In normal phase chromatography, a polar bonded stationary phase is generally employed, and the surface of the silica gel is bonded to a polar organic group, and the name of the bonded phase is determined by the bonded group. The most commonly used are cyano (-CN), amino (-NH2), and diol (DIOL) bonded phases. The mobile phase generally uses a nonpolar or weakly polar organic solvent having a lower polarity than the bonded phase, such as a hydrocarbon solvent, or a certain amount of a polar solvent (such as an alcohol, acetonitrile, etc.) to adjust the washing of the mobile phase. De-strength. Usually used to separate polar compounds. The separation mechanism of normal phase chromatography is generally considered to belong to the partition chromatography. The distribution ratio of the components increases with the increase in polarity, but decreases as the polarity of the polar modifier in the mobile phase increases (or increases in concentration). At the same time, the greater the polarity of the polar bonded phase, the greater the retention of the component. This method is mainly used to separate isomers, compounds with different polarities, especially for separating different types of compounds.
In reversed-phase chromatography, a non-polar bonded stationary phase is generally used, such as silica gel-C18H37 (ODS or C18 for short) silica gel-phenyl, etc., with a strong polar solvent as the mobile phase, such as methanol/water, acetonitrile/water. , water and inorganic salt buffers, etc.
The difference between normal phase chromatography and reversed phase chromatography:
In essence, the filler (stationary phase) is different. The positive phase column packing has strong polarity and the elution order is weak to strong; the reverse phase column packing has weak polarity and the elution order is strong to weak.
1. Normal phase chromatography
The stationary phase for normal phase chromatography is typically silica gel (Silica) and other bonded phase fillers having polar functional amine groups such as (NH2, APS) and cyano groups (CN, CPS). Since the silicon hydroxy group (SiOH) or other polar groups on the surface of the silica gel are relatively polar, the order of separation is based on the polarity of each component in the sample, that is, the component with weak polarity is first washed out. Column. The phase of the mobile phase used in normal phase chromatography is relatively lower than that of the stationary phase, such as Hexane, Methylene Chloride, and the like.
2, reversed phase chromatography
The packing for reversed-phase chromatography is usually based on silica gel, and a bonded phase having a relatively weakly polar functional group is bonