from the middle to both sides of the TLC plate. Therefore, the amount of solvent rising on both sides is greater than in the middle. Therefore, if the same compound is used on the same TLC plate, the Rf value of the resulting stain will be higher on both sides and slightly lower in the middle. This phenomenon is called the "edge effect." Especially when using mixed solvents, edge effects are more likely to occur. (2) Saturated expansion tank: Surround the three inner walls of the development tank with filter paper. The opposite side of the thin-layer plate is not surrounded by filter paper. Add the development solvent so that the solvent rises along the filter paper. When the filter paper is completely wetted by the solvent, the solvent evaporates from the surface of the filter paper and the tank is saturated with the development solvent vapor. At this time, the thin-layer plate is placed for development. Since the tank is filled with solvent vapor, the solvent on the TLC plate no longer evaporates, thus eliminating edge effects. Expansion in a saturated tank is faster than expansion in an unsaturated tank, and the reproducibility of the Rf value is also better. (2) Horizontal expansion method: There are two commonly used horizontal expansion methods. One is as shown in Figure 4-16. In a flat glass tank, a glass rod with filter paper is placed on one end, and a glass rod without filter paper is placed on the other end. The lid is immediately placed. At this time, the unfolding solvent flows along the filter paper to the thin layer plate. This method has few applications at present. The other is the annular thin layer expansion method. The origin point is in the middle of the thin layer plate. Through the capillary action of cotton, the solvent rises above the thin layer and expands in a ring shape. (3) Wedge expansion method: Use a stainless steel knife to scrape the surface of the thin layer plate into a wedge shape. When unfolding, after the solvent passes through the origin, it spreads radially. As the unfolding solvent spreads, the separated compounds form an arc shape. This expansion technology has a large separation capacity and is suitable for mixtures with close Rf values ​​that are difficult to separate. (4) Two-way expansion method: The bidirectional development method commonly used in paper chromatography is very successful for separating complex natural mixtures. This development method is also suitable for thin layer chromatography. In two-way expansion, standard thin-layer boards of 20 × 20 cm are usually used. Place the sample on a corner of the TLC plate, 2 cm away from both sides, and develop it once with solvent I according to the general upward method. Take out the TLC plate, evaporate the solvent, and turn it 90° clockwise, that is, use solvent II to develop it again on the side perpendicular to the original development direction. Due to the different separation efficiencies of the two solvents, the separated mixture will be distributed on a square thin layer plate. (5) Gradient expansion method: The gradient elution method in column chromatography can also be used in the development of thin layer chromatography. This development method continuously changes the percentage composition of the mixed solvent during the development process, thus improving the separation efficiency and eliminating the tailing phenomenon that occurs with other development methods. (3) Add another solvent II at a constant speed and mix quickly with the stirrer (2). Excessive solvent overflows from the drain pipe (4). In this case, the percentage composition of the solvent in the development tank will continue to change evenly. (6) Staged expansion method: This development method is suitable for separating compounds with relatively large differences in polarity. The specific method is to use two solvents of different polarities to develop in two times. The first expansion distance is shorter, about 5-6 cm, while the second expansion distance is longer, about 10-12 cm. In the first development, if a strong polar solvent is used, the non-polar compounds move with the solvent to the liquid front, while the polar compounds are separated during this development. When a non-polar solvent is used for the second development, the non-polar compounds will be separated on the upper part of the thin layer plate, while the polar compounds will remain unchanged within the first development distance. (7) Multi-level expansion method: That is, the same developing solvent is developed multiple times on a thin layer plate. This method is also a method used in paper chromatography. It is used to separate mixtures that are difficult to separate after one expansion and have low Rf values. 4. Color development Color development is an important step in thin layer chromatography. After the thin layer is developed, the Rf value of the separated compound and its separation status are observed through color development technology, which is the basis for qualitative and quantitative thin layer work. Commonly used color development methods include the following categories:: (1) Physical color development method: In addition to the direct identification of colored compounds (such as pigments, etc.) under visible light irradiation, in the color development of thin layer chromatography, ultraviolet light is a major verification light source. Commonly used are short-wave ultraviolet (wavelength 254 nanometers) and long-wave ultraviolet (wavelength 365 nanometers). Methods applying ultraviolet detection can be divided into three categories:: (1) Make the separated compound display fluorescence under ultraviolet excitation. This method is mainly used to develop substances that can display fluorescence under ultraviolet excitation. The method is to directly irradiate the unfolded thin layer plate under long-wave ultraviolet light. At this time, the separated compound will show fluorescent spots on a black background. Use a dissecting needle to mark around the spot for observation. For example, the determination of aflatoxin compounds belongs to this type of method. (2) Make thin-layer plates containing fluorescent substances fluoresce under ultraviolet excitation. This method mainly develops the color of separated compounds that have a certain ability to absorb short-wave ultraviolet rays. Since this type of compound absorbs ultraviolet light, the fluorescence of the stained part is quenched and the spots appear black. At the same time, the parts other than the stained spots show fluorescence due to the excitation of ultraviolet rays. This method is called fluorescence quenching method. [29] The thin layers containing fluorescent substances used include silica gel GF254, alumina GF254, diatomite GF254, etc. (3) Prompt the separated compounds to react with chemical developers under ultraviolet irradiation to produce stains. For example, organochlorine pesticides can produce black spots under short-wave ultraviolet irradiation after spraying silver nitrate developer. (2) Chemical color development method ； The chemical color development method uses the separated compounds to chemically react with chemical color development reagents on a thin layer plate to produce colored compounds and show the location of color spots. The method of chemical color development is usually the spray method, that is, the color development reagent is sprayed on the thin layer plate to cause a chemical reaction. At present, when spraying with spray instruments commonly used in various laboratories, the distance between the sprayer and the thin-layer plate must be appropriate, and the pressure must be appropriate so that the sprayed droplets are evenly distributed on the thin-layer plate. If it is too close to the thin-layer plate, the pressure is too high or the droplets are uneven, the surface of the thin-layer plate can be damaged and the test will fail. Chemical color development is the most commonly used method in thin layer color development. Many chromogenic reagents are toxic, and some are highly corrosive. Therefore, the spray operation should be carried out in a gas cabinet, and the operator should have protective measures, such as gas masks, rubber gloves, etc. (3) Enzyme chemical color development method: Using enzyme inhibition technology, enzyme chemical reactions are performed on thin-layer plates to display color spots. There are two types of this method. One is that the substance to be tested itself is an enzyme compound, such as amylase separated in a thin layer. After unfolding, starch solution can be sprayed, and after incubation, iodine solution can be sprayed to obtain white spots on a blue background. The other type is that the separated substances have the effect of inhibiting enzymes. For example, organophosphate pesticides can inhibit cholinesterase. Therefore, on the thin layer plate where organophosphate compounds are separated, spray a solution containing cholinesterase and an enzyme hydrolysis matrix (a reagent that can be hydrolyzed by enzymes and produce colored substances). After incubation at 37°C for 30 minutes, enzymatic hydrolysis is carried out. At the spots of organophosphorus compounds, the enzyme is inhibited and colorless, and the background shows a certain color. (4) Biochromogenic method: In the thin layer chromatography separation of antibiotics, the inhibitory effect of antibiotics on microorganisms is used to perform spectral identification. 9. Rf value and its influencing factors Rf value (ratio shift value) is an important basis for qualitative thin layer chromatography. Under specific chromatographic conditions, the Rf value of the separated compound is related to the characteristics of the substance, that is, the Rf value of the compound is a physical constant of the substance. As the basis for qualitative determination, the Rf value must first indicate the chromatographic conditions, otherwise the Rf value will lose its meaning as the basis for qualitative determination. Chromatographic conditions mainly include adsorbent, development solvent and development method. There are many factors that affect the Rf value in thin layer chromatography, resulting in poor reproducibility of the Rf value, and the error can reach ±0.05 or greater. Therefore, the accuracy is often poor based on the Rf value in the chromatography manual, so the standard control method is often used in practical work. This method is to add the sample solution and the standard solution of the compound to be tested at the same time on the same thin layer plate. After development, if the Rf value of the compound to be tested is the same as the Rf value of the standard material, it can be considered that the compound to be tested is the same as the standard material. Since this method performs chromatographic separation on the same thin layer plate with the same conditions, the results are accurate and reliable. Of course, it is best to use two developing solvent systems and use standard control methods to develop on two thin layer plates to avoid accidental errors. Since the reproducibility of the Rf value is difficult to control, someone proposed the Rs value method. This method is: In addition to dotting the sample solution on the TLC plate, add a standard pigment, such as cream yellow, Sudan red, orange G, etc., or dot the homolog of the separated compound. The ratio of the migration distance of the two after expansion is the Rs value. Although the Rf value is an important basis for the qualification of thin layer chromatography, there are many factors that affect the Rf value. Therefore, in order to obtain better reproducibility of the Rf value, the following factors should be paid attention to: (1) Thickness of thin layer: Experiments have confirmed that the thickness of the thin layer has a certain impact on the expansion speed of the solvent. Using n-hexane-ethyl acetate (99+1) as the solvent, it was expanded on several silica gel plates with different thicknesses. As a result, the expansion distance of each plate was different within the specified time. ； The thickness is within 250 microns. The thinner the layer, the slower the expansion speed. Therefore the thickness will affect the Rf value of TLC. As for thicknesses above 250 microns, the effect of the thickness of the thin layer on the Rf value is related to the type of expansion groove used. To sum up, it can be considered that if the thickness of