Classification of the concept and principle of max

2022-08-01
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The concept, principle and classification of chromatography

a technique for separating substances in two phases by using the differences of physical and chemical properties (adsorption force, molecular shape, size, molecular polarity, molecular affinity and partition coefficient) of each component in the mixture

at the beginning of this century (1903), botanist Er discovered and used this technology to prove that the leaves of plants contain not only chlorophyll, but also other pigments. In fact, he used adsorption chromatography. Now, stratigraphy has become one of the effective separation and analysis tools in biochemistry, molecular biology and other disciplines

II. Principle

specific process: separable substances: sugars, organic acids, amino acids, nucleotides

III. classification

1 Classification according to the physical state of the two phases

gas chromatography: gas-liquid chromatography (the stationary phase is liquid) and gas-solid chromatography (the stationary phase is solid), and the mobile phase is gas

liquid chromatography: liquid-liquid chromatography (the stationary phase is liquid) and liquid-solid chromatography (the stationary phase is solid), and the mobile phase is liquid

2. Classification by chromatography

column chromatography: the stationary phase is installed in the column to make the sample move in one direction and separate

thin layer chromatography: the stationary phase with appropriate viscosity is evenly spread on the thin plate, and the mobile phase is used for development after sampling

paper chromatography: filter paper is used as the carrier of liquid, and mobile phase is used to develop after sampling

3. Classification according to the principle of chromatography: common classification method

(1) adsorption chromatography: the stationary phase is a solid adsorbent, which can be separated by different adsorption strengths of different solutes

(2) partition chromatography: different partition coefficients in two phases are used

partition coefficient: when a solute is distributed in two given immiscible solvents, after reaching equilibrium at a certain temperature, the concentration ratio of the solute in the two phases is a constant, that is, the partition coefficient (KD)

kd = Ca/cb

ca and CB represent the concentration of a substance in the immiscible two phases, phase a (mobile phase) and phase B (static phase) respectively

(3) ion exchange chromatography: the stationary phase is an ion exchanger, and each component is different from its affinity

(4) gel chromatography: the stationary phase is a porous gel, which is separated according to the molecular weight difference of solute in the mobile phase

(5) affinity chromatography: use the surface of the stationary phase carrier to couple ligands with special affinity, Reversible specific binding with solute molecules for separation

(6) metal chelation chromatography

(7) hydrophobic chromatography

(8) reverse phase chromatography

IV. gel chromatography (also known as gel filtration or molecular sieve)

gel chromatography refers to the technology that I do not refuse when the mixture flows through the chromatographic column of the stationary phase with the mobile phase, each component in the mixture is classified according to its molecular size

1. Principle

the stationary phase of gel chromatography is gel. Gel is an uncharged material with three-dimensional porous structure. Each particle of gel has many fine pores, just like a sieve

common gel include agarose gel, polyacrylamide gel, dextran gel, etc

dextran gel medium is composed of polydextran and epichlorohydrin. Sephadex G series is the most commonly used Sephadex gel chromatography medium. Different types of gel are represented by G, and G represents the degree of crosslinking, from G10 to G100. The numbers after "g" indicate the water absorption of each 10g of dry glue. Gel with different "g" values can be selected according to the molecular weight of the mixture to be separated

2. Separation process:

the sample moves down with the flow of eluent

3 Main factors affecting gel column chromatography (1) selection and filling of chromatographic column the size of chromatographic column should be determined according to the amount of separated samples and the requirements for resolution. After the gel column is filled, it shall be uniform without lines and bubbles by visual inspection

(2) selection of mobile phase eluent: it is a buffer containing a certain concentration of salt to prevent possible adsorption of gel. Its selection mainly depends on the sample to be separated. Generally speaking, as long as the eluting substance can be dissolved, the buffer that does not denature it can be used for gel chromatography

(3) amount of sample added: the amount of sample added shall be determined according to the specific experiment: generally, the amount of sample added in the stage separation is about 1% - 5% of the volume of the gel column bed, while the amount of sample added in the group separation is about 10% - 25% of the volume of the gel column bed

(open aluminum alloy window handle qb/t 3886 ⑴ 9994) gel regeneration: the regeneration of dextran gel is treated with the mixture of NaOH (0.2m) and NaCl (0.5m)

v. ion exchange chromatography

ion exchange chromatography is a separation method that uses reversible ion exchange reaction between ion exchange groups coupled to the stationary phase and ionic compounds dissociated from the mobile phase with low access threshold

1. Principle

ion exchange chromatography is a technology that uses different affinity of ion exchanger for various ions to separate various ions in the mixture. Its main feature is that it depends on the attraction between particles with opposite charges

the stationary phase of ion exchange chromatography is an ion exchanger loaded with a large amount of charge, and the mobile phase is an electrolyte solution with a certain pH value and ionic strength

ion exchangers are divided into:

anion exchangers are positively charged and combined with negatively charged components in the mixture by anion exchange chromatography

the cation exchanger is negatively charged and combines with the positively charged components in the mixture for cation exchange chromatography

common ion exchangers include: ion exchange resin, ion exchange cellulose, etc

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