Principle of high performance liquid chromatography Application and principle analysis of high performance liquid chromatography
High performance liquid chromatography (HPLC) is one of the effective methods to separate, analyze and purify organic compounds (including inorganic substances that can be converted into organic compounds through chemical reactions).
About 80% of known organic compounds can be separated and analyzed by high performance liquid chromatography, and this method has mild conditions and no damage to the sample, especially suitable for organic compounds and biological substances with high boiling point, difficult gasification and volatilization and poor thermal stability. HPLC system generally consists of infusion pump, sampler, chromatographic column, detector, data recording and processing device, etc.
Infusion pump, chromatographic column and detector are key components. Some instruments include gradient elution device, on-line degasser, automatic sampler, column or protection, column temperature controller and so on. Modern HPLC instruments also have a microcomputer control system for automatic control and data processing.
The preparative HPLC instrument is also equipped with an automatic fraction collection device. At present, the common HPLC instrument manufacturers in foreign countries are Waters Company, Agilent Company (formerly HP Company) and Shimadzu Company, and there are Shanghai Wufeng Scientific Instrument Co., Ltd., Shanghai Hegong Scientific Instrument Co., Ltd., Dalian Yiyuan Jung Su Company, Beijing Innovation Tongheng Company and Beijing Wen Branch in China.
I. infusion pump 1. Structure and performance of pump Infusion pump is one of the most important components in HPLC system. The performance of the pump directly affects the overall quality and the reliability of the analysis results.
The infusion pump should have the following properties: ① the flow rate is stable, and its RSD should be less than 0.5%, which is related to the accuracy of qualitative and quantitative analysis; (2) The flow range is wide, the analytical type should be continuously adjusted within the range of 0. 1~ 10ml/min, and the preparative type should reach100ml/min; ③ The output pressure is high, which should generally reach 150 ~ 300 kg/cm2: ④ The hydraulic cylinder is small; ⑤ Good sealing performance and corrosion resistance. There are many kinds of pumps, which can be divided into constant pressure pump and constant flow pump according to the nature of infusion.
Constant flow pump can be divided into spiral jet pump, plunger reciprocating pump and reciprocating pump. Constant pressure pump is affected by column yin, and the flow rate is unstable; Screw pump cylinder is too big, these two pumps have been eliminated. At present, the plunger reciprocating pump is the most widely used.
The cylinder volume of plunger reciprocating pump is small, up to 0. 1ml, which is convenient for cleaning and replacing mobile phase, especially suitable for recirculation and gradient elution. Changing the motor speed can easily adjust the flow rate, and the flow rate is not affected by the column pressure; The pump pressure can reach 400 kg /CM2. The main disadvantage of ADW is the large output pulse, which is now overcome by double pump system.
According to the connection mode, double pumps can be divided into parallel type and series type. Generally speaking, the flow reproducibility of parallel pumps is good (RSD is about 0. 1%, and that of series pumps is 0.2~0.3%), but the failure probability is high (because there are more check valves) and the price is expensive. Second, the general HPLC analysis of the sampler The commonly used six-way injection valve (the 7725 and 7725I models of American RHEODYNE company are the most common), and its key components are composed of a circular gasket (rotor) and a fixed base (stator).
High pressure resistance (35~40MPA), accurate sampling, good repeatability (0.5%) and convenient operation. There are two sampling methods for six-way valve: partial filling method and complete filling method.
(1) When injecting samples by partial liquid filling method, the volume of injection should not exceed 50% (at most 75%) of the volume of quantitative loop, and the volume of each injection should be accurate and the same. The accuracy and repeatability of this method depend on the proficiency of the sampler, and it is easy to produce peak broadening caused by sampling.
(2) When the sample is injected by completely filling liquid, the sample volume should be not less than 5~ 10 times or at least 3 times of the volume of the quantitative ring, so as to completely replace the internal phase and mobile phase of the quantitative ring, eliminate the pipe wall effect and ensure the accuracy and repeatability of the sample injection. 3. Column chromatography is a means of separation and analysis, and separation is the core, so the column responsible for separation is the heart of the chromatographic system.
The requirements for chromatographic column are high column efficiency, good selectivity and fast analysis speed. Porous silica gel, silica gel-based binding phase, alumina, organic polymer microspheres (including ion exchange resin), porous carbon, etc. Commercially available for HPLC, it usually has a particle size of 3, 5, 7, 654, 38 00 μ m, etc. The theoretical value of column efficiency can reach 5160000/m. ..
