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Chromatography, also known as chromatographic analysis, chromatographic analysis and chromatography, is a separation analysis method, which is widely used in analytical chemistry, organic chemistry, biochemistry and other fields. Chromatography uses the selective distribution of different substances in different phases to elute the mixture in the mobile phase. Different substances in the mixture will move along the stationary phase at different speeds, and finally achieve the separation effect. Chromatography originated in the early 20th century, and developed rapidly after 1950' s, and an independent third-level discipline-chromatography appeared. In history, two chemists won the Nobel Prize in chemistry for their outstanding contributions in the field of chromatography. In addition, chromatographic analysis method also played a key role in the 12 research project which won the Nobel Prize in chemistry.

catalogue

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* 1 history

O 1. 1 Source of chromatogram

O 1.2 the appearance of partition chromatography and the popularization of chromatographic methods.

O 1.3 gas chromatography and the emergence of chromatographic theory

O 1.4 high performance liquid chromatography

* 2 principle

O 2. 1 adsorption chromatography

O 2.2 partition chromatography

O 2.3 ion exchange chromatography

O 2.4 gel chromatography

* 3 chromatographic theory

O 3. 1 retention time theory

O 3.2 tray theory based on thermodynamics

O 3.3 Van Deemter equation based on dynamics

* 4 Basic techniques and methods

* 5 Applications

* 6 development direction

Study on o 6. 1 new stationary phase

Study on detection method of o 6.2

O 6.3 expert system

O 6.4 New chromatographic method

* 7 See also.

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history

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Origin of chromatography

Chromatography originated in the early 20th century. 1906, Russian botanist Mikhail Tswett filled a vertical glass tube with calcium carbonate and eluted the plant pigment extract with petroleum ether. After a period of elution, the plant pigment was separated in the calcium carbonate column and dispersed from one color band to several parallel color bands. Because this experiment separated the mixed plant pigments into different bands, Tsvet named this method хроматограия, which was finally accepted by English and other pinyin languages and became the name of chromatography. Chromatogram is also a free translation of this word in Chinese

Tswett is not a famous scientist. Shortly after his research on chromatography was published in Russian academic journals, World War I broke out and normal academic exchanges in Europe were forced to stop. These factors made chromatography unknown to academic circles for more than ten years after its advent. It was not until 193 1 that Kuhn of the Emperor William Institute in Berlin applied Tswett's method to the study of lycopene and lutein that Kuhn's research was widely recognized and accepted by the scientific community. After that, chromatography with alumina as stationary phase was widely used in the separation of colored substances, which is today's adsorption chromatography.

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The appearance of partition chromatography and the popularization of chromatographic methods.

Separation of composition of printer black ink by TLC

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Separation of composition of printer black ink by TLC

1938, archer john porter martin and richard laurence millington synge prepared to use the solubility difference of amino acids in water and organic solvents to separate different kinds of amino acids. Martin designed a countercurrent extraction system to separate vitamins in the early days, and Martin and Singh prepared to separate amino acids with two countercurrent solvents, but both failed. Later, they absorbed water on solid silica gel and washed it with chloroform, and successfully separated amino acids, which is now commonly used partition chromatography. After success, Martin and Singer's methods were widely used in the separation of various organic substances. 1943, Martin and Singer invented paper chromatography in steam saturated environment.

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The emergence of gas chromatography and chromatographic theory

1952, Martin and James put forward the idea of chromatographic separation with gas as mobile phase. They used silicone oil adsorbed by diatomite as stationary phase and nitrogen as mobile phase to separate several volatile organic acids with small molecular weight.

The appearance of gas chromatography has further developed chromatographic technology from the initial qualitative separation method to the quantitative determination method with separation function, which greatly stimulated the development of chromatographic technology and theory. Compared with the early liquid chromatography, the chromatography with gas as mobile phase requires higher equipment, which promotes the mechanization, standardization and automation of chromatography technology. Gas chromatography needs special and more sensitive detection devices, which promotes the development of detectors; The standardization of gas chromatography makes chromatographic theory form tray theory and Van Deemter equation, which play an important role in chromatographic theory. The concepts of retention time, retention index and peak width are all formed in the process of studying gas chromatographic behavior.

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high efficiency liquid chromatography

In the1960s, the theory and method of gas chromatography were reintroduced into the classical liquid chromatography to separate protein, nucleic acids and other macromolecular substances that are not easy to evaporate. 1At the end of 1960s, Cauquelin, Haber, Horvas, Puheis, Pusker and others developed the world's first high performance liquid chromatograph, which opened the era of high performance liquid chromatography. High performance liquid chromatography (HPLC) uses the stationary phase with finer particle size to fill the chromatographic column, increases the number of plates of the chromatographic column, and drives the mobile phase with high pressure, so that the separation work of classical liquid chromatography, which takes several days or even months, can be completed in several hours or even dozens of minutes.

197 1 year, Kauklin and others published Modern Practice of Liquid Chromatography, which marked the formal establishment of high performance liquid chromatography. In the following time, HPLC became the most commonly used separation and detection means, and was widely used in organic chemistry, biochemistry, medicine, drug development and detection, chemical industry, food science, environmental monitoring, commodity inspection and legal inspection. At the same time, HPLC has greatly promoted the development of stationary phase materials, detection technology, data processing technology and chromatographic theory.

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principle

The essence of chromatographic process is that molecules of substances to be separated are evenly distributed between stationary phase and mobile phase. Different substances are distributed differently between the two phases, which makes them move at different speeds with the mobile phase. With the movement of the mobile phase, different components in the mixture are separated from each other on the stationary phase. According to the separation mechanism of substances, they can be divided into adsorption chromatography, partition chromatography, ion exchange chromatography, gel chromatography, affinity chromatography and so on.

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Adsorption chromatography

Adsorption chromatography uses the difference of adsorption capacity between Chinese and western substances on stationary phase to separate the mixture. The chromatographic process of adsorption chromatography is a process in which mobile phase molecules and substance molecules compete for the adsorption center of stationary phase.

The distribution coefficient expression of adsorption chromatography is as follows:

K_a =\frac{[X_a]}{[X_m]}

Where [Xa] represents the molecular content of the component adsorbed at the active center of the stationary phase, and [Xm] represents the molecular content of the component dissociated in the mobile phase. The distribution coefficient is of great significance for calculating the retention time of the components to be separated.

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partition chromatography

Partition chromatography uses the solubility difference between stationary phase and mobile phase to achieve separation. The stationary phase of partition chromatography is generally a liquid solvent, which is distributed on the surface of chromatographic column or carrier by mapping, bonding and adsorption. Partition chromatography process is essentially a process in which component molecules constantly reach the dissolution equilibrium between stationary phase and mobile phase.

Partition chromatography's narrow distribution coefficient is expressed as follows:

k = \ frac { C _ s } { C _ m } = \ frac { X _ s/V _ s } { X _ m/V _ m }

In the formula, Cs represents the solubility of component molecules in stationary phase liquid, and Cm represents the solubility of component molecules in mobile phase.

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ion exchange chromatography

Ion exchange chromatography uses the amazing ion exchange ability between the separated components and the stationary phase to achieve separation. The stationary phase of ion exchange chromatography is generally ion exchange resin, and there are many ionizable active centers in the molecular structure of the resin. Ions in the components to be separated will exchange with these active centers to form an ion exchange equilibrium, thus forming a distribution between the mobile phase and the stationary phase. The inherent ions in the stationary phase and the ions in the components to be separated compete for the ion exchange center in the stationary phase, and move with the movement of the mobile phase, and finally achieve separation.

The distribution coefficient of ion exchange chromatography is also called the selection coefficient, and its expression is:

K_s=\frac{[RX^+]}{[X^+]}

Where [RX+] represents the ion concentration bound to the active center of the ion exchange resin, and [X+] represents the ion concentration dissociated in the mobile phase.

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Gel chromatography

The principle of gel chromatography is special, similar to molecular sieve. After entering the gel chromatography, the components to be separated will enter or not enter the pores of the stationary phase gel according to the molecular weight difference, and the molecules that cannot enter the gel pores will be quickly eluted by the mobile phase, while the molecules that can enter the gel pores will take longer to flow out of the stationary phase after washing, thus realizing the separation of components according to the molecular weight difference. Adjusting the crosslinking degree of gel used as stationary phase can adjust the size of gel pores; Changing the solvent composition of mobile phase will change the swelling state of stationary phase gel, and then change the pore size to obtain different separation effects.

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Chromatographic theory

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Retention time theory

Retention time is the time required for the sample to flow out of the chromatographic column. Different substances will have different retention times when eluting with different mobile phases on different chromatographic columns, so retention time is one of the more important parameters in chromatographic analysis.

Retention time is determined by the partition coefficient of substances in chromatography:

TR = t0( 1+kv/virtual machine)

TR represents the retention time of the substance, and t0 is the dead time of the chromatographic system, that is, the time from the mobile phase entering the chromatographic column to flowing out of the chromatographic column is determined by the pore size of the chromatographic column, the flow rate of the mobile phase and other factors. K is the distribution coefficient, and VsVm represents the volumes of stationary phase and mobile phase. This formula, also known as chromatographic process equation, is one of the most basic formulas in chromatography.

In thin-layer chromatography, there is no process of samples entering and leaving the stationary phase, so people use the specific shift value to represent the chromatographic behavior of substances. The specific shift value is a concept corresponding to the retention time, which is the ratio of the moving distance of the sample point to the moving distance of the mobile phase front in the chromatographic process. Like retention time, the specific displacement value is also determined by the distribution coefficient of substances in chromatography:

R_f=\frac{V_m}{V_m+KV_s}

Where Rf is the specific shift value, K is the chromatographic partition coefficient, and VsVm is the volume of stationary phase and mobile phase.

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Tray theory based on thermodynamics

Tray theory is the basic theory of chromatography. Tray theory regards the chromatographic column as a fractionator, and the components to be separated move between trays in the fractionator. In each tray, the component molecules form an equilibrium between the stationary phase and the mobile phase. With the flow of mobile phase, component molecules move from one tray to the next and form a new equilibrium. The more plates in the chromatographic column, the better the separation effect.

According to the tray theory, the concentration of the components to be separated flows out of the chromatographic column in a binomial distribution with time. When the number of plates in the chromatographic column is high, the binomial distribution tends to be normal. The relationship between component concentration and time on the outflow curve can be expressed as:

e^{-\frac{(t-t_r)^2}{2\sigma^2}}

This equation is called the outflow curve equation, where Ct is the component concentration at time t; C0 is the total concentration of components, that is, the peak area; σ is the half-peak width, that is, the standard deviation of normal distribution; TR is the retention time of the component.

According to the equation of outflow curve, the theoretical plate height of chromatographic column is defined as the variance of chromatographic peak per unit column length:

H=\frac{\sigma^2}{L}

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.

Tray theory is based on thermodynamic approximation theory. The real chromatographic column has no isolated tray, which can not fully meet the premise of tray theory. For example, the tray theory holds that the substance components can quickly establish an equilibrium between the mobile phase and the stationary phase, and the substance components will not spread radially when moving along the chromatographic column, which is not in line with the actual situation of the chromatographic column. Therefore, although the tower plate theory can well explain the peak type and peak height of chromatographic peaks and objectively evaluate the column efficiency of chromatographic columns, it can not well explain some phenomena related to kinetic processes, such as the deformation of chromatographic peak type, the relationship between the number of theoretical towers and the flow rate of mobile phase, etc.

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Van Dimt Equation Based on Dynamics

Van Deemter equation is a modification of tray theory, which is used to explain the reasons of chromatographic peak expansion and column efficiency reduction. Based on thermodynamics, tray theory introduces some assumptions that are not in line with the actual situation, while Van Deemter equation establishes a set of empirical equations to correct the errors of tray theory.

Van Deemter equation attributed the change of peak shape to the change of theoretical tray height, which was caused by many reasons, including vortex diffusion, longitudinal diffusion and mass transfer impedance.

Due to the uneven packing of stationary phase in the chromatographic column, the same component will pass through the chromatographic column along different paths, which will lead to the peak expansion and the column efficiency decline. This is called eddy diffusion.

Longitudinal diffusion is caused by concentration gradient, and the components concentrated in a certain area of the chromatographic column will diffuse along the radial direction driven by concentration gradient, which will widen the peak deformation and reduce the column efficiency.

Mass transfer impedance is mainly affected by the rate of reaching distribution equilibrium. In practical systems, the equilibrium of component molecules between stationary phase and mobile phase requires the process of molecular adsorption, desorption, dissolution and diffusion, which is called mass transfer process, and the factor that hinders this process is called mass transfer impedance. In an ideal state, when the mass transfer impedance of the chromatographic column is zero, the equilibrium between the mobile phase and the stationary phase of the component molecules will soon reach. In the actual system, the mass transfer impedance is not zero, which leads to the diffusion of chromatographic peaks and the decrease of column efficiency.

In gas chromatography, the form of Van Deemter equation is:

h = A++C

Where h is the number of trays, a is the eddy diffusion coefficient, b is the longitudinal diffusion coefficient, c is the mass transfer impedance coefficient, and μ is the mobile phase velocity.

In high performance liquid chromatography, because the viscosity of mobile phase is much higher than that of gas chromatography, the influence of vertical diffusion on the peak type is very small and can be ignored, so the Van Deemter equation is in the form of:

H = A + Cμ

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Basic techniques and methods

The commonly used methods of chromatography are column chromatography, thin layer chromatography, gas chromatography and high performance liquid chromatography.

Column chromatography is the most primitive chromatographic method. In this method, the stationary phase is injected into a glass tube with cotton or filter paper at the lower end, and the sample-saturated stationary phase powder is coated on the top of the glass tube and eluted with mobile phase. There are two common elution methods, one is to use the gravity of the solvent itself from top to bottom, and the other is to use capillary action from bottom to top. There are also two different methods to collect the separated pure components. One method is to directly receive the flowing solution at the column end, and the other method is to mechanically separate each band after the stationary phase is dried, and soak the stationary phase in a suitable solvent to extract the component molecules. Column chromatography is widely used to separate mixtures, including organic synthetic products, natural extracts and biological macromolecules.

Thin layer chromatography is a widely used chromatographic method. In this chromatographic method, the stationary phase is spread on a metal or glass plate to form a thin layer, and the sample is placed at one end of the plate by capillary, pen or other tools, and then the point end is immersed in the mobile phase, and the mobile phase solvent spreads the sample along the plate by capillary action. Thin-layer chromatography (TLC) has low cost and simple operation, and is used for rough measurement of samples and detection of organic synthesis reaction progress.

Gas chromatography is a highly mechanized chromatographic method. The gas chromatography system consists of gas source, chromatographic column and chromatographic column box, detector and recorder. The gas source is responsible for providing the carrier gas needed for chromatographic analysis, that is, the mobile phase, which needs purification and constant pressure treatment. The chromatographic column of gas chromatography is generally very thin in diameter and very long in length. According to the structure, it can be divided into packed column and capillary column. The packed column is short and thick, with a diameter of about 5mm and a length of 2-4m. The shell material is generally stainless steel, and the interior is filled with stationary phase filler. The capillary column is made of glass or timely, with an inner diameter of less than 0.5 mm and a length of tens of meters to 100 meters, and the column is filled with packing or liquid phase stationary phase. Column box is a device for protecting chromatographic column and controlling column temperature. In gas chromatography, the column temperature often has a great influence on the separation effect. To achieve the separation effect, it is often necessary to control the temperature by program, so the column box plays a very important role. Detector is a new equipment brought by gas chromatography to chromatographic analysis. In classical column chromatography and thin-layer chromatography, the separation and detection of samples are carried out separately, while gas chromatography realizes the combination of separation and detection. With the development of technology, more than 30 different types of gas chromatography detectors have appeared. A recorder is a device for recording chromatographic signals. Early gas chromatography was recorded by recording paper and recorder. Now, the recording work has been completed by computer, and the data can be processed in real time by chemometrics. Gas chromatography is widely used for quantitative analysis of complex components with small molecular weight.

High performance liquid chromatography is the most widely used chromatographic analysis method at present. HPLC system consists of mobile phase liquid storage bottle, infusion pump, syringe, chromatographic column, detector and recorder. Its overall composition is similar to that of gas chromatography, but many adjustments have been made according to the characteristics that the mobile phase is liquid. The infusion pump of HPLC requires constant and stable infusion volume; The sampling system requires convenient sampling and strict switching; Because the viscosity of liquid mobile phase is much higher than that of gas, in order to reduce the column pressure, the chromatographic column of HPLC is generally thicker and the length is much smaller than that of gas chromatographic column. HPLC is widely used in almost all quantitative and qualitative analysis fields.

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App application

The application of chromatography can be divided into two categories according to the purpose: preparative chromatography and analytical chromatography.

The purpose of preparative chromatography is to separate the mixture and obtain a certain amount of pure components, including the purification of organic synthetic products, the separation and purification of natural products and the preparation of deionized water. Compared with the purification and separation technology before chromatography, such as recrystallization, chromatography can separate the mixture in one step, but the yield of chromatographic separation and purification is limited, which is only suitable for laboratory application.

The purpose of analytical chromatography is to determine the nature and content of each component in the mixture quantitatively or qualitatively. Qualitative analysis chromatography includes thin layer chromatography, paper chromatography and so on. Quantitative analysis chromatography includes gas chromatography and high performance liquid chromatography. The application of chromatography in the analysis field integrates the separation and determination processes, reduces the difficulty of mixture analysis and shortens the analysis period. It is the mainstream analysis method at present. In People's Republic of China (PRC) Pharmacopoeia and China Pharmacopoeia, the quality of more than 600 kinds of chemically synthesized drugs and 400 kinds of traditional Chinese medicines are controlled by HPLC.

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development direction

Chromatography is the most widely used and fastest developing research field in analytical chemistry, and new technologies and methods emerge one after another.

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Study on New Stationary Phase

Stationary phase and mobile phase are the protagonists of chromatography, and the research of new stationary phase continuously expands the application field of chromatography, such as chiral stationary phase, which enables chromatography to separate and determine chiral compounds; The reversed-phase stationary phase has no dead adsorption and can simply separate and determine biopharmaceuticals such as plasma.

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Research on detection method

Detection method is also one of the hot spots in chromatographic research. People constantly update the sensitivity of detectors to make chromatographic analysis more sensitive. People also introduce other spectral techniques into chromatography, such as chromatography-mass spectrometry, chromatography-infrared spectroscopy, chromatography-ultraviolet spectroscopy and so on. The structure of the compound was determined at the same time of separation. The development of chromatographic detector is also accompanied by the development of data processing technology. After calculating and processing the detected data, experimenters can get more information.

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Expert system

Expert system is the product of the combination of chromatography technology and information technology. Because it is necessary to choose different conditions such as mobile phase, stationary phase and pretreatment method according to the research content, it needs a lot of practical experience. Chromatographic expert system is a program that simulates the thinking mode of chromatographic experts to help chromatographic users. The expert system knowledge base stores a large number of practical experiences of chromatographic experts, which can help users in column system selection, sample processing methods, chromatographic separation conditions selection, qualitative and quantitative results analysis and so on.

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New chromatographic method

New chromatographic methods are also one of the hot spots in chromatographic research. High performance capillary electrophoresis (HPCE) is a new chromatographic method that has been studied most at present. This method does not distinguish between mobile phase and stationary phase, but relies on the drive of external electric field to make charged ions move in the direction of electric field in capillary, and different ions are separated from each other due to the differences of charged state, mass and morphology of ions. High-performance capillary electrophoresis (HPCE) has no factors that reduce column efficiency, such as mass transfer impedance and eddy diffusion, which exist in HPLC method, and vertical diffusion is also inhibited by the existence of electric double layer on capillary wall, so it can reach a high theoretical plate number and has excellent separation effect.

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see

* Analytical chemistry

* High performance liquid chromatography

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