Main contents:
1.？ Concepts and types of instrumental analysis technology
2.？ Instrumental analysis methods in metabonomics
A) spectral analysis b) chromatographic analysis
3.？ Analytical instrument combination technology (GC-MS, LC-MS, ...)
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1. Concepts and types of instrumental analysis technology
1. 1 What is instrumental analysis technology?
Modern instrumental analysis technology:? A method of obtaining information on chemical composition, composition content and chemical structure of a substance by measuring the parameters of physical or physicochemical properties of the substance and their changes with relatively complex or special instruments and equipment.
1.2? Classification of instrumental analysis techniques
Spectral analysis: ultraviolet-visible spectrum (UV), nuclear magnetic resonance spectrum (NMR), mass spectrometry (MS )…… ... ...
B. chromatographic analysis: liquid chromatography, gas chromatography ...
C. Phase analysis: magnetic separation analysis, specific gravity analysis, X-ray structure analysis ...
Elemental analysis: electron microscope energy spectrum analysis (EDS), inductively coupled plasma mass spectrometry (ICP-ms). ...
2.？ Instrumental analysis methods in metabonomics
First, the spectrum analysis method
Principle: Wave-particle duality of light.
Interaction between light and matter:
? Molecular energy level: molecules are in a specific state and have certain energy; ?
Molecular energy level transition: the transition of molecular absorption energy from low energy level to high energy level;
? Molecular spectrum: the absorption energy of molecules jumps, and the wavelength of absorbed photons and the intensity of absorbed signals are molecular absorption spectrum, otherwise it is analytical emission spectrum.
Spectral classification: molecular translation, molecular rotation, chemical bond vibration, electronic energy level transition, electronic spin, isotope nuclear spin.
1? Nuclear magnetic resonance spectroscopy (NMR)
Nuclear Magnetic Resonance (NMR): A spectroscopic method to study the nuclear energy levels and transitions in a magnetic field. -Molecular structure and intermolecular relationship
Basic principles:
? Spin motion of nuclei: only nuclei with non-zero spin can interact with external magnetic field and have the conditions of nuclear magnetic resonance;
? Spatial quantization of momentum moment and magnetic moment of nucleus (the behavior of nucleus in external magnetic field): Under the excitation of strong magnetic field, the energy of some nuclei with certain magnetism can be divided into two or more energy levels. ?
? Generation of * * * vibration in nuclear magnetic resonance: At this time, when an energy is added to make it exactly equal to the difference between two adjacent energy levels after splitting, the nucleus may absorb energy (called * * * vibration absorption).
From low-energy state to high-energy state, the absorbed energy is equivalent to electromagnetic wave (belonging to radio wave category, radio frequency) with frequency range of 0. 1~ 100MHz. So nuclear magnetic resonance is to study the absorption of radio frequency energy by magnetic nuclei.
1. 1? Relaxation process-spectral line width
Longitudinal relaxation (spin-lattice relaxation): the process of energy exchange between high-energy nuclei and the surrounding environment (solid lattice, solvent molecules in liquid, etc.). ).
Transverse relaxation (spin-spin relaxation): the process of transferring energy from a high-energy nucleus to an adjacent low-energy nucleus.
The influencing factors of spectral line width: relaxation time, uniformity of instrument magnetic field and paramagnetic materials.
1.2? Chemical shift δ
Chemical shift: the nucleus is shielded by different electrons in different chemical environments, and its * * * vibration frequency is different, and there will be * * * vibration absorption peaks in different frequency bands of NMR spectrum or different magnetic fields.
The expression method of chemical shift: relative shift, which takes the * * * vibration peak (TMS) of a substance as the origin, and sorts the relative distance between the * * * vibration peak and the origin of protons in the sample. -Unification of data.
1.3? Nuclear magnetic vibration instrument
Musical instrument classification:?
? Radio frequency: 60 MHz, 100 MHz, 600 MHz ...
? Type of magnet: permanent magnet, electromagnet, superconducting magnet ...
? Radio frequency source: continuous wave, pulse Fourier transform ...
Instrument structure:
2. Mass spectrometry analysis
Mass spectrometry is a method to detect moving ions (charged atoms, molecules or molecular fragments, molecular ions, isotope ions, fragment ions, rearranged ions, multi-charged ions, metastable ions, negative ions and ions generated by ion-molecule interaction) by using electric and magnetic fields.