General analysis only needs the column efficiency of 5000 plates; For homologous analysis, as long as 500; For substances that are difficult to separate, columns as high as 20000 can be used, so the column length of about 10 ~ 30cm can generally meet the needs of complex mixture analysis. Column efficiency is affected by factors inside and outside the column. In order to achieve the best efficiency of the tower, there should be no reasonable tower structure (reducing the dead volume outside the packed bed as much as possible) and packing technology except that the dead volume outside the tower should be small.
Even with the best packing technology, the packing situation in the center of the tower and along the pipe wall is always different. The part close to the pipe wall is loose, which is easy to produce gully flow and has a fast flow rate, which affects the manifold of the flushing agent and broadens the spectral band. This is the wall effect. The tube wall area is about 30 times the diameter and thickness of the material inward from the tube wall.
In the general liquid chromatography system, the effect outside the column is far greater than the wall effect on the column efficiency. Fourth, detectors HPLC detectors are divided into two categories: general detectors and special detectors.
1. Universal detector can continuously measure all characteristic changes of chromatographic column effluent, and usually adopts differential measurement method. Such detectors include refractive index detectors, dielectric constant detectors, conductivity detectors, etc. Universal detector has a wide range of applications, but its response to mobile phase is easily affected by temperature changes, mobile phase and composition changes, with large noise and drift and low sensitivity, so gradient elution cannot be used. 2. The special detector is used to measure the change of some characteristics of the separated sample components.
This detector is sensitive to some physical or chemical properties of the components in the sample, which are not available in the mobile phase, or at least will not be displayed under the operating conditions. Such detectors include ultraviolet detectors, fluorescence detectors, radioactive detectors, etc.
What is the working principle of HPLC?
Working principle of high performance liquid chromatograph; The high-pressure pump sends the mobile phase of the liquid storage tank into the chromatographic column through the sampler and then flows out from the outlet of the detector. At this point, the whole system is full of mobile phase. When the sample to be separated enters from the sampler, the mobile phase flowing through the sampler brings it into the chromatographic column for separation. After separation, different components enter the detector in turn, and the recorder records the signals entering the detector to obtain the liquid chromatogram.
High performance liquid chromatography refers to the theory of gas chromatography developed on the basis of classical chromatography. Technically, the mobile phase is transported by high pressure, and the chromatographic column is filled with small particle size filler in a special way, so that the column efficiency is much higher than that of classical liquid chromatography (the number of plates per meter can reach tens of thousands or hundreds of thousands), and a highly sensitive detector is connected behind the column to continuously detect the effluent.
Extended data
High-performance liquid chromatograph is equipped with high-pressure binary pump or low-pressure quaternary pump. The stroke volume of the pump and the volume of the mixer will affect the baseline noise level of chromatography, especially in gradient elution. Generally, the smaller the stroke volume of the pump, the larger the volume of the mixer, the smaller the pulse caused by infusion, the higher the response ability to gradient change and the smoother the baseline.
When using binary pump, it should be noted that when the proportion of a mobile phase in binary mixing is less than 5%, especially when normal phase elution is used for chiral resolution of some pharmaceutical intermediates and final products, it is best to use single pump premixing method. Avoid the peak related to the stroke on the chromatographic baseline, because the pumping accuracy of the pump is relatively poor at low ratio.
Reference source; Sogou encyclopedia-high performance liquid chromatograph
Basic working principle of high performance liquid chromatograph
Basic working principle of high performance liquid chromatograph
The high performance liquid chromatography system consists of several parts, such as reservoir, pump, sampler, chromatographic column, detector and recorder. The mobile phase in the reservoir is pumped into the system by the high-pressure pump, and the sample solution enters the mobile phase through the sampler, and is loaded into the chromatographic column (stationary phase) by the mobile phase. Because the distribution coefficients of each component in the sample solution are different in the two phases, when they move relatively in the two phases, after repeated adsorption-desorption distribution process, the moving speeds of each component are very different, and they are separated into single components and flow out of the column in turn. When passing through the detector, the sample concentration is converted into an electrical signal for transmission.
What is the principle of high performance liquid chromatography?
