Tomography concept, principle, classification
First, the concept
A technique in which a substance is separated in two phases by utilizing the difference in physical and chemical properties (adsorption, molecular shape, size, molecular polarity, molecular affinity, and partition coefficient) of each component in the mixture.
At the beginning of this century (1903), the Russian botanist MCJber discovered and used this technique to prove that the leaves of plants not only have chlorophyll, but also other pigments, in fact he uses adsorption chromatography. Now the layer system has become one of the effective separation and analysis tools in biochemistry, molecular biology and other disciplines.
Second, the principle
Specific process:
Separable substances: sugars, organic acids, amino acids, nucleotides.
Third, 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 a 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 a liquid.
2. Sort by chromatography
Column chromatography: The stationary phase is packed in a column and the sample is moved in one direction to separate.
Thin layer chromatography: A stationary phase of appropriate viscosity is evenly spread on a thin plate, and after spotting, it is spread with a mobile phase.
Paper chromatography: filter paper was used as a carrier for the liquid, and the mobile phase was developed after spotting.
3. Classification according to chromatographic principles: common classification
(1) Adsorption chromatography: The stationary phase is a solid adsorbent, which is separated by different adsorption strengths of different solutes.
(2) Distribution chromatography: The distribution coefficients in the two phases are different.
Partition coefficient: When a solute is distributed in two given mutually incompatible solvents, the concentration ratio of the solute in the two phases is constant after reaching equilibrium at a certain temperature, ie, the partition coefficient (Kd).
Kd=Ca / Cb
Ca and Cb represent the concentrations of a substance in mutually incompatible two phases, phase A (moving phase) and phase B (static phase), respectively.
(3) Ion exchange chromatography: the stationary phase is an ion exchanger, and the components have different affinity
(4) Gel chromatography: the stationary phase is a porous gel, which is separated according to the difference in molecular weight of the solute in the mobile phase.
(5) Affinity chromatography: using a surface of a stationary phase carrier to couple a ligand with a special affinity, and reversible specific binding to the solute molecule for separation
(6) Metal chelate chromatography
(7) Hydrophobic chromatography
(8) Reversed phase chromatography
Fourth, gel chromatography (also known as gel filtration or molecular sieve)
Gel chromatography refers to a technique in which the components of a mixture are classified according to their molecular sizes when the mixture flows through the stationary phase of the column with the mobile phase.
Principle
The stationary phase of the gel chromatography is a gel. A gel is an uncharged substance having a three-dimensional porous network structure. Each particle of the gel has many fine pores inside it, just like a sieve.
Commonly used gels are agarose gel, polyacrylamide gel, dextran gel and the like.
The glucan gel medium is formed by interlacing polyglucan and epoxy green propane. The most commonly used glucan gel chromatography medium is the Sephadex G series, different types of gels are represented by G, and G represents the degree of crosslinking, from G10 to G100. The number after "G" indicates the amount of water per 10 g of dry glue, and gels of different "G" values ​​may be selected depending on the molecular weight of the mixture to be separated.
2. Separation process:
The sample moves down with the flow of the eluent
3. Main factors affecting gel column chromatography
(1) Selection and filling of chromatography columns
The size of the column should be based on the amount of sample separated and the resolution requirements. After filling the gel column, it should be observed with the naked eye, uniform, no grain, no bubble.
(2) Mobile phase - Selection of eluent: It is a buffer containing a certain concentration of salt in order to prevent the gel from adsorbing. The choice depends mainly on the sample to be separated. Generally, as long as it can dissolve the eluted material, the buffer which does not denature it can be used for gel chromatography.
(3) Sample loading amount: The amount of sample loading should be determined according to the specific experiment: the general sample separation is about 1%-5% of the volume of the gel column bed, and the sample loading amount is about 10%-25% of the volume of the gel bed.
(4) Gel regeneration: Dextran gel regeneration was treated with a mixture of NaOH (0.2 M) and NaCl (0.5 M).
5. Ion exchange chromatography
Ion exchange chromatography is a separation method that utilizes a reversible ion exchange reaction between an ion exchange group coupled to a stationary phase and an ionic compound dissociated from a mobile phase.
Principle
Ion exchange chromatography is a technique in which the affinity of an ion exchanger for various ions is different, thereby separating various ions in the mixture. Its main feature is the role of attraction between particles with opposite charges.
The stationary phase of ion exchange chromatography is an ion exchanger carrying a large amount of charge, and the mobile phase is an electrolyte solution having a certain pH and a certain ionic strength.
Ion exchangers are classified according to their charge properties:
The anion exchanger is positively charged and is combined with anion exchange chromatography in the negatively charged component of the mixture.
The cation exchanger is negatively charged and combines with the positively charged component of the mixture for cation exchange chromatography.
Common ion exchangers are: ion exchange resin, ion exchange cellulose, etc.
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