the thin layer is between 250-500 microns and a fully saturated expansion groove is used, the thickness will have little effect on the Rf value. (2) Expansion method: The development methods of thin layer chromatography have been described in the third section of Chapter 4, among which there are three main types: unsaturated development, saturated development and sandwich development. The Rf value varies with different development methods. The general situation is that the Rf value of saturated development is lower than that of unsaturated development. This is because in the saturated development tank, the solvent evaporates very little from the thin layer plate, which reduces the amount of solvent flowing on the thin layer plate and speeds up the development speed. On the other hand, in the unsaturated development tank, a large amount of solvent on the thin layer plate volatilizes, and the solvent consumption increases, so the Rf value is high. During the solvent vapor equilibrium process of the thin-layer plate in the saturated expansion tank, the adsorbent adsorbs a certain amount of solvent and at the same time expels a small amount of water, which increases the activity of the thin-layer plate and also causes the Rf value to decrease. Unsaturated expansion grooves often form "edge effects" and affect the Rf value, which has been mentioned in the previous chapter. However, Zeeuw proposed that the separation efficiency of the unsaturated expansion tank is better than that of the saturated tank, and believed that good Rf value reproducibility can also be obtained under strict control of development conditions. He conducted a series of experiments using homologs of barbiturate to confirm the above-mentioned advantages of unsaturated expansion grooves. (3) Relative humidity: There are also some literature reports on the impact of the relative humidity in the expansion tank on the Rf value. Therefore, when operating thin layer chromatography, special attention should be paid to the influence of atmospheric relative humidity. If the operation is in an environment with high humidity, the sampling should be done very quickly. Try not to expose the thin layer plate to the air for too long, so as to prevent the moisture in the air from changing the activity of the thin layer plate and causing changes in the Rf value. (4) Temperature: The effect of temperature on Rf value during thin layer chromatography operation is generally not serious (5) Development solvent: The composition of the developing solvent should be fixed, so its ratio should be very accurate during preparation, otherwise the reproducibility of the Rf value will be affected. Special attention should be paid to the purity of the developing solvent. For example, the water content in the solvent will have a certain impact on the Rf value, because changes in water content will affect the polarity of the developing solvent. Generally, analytically pure reagents can be used as solvents, and they can be refined before use if necessary. (6) Activity of thin layer plate: It is certain that the activity has an impact on the Rf value, so the activation temperature and time must be strictly controlled. The activated TLC should not be exposed to the air or left in a dryer for too long. In addition to the above-mentioned factors that affect the Rf value, other factors such as the viscosity of the adsorbent, the type of binder, and the amount of spotting have some impact on the Rf value of thin layer chromatography, and should be paid attention to. 10. Thin-layer chromatography quantification For a long time, thin-layer chromatography has been considered a semi-quantitative technical method in terms of content determination, because it still has shortcomings compared with other micro-quantitative techniques. However, in recent years, due to the continuous development of instruments and technology, thin layer quantification has reached a certain level. The quantitative methods of thin layer chromatography can be divided into two types. One is to elute the separated compound from the thin layer plate and then measure it with other methods. The other is to measure directly on the thin layer plate, such as area method, scanning optical density method, etc. (1) Elution method This method is to peel off the adsorbent in the spot area of ​​the separated compound after development from the thin layer plate, then dissolve it with an appropriate solvent, extract the separated compound, and then measure it using other content determination methods. Commonly used measurement methods include spectrophotometry, and others such as polarography, coulomb titration, gas chromatography, etc. are also used. Strictly speaking, thin layer chromatography only plays a separation role here, and content determination relies on other analytical techniques, so in this method, the main elution technology of the separated compounds is. First, it is necessary to determine the location of the stain. One of the commonly used methods is the ultraviolet color development method. Fluorescence method or fluorescence quenching method can be used. After color development, use a dissecting needle to mark the location of the stain, and then remove it for elution. Although the fluorescence method is simple and convenient, sometimes ultraviolet rays may cause the separated compounds to decompose, resulting in lower results. Therefore, the standard material control method is more reliable. That is, the standard material is spotted on the thin layer plate of the compound to be separated at the same time. After unfolding, the standard material develops color, while the substance to be tested does not develop color. Then, the spot position of the compound to be separated is determined at a position parallel to the standard spot, and the eluent is removed. Commonly used techniques for spot separation and elution from TLC plates are: Take a dropper, add glass wool, connect the tip to the air pump, and place the other end at the spot position of the TLC sample. Use a dissecting needle to carefully stir the adsorbent and try to make it completely sucked into the dropper. Then remove the dropper, dissolve and extract with an appropriate solvent, and collect the extract in a centrifuge tube for later use. This method often causes the sample solution to produce opalescence or turbidity due to the inability of glass wool filtration to remove fine particles of the adsorbent, which will cause certain errors when using photometric measurements. Therefore, the effect of membrane filtration or centrifugal sedimentation will be better than that of glass wool. The elution method still has the following shortcomings:: (1) When compounds are strongly adsorbed by adsorbents, quantitative recovery is often not satisfactory. ； (2) Impurities on the adsorbent may cause interference ； (3) The operation is cumbersome (2) Area method The area method is a direct quantitative method on the thin layer plate. Compared with the elution method, it simplifies the cumbersome elution operation and directly measures the content based on the relationship between the spot area and the substance concentration on the thin layer plate, which has certain advantages. One of the simplest methods is the visual comparison method. On the same thin layer plate, add the sample solution to be tested and a series of standard material solutions with different contents, and develop them. After color development, compare the sample spot area with the standard spot area to estimate the content of the compound to be tested. This method can obtain more accurate results when used on paper chromatography, but when applied on thin layer chromatography, the error is large, and it is rarely used at present. 11. Extraction and purification of samples In food hygiene analysis, the components to be measured often exist in extreme amounts in the samples, and the composition of these samples is very complex. Therefore, before thin layer chromatography analysis is performed, it is first necessary to go through a process of extraction, purification and concentration of the components to be measured to facilitate spot analysis. This process can not only eliminate interference in the measurement, but also improve the sensitivity of the measurement. For example, if 100 grams of sample is extracted, purified and then concentrated to 1 ml, the sensitivity can be increased by 100 times. Therefore, for thin layer chromatography analysis of food, sample extraction and purification are very important. (1) Sample extraction: Sample extraction is to use a solvent to extract the components to be measured from the sample into the extraction solvent, while most of the non-measurement components are not extracted. The method of extraction depends on the nature of the sample and the components to be measured. For example, solid samples mostly use the impregnation method, Soxhlet extraction method, and elution method, while liquid samples mostly use the liquid-liquid extraction method. The choice of extraction solvent depends on the polarity of the component to be measured and its solubility and properties in the extraction solvent. For example, when measuring pesticide residues in food, various organic solvents are often used. For less polar pesticides, such as organochlorines, parathion, malathion, etc., use non-polar solvents such as hexane, petroleum ether, etc. ； For pesticides with strong polarity, such as dimethoate, trichlorfon and other organophosphorus pesticides, use methylene chloride, chloroform or ethyl acetate of a certain polarity to be extracted as a solvent. Commonly used extraction methods include the following categories:: 1 Dip method: Place the fully crushed dry sample into a ground-mouth Erlenmeyer flask, add the selected extraction solvent, plug the bottle, place it on a oscillator to shake for a certain period of time, and then filter or centrifuge to separate the solvent. For samples containing a certain amount of moisture, such as meat, fruits, vegetables, etc., a certain amount of anhydrous sodium sulfate can be added to the sample, fully ground in a mortar, and then transferred to a ground-mouth Erlenmeyer flask for extraction. This type of sample can also be mixed with the extraction solvent in a tissue masher and fully chopped at a high speed of 20,000 rpm to increase the contact area between the extraction solvent and the sample, so that the component to be measured can be extracted from the sample in a shorter time. The tissue masher mixing and impregnation method has higher extraction efficiency than the direct shaking method. However, emulsification often occurs, and the extraction solvent must be separated with a high-speed centrifuge. 2 Soxhlet extraction method: This method uses a Soxhlet extractor to repeatedly extract the component to be measured in the sample with a small amount of solvent. When the extraction solvent is heated, the solvent continues to evaporate and condenses into a liquid on the wall of the condenser tube of the extractor, which is dropped into the sample tube for extraction. When the solvent in the sample tube reaches a certain height, it automatically flows into the extraction bottle due to the siphon effect, and the extraction is repeated many times. The advantage of this method is that the sample is continuously impregnated with fresh solvent, the extraction is more complete, and the operation is simple. In particular, the concentrated solution can be directly concentrated in the instrument after extraction. The disadvantage is that the extraction time is long, usually 4-10 hours or more. 3 Elution method: Mix the ground sample with deactivated silica gel (burned at above 600°C for several hours) or Florisite, put it into a chromatographic tube, and use the selected elution solvent to flow through the chromatographic column for elution. Fats, pigments, and insoluble impurities are adsorbed or retained by the silica gel or Florisite, and the components to be measured are eluted with the solvent. The advantage is that it avoids the emulsification phenomenon that occurs in the dipping method. 4 Extraction method: For the extraction of liquid samples, none of the above methods are applicable. Therefore, liquid-liquid extraction method was used. The selection of extraction solvent can be determined based on the distribution coefficient of the extracted components in the organic solvent phase and the aqueous phase. 