Principle: The mobile phase in the reservoir is pumped into the system by a high-pressure pump, and the sample solution enters the mobile phase through the sampler, which is loaded into the chromatographic column (stationary phase). Because the distribution coefficients of each component in the sample solution are different in the two phases, when they move relatively in the two phases, the moving speeds of each component are very different after repeated adsorption-desorption distribution processes.
The separated components flow out from the column in turn, and when passing through the detector, the sample concentration is converted into electrical signals and transmitted to the recorder, so that the data can be printed out in the form of atlas for researchers to analyze. Extended data:
High performance liquid chromatography (HPLC) is also called high-pressure liquid chromatography, high-speed liquid chromatography, high-resolution liquid chromatography and modern column chromatography.
① High pressure: the mobile phase is a liquid, which is subject to great resistance when flowing through the chromatographic column. In order to pass through the chromatographic column quickly, high pressure must be applied to the carrier liquid. ② Fast speed: the analysis speed is fast, and the flow rate of carrier liquid is fast, which is much faster than that of classical liquid chromatography. It usually takes 15~30 minutes to analyze a sample, and some samples can even be completed within 5 minutes, generally less than 1 hour.
③ High efficiency: high separation efficiency. Stationary phase and mobile phase can be selected to achieve the best separation effect, which is many times higher than that of industrial distillation column and gas chromatograph.
④ High sensitivity: the ultraviolet detector can reach 0.0 1ng, and the sample volume is in the order of μ L. ⑤ Wide application range: HPLC can analyze more than 70% of organic compounds, especially those with high boiling point, macromolecules, strong polarity and poor thermal stability, showing advantages.
⑥ The chromatographic column can be reused: one chromatographic column can separate different compounds. ⑥ Small sample size and easy recovery: the sample will not be destroyed after passing through the chromatographic column, and a single component can be collected or prepared. In addition, HPLC has many advantages, such as the chromatographic column can be reused, the sample is not destroyed, and it is easy to recover, but it also has some disadvantages. Compared with gas chromatography, high performance liquid chromatography has its own advantages and complements each other.
The disadvantage of high performance liquid chromatography is that it has "extracolumn effect". In any dead space (syringes, column connectors, connecting tubes and detection cells, etc.). From injection to detector, if the flow pattern of mobile phase changes, any diffusion and retention of separated substances will significantly lead to the broadening of chromatographic peaks and the decline of column efficiency.
The sensitivity of HPLC detector is not as good as that of gas chromatography detector. The chromatographic column used in HPLC is very thin (1~6 mm), and the particle size of the stationary phase is also very small (several microns to dozens of microns), so the flow of mobile phase in the column is greatly hindered. Under normal pressure, the flow rate of mobile phase is very slow, the column efficiency is low and time-consuming
In order to achieve rapid and effective separation, great pressure must be applied to the mobile phase to accelerate its flow in the column. Therefore, high-pressure infusion must use high-pressure pump.
High pressure and high speed are one of the characteristics of high performance liquid chromatography. The high-pressure pump used in HPLC should meet the following conditions: a. Constant flow, no pulsation and wide adjustment range (generally1~10 ml/min); B. it can resist solvent corrosion; C. there is a high infusion pressure; For general separation, the pressure of 60 *10.5pa is enough, and for efficient separation,150 ~ 300 *10.5pa is required.
(1) Reciprocating piston pump When the plunger is pushed into the cylinder, the one-way valve at the outlet (upper part) of the pump head is opened, and the one-way valve (lower part) where the mobile phase enters is closed, so a small amount of fluid is output. On the contrary, when the plunger is pulled outward, the one-way valve at the entrance of the mobile phase is opened, while the one-way valve at the exit is closed, and a certain amount of mobile phase is sucked into the cylinder by its liquid reservoir.
The characteristic of this pump is that it is not affected by the slight change of resistance of the rest of the whole chromatographic system, and it continuously provides a constant volume of mobile phase. ⑵ The working principle of pneumatic amplification pump is that low-pressure gas with pressure of p 1 pushes large-area (SA) piston A, and then small-area (SB) piston B outputs liquid with pressure increased to p2.
The multiple of pressure increase depends on the area ratio of the two pistons A and B. If the area ratio of A and B is 50: 1, the gas with pressure of 5 * Pa can get the output liquid with pressure of 250*Pa. This is a constant pressure pump.
References:
Baidu Encyclopedia-HPLC.
What is the working principle of HPLC instrument?
