1. Liquid-solid chromatography uses solid adsorbents, and the separation principle of separated components on the chromatographic column is based on the different adsorption forces of fixed relative components. The separation process is an adsorption-desorption equilibrium process. The commonly used adsorbents are silica gel or alumina, the particle size of which is 5 ~10 μ m. It is suitable for separating components with molecular weight of 200~ 1000, most of which are used for nonionic compounds, and ionic compounds are easy to tail. Often used to separate isomers.
2. Liquid-liquid chromatography uses a stationary phase, which is formed by coating a specific liquid substance on the surface of a carrier or chemically bonding it to the surface of the carrier. The principle of separation is to separate the separated components according to their solubility in mobile phase and stationary phase. The separation process is a distribution equilibrium process.
Coating fixation has good inertia; The mobile phase must be saturated with stationary phase in advance to reduce the loss of stationary phase from the carrier surface; The change of temperature and the abnormal difference of mobile phases in different batches cause the change of chromatographic column; In addition, the stationary phase in the mobile phase also complicates the separation and collection of samples. Because the loss of stationary liquid is inevitable, coated stationary phases are rarely used now. At present, chemically bonded stationary phases, such as C 18, C8, amino column, cyano column and phenyl column, are widely used.
According to the different polarities of stationary phase and mobile phase, liquid-liquid chromatography can be divided into normal phase chromatography (NPC) and reverse phase chromatography (RPC).
Normal phase chromatography adopts polar stationary phase (such as polyethylene glycol, amino and nitrile bonded phase); The mobile phase is a relatively nonpolar hydrophobic solvent (alkanes such as n-hexane and cyclohexane), and ethanol, isopropanol, tetrahydrofuran and chloroform are often added to adjust the retention time of the components. It is often used to separate compounds with medium polarity and strong polarity (such as phenols, amines, carbonyl groups and amino acids).
Generally, nonpolar stationary phases (such as C 18, C8) are used for reversed-phase chromatography. The mobile phase is water or buffer, and water-soluble organic solvents such as methanol, acetonitrile, isopropanol, acetone and tetrahydrofuran are often added to adjust the retention time. It is suitable for separating nonpolar and weakly polar compounds. RPC is the most widely used method in modern liquid chromatography. According to statistics, it accounts for about 80% of the whole HPLC application.
With the rapid development of column packing, the application scope of reversed-phase chromatography has gradually expanded, and it has been applied to the analysis of some inorganic samples or easily dissociated samples. In the process of analysis, in order to control the dissociation of samples, buffer is often used to control the pH value of mobile phase. However, it should be noted that the pH value used for C 18 and C8 is usually 2.5~7.5(2~8). If the pH value is too high, the silica gel will be dissolved, and if the pH value is too low, the bonded alkyl will fall off. It is reported that the new commodity column can be operated in the range of pH 1.5~ 10.
Comparison table of normal phase chromatography and reverse phase chromatography
Normal phase chromatography reversed phase chromatography
The polarity of stationary phase is high ~ medium ~ low.
The polarity of mobile phase is low ~ medium ~ high.
The elution order of components is small polarity, washed out first, and polarity washed out first.
As can be seen from the above table, when the polarity is medium, there is no obvious boundary between normal phase chromatography and reverse phase chromatography (such as amino bonded stationary phase).
3. The stationary phase of ion exchange chromatography is ion exchange resin, usually a polymer skeleton formed by crosslinking styrene and divinyl, and carboxyl, sulfonic acid group (called cation exchange resin) or quaternary amino group (anion exchange resin) is attached to the aromatic ring at the end of the surface. The separation principle of that component separated on the chromatographic column is that the ionizable ion on the resin are reversibly exchanged with the ions with the same charge in the mobile phase and the ions of the teste component, and are separated according to the different charge attraction of each ion and ion exchange group.
Buffer is often used as the mobile phase of ion exchange chromatography. The retention time of separated components in ion exchange column is not only related to the interaction between component ions and ion exchange groups on resin, but also affected by pH value and ionic strength of mobile phase. The pH value can change the dissociation degree of the compound, and then affect its interaction with the stationary phase. When the concentration of mobile phase salt is high, the ionic strength is high, which is not conducive to the dissociation of the sample and leads to the rapid outflow of the sample.
Ion exchange chromatography is mainly used to analyze organic acids, amino acids, peptides and nucleic acids.
4. Ion pair chromatography, also called even ion chromatography, is a branch of liquid-liquid chromatography. It is based on the formation of neutral ion-pair compounds by the ions of the tested components and ion-pair reagents, and the solubility in nonpolar stationary phase is increased, thus improving its separation effect. It is mainly used to analyze acid and alkali substances with high ionic strength.
The commonly used ion pair reagents for analyzing alkaline substances are alkyl sulfonates, such as sodium pentane sulfonate and sodium octane sulfonate. In addition, perchloric acid and trifluoroacetic acid can also form strong ion pairs with various alkaline samples.
Tetrabutyl quaternary ammonium salts, such as tetrabutyl ammonium bromide and tetrabutyl ammonium phosphate, are usually used to analyze acidic substances.
ODS column (C 18) is often used in ion pair chromatography. The mobile phase is methanol-water or acetonitrile-water, and 3~ 10 mmol/L ion pair reagent is added to the water, and the separation is carried out within a certain pH range. The retention time of the tested components is related to the nature and concentration of ion pairs, the composition of mobile phase, its pH value and ionic strength.
5. The stationary phase of exclusion chromatography is a porous filler with a certain pore size, and the mobile phase is a solvent that can dissolve the sample. Compounds with small molecular weight can enter the pores and stay for a long time; Compounds with large molecular weight can't enter the pores and flow out directly with the mobile phase. It uses the difference of molecular sieve's repulsion ability to different molecular weight components to complete the separation. It is often used to separate macromolecular compounds, such as tissue extracts, peptides, protein, nucleic acids and so on.
Basic principles of chromatography
According to the differences of chemical and physical properties of each component in the sample mixture, each component is divided into two immiscible phases to varying degrees. When the two phases move relatively, the components are redistributed repeatedly in the two phases, resulting in the separation of the mixture.
Of the two phases, the stationary phase is fixed; Mobile phase.
Classification:
Mobile phase-gas phase as mobile phase-gas chromatography-stationary phase as liquid gas chromatography.
Gas-solid chromatography with solid as stationary phase
-Liquid chromatography with liquid as mobile phase-Liquid chromatography with liquid as stationary phase.
Liquid-solid chromatography with solid as stationary phase
When the mobile phase is a liquid working near its critical temperature and pressure-supercritical chromatography
According to the attachment mode of stationary phase
-stationary phase in cylindrical column chromatography
-stationary phase coated on glass or metal plate-thin film chromatography (plate chromatography)
-Liquid stationary phase coated on paper-Paper chromatography (plate chromatography)
According to the separation mechanism
—partition chromatography—The distribution coefficients of sample components are different.
Adsorption chromatography-sample components have different adsorption forces on the surface of stationary phase.
-Size exclusion chromatography-Separation of sample components by molecular size using stationary phases with different pore sizes.
-Ion exchange chromatography-Different ions have different forces with opposite charges on stationary phase.
According to polarity
-mobile phase polarity > stationary phase polarity-reversed phase chromatography
-polarity of mobile phase