2. Purification of the extraction solution. The sample extraction solution often contains various non-measurement components, such as fat, protein, pigment, wax, etc., which will be interfered by these substances during thin layer chromatography analysis. Therefore, properly processing the sample extract to remove interfering substances without causing loss of the components to be measured is called purification. Purification methods vary depending on the nature of the sample. In thin layer chromatography analysis of different samples. There are different purification methods for sample extraction solutions, and they have corresponding regulations in the analysis methods, which will not be described here. Section 3 Mass Spectrometry Analysis Mass spectrometry is a physical analysis method that converts samples into moving gaseous ions, separates them according to their mass-to-charge ratio (M/Z), and records their information. The results obtained are expressed in a spectrum, the so-called mass spectrum (also known as Mass Spectrum). Based on the information provided by the mass spectrum, qualitative and quantitative analysis of various organic and inorganic substances, structural analysis of complex compounds, determination of various isotope ratios in samples, and structure and composition analysis of solid surfaces can be carried out. Mass spectrometry is an analysis method that ionizes the substance to be measured, separates it according to the mass-to-charge ratio of the ions, and measures the intensity of various ion spectrum peaks to achieve the purpose of analysis. Mass is one of the inherent characteristics of substances. Different substances have different mass spectra - mass spectra. Using this property, qualitative analysis (including molecular mass and related structural information) can be carried out. ； The peak intensity is also related to the content of the compound it represents and can be used for quantitative analysis. A mass spectrometer generally consists of four parts: Sampling system - according to the needs of the ionization method, the sample is sent to the appropriate part of the ion source ； Ion source - used to ionize sample molecules to generate ions and converge the generated ions into an ion beam with a certain energy and geometry. ； Mass analyzer - uses the effect of electromagnetic fields (including magnetic fields, combinations of magnetic fields and electric fields, high-frequency electric fields, and high-frequency pulsed electric fields, etc.) to separate ions of different mass-to-charge ratios in the ion beam from the ion source according to spatial position, time sequence, or whether the motion track is stable or not. ； Detector - used to receive, detect and record the separated ion signals. Under normal circumstances, the sample introduction system introduces the analyte into the ion source (10-6~10-8mmHg) without destroying the vacuum of the system. After ionization, it is separated and then detected by the mass analyzer. ； The computer system controls the instrument, collects and processes data, and compares the mass spectra to spectra in a database. 1. Sampling system and connections * * The technique of introducing samples into the mass spectrometer can be divided into two methods: direct injection and through an interface. 1. Direct injection At room temperature and normal pressure, gaseous or liquid samples can be introduced into the ion source in the form of a neutral flow through an adjustable nozzle device. Volatile substances adsorbed on solids or dissolved in liquids can be enriched through a headspace analyzer, captured using an adsorption column, desorbed using a programmed temperature rise, and introduced into a mass spectrometer through a capillary tube. For solid samples, a commonly used injection rod is introduced directly. The sample is placed in a small crucible on top of the injection rod and introduced by heating in a vacuum environment near the ion source, or by desorbing the sample from a rapidly heated wire in an ionization chamber or using laser-assisted desorption. This method can be combined with electron bombardment ionization, chemical ionization and field ionization, and is suitable for the analysis of poorly thermally stable or refractory substances. At present, the rapid development of mass spectrometry sampling systems is a variety of liquid chromatography/mass spectrometry interfaces. * * Technology is used to introduce the chromatographic effluent into the mass spectrometer, and then ionize it for mass spectrometry analysis. Main technologies include various spray techniques (electrospray, thermal spray and ion spray) ； Transmission devices (particle beams) and particle-induced desorption (fast atom bombardment), etc. 2. The chromatography mobile phase with the sample in the electrospray interface is sprayed through a needle tip nozzle with a high voltage of several thousand volts, generating charged droplets. After the solvent is removed by drying gas, the charged ions directly enter the mass analyzer through the capillary or small hole. The traditional electrospray interface is only suitable for systems with a mobile phase flow rate of 1 to 5 μl/min, so the electrospray interface is mainly suitable for micro-column liquid chromatography. At the same time, because ions can be multi-charged, the mass-to-charge ratio of polymer substances falls into the analysis range of most quadrupole or magnetic mass analyzers (mass-to-charge ratio is less than 4000), so that substances with molecular weights up to several hundred thousand Daltons (Da) can be analyzed. 3. Thermal spray interface The analyte present in the volatile buffer mobile phase (such as ammonium acetate solution) is introduced into the ion source through a narrow tube and heated at the same time. The solvent is removed in the narrow tube and the analyte enters the gas phase. The neutral molecules can be ionized by chemical ionization by reacting with buffer ions (such as NH4+) in the gas phase, and then introduced into the mass analyzer. The thermal spray interface is suitable for liquid flow rates up to 2ml/min, and is suitable for mobile phases containing a large amount of water, and can be used to determine various polar compounds. Since higher temperature heating is required when the solvent evaporates, the analyte may be thermally decomposed. 4. Based on the electrospray interface, the ion spray interface uses gas-assisted spraying to increase the mobile phase flow rate to 1ml/min. Mobile phase systems used in electrospray and ion spray techniques contain buffers that must be volatile. 5. The particle beam interface converts the chromatographic effluent into an aerosol, and removes the solvent in the desolvation chamber. The neutral analyte molecules obtained are introduced into the ion source, and are ionized using electron bombardment or chemical ionization. The mass spectrum obtained is a classic electron bombardment ionization or chemical ionization mass spectrum. The former contains rich molecular structure information of the sample. However, the particle beam interface has certain limitations on the polarity, thermal stability and molecular weight of the sample, and is most suitable for the determination of small organic molecules with a molecular weight of less than 1000 Da. 6. Desorption technology combines micro-column liquid chromatography with particle-induced desorption technology (fast atom bombardment, liquid-phase secondary particle mass spectrometry). The generally used flow rate is between 1 and 10 μl/min, and a trace amount of difficult-to-volatile liquid (such as glycerol) must be added to the mobile phase. The mixed liquid flows through a capillary tube onto a metal target placed in the ion source. The liquid film formed after the solvent evaporates is bombarded by high-energy atoms or ions and ionized. The obtained mass spectrum is similar to that of fast atom bombardment or liquid phase secondary ion mass spectrometry, but the background is different. * * reduce. 2. Ion source The performance of the ion source determines the ionization efficiency and, to a large extent, the sensitivity of the mass spectrometer. There are two common ionization methods:: One is that the sample is ionized in the form of a gas in the ion source, and the other is that charged ions are sputtered from a solid surface or solution. In many cases injection and ionization are performed simultaneously. 1. After electron impact ionization (EI), the vaporized sample molecules enter the ionization chamber and are bombarded by the electron flow emitted and accelerated by the tungsten or rhenium filament to generate positive ions. The ionization chamber pressure is maintained at 10-4~10-6 mmHg. The energy of the bombarding electron is greater than the ionization energy of the sample molecules, causing the sample molecules to ionize or fragment. Electron impact mass spectrometry can provide the richest structural information of organic compounds, has good reproducibility, and has the most complete research on its cracking rules. A standard spectral library of tens of thousands of organic compounds has been established for search. Its disadvantage is that it is not suitable for samples that are difficult to volatilize and have poor thermal stability. 2. Chemical ionization (CI) introduces a reaction gas of a certain pressure into the ionization chamber, and the reaction gas is ionized or cracked under the action of an electron flow with a certain energy. The generated ions further react with the reagent gas molecules or react with the sample molecules to ionize the sample molecules through proton exchange. Commonly used reaction gases are methane, isobutane and ammonia. Chemical ionization usually produces quasi-molecular ions. If the proton affinity of the sample molecule is greater than the proton affinity of the reaction gas, [M+H]+ is generated, and vice versa, [MH]+ is generated. According to the different reaction gas pressures, chemical ionization sources are divided into three types: atmospheric pressure, medium pressure (0.1~10mmHg) and low pressure (10-6mmHg). The atmospheric pressure chemical ionization source is suitable for coupling chromatography and mass spectrometry, and the detection sensitivity is 2 to 3 orders of magnitude higher than that of general chemical ionization sources. The low-pressure chemical ionization source can analyze difficult-to-volatile samples at lower temperatures and can use difficult-to-volatile reaction reagents, but it can only be used with Fourier transform mass spectrometers. 3. Fast atom bombardment (FAB) disperses the sample in a matrix (commonly used high boiling point solvents such as glycerol) to form a solution, coats it on the metal target and sends it to the FAB ion source. A beam of inert gas neutral atoms (such as xenon) accelerated by a strong electric field is directed at the sample on the target and bombarded. The associated ions present in the matrix and the sample ions produced by fast atom bombardment are sputtered into the gas phase and enter the mass analyzer under the action of the electric field. If an inert gas ion beam (such as cesium or argon) is used to replace the neutral atom beam for bombardment, the resulting mass spectrum is called liquid secondary ion mass spectrometry (LSIMS). The advantage of this method is that it has strong ionization ability and can be used for samples with strong polarity, low volatility, poor thermal stability and large relative molecular mass, as well as samples for which EI and CI are difficult to obtain meaningful mass spectra. FAB is easier to obtain stronger molecular ions or quasi-molecular ions than EI ； One advantage that is different from CI is that the mass spectrum obtained has more fragment ion peak information, which is helpful for structural analysis. The disadvantage is that the sensitivity to non-polar samples is reduced, and the matrix produces more interference peaks in the low mass region (below 400). FAB is a surface analysis technology that requires attention to the sample handling process to optimize surface conditions. The adduction of sample molecules with alkali metal ions, such as [M+Na] and [M+K], helps to form ions. This phenomenon contributes to the ionization of biomolecules. Therefore, treating the sample surface with sodium chloride solution helps to increase the yield of adduct ions. Heating the sample during analysis can also help improve yields. During the FAB ionization process, positive and negative ions can be generated simultaneously, and both ions can be analyzed by mass spectrometry. Sample molecules with strong electron capture structures, especially halogen atoms, can generate a large number of negative ions. Negative ion mass spectrometry has been successfully used for the analysis of pesticide residues. 