High performance liquid chromatography (HPLC) is a theory that introduces gas chromatography on the basis of classical chromatography. Technically, the mobile phase is changed to high-pressure transportation (the maximum transportation pressure can reach 4.9? 107 pa)； The chromatographic column is filled with small particle size filler in a special way, which makes the column efficiency much higher than that of classical liquid chromatography (the number of plates per meter can reach tens of thousands or hundreds of thousands); At the same time, a highly sensitive detector is connected behind the column, which can continuously detect the effluent.
Features 1. High pressure: liquid chromatography takes liquid as the mobile phase (called carrier liquid), and the liquid flows through the chromatographic column, which is subject to great resistance. In order to pass through the chromatographic column quickly, high pressure must be applied to the carrier liquid. Generally, it can reach 150~350* 105Pa.
2. High speed: the flow rate of mobile phase in the column is much faster than that of classical chromatography, which can generally reach 1~ 10ml/min. The analysis time of high performance liquid chromatography is much less than that of classical liquid chromatography, generally less than1h.
3. High efficiency: Recently, many new stationary phases have been developed, which greatly improves the separation efficiency. 4. High sensitivity: High sensitivity detector has been widely used in high performance liquid chromatography, which further improves the sensitivity of analysis.
For example, the sensitivity of fluorescence detector can reach10-11g. In addition, the sample volume is small, usually a few microliters.
5. Wide scope of application: Comparison between gas chromatography and high performance liquid chromatography: Although gas chromatography has the advantages of good separation ability, high sensitivity, fast analysis speed and convenient operation, it is difficult to analyze substances with high boiling point or poor thermal stability by gas chromatography due to technical conditions. High performance liquid chromatography only requires that the sample can be made into solution without gasification, so it is not limited by the volatility of the sample.
In principle, high-performance liquid chromatography can be used to separate and analyze organic compounds with high boiling point, poor thermal stability and large relative molecular weight (above 400) (these substances account for almost 75% ~ 80% of the total organic compounds). According to statistics, about 20% of the known compounds can be analyzed by gas chromatography and 70~80% by liquid chromatography.
According to the properties of its stationary phase, HPLC can be divided into high performance gel chromatography, hydrophobic high performance liquid chromatography, reversed-phase high performance liquid chromatography, high performance ion exchange liquid chromatography, high performance affinity liquid chromatography and high performance focused liquid chromatography. The principle of separating or analyzing various compounds by different types of high performance liquid chromatography is basically similar to that of the corresponding ordinary liquid chromatography.
The difference is that HPLC is sensitive, rapid, high resolution and good repeatability, and it must be carried out in a chromatograph. Main types and separation principles of high performance liquid chromatography According to different separation mechanisms, high performance liquid chromatography can be divided into the following main types: 1. Liquid-liquid partition chromatography and chemical bonding phase chromatography. Both mobile phase and stationary phase are liquids.
Mobile phase and stationary phase are immiscible with each other (polarity is different to avoid the loss of stationary liquid), and there is an obvious interface. When the sample enters the chromatographic column, the solute is distributed between the two phases.
When equilibrium is reached, it obeys the following formula: in the formula, CS is the concentration of solute in the stationary phase; Cm-concentration of solute in mobile phase; Vs- volume of stationary phase; VM- volume of mobile phase. LLPC is similar to GPC, that is, the separation order depends on k, and the greater the k, the greater the retention value of the components. However, there are also differences. In generalized predictive control, the flow has little influence on K, while the LLPC flow has great influence on K. ..
A. Normal phase liquid chromatography: the polarity of mobile phase is less than that of stationary phase. B. reversed-phase liquid chromatography: the polarity of mobile phase is greater than that of stationary phase.
C. Disadvantages of liquid-liquid partition chromatography: Although the polarity requirements of mobile phase and stationary phase are completely different, a small amount of stationary phase is still dissolved; The mechanical impact of mobile phase passing through chromatographic column will cause the loss of stationary liquid. The chemically bonded stationary phase (see below) developed in the late 1970s can overcome the above shortcomings.
Now it is widely used (70~80%). 2. The mobile phase of liquid-solid chromatography is liquid, and the stationary phase is adsorbent (such as silica gel and alumina). ).
