Principle of chromatographic separation

According to the separation mechanism, HPLC can be divided into liquid-solid adsorption chromatography, liquid-liquid partition chromatography (normal phase and reverse phase), ion exchange chromatography, ion pair chromatography and molecular exclusion chromatography.
1. Solid adsorbent is used in liquid-solid chromatography, and the separation principle of separated components on chromatographic column is based on the different adsorption capacity of fixed relative components. The separation process is an equilibrium process of adsorption and desorption. The commonly used adsorbent is silica gel or alumina with particle size of 5-10 μ M. It is suitable for separation of components with molecular weight of 200 ~ 1000. Most of them are used for non-ionic compounds, and ionic compounds are prone to tailing. It is often used to separate isomers.
2. Liquid liquid chromatography (HPLC) uses a fixed phase formed by coating a specific liquid substance on the surface of the support or chemically bonding to the surface of the support. The separation principle is based on the different solubility of the separated components in the mobile phase and the stationary phase. The separation process is a process of distribution balance.
The results show that coating fixation has good inertness; the mobile phase must be saturated with stationary phase in advance to reduce the loss of stationary phase from the surface of support; the change of temperature and the difference of mobile phase of different batch often cause the change of column; in addition, the stationary phase existing in the mobile phase also complicates the separation and collection of samples. As coated stationary phase is difficult to avoid the loss of stationary liquid, it has been rarely used. Chemical bonded stationary phases, such as C18, C8, amino, cyano and phenyl, are widely used.
According to the polarity of stationary phase and mobile phase, liquid chromatography can be divided into normal phase chromatography (NPC) and reverse phase chromatography (RPC).
In normal phase chromatography, polar stationary phase (such as polyethylene glycol, amino and nitrile bonded phase) is used; the mobile phase is relatively non-polar hydrophobic solvent (such as n-hexane, cyclohexane), and ethanol, isopropanol, tetrahydrofuran, trichloromethane are often added to adjust the retention time of components. It is often used to separate compounds with medium polarity and strong polarity (such as phenols, amines, carbonyls and amino acids).
Generally, non-polar stationary phases (such as C18, C8) are used in reversed-phase chromatography; the mobile phase is water or buffer solution, and organic solvents such as methanol, acetonitrile, isopropanol, acetone, tetrahydrofuran and other water-soluble organic solvents are often added to adjust the retention time. It is suitable for separating non-polar and weak polar compounds. RPC is the most widely used in modern liquid chromatography. According to statistics, it accounts for about 80% of the application of HPLC.
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