4. Field ionization (FI) and field desorption (FD) The FI ion source consists of an anode and a cathode that are very close together. After a high voltage is applied between the two electrodes, a strong electric field of up to 10+7~10+8V/cm is generated near the anode. The gaseous sample molecules approaching the anode are ionized to form positive molecular ions, which are then accelerated into the mass analyzer. For liquid samples (solid samples are first dissolved in the solvent), FD can be used to achieve ionization. The metal wire is immersed in the sample liquid, and after the solvent evaporates, the metal wire is used as an emitter and sent to the ion source. The energy required for sample desorption is provided through a weak current, and the sample molecules diffuse to the high-field-strength emission area and achieve ionization. FD is suitable for compounds that are difficult to gasify and have poor thermal stability. Both FI and FD can easily obtain molecular ion peaks. 5. Atmospheric pressure ionization source (API) API is the most commonly used ionization method for liquid chromatography/mass spectrometry. There are three common atmospheric pressure ionization sources:: Atmospheric pressure electrospray (APESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). Electrospray ionization is the process of forming ions from charged droplets after solvent removal, and is suitable for samples or polar compounds that easily form ions in solution. Due to its multi-charge capability, it can analyze a wide range of molecular weights and can be used not only for small molecule analysis, but also for peptide, protein and oligonucleotide analysis. APCI is an ionization technology that uses corona discharge to ionize gas phase samples and mobile phases under atmospheric pressure. It requires the sample to have a certain volatility and is suitable for non-polar or low or medium polar compounds. Since multiply charged ions are rarely formed, the molecular weight range analyzed is limited by the mass range of the mass analyzer. APPI is an ionization technology that uses ultraviolet lamp to replace the corona discharge of APCI and uses photochemistry to ionize the sample in the gas phase. It is suitable for non-polar compounds. Since the atmospheric pressure ionization source is independent of the mass analyzer in a high vacuum state, switching between different atmospheric pressure ionization sources is very convenient. 6. Matrix-assisted laser desorption ionization (MALDI) coats a sample dissolved in an appropriate matrix on a metal target, and ionizes the sample by irradiating it with high-intensity ultraviolet or infrared pulse laser. This method is mainly used for the analysis of macromolecules with a mass up to 100,000 Da, and is limited to use as an ion source for a time-of-flight analyzer. 7. Inductively coupled plasma ionization (ICP) plasma is composed of free electrons, ions and neutral atoms or molecules. It is generally an electrically neutral gas with an internal temperature as high as several thousand to 10,000 degrees. The sample is carried by the carrier gas through the center of the plasma torch, is quickly evaporated and ionized, and is introduced into the mass analyzer through the ion extraction interface. The sample is completely evaporated and dissociated at extremely high temperatures, with a high percentage of ionization, resulting in high detection sensitivity for almost all elements. Since the molecular structure of the compound has been destroyed under this condition, ICP is only suitable for elemental analysis. 3. Mass analyzer The mass analyzer separates charged ions according to their mass-to-charge ratio and is used to record the mass number and abundance of various ions. The two main technical parameters of a mass analyzer are the range of mass-to-charge ratios that can be determined (mass range) and resolution. 1. The ions generated in the ion source of the sector magnetic analyzer are focused by the sector magnetic field and the slit to form an ion beam. After ions leave the ion source, they enter a magnetic field perpendicular to their direction of travel. Under the action of a magnetic field, ions with different mass-to-charge ratios deflect in different directions, causing the ion beam to diverge. Since ions with different mass-to-charge ratios have their own unique radius of curvature of movement in a sector-shaped magnetic field, by changing the magnetic field intensity, the ions passing through the slit exit in sequence are detected, thereby achieving spatial separation of ions and forming a mass spectrum. 2. The quadrupole analyzer is so named because it consists of four parallel rod-shaped electrodes. The ion beam is focused on an axis parallel to the rod electrode. A fixed direct voltage (DC) and a radio frequency voltage (RF) are applied to the rod electrode, with opposite potentials between the two pairs of electrodes. For a given DC and radio frequency voltage, ions with a specific mass-to-charge ratio move stably in the axial direction, while ions with other mass-to-charge ratios collide with the electrode and are annihilated. By changing DC and RF at a fixed slope, the mass spectrum scanning function can be realized. Quadrupole analyzers have high sensitivity for selected ion analysis. 3. The ion trap analyzer consists of two end cap electrodes and a quadrupole-like ring electrode between them. The end cap electrode applies DC voltage or ground, and the ring electrode applies radio frequency voltage (RF). By applying appropriate voltage, a potential energy trap (ion trap) can be formed. Depending on the size of the RF voltage, the ion trap can capture ions in a certain mass range. The ion trap can store ions. After the ions accumulate to a certain number, the RF voltage on the ring electrode is increased. The ions leave the ion trap in order from high to low mass and are detected by the electron multiplication monitor. At present, ion trap analyzers have been developed to analyze ions with mass-to-charge ratios up to several thousand. The ion trap still has high sensitivity in full scan mode, and a single ion trap can realize the function of multi-stage mass spectrometry (MSn) through time series settings. 4. Time-of-flight analyzer ions with the same kinetic energy but different masses are separated due to their different flight speeds. If the ion flight distance is fixed, the flight time of ions with different masses is different. The flight time of ions with small mass is short and reaches the detector first. The flight time of various ions is proportional to the square root of the mass-to-charge ratio. Ions are introduced into the mass spectrometer in the form of discrete packets, which allows the origin of the flight to be unified and the time of flight measured sequentially. Ion packets are generated continuously through a pulse or grid system, but are only introduced into the flight tube at a specific time. The newly developed time-of-flight analyzer has a large mass analysis range and high mass resolution, and is especially suitable for the analysis of biological macromolecules such as proteins. 5. Fourier transform analyzer In a magnetic field of a certain intensity, ions move in a circular motion, and their orbits are limited by the resonant transformation electric field. When the frequency of the transformed electric field is the same as the cyclotron frequency, the ions accelerate steadily, the radius of the orbit becomes larger and larger, and the kinetic energy becomes larger and larger. When the electric field disappears, the orbiting ions generate alternating currents on the electrodes. Analysis of the signal frequency yields the ion mass. The time and corresponding frequency spectrum are transformed into a mass spectrum using a computer through Fourier transform. The advantage is that the resolution is very high and the mass-to-charge ratio can be accurate to one thousandth of a dalton. 4. Tandem mass spectrometry and hyphenated technology 1. Tandem mass spectrometry Two or more mass spectrometers are connected together, which is called tandem mass spectrometry. The simplest tandem mass spectrometry (MS/MS) consists of two mass spectrometers connected in series. The first mass analyzer (MS1) pre-separates or energy-modifies the ions, and the second-stage mass analyzer (MS2) analyzes the results. The most common tandem mass spectrometer is the triple quadrupole tandem mass spectrometer. The first and third-stage quadrupole analyzers are MS1 and MS2 respectively. The function of the second-stage quadrupole analyzer is to bombard each peak obtained from MS1 to fragment the precursor ions and enter MS2 for further analysis. Now there are tandem mass spectrometers composed of a variety of mass analyzers, such as quadrupole-time-of-flight tandem mass spectrometry (Q-TOF) and time-of-flight-time-of-flight (TOF-TOF) tandem mass spectrometry. * * Expanded application scope. The ion trap and Fourier transform analyzer can realize time series multi-level mass spectrometry scanning function in different time sequences. The most basic functions of MS/MS include the ability to illustrate the relationship between the precursor ions in MS1 ​​and the product ions in MS2. According to the scanning modes of MS1 and MS2, such as product ion scanning, precursor ion scanning and neutral fragment loss scanning, the relationship between ions of different mass numbers can be found out. Fragmentation of precursor ions can be achieved by: Collision-induced dissociation, surface-induced dissociation and laser-induced dissociation. Dissociation without excitation is called metastable decomposition. MS/MS has many advantages in mixture analysis. When mass spectrometry is coupled with gas chromatography or liquid chromatography, identification can be performed even if the chromatography fails to completely separate the substances. MS/MS can select precursor ions from the sample for analysis without interference from other substances. MS/MS has many applications in the pharmaceutical field. Product ion scanning can obtain qualitative information on the parent ions of the main components of drugs, impurities and other substances, which is helpful for the identification of unknown substances and can also be used to identify the amino acid sequences of peptides and proteins. In pharmacokinetic studies, multiple reaction monitoring (MRM) mode can be used to eliminate interference when quantitatively analyzing low-concentration samples in biologically complex matrices. If a specific ion in a drug is analyzed, and signals from other compounds in the matrix may obscure the detection signal, using MS1/MS2 to selectively monitor the fragments of the specific ion can eliminate interference. MRM can also quantitatively analyze multiple compounds simultaneously. In drug metabolism studies, in order to find molecules with the same structural characteristics as pre-metabolized substances, neutral fragment loss scanning can be used to find all ions that have lost the same functional group, such as carboxylic acids losing neutral carbon dioxide. If the missing fragment is in the form of an ion, a precursor ion scan will find all ions missing this fragment. 