This separation is carried out according to the different adsorption of substances. The mechanism is that when the sample enters the chromatographic column, solute molecules (X) and solvent molecules (S) compete to adsorb the active centers on the surface of the adsorbent (when no sample is injected, all active centers of the adsorbent adsorb S), which can be expressed as: XM+NSA = = = = XA+NSM: XM-solute molecules in the mobile phase; Sa-solvent molecules in stationary phase; Solute molecules in Xa-stationary phase; Sm-solvent molecules in mobile phase.
When the competitive adsorption reaction reaches equilibrium, K=[Xa][Sm]/[Xm][Sa] where k is the adsorption equilibrium constant. [Discussion: The greater the k, the greater the retention value.
3. Ion exchange chromatography) IEC uses ion exchanger as stationary phase. IEC is based on reversible exchange between ionizable ions on ion exchange resin and solute ions with the same charge in mobile phase, and these ions are separated according to their different affinities with the exchanger.
Take anion exchanger as an example, the exchange process can be expressed as: X- (in solvent)+(resin -r4n+cl-) = (resin -R4N+ X-)+Cl- (in solvent) When the exchange reaches equilibrium: KX = [-R4N+X-] [Cl-]/[-R4N. [Cl-] [Discussion: Relationship between DX and Retention Value] Any substance that can be ionized in a solvent can usually be separated by ion exchange chromatography. 4. Ion-pair chromatography Ion-pair chromatography is to add one (or more) ions (called counterions or counterions) with opposite charges to solute molecules in the mobile phase or stationary phase, so that they can combine with solute ions to form hydrophobic ion-pair compounds, thus controlling the retention behavior of solute ions.
Its principle can be expressed by the following formula: X+ water phase +Y- water phase = = X+Y- organic phase: X+ water phase-organic ions (or cations) to be separated in mobile phase; Y- water phase-ion pairs with opposite charges in mobile phase (such as tetrabutyl ammonium hydroxide, cetyltrimethyl ammonium hydroxide, etc.). ); X+y type.
Application and working principle of liquid chromatograph
Working principle: the mobile phase flows through the injection valve through the infusion pump, mixes with the sample solution, flows through the chromatographic column, and is adsorbed and separated in the chromatographic column. Finally, the components are converted into electrical signals by the detector, and the corresponding sample peaks appear on the chromatographic workstation.
The use of liquid chromatography: firstly, the sample is pretreated, then the sample is injected, after the sample is injected, the injection port is cleaned, after each analysis, the channel is cleaned, and finally the instrument is closed. Extended data:
The basic concepts such as retention value, plate number, plate height, resolution and selectivity used in liquid chromatography are consistent with those used in gas chromatography.
The basic theory of liquid chromatography: tray theory and rate equation are basically the same as those of gas chromatography, but because gas chromatography uses liquid as mobile phase, the properties of liquid and gas are different. In addition, the instruments, equipment and operating conditions used in liquid chromatography are also different from those used in gas chromatography, so there are certain differences between liquid chromatography and gas chromatography.
There are mainly the following aspects: ① Different operating conditions and scope of application. For gas chromatography, it is a heating operation. It can only analyze the substances that can vaporize without decomposition at the operating temperature, and it is difficult to separate and analyze high-boiling compounds, nonvolatile substances, thermally unstable compounds, ionic compounds and polymers, which limits its application to some extent. According to statistics, only about 20% of organic matter can be analyzed by gas chromatography.
Liquid chromatography operates at room temperature and is not limited by the volatility and thermal stability of the sample. It is very suitable for the separation and analysis of substances with relatively large molecular weight, ionic compounds, polymers and other substances that are difficult to vaporize, volatilize or are sensitive to heat, accounting for about 70%~80% of organic matter. (2) Liquid chromatography can accomplish difficult separation. A. The mobile phase carrier gas in gas chromatography is chromatographic inert, basically does not participate in the distribution equilibrium process, and has no affinity with sample molecules, and sample molecules mainly interact with stationary phases.
In liquid chromatography, mobile phase liquid also competes with stationary phase for sample molecules, which increases the factors to improve selectivity. Two or more liquids with different proportions can also be selected as the mobile phase to improve the separation selectivity.
B there are many kinds of stationary phases in liquid chromatography, such as ion exchange chromatography and exclusion chromatography, so there are many choices for analysis. Gas chromatography is impossible. C. Liquid chromatography is usually operated at room temperature, and lower temperature is generally beneficial to the selection of chromatographic separation conditions.