Application: chemical analysis, qualitative and quantitative analysis of compound structure ...
2. 1? Reasons for adopting mass spectrometry
By measuring the precise mass of ions, the compound composition of ions can be determined. Because the accurate mass of nuclides is decimal, no two nuclides will have the same mass, and no one nuclide has an integral multiple of the other.
2.2? fundamental principle
The sample enters the mass spectrometer. In the ion source of mass spectrometer, compounds are bombarded by electrons and ionized into molecular ions and fragment ions. These ions are separated in the order of mass-to-charge ratio in the mass analyzer, and the mass spectra of the compounds can be obtained after being detected by the electron multiplier.
2.3? mass chromatogram
Abscissa: mass-to-charge ratio?
Ordinate: ionic strength?
Absolute Intensity of Ions: Sample Size and Instrument Sensitivity?
Relative strength of ions: correlation of sample molecular structure?
The mass spectra of the same sample are the same under certain ionization conditions. This is the basis of organic qualitative analysis by mass spectrometry.
2.4? Main structure of mass spectrometer
Second, the chromatographic analysis method
A separation analysis method; ?
Features: there are two phases-stationary phase and mobile phase, and the two phases move relatively.
Separation principle: when the mixture carried in the mobile phase passes through the stationary phase, it will interact with the stationary phase. Due to the different nature and structure of each component in the mobile phase, the degree of interaction with the stationary phase is also different. Therefore, under the same driving force, the retention time of different components in the stationary phase is longer or shorter, so they are left out of the stationary phase in different order.
Factors affecting the separation of 1. 1 components
Basic theory of 1.2 chromatography
A. Chromatographic distribution equilibrium
Chromatographic distribution equilibrium: the process of adsorption, desorption or dissolution and volatilization of components between stationary phase and mobile phase-distribution process. At a certain temperature, when the distribution between two phases reaches equilibrium, the concentration ratio of each component is called the distribution coefficient; The mass ratio is called the distribution ratio.
Partition coefficient: the essence of chromatographic separation.
B. Tray theory
Tray theory derives the equation of outflow curve with the help of the concept of tray theory in chemical industry.
Comparing a chromatographic column to a distillation column, imagine that a continuous chromatographic column is composed of many small segments. In each small segment, part of the space is occupied by the stationary phase and the other part is filled by the mobile phase. The components enter the chromatographic column with the mobile phase and are distributed between the two phases. It is assumed that each component can quickly reach the distribution equilibrium in the two phases in each short section, which is called theoretical plate and the length of theoretical plate is called theoretical plate height H. After many times of distribution equilibrium, the component with small distribution coefficient leaves the distillation column first, and the component with large distribution coefficient leaves the distillation column. Because the number of trays in the chromatographic column is quite large, even if the component distribution coefficient is only slightly different, good separation effect can still be obtained.
A) Relationship between component concentration and time: outflow curve equation
Explained problems: chromatographic peak type and maximum concentration position.
B) The theoretical plate height of chromatographic column is the variance of chromatographic peak per unit column length.
The lower the theoretical tray height, the higher the number of trays per unit length, and the better the separation effect.
The factors that determine the theoretical tray height are: the material of stationary phase, the uniformity of chromatographic column, the physical and chemical properties of mobile phase and the flow rate of mobile phase.
C) theoretical plate number &; Effective plate number
Effective plate number: depends on the type and properties of stationary phase (particle size, particle size distribution, etc.). ), packing conditions, column length, type and flow rate of mobile phase, and properties of substances used to determine column efficiency.
D) characteristics and shortcomings of tray theory
The theoretical equation of tray accords with the chromatographic outflow curve, which provides guidance for chromatographic theory.
When the column efficiency index is measured by the number of (effective) trays and the height of (effective) trays, specific substances should be specified (different substances have different distribution coefficients on the same chromatographic column). ?
The essence of influencing column efficiency is not clear.
? Column efficiency cannot represent the actual separation effect of separated components; ?
? It is impossible to point out the factors affecting the column efficiency and the ways to improve the column efficiency. ?
Can't explain the influence of carrier gas rate on theoretical plate number?
? It is impossible to explain the experimental results of different column efficiencies of the same chromatographic column at different carrier gas flow rates.
C. rate theory
According to the rate theory, a single component molecule must undergo tens of millions of transfers between the stationary phase and the mobile phase in the chromatographic column. Coupled with factors such as molecular diffusion and movement path, its movement in the column is highly irregular and random, and its speed with the mobile phase in the column is uneven.
A) Van Dymer equation (Van Dymer rate theory equation)
B) eddy current diffusion term
F) Measures to improve chromatographic column efficiency
Selecting a uniform filler with smaller particles;
Under the premise of not increasing the viscosity of the stationary liquid too much, operate at the lowest column temperature as much as possible;
Use the stationary liquid with the lowest actual concentration; ?
Carrier gas with larger molecular weight; ?
Select the best carrier gas flow rate; Cylindrical shape with smaller inner diameter and larger radius of curvature is adopted.
D. degree of separation
Separation degree: describes the actual separation efficiency of substances (tray theory and rate theory are difficult to describe).
Influencing factors of resolution: thermodynamic factors (retention value) and kinetic factors (peak width and column efficiency). ?
What is the degree of separation?
① High column efficiency, large distribution coefficient and complete separation; ?
② The distribution coefficient is not very large, the column efficiency is high, the peak is narrow, and the separation is basically complete; ?
③ Low column efficiency and large distribution coefficient, but poor separation; ?
④ The distribution coefficient is small, the column efficiency is low and the separation effect is poor.
1.3? gas chromatography
Chromatographic analysis method with gas as mobile phase;
It is suitable for the separation and analysis of samples that are easy to evaporate, stable, difficult to decompose and difficult to react, especially for the separation of homologues and isomers.
A) the basic principle of gas chromatography
B) gas chromatograph and its main structure
1.4? High performance liquid chromatography (HPLC)
Chromatography with liquid as mobile phase; ?
It is suitable for separating ion samples with high boiling point and thermal instability. ?
Classical liquid chromatography: separation means; High performance liquid chromatography: separation and analysis.
Basic principle: In the process of chromatography, different components exchange between two phases which are relatively moving and insoluble, the relatively static phase is the stationary phase, and the relatively moving phase is the stationary phase. Different components are separated by continuous mass exchange between two phases by using tiny differences in adsorption, distribution, ion exchange, affinity or molecular size.
Four types: adsorption chromatography, partition chromatography, gel chromatography and ion chromatography.
A) main features and structure of liquid chromatograph
3. Analytical instrument combination technology (gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry ..............................................................................).
3. 1 Overview of analytical instrument combination technology
Analytical instrument combination technology: the technology of combining two or more analytical instruments to obtain a fast and effective analytical tool. ?
The biggest feature of chromatography is that it can separate complex mixtures into single components, but its qualitative and structural abilities are poor.
The structure of pure components can be easily determined by mass spectrometry and other techniques.
The combination of analytical instruments forms a complete analysis of mixed substances.
3.2 Important issues in the combination of analytical instruments
Interface problem, its general requirements are as follows: Effective sample transfer can be carried out; ?
? Sample transfer should have good reproducibility; ?
? It is easy to meet the arbitrary operation modes and conditions of the two instruments; ?
? The sample will not change chemically through the interface; ?
? Does not affect the efficiency of the instrument; ?
? The sample passes through the interface as quickly as possible; ?
? The interface itself is simple, convenient and reliable.
3.3 Gas chromatography-mass spectrometry (GC-MS)
It combines the advantages of gas chromatography and mass spectrometry, with high resolution of gas chromatography and high sensitivity and strong resolution of mass spectrometry.
Suitable for small molecules, volatility, thermal stability and vaporized compounds;
The spectrogram obtained by electron bombardment (EI) can be compared with the standard spectrogram library.
3.4 high performance liquid chromatography-mass spectrometry (HPLC-MS) technology
LC-MS can solve the following problems:
? Will not send compound analysis and determination; ?
? Analysis and determination of polar compounds;
? Analysis and determination of thermally unstable compounds;
? Analysis and determination of high molecular weight compounds (including protein, peptides, polymers, etc.). ); ?
? If there is no commodity, the spectrum library can be compared and queried, and you can only build your own library or analyze the spectrum yourself. ?
Important problems to be solved:?
? Influence of liquid chromatography process on working conditions of mass spectrometry:
? Influence of mass spectrometry ion source temperature on liquid chromatography analysis source.
3.5? Gas chromatography-mass spectrometry. Main structure of liquid chromatography-mass spectrometry instrument