2. Hydraulic technology chromatography can be used as a sample introduction device for mass spectrometry and perform preliminary separation and purification of samples. Therefore, chromatography/mass spectrometry technology can separate and analyze complex systems. Because chromatography can obtain the retention time of the compound, and mass spectrometry can give the molecular weight and structural information of the compound, it is very effective for the identification and determination of compounds in complex systems or mixtures. Among these hyphenated technologies, chip/mass spectrometry (Chip/MS) shows good promise, but is still immature, while gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry have been widely used in drug analysis. (1) Gas Chromatography/Mass Spectrometry (GC/MS) The effluent from the gas chromatograph is already in the gas phase and can be directly introduced into the mass spectrometer. Since the working pressures of gas chromatography and mass spectrometry differ by several orders of magnitude, various gas separators were used between them at the beginning of the coupling to account for the difference in working pressure. With the advent of capillary gas chromatography and the use of high-speed vacuum pumps, gas chromatography effluents can now be directed into mass spectrometers. (2) Liquid Chromatography/Mass Spectrometry (HPLC/MS) The interface of liquid chromatography/mass spectrometry has been discussed before. It is mainly used to analyze substances that cannot be analyzed by GC/MS, or have poor thermal stability, strong polarity and high molecular weight, such as biological samples (drugs and their metabolites) and biological macromolecules (peptides, proteins, nucleic acids and polysaccharides). (3) Capillary electrophoresis/mass spectrometry (CE/MS) and chip/mass spectrometry (Chip/MS). Capillary electrophoresis (CE) is suitable for separation and analysis of extremely small amounts of samples (nl volume) and for specific purposes (such as separation of chiral enantiomers, etc.). The CE effluent can be directed into the mass spectrometer, or an auxiliary mobile phase can be added to match the mass spectrometer. Microfluidic chip technology is a miniaturized technology that has developed rapidly in recent years and can realize various laboratory technologies such as separation, filtration, and derivatization on a single chip. It has the advantages of high throughput and miniaturization. It has also realized the coupling of chip and mass spectrometry, but it has not yet been commercialized. (4) Supercritical fluid chromatography/mass spectrometry (SFC/MS). SFC, which commonly uses supercritical fluid carbon dioxide as the mobile phase, is suitable for the separation and analysis of small and medium polar substances. The combination of SFC and MS can be achieved through a separator between the chromatographic column and the ion source. (5) Plasma emission spectrometry/mass spectrometry (ICP/MS) uses ICP as an ion source and MS to achieve combined use, and is mainly used for elemental analysis and elemental form analysis. 5. Data processing and application The detector is usually a photomultiplier or an electron multiplier. The collected signal is amplified and converted into a digital signal, and the mass spectrum is obtained after computer processing. The number of mass spectrum ions is expressed by abundance, that is, the number of ions with a certain mass-to-charge ratio. Since the "quantity" of a specific ion cannot be measured, relative abundance is generally used to express its intensity, that is, the strongest peak is called the base peak, and the abundance of other ions is expressed as a percentage relative to the base peak. Within the mass range measured by the mass spectrometer, the mass spectrum consists of the mass-to-charge ratio of the ions and their relative abundance. In LC/MS and GC/MS, the chromatographic total ion chromatogram is commonly composed of the chromatographic retention time of each analyte and the relative intensity of its ions obtained from mass spectrometry. A fixed mass-to-charge ratio can also be determined, and selected ion monitoring (SIM) can be performed on the entire chromatographic effluent to obtain a selected ion chromatogram. The ability of a mass spectrometer to separate ions is called resolution, which is usually defined as the ratio of the average mass of two peaks to their mass difference when the valley height of the two peaks is 10% of the peak height for two adjacent peaks with the same height. For low, medium and high-resolution mass spectrometry, it refers to the resolution of 100 to 2000, 2000 to 10000 and above 10000 respectively. The main applications of mass spectrometry in the pharmaceutical field are qualitative identification, quantitative analysis and structural analysis of drugs. If a neutral molecule loses or gains an electron, the mass-to-charge ratio of the molecular ion is the same as the molecule's mass number. High-resolution mass spectrometry can be used to obtain the accurate mass of an ion and then calculate the molecular formula of the compound, or peak matching with a reference can confirm the molecular weight and formula. Various chemical bonds of molecular ions are broken to form fragment ions, from which the fragmentation method can be inferred and corresponding structural information can be obtained. Mass spectrometry is used for quantitative analysis and has high selectivity, precision and accuracy. Compounds are separated and purified by direct injection or using gas chromatography and liquid chromatography before being introduced into the mass spectrometer. Mass spectrometry quantitative analysis uses external standard method or internal standard method, the latter is more accurate than the former. Internal standards in quantitative analysis can use similar structural substances or isotopic substances. The former is low in cost, but its precision and accuracy are higher by using isotopic substances. When using isotope substances as internal standards, it is required that the isotope substances are not lost during the injection, separation and ionization processes. When using FAB mass spectrometry and LC/MS (thermal spray and electrospray) for quantitative analysis, stable isotope internal standards are generally required. The relative abundances of analytes and internal standard ions are determined using selected ion monitoring (monitoring only specific ions of the analyte and internal standard). Compared with full-range scanning, selected ion monitoring increases selectivity and sensitivity due to the long ion current integration time. The chromatographic peak area or peak height ratio of the analyte and the internal standard is used to derive a calibration curve, and then the chromatographic peak area or its amount of the analyte in the sample is calculated. EI: Electron Impact Electron impact mass spectrometry has the characteristics of fast analysis, high sensitivity, and the information provided is directly related to its structure. Combined with gas chromatography, it has become one of the most powerful and reliable analytical tools for rapid identification of the composition of complex mixtures and has been widely used. An analytical method in which the sample is ionized to form a mass spectrum. When gas (or substances that can be converted into gas) molecules are bombarded by electrons at low pressure, various positively charged ions are produced, and then the cations are separated in order of mass through a stable magnetic field to form a regular mass spectrum, which is then detected with a detector to perform qualitative and quantitative analysis. 1. Analysis experience (1) In the positive ion mode, the sample is mainly detected as +, +, + quasi-molecular ions ； In the negative ion mode, the samples are mostly detected as –, – quasi-molecular ions. (2) In the positive ion mode, the sample will also have peaks such as M-1(MH), M-15(M-CH3), M-18(M-H2O), M-20(M-HF), M-31(M-OCH3), etc. The molecular ion peak should have a reasonable mass loss. That is, it is impossible and unreasonable to have a mass difference of 4-13, 21-26, 37-, 50-53, 65, 66 compared to the molecular ion. Otherwise, the peak with the largest mass number is not the molecular ion peak, because an organic compound molecule cannot lose 4 to 13 hydrogens and continue to bond. If the bond is broken, the smallest fragment lost should be CH3, and its mass is 15 mass units. (3) The molecular ion peak should be an odd electron ion, and its mass number should comply with the nitrogen rule: Among organic compounds, those containing an even number of nitrogen atoms or no nitrogen atoms must have an even molecular mass. On the contrary, those containing an odd number of nitrogen atoms must have an odd relative molecular mass. This is the nitrogen rule. The application of nitrogen rules will be beneficial to the judgment of molecular ion peaks and the inference of molecular formulas. After the elemental composition of a compound is determined through elemental analysis, if the mass of the highest mass ion does not comply with the nitrogen rules, then the ion must not be a molecular ion. If a certain ion peak completely complies with the above three judgment principles, then this ion peak may be a molecular ion peak; if one of the three principles does not comply with it, this ion peak is definitely not a molecular ion peak. It should be noted that some compounds are prone to M-1 peaks or M+1 peaks. Below are the mass spectra of common organic compounds: M-15 (.CH3) M-27 (CHNH2.CHCH2) M-32 (CH3OH,S,O2) M-16 (O) M-28 (CO,CH2CH2) M-33 (CH3+H2O) M-17 (.OH,NH3) M-29 (CHO,C2H5) M-34 (H2S) M-18 (H2O) M-30 (CH2O,NO) M-35 (Cl) M-26 (CN,HCCH) M-31 (OCH3,CH2OH) M-36 (2H2O,HCl) 2. Basic Principle 2.1 The basic principle of mass spectrometry analysis is to vaporize the sample molecules to be measured, bombard the gaseous molecules with an electron beam with a certain energy, causing them to lose one electron and become positively charged molecular ions. The molecular ions may also break into various fragment ions. Under the combined action of the electric field and magnetic field, all the positive ions are arranged in order according to the mass-to-charge ratio ( ) to obtain the spectrum. Since most ions carry only one positive charge, z=1, the mass-to-charge ratio is the mass of the ion. The spectrum gives the mass of various fragments. By splicing these fragments together, the original structure can be obtained. A mass spectrometer is a device that uses the principle of electromagnetism to generate positively charged moving ions from gas molecules and separate them in the electromagnetic field according to their mass-to-charge ratio. Taking a linear single-focus mass spectrometer as an example to illustrate the basic principles of mass spectrometry, the instrument structure is shown in Figure 2-1. The sample enters the ion source from the injector, and positive ions are generated in the ion source. Positive ions accelerate into the mass analyzer, and the mass analyzer separates them according to their different mass-to-charge ratios. The separated ions enter the detector one after another, and the detector obtains the ion signal. The amplifier amplifies the signal and records it on the readout device. Figure 2-1 Schematic diagram of a single-focus mass spectrometer. After ionization, the ion enters the magnetic field through the accelerator. Its kinetic energy is related to the acceleration voltage and charge z, namely (2-1) where z is the number of charges, e is the elementary charge (e=1.60×10-19C), U is the acceleration voltage, m is the mass of the ion, and v is the speed of the ion after being accelerated. Charged particles with speed v enter the electromagnetic field of the mass spectrometer. Due to the action of the magnetic field, the ions move in an arc. At this time, the centripetal force Bzv experienced by the ions is equal to the centrifugal force of motion, and we get (2-2), where: R is the radius of the ion arc motion, and B is the magnetic field strength. From formulas (2-1) and (2-2), we can get the relationship between ion mass-to-charge ratio and motion orbit curve radius R: (2-3) (2-4) Formulas (2-3) and (2-4) are called mass spectrometry equations, which are the basic formulas of mass spectrometry analysis and the main basis for designing mass spectrometers. It can be seen from formula (2-3) that the mass-to-charge ratio m/z of ions is proportional to the square of the radius R of the curve of ions moving in the magnetic field. If the accelerating voltage U and magnetic field strength B are both constant, ions of different m/z will be separated from each other in the mass analyzer due to different curve radii of motion, and the relative intensity of the ions of their respective m/z will be recorded. Qualitative and structural analysis of substances based on the position of mass spectrum peaks ； Quantitative analysis is performed based on peak intensity. In essence, mass spectrum is not a wave spectrum, but a mass spectrum of charged particles of matter. 2.2 Mass Spectrum Mass spectra are represented by bar graphs. Each line represents a peak and represents an ion. The abscissa is the value of the ion mass-to-charge ratio ( ), and the ordinate is the relative intensity, that is, the ratio of each peak to the highest peak (called the base peak). In literature reports, mass spectrum tables are often used instead of mass spectra. 2.3 Various types of mass spectrum peaks (1) Molecular ion peaks: Molecules are bombarded by electron flow and lose one electron to obtain molecular ions. The peak that appears on the spectrum is usually the rightmost peak. If the molecular ion peak on the mass spectrum can be correctly identified, the relative molecular mass of the measured substance can be read directly from the spectrum. When judging the molecular ion peak, attention should be paid to the nitrogen rule, that is, the relative molecular mass of organic matter containing no nitrogen or an even number of nitrogens is an even number, and the relative molecular mass of an organic matter containing an odd number of nitrogens is an odd number, and the molecular ion must be an odd electron ion. (2) Isotope peaks are common in organic matter. C, H, O, N, S, Cl, Br, I, etc. all have isotopes. Therefore, some small peaks of M+1 and M+2 can often be seen next to the molecular ion peak, which can be used to infer the molecular formula. The more common ones are: 32S, 34S 35Cl, 37Cl 79Br, 81Br (3) Under the action of electron flow, the fragment ion peak breaks the bonds of the molecules, forming ions with smaller masses. These breaks proceed according to certain rules and play an important role in determining the structure. They are important for learning. * the focus. 2.4 Trans (1) representation method of fragmentation of organic matter. The arrow indicates the transfer of a pair of electrons. ； , fishhook represents an electron transfer, odd electron cation, radical ion ； , even electron ions ； , free radicals, uncharged. (2) The cracking type is simple cracking such as: α-Cracking, often occurs in compounds with functional groups.: β-crack example: The cleavage small molecules that cause the detachment of neutral small molecules refer to H2O, H2S, CH3CO2H, CH3OH, CO, HCN, etc. The cleavage of detached small molecules is often accompanied by rearrangement. example: Machafferty rearrangement general formula: Similarly, aldehydes, alkenes, amides, nitriles, esters, and aromatic compounds can all occur.: 2.5 Mass spectrum of main organic compounds (1) Alkanes take n-hexane as an example. The m/e of is 86, which is the molecular mass of n-hexane. There are also a series of peaks minus CnH2n+1, such as 71, 57, 43, and 29, among which m/e 57 is the strongest peak and m/e 43 is the second strongest peak. When the isomeric form changes, the mass spectrum signal is clearly different. See the following table for the strongest peak of the compound 57 57 43. It is difficult to form and can only be stably generated in the presence of more σ-p hyperconjugation. (2) Alkenes The mass spectrum of alkenes is similar to that of alkanes, with a series of peaks and the β-fragmentation product peaks mentioned before. For example, (3) the molecular ion peaks of alcohols and alcohols are weak, but there are no peaks for tertiary alcohols. First, various fragmentation occurs, such as ①, the broken line is shown as the broken line, and the alkyl ion peak is obtained. ② ③ Most primary alcohols have m/e 18 peaks and remove H2O. (4) The mass spectrum peaks of ethers are also weak: α-cleavage β-cleavage (5) Aldehyde has the same obvious M peak as usual. When α-fracture breaks, the large group is lost first. Aldehydes often have characteristic peaks m/e 28 and M-1 peak (6) The fragmentation mode of carboxylic acids and their derivatives is: (7) Aromatic hydrocarbons are characterized by strong M peaks. Alkyl-substituted benzene most commonly undergoes β-fragmentation, resulting in a base peak of 91. There are also α-fragments: 3. Mass spectrometer instrument A mass spectrometer usually consists of six parts: Vacuum system, sampling system, ion source, mass analyzer, ion detector and computer automatic control and data processing system. Now, taking a sector-shaped magnetic field single-focus mass spectrometer as an example (Figure 2-1), we will discuss the working principles of each main component. (1) In high vacuum system mass spectrometry analysis, in order to reduce the background and reduce collisions between ions or between ions and molecules, the ion source, mass analyzer and detector must be in a high vacuum state. The vacuum degree of the ion source is 10-4~10-5Pa, and the mass analyzer should maintain 10-6Pa, which requires a very stable vacuum degree. Generally, a mechanical pump or molecular pump is used to pre-evacuate, and then a high-efficiency diffusion pump is used to pump to high vacuum. (2) Sampling system There are various mass spectrometry sampling systems, generally there are three methods:: 1. This injection method is used for indirect injection of general gas or volatile liquid samples. The sample enters the sample container, the temperature is adjusted to evaporate the sample, and the sample vapor diffuses into the ion source through the leak hole by relying on the pressure difference. 2. For direct injection of high boiling point test liquids and solid samples, a probe or direct sample injector can be used to send them into the ion source, and the temperature can be adjusted to vaporize the sample. 3． In chromatography injection chromatography-mass spectrometry instruments, the effluent components after chromatographic separation are directly introduced into the ion source through the interface component. (3) Ion source The function of the ion source is to ionize the sample molecules or atoms, and also has the function of focusing and collimating, so that the ions can be converged into an ion beam with a certain geometric shape and energy. The structure and performance of the ion source have a great influence on the sensitivity and resolution of the mass spectrometer. Commonly used ion sources include electron bombardment ion source, chemical ionization source, high-frequency spark ion source, ICP ion source, etc. The first two are mainly used for organic matter analysis, and the latter two are used for inorganic matter analysis. Currently, the most commonly used ion source is the electron bombardment ion source (Figure 3-2). Figure 3-2 Electron bombardment ion source (4) Mass analyzer The function of the mass analyzer is to separate and focus the ions generated by the ion source according to their m/z size. There are many types of mass analyzers, the common ones include single focusing mass analyzers, dual focusing mass analyzers, quadrupole mass filters, etc. (1) Single-focus mass analyzer The single-focus mass analyzer is shown in Figure 7-1. Its main component is a circular pipe with a certain radius, and a sector magnet is installed in the vertical direction to generate a uniform and stable magnetic field. The ion beam injected from the ion source changes from linear motion to arcuate motion under the action of the magnetic field. Ions with different m/z have different motion curve radii R and are separated by the mass analyzer. Since the positions of the exit slit and the ion detector are fixed, that is, the curve radius R of the arc-shaped motion of the ions is fixed, it is generally used to continuously change the acceleration voltage or magnetic field intensity so that ions with different m/z pass through the exit slit in sequence and reach the ion detector in an arc-shaped motion with a radius of R. From formula (3-3), if the accelerating voltage U is fixed and the magnetic field intensity B is continuously changed, which is called magnetic field scanning, then ； If the magnetic field intensity B is fixed and the accelerating voltage U is continuously changed, it is called electric field scanning. Regardless of magnetic field scanning or electric field scanning, ions with the same m/z can be converged into an ion beam, that is, directional focusing. Since the resolution of the instrument is improved by increasing the accelerating voltage, it is advisable to use the highest possible accelerating voltage. When U is taken as a fixed value, the m/z and relative intensity of the ions are sequentially recorded through magnetic field scanning to obtain the mass spectrum. The single-focus mass analyzer has a simple structure and is easy to operate, but has low resolution. (2) Dual-focusing mass analyzer In a single-focusing mass analyzer, the ions generated by the ion source have different initial energies when being accelerated, that is, different velocities. Even if the ions have the same mass-to-charge ratio, they cannot all be focused on the detector in the end, resulting in low instrument resolution. In order to improve the resolution, a dual-focus mass analyzer is usually used, that is, an electrostatic analyzer is added before the magnetic analyzer, as shown in Figure 7-3. The ions are affected by the electrostatic analyzer and change into circular motion. When the electric field force experienced by the ions is balanced with the centrifugal force of the ion motion, the radius R of the deflection of the ion motion has the following relationship with its mass-to-charge ratio m/z, motion speed v and the electric field strength E of the electrostatic field.: (3-5) It can be seen from equation (3-5) that when the electric field intensity is constant, R depends on the speed or energy of the ions. Therefore, the electrostatic analyzer separates and focuses ions with the same mass but different velocities, that is, it has the function of velocity separation and focusing. Then, it enters the magnetic analyzer through the slit, and then focuses in the m/z direction. This type of analyzer that achieves dual focusing of speed and direction at the same time is called a dual focusing analyzer. A mass spectrometer with a dual-focusing mass analyzer is called a dual-focusing mass spectrometer. Figure 3-3 Dual focusing mass analyzer (3) Quadrupole mass filter The quadrupole mass filter is composed of four parallel cylindrical metal poles. The opposite poles are connected diagonally to form two sets of electrodes. As shown in Figure 7-4, a DC voltage Ude and a radio frequency AC voltage Urf of equal value and opposite direction are applied between the two electrodes. The space enclosed by the four poles generates a hyperbolic electric field. Accelerated ions incident from the ion source pass through the hyperbolic electric field of the quadrupole and are affected by the electric field. Only the selected m/z ions stably pass through the quadrupole mass filter at a limited frequency. Other ions hit the pole and are sucked out and cannot pass through the quadrupole mass filter, thus achieving the role of "filter". In fact, under certain conditions, the detected ion (m/z) has a linear relationship with voltage. Therefore, changing the DC and RF AC voltage can achieve the purpose of mass scanning. This is the working principle of the four-pole mass filter. Because the quadrupole mass filter has a compact structure and fast scanning speed, it is suitable for chromatography-mass spectrometry instruments. Figure 3-4 Quadrupole mass filter (5) Ion detector and recording system The commonly used ion detector is an electrostatic electron multiplier, and its working principle is shown in Figure 3-5. Electron multipliers generally consist of a conversion pole, 10 to 20 dynodes and a collector. Ions of a certain energy bombard the cathode, causing electron emission. Under the action of the electric field, the electrons bombard the next-level electrode in turn and are amplified. The amplification factor of the electron multiplier is generally 105 to 108. The passage time of electrons in the electron multiplier is very short, and the electron multiplier can be used to achieve highly sensitive and rapid measurement. However, the electron multiplier has a mass discrimination effect, and the gain will gradually decrease as the use time increases. Tunneling electron multipliers are often used in modern mass spectrometers. Its working principle is similar to that of electron multipliers. Because of its small size, multiple tunneling electron multipliers can be connected in series to detect multiple ions with different m/z at the same time. * * Improve analysis efficiency. The current detected by the ion detector is amplified by an amplifier and quickly recorded on photosensitive recording paper with a recorder, or the results are processed by a computer. Figure 3-5 Electrostatic electron multiplier 5. Data processing and application The detector is usually a photomultiplier or an electron multiplier. The collected signal is amplified and converted into a digital signal, and the mass spectrum is obtained after computer processing. The number of mass spectrum ions is expressed by abundance, that is, the number of ions with a certain mass-to-charge ratio. Since the "quantity" of a specific ion cannot be measured, relative abundance is generally used to express its intensity, that is, the strongest peak is called the base peak, and the abundance of other ions is expressed as a percentage relative to the base peak. Within the mass range measured by the mass spectrometer, the mass spectrum consists of the mass-to-charge ratio of the ions and their relative abundance. In LC/MS and GC/MS, the chromatographic total ion chromatogram is commonly composed of the chromatographic retention time of each analyte and the relative intensity of its ions obtained from mass spectrometry. A fixed mass-to-charge ratio can also be determined, and selected ion monitoring (SIM) can be performed on the entire chromatographic effluent to obtain a selected ion chromatogram. The ability of a mass spectrometer to separate ions is called resolution, which is usually defined as the ratio of the average mass of two peaks to their mass difference when the valley height of the two peaks is 10% of the peak height for two adjacent peaks with the same height. For low, medium and high-resolution mass spectrometry, it refers to the resolution of 100 to 2000, 2000 to 10000 and above 10000 respectively. The main applications of mass spectrometry in the pharmaceutical field are qualitative identification, quantitative analysis and structural analysis of drugs. If a neutral molecule loses or gains an electron, the mass-to-charge ratio of the molecular ion is the same as the molecule's mass number. High-resolution mass spectrometry can be used to obtain the accurate mass of an ion and then calculate the molecular formula of the compound, or peak matching with a reference can confirm the molecular weight and formula. Various chemical bonds of molecular ions are broken to form fragment ions, from which the fragmentation method can be inferred and corresponding structural information can be obtained. Mass spectrometry is used for quantitative analysis and has high selectivity, precision and accuracy. Compounds are separated and purified by direct injection or using gas chromatography and liquid chromatography before being introduced into the mass spectrometer. Mass spectrometry quantitative analysis uses external standard method or internal standard method, the latter is more accurate than the former. Internal standards in quantitative analysis can use similar structural substances or isotopic substances. The former is low in cost, but its precision and accuracy are higher by using isotopic substances. When using isotope substances as internal standards, it is required that the isotope substances are not lost during the injection, separation and ionization processes. When using FAB mass spectrometry and LC/MS (thermal spray and electrospray) for quantitative analysis, stable isotope internal standards are generally required. The relative abundances of analytes and internal standard ions are determined using selected ion monitoring (monitoring only specific ions of the analyte and internal standard). Compared with full-range scanning, selected ion monitoring increases selectivity and sensitivity due to the long ion current integration time. The chromatographic peak area or peak height ratio of the analyte and the internal standard is used to derive a calibration curve, and then the chromatographic peak area or its amount of the analyte in the sample is calculated. To analyze the mass spectrum of an unknown sample, roughly follow the following procedure. (1) Analyze the molecular ion region (1) Mark the mass-to-charge ratio of each peak, paying special attention to the peaks in the high mass-to-charge ratio region. (2) Identify molecular ion peaks. First, assume a molecular ion peak in the high mass-to-charge ratio region, determine whether the relationship between the assumed molecular ion peak and adjacent fragment ion peaks is reasonable, and then determine whether it complies with the nitrogen law. If both are consistent, it can be considered as a molecular ion peak. (3) Analyze the relative intensity ratio of isotope peak clusters and the Dm value between peaks to determine whether the compound contains elements such as C1, Br, S, Si, and elements without isotopes such as F, P, and I. (4) Derive the molecular formula and calculate the degree of unsaturation. The precise molecular weight is determined by a high-resolution mass spectrometer or the molecular formula is calculated from the relative intensities of clusters of isotope peaks. If both are difficult to achieve, it can be deduced from the missing fragments of the molecular ion peak and the main fragment ions, or combined with other methods. (5) Understand the information of molecular structure from the relative intensity of molecular ion peaks. The relative intensity of the molecular ion peak is determined by the structure of the molecule. The greater the structural stability, the greater the relative intensity. For a compound with a molecular weight of about 200, if the molecular ion peak is a base peak or a strong bee, there are fewer fragment ions in the spectrum, indicating that the compound is a highly stable molecule and may be an aromatic hydrocarbon or a condensed ring compound. For example: The naphthalene molecular ion peak m/z 128 is the base peak, and the anthraquinone molecular ion peak m/z 208 is also the base peak. The molecular ion peak is weak or does not appear, and the compound may be multi-branched hydrocarbons, alcohols, acids, etc. (2) Analyze fragment ions (1) Understand possible structural information from characteristic ion peaks and missing neutral fragments. If a series of CnH2n+1 peaks appear in the mass spectrum, the compound may contain long-chain alkyl groups. If weak fragment ions such as m/z 77, 66, 65, 51, 40, and 39 appear or partially appear, it indicates that the compound contains a phenyl group. If m/z 91 or 105 is a base peak or a strong peak, it indicates that the compound contains benzyl or benzoyl groups. If the base peak or strong peak in the mass spectrum appears in the middle of the mass-to-charge ratio, and there are few other fragment ion peaks, the compound may have a stable structure of two parts, connected by weak bonds that are easily broken. (2) Comprehensively analyze all the information obtained above, combine the molecular formula and degree of unsaturation, and propose the possible structure of the compound. (3) Analyze the fragmentation mechanism of the deduced possible structure to see if it is consistent with the mass spectrum, determine its structure, and further interpret the mass spectrum, or compare it with the standard spectrum, or cooperate with other spectra (1H NMR, 13C NMR, IR) to confirm the structure. Section 4 Organic Mass Spectrometry and Related Knowledge Mass spectrometry has high sensitivity in the structural identification of organic compounds and can give the molecular weight and molecular formula of the compound. 3 W' }) }/ Mass spectrum is a spectrum in which compound molecules are "bombarded" by electron flow or acted by strong electric fields and other methods under vacuum conditions, and are ionized into ions. At the same time, certain chemical bonds are regularly broken, generating positively charged ions with different masses. These ions are collected and recorded according to their mass-to-charge ratio, m/z (the ratio of the ion mass m to the number of charges z it carries). The mass spectrum records the ions of various mass-to-charge ratios collected after ionization and their relative abundance (or intensity). 4.1 Mass spectrometer The mass spectrometer mainly consists of a high vacuum system, a sampling system, an ion source, an accelerating electric field, a mass analyzer, a detection and recording system. High vacuum system: The pressures of the ion source and mass analyzer are usually (10-4-10-5 and 10-5-10-6Pa) respectively. Sampling system: Under vacuum conditions, a small amount of solid and liquid samples with higher boiling points can be sent into the ion source through the injection push rod and heated and vaporized there. Low-boiling point samples enter the ion source after being vaporized in the gas reservoir. Gas samples can enter the ion source through the gas reservoir. Ion source: the ionization place of sample molecules. 9 D+ {' B. I * l-B * I+ s1 R8 D7 t Mass analyzer: Various ions are separated and focused in the mass analyzer according to their mass-to-charge ratio (m/z)." K9 ^1 b' B! |6 P detection and recording system: The ion beams separated by the mass balancer pass through the exit slits successively according to the mass-to-charge ratio and arrive at the collection pole. Their signals are amplified and recorded on photosensitive paper with a recorder or sent to the data processing system, which is processed by the computer to obtain various processing results. # j$ \% m9 k7 F * Y: K Resolution (R) is an important indicator of mass spectrometer performance. High-resolution mass spectrometers can measure the precise mass of ions. ) }" B1 y6 N) C9 j; C 4.2 Ionization methods There are many ionization methods. The main ones used in organic mass spectrometers are electron bombardment, chemical ionization, field ionization and field desorption, fast atom bombardment, etc. Electron Impact (EI) (`' U1 E# {- _4 |4 F, z Use an electron beam with an energy of about 70eV to interact with the vaporized sample molecules to ionize the valence electrons or non-bonding electrons (such as the lone pair of O and N) in the molecule (M), and lose one electron to generate a positively charged molecular ion (M + e M+. + 2e). Further fragmentation generates positively charged fragment ions with different mass-to-charge ratios. When some compounds are ionized with EI, the intensity of the molecular ion peak is weak or even does not appear, but more fragment ions are produced. 4.3 The mass analyzer generates positively charged ions with mass m and charge z in the ionization chamber. After being accelerated by the accelerating voltage V, the speed of the ion is, and its kinetic energy is: m 2/2 = zV. The magnetic field force Bz on the ion is (pointing along the radius to the center of the circle) is equal in size and opposite to the centrifugal force m 2/R of ion motion: m 2/R =Bz m/z = B2R2/2V 8 C/ W/ l& @. `8 T If the V value remains unchanged and the magnetic field strength B changes continuously from small to large, then the m of the ions received through the collection slit /z also changes from small to large, which is called magnetic field scanning. Usually the highest possible accelerating voltage is used to improve the resolution and sensitivity of the instrument. The magnetic field scanning is used to receive and record the intensities of ions of different m/z in sequence to obtain a mass spectrum. In the mass spectrum, the ordinate in the figure is the relative intensity of the ion, and the abscissa is the mass-to-charge ratio of the ion. 5 w+ C. " L) e5 d, O1 e) x8 r; b3 L3 p" L! z8 @1 ~0 ~ 4.4 Mass spectrometry terminology Base peak: the peak with the largest ion intensity in the mass spectrum, its relative intensity or relative abundance is specified as 100. Mass-to-charge ratio: the ratio of the mass of the ion to the number of charges it carries, expressed in m/z. m is the sum of the number of protons and the number of neutrons in the nuclei of the isotopes of the elements that make up the ion, such as H1, C12,13, N14,15, O16,17,18, Cl, 35, Accurate mass such as 37: The mass of the ion in the low-resolution mass spectrum is an integer, and the high-resolution mass spectrum gives the accurate mass of the molecular ion or fragment ion, and its significant digits depend on the resolution of the mass spectrometer. The calculation of the accurate mass of the molecular ion or fragment ion is based on the accurate atomic weight. The accurate mass and abundance of the natural isotopes of some elements are shown in P188: Table 5.1. The precise molecular weights of CO, N2, and C2H4 calculated from the data in the table are 27.9949, 28.0062, and 28.0313, so as long as their precise (percentile) molecular weights are measured, these molecular ions can be distinguished. 