③ Because the diffusion coefficient of liquid is less than that of gas 105 times, the mass transfer rate of solute in liquid phase is slow, and the extracolumn effect is particularly important; In gas chromatography, the expansion caused by the area outside the column can be ignored. ④ In liquid chromatography, sample preparation is simple, sample recovery is relatively easy and quantitative, which is suitable for batch preparation. However, liquid chromatography still lacks a universal detector, which is complex and expensive.
In practical application, these two technologies complement each other. To sum up, liquid chromatography has the advantages of high column efficiency, high selectivity, high sensitivity, fast analysis speed, good repeatability and wide application range. This method has become one of the main means of modern analysis technology.
At present, it has been widely used in chemistry, chemical engineering, medicine, biochemistry, environmental protection, agriculture and other scientific fields. High performance liquid chromatography is widely used, covering almost all fields of quantitative and qualitative analysis.
(1) HPLC only requires that the sample can be made into a solution, which is not limited by the volatility of the sample. The mobile phase can be selected in a wide range, and there are many kinds of stationary phases, which can separate thermally unstable and nonvolatile, dissociated and undissociated substances with various molecular weight ranges. Combined with sample pretreatment technology, HPLC can achieve high resolution and high sensitivity, and can simultaneously separate and determine substances with very similar properties, and can separate trace components in complex mixtures.
With the development of stationary phase, the separation of biochemical substances can be completed under the condition of fully maintaining its activity. (2) Biochemical analysis has been widely used in biochemistry, food analysis, medical research, environmental analysis, inorganic analysis and other fields because of the advantages of high resolution, high sensitivity, high speed, reusable chromatographic column and easy collection of effluent components, and has become the most promising method to solve biochemical analysis problems.
(3) Instrument combination The combination of high performance liquid chromatography and structural instruments is an important development direction. High performance liquid chromatography-mass spectrometry (HPLC-MS) technology has been highly valued, such as the analysis of carbamate pesticides and polynuclear aromatic hydrocarbons. HPLC-IR spectroscopy technology has also developed rapidly, such as the determination of hydrocarbons in water in environmental pollution analysis, which has a new development reference for environmental pollution analysis.
Baidu encyclopedia-liquid chromatography.
What is the principle of liquid chromatograph? What is this for?
Principle of liquid chromatograph: the mobile phase in the reservoir is pumped into the system by a high-pressure pump, and the sample solution enters the mobile phase through the sampler, and is loaded into the chromatographic column (stationary phase) by the mobile phase. Because the distribution coefficients of each component in the sample solution are different in the two phases, when they move relatively in the two phases, after repeated adsorption-desorption distribution process, the moving speeds of each component are very different, and they are separated into single components and flow out of the column in turn. When passing through the detector,
It is mainly used to separate the mixture of high boiling point and difficult gas phase compounds by chromatographic column. Used in biochemistry, biomedicine, environmental chemistry, petrochemical and other departments.
Extended data liquid chromatograph can be divided into liquid-liquid chromatography (LLC) and liquid-solid chromatography (LSC) according to whether the stationary phase is liquid or solid. Modern liquid chromatograph consists of high-pressure infusion pump, sampling system, temperature control system, chromatographic column, detector and signal recording system.
Compared with the classical liquid column chromatography device, it has the characteristics of high efficiency, rapidity and sensitivity. High performance liquid chromatograph mainly includes sampling system, sampling system, separation system, detection system and data processing system.
The injection system generally uses diaphragm injector or high-pressure injection chamber to complete the injection operation, and the injection quantity is constant. This is beneficial to improve the repeatability of analytical samples.
Infusion system This system includes three parts: high-pressure pump, mobile phase storage tank and gradiometer. The general pressure of high-pressure pump is l.47~4.4X 10Pa, and the flow rate is adjustable and stable. When the high-pressure mobile phase passes through the chromatographic column, it can reduce the diffusion effect of the sample in the column and accelerate its moving speed, which is conducive to improving the resolution, recovering the sample and maintaining the biological activity of the sample.
Separation system This system includes chromatographic column, connecting tube and thermostat. Generally, the length of chromatographic column is 10~50cm (connecting tube can be added between two columns when they are used together), the inner diameter is 2~5mm, the material is high-quality stainless steel, thick-walled glass tube or titanium alloy, and the stationary phase is used, and the particle size is 5~ 10μm (composed of matrix and stationary liquid).