2. Ions in the mass spectrum 0 ~3 ~9 }. w1 B3 Y ( n% s/ k4 k! p Molecular ion: A positively charged ion generated by the loss of an electron from a sample molecule, recorded as M+. Molecular ion is the origin of all ions in the mass spectrum, and its corresponding peak in the mass spectrum is the molecular ion peak. Fragment ions: In a broad sense, fragment ions are all ions produced by the fragmentation of molecular ions. 9 |8 V: H7 T0 Q5 Z Rearrangement ion: An ion produced by a rearrangement reaction, whose structure is not the same as that of the original molecule. In the rearrangement reaction, the breaking and formation of chemical bonds occur at the same time, and neutral molecules or fragments are lost. ( Y * B2 | Z; ~) _- D6 c6 E) I Parent ion and product ion: Any ion (molecular ion or fragment ion) is further fragmented to form an ion with a smaller mass-charge ratio. The former is called the parent ion (or precursor ion) of the latter, and the latter is called the product ion of the former. Odd-electron ions and even-electron ions (represented by OE+. and EE+ respectively): ions with unpaired electrons are odd-electron ions, such as M+., A+., B+...; ions without unpaired electrons are even-electron ions, such as D+, C+, E+...; molecular ions are odd-electron ions. In mass spectrometry analysis, odd-electron ions are more important. $ M2 `$ M6 K( t% V6 u/ M Multi-charged ions: ions formed by losing more than one electron from a molecule are called multi-charged ions. In the mass spectrum, doubly-charged ions appear at 1/2 the mass of singly-charged ions. , O0 L6 s3 N6 f( C Quasi-molecular ions: Using the CI ionization method, ions that are 1 mass unit more (or less) than the molecular weight are often obtained, called quasi-molecular ions, such as (MH)+, (M - H)+. M + 1 appears in the mass spectrum of ether compounds. The peak is (MH)+. Metastable ions: The ions generated and recorded from the ion source outlet before reaching the detector are called metastable ions. The time it takes for ions to reach the detector from the ion source is about 10-5 seconds (varies with the instrument and experimental conditions). Stable ions with a lifespan greater than 10-5 seconds are enough to reach the detector, while ions with a lifespan less than 10-5 seconds may fragment (M1+ M2+ + neutral fragments). The M2+ generated by fragmentation before the mass analyzer has a different deflection in the magnetic analyzer because its kinetic energy is smaller than the M2+ generated by the ion source, with low intensity, relative to the apparent mass m * (spanning 2-3 mass units) is recorded, and its m/z is generally not an integer.m * The relationship between m1 and m2 (the masses of M1 and M2 ions respectively) is: m * = m 22/ m1 In mass spectrometry analysis, m can be used * To determine the "mother-child" relationship between m1 and m2. For example: In the mass spectrum of phenethylidene, there are m/z134, 105, 77, 56.47 plasma bees, and 56.47 is a metastable ion peak. From 56.47=772/105, it can be seen that the m/z77 ion is generated by the fragmentation of the m/z105 ion and the loss of CO. The MS spectrum of phenethylidene: I- S0 b( f3 r V5 }4 r! h 5.2 Molecular ions and molecular formulas 5.2.1 Identification of molecular ion peaks) _0 u% { * A: P2 l7 { The molecular ion peak must be the ion peak with the highest mass-to-charge ratio (except for isotope ions and quasi-molecular ion peaks); it must be an odd-electron ion peak; the molecular ion can reasonably lose neutral fragments (free radicals or neutral molecules), and has a reasonable relationship with its adjacent fragment ions with smaller mass-to-charge ratios. See P190 for the reasonably lost neutral fragments and possible structural sources of the compound by M+: Table 5.2. The data in the table is helpful for identifying the molecular ion peak and inferring the type of the compound. Nitrogen law: Most of the elements that make up organic compounds, in terms of their naturally abundant isotopes, even-numbered elements have even-numbered valences (such as 12C is 4-valent, 16O is 2-valent, 32S is 2-, 4-, or 6-valent, 28Si is 4-valent, etc.). Odd-number qualities A large amount of elements has an odd valence (such as 1H, 35C1, 79Br is 1 valence, 31P is 3 valence, 5 valence, etc.). Only N is abnormal, the mass number is an even number (14), and the valence is an odd number (3 valence, 5 valence). From this, the following law is derived, which is called the nitrogen law. # o T3 y3 ^6 B$ Q. y1 O "In organic compounds, the molecular weight of compounds that do not contain nitrogen or contain an even number of nitrogens must be an even number (the mass-to-charge ratio of the molecular ion is an even number), and the molecular weight of the compound that contains an odd number of nitrogens must be an odd number. Conversely, the molecular ion peak with an even number of mass-to-charge ratios does not contain nitrogen or contains an even number of nitrogens." 5.2.2 Relative intensity of the molecular ion peak 3 d# | z' { * u# + t: t: p The relative intensity of the molecular ion peak can be summarized as follows:' X2 {0 c6 b5 b4 v9 R' ] 8 g (1) Aromatic compounds > Conjugated polyenes > Alicyclic compounds > Low-molecular linear alkanes > Certain sulfur-containing compounds. (2) The molecular ion peaks of linear compounds such as esters, acids, aldehydes, amides, halides and other compounds are usually visible. (3) Aliphatic alcohols, amines, nitrites, nitrates, nitro compounds, nitriles and multi-branched compounds are easy to crack, and the molecular ion peaks are usually weak or do not appear. 5.2.3 Derivation of molecular formula 3 B) m5 w2 l8 L. l' x * y6 J 1. Low-resolution mass spectrometry 4 p3 @7 O# S+ _: t) N; s+ j (1) Isotope peak clusters and their relative abundance. In mass spectra, molecular ion or fragment ion peaks are often accompanied by peaks that are 1, 2... mass units larger than their mass-to-charge ratio. Relative to M+., it can be recorded as (M + 1), (M + 2) Peaks, these peaks are called isotope peak clusters. The relative intensity of an isotope peak cluster is determined by the number of isotope atoms and its natural abundance. Table 5.3 gives the calculated value of common isotopes relative to the light isotope (A) with the largest natural abundance when the abundance is 100. 6?! Y' ] ) g, q1 h. p( { Relative abundance of natural isotopes of common elements CHNOF Si PS Cl Br IA 100 100 100 100 100 100 100 100 100 100 1000 G! _0m% ] $ n6 A9 B A+1 1.1 0.016 0.37 0.04 - 5.1 - 0.8 - - - A+2 - - - 0.2 - 3.4 - 4.4 32.5 98.0 -0 b: K) }0 o! z- B' k9 q' Z * C A compound composed of C, H, N, and O elements. The general molecular formula is: CxHyNzOw (x, y, z, w are the number of atoms of C, H, N, and O respectively). The relative intensity of its isotope peak cluster can be calculated by the following formula: 9 J& L& V0 m1 S RI(M+1)/RI(M) 100 = 1.1x + 0.37zl/ l0 S9 m+ u6 M$ ~, l RI(M+2)/RI(M) 100 = (1.1x)2/200 + 0.2w If the compound contains chlorine or bromine, the relative abundance of its isotope peak cluster is calculated according to the coefficient of the expansion of (a+b)n. If the two coexist, then it is calculated as (a + b)m(c + d) Calculation of coefficients of the expansion of n. m, n are the number of chlorine and bromine atoms in the molecule, a, b and c, d are numerically 3,1 and 1,1 of the isotope relative abundance ratio respectively. If the molecule contains two chlorine atoms, the relative abundance ratio of the isotope peak cluster M M+2) M+4)=9:6: 1 m=2 between two adjacent peaks." s! r0 u * r0 Q: N6 Q! MS Figure 6 of X/ t (E)-1,2-dichloroethylene ] ! @ G: Q. E1 Z3 F8 z w9 N5 R (2) Use the relative abundance of isotope peak clusters to deduce the molecular formula of a compound. Before deriving the molecular formula of a compound, it is necessary to first identify the molecular ions and the isotope peak clusters they constitute. Read out the relative intensities of isotope peaks such as M+., M+1, M+2 from a table or spectrum, and then use (5.7) and (5.8) formulas Calculate the number of C, N, and O, and calculate the number of hydrogen atoms by combining the mass-to-charge ratio of M+. and the number of C, N, and O. The calculated number of carbon atoms is often an approximate value, usually based on the m/z of M+. and the number of atoms of other elements. The smaller the relative intensity of M+., the greater the calculation error. Example 1_The mass spectrum data and figures of compound A are shown in Table 5.4 and Figure 5.6 on page 193. Its molecular formula is deduced. - ~ V: d6 s: p! Assume z=1, then x=(6.1 – 0. 37)/1.1 5. If the molecular formula is C5N, its formula weight is greater than 73, which is obviously unreasonable. D1 H Example 2 The mass spectrum of compound B is shown in P194: Figure 5.7 and Table 5.5, and its molecular formula is deduced. 4 j& N( s6 x' ~, i * _ Solution: Let 97 be the molecular ion peak. Since the relative intensity ratio of 97 and 98 is about 2:1, it neither conforms to the isotope relative abundance ratio of C, H, N, O, S compounds nor the relative abundance of the isotope peak cluster composed of different C1 and Br atoms in Figure 5.5. Therefore, 97 may not be the molecular ion peak. Let 98 be the molecular ion peak. # q; m% u- N7 ] 7 |0 om/z 98(56) M+., 99(7.6) M+1,100 (2.4) M+2, then RI(M+1)/RI(M)×100=13.6, RI(M+2)/RI(M)×100=4.35 |% |( Q$ n" k. F. N Judging from the relative intensity of (M+2) 4.3, the molecule of compound B contains a sulfur atom. Example 3 The mass spectrum of compound C is shown on page 195: Figure 5.8 and Table 5.6. The molecular formula is deduced / d" f" V3 i3 B( B) s$ }6 ] 3 e/ G Solution: The relative intensity ratios of 164 and 166, and 135 and 137 are all approximately 1:1, so it is believed that 164 is the molecular ion peak of compound C. It contains one bromine atom, no nitrogen or an even number of nitrogens. If the elemental composition of fragment ion 85 can be deduced, its molecular formula can be known., f/ 4 B * M" J 5.3 Fragmentation reactions in organic mass spectrometry 5.3.1 Experimental methods for studying fragmentation reactions in organic mass spectrometry The fragmentation reaction in organic mass spectrometry refers to the reaction in which ions (including molecular ions and fragment ions) are further fragmented to generate fragment ions with smaller mass-charge ratios. The commonly used methods for studying fragmentation reactions in organic mass spectrometry are the metastable ion method and isotope labeling method. - M; B& b, q% C. v( T/ B Metastable ion method: if m * =m22/m1, then M1 is the parent ion of M2, which means that M2 is produced by the fragmentation of M1. Analyzing these ion pairs is helpful for understanding the mass spectrometry fragmentation reaction process, the possible connection sequence of the structural units of the ions, etc. For example: In the mass spectrum of ethyl hexanoate, peaks such as 144 (M+.), 115, 99, 88 (100), 71, and 43 appear, and there is a metastable ion peak at 53.7. Is 88 produced by M+., or is it produced by the cleavage of 115 and is produced by m * It can be determined. Since 53.7=882/144, the 88 base peak is an odd electron ion generated by the direct loss of 56 mass units of the molecular ion. 6 g7 C9 S/ N * _- X7 The cleavage reaction caused by single electron transfer is called homolysis, and the cleavage reaction caused by double electron transfer is called heterolysis. (1) The cleavage reaction initiated by the free radical position 9 X. p+ _$ W6 n The driving force for the cleavage reaction at the free radical position comes from the strong tendency of free radicals to pair electrons, which can easily cause splitting at the free radical position, accompanied by the breakage of original chemical bonds and the formation of new chemical bonds. / D) }7 R, q! b% _- z% z When a molecule is ionized, the free radical or charge position preferentially occurs on the electron with the lowest ionization potential. In organic compounds, the order of ionization potential (I) from small to large is: non-bonding n electrons (unbonded electrons of heteroatoms such as O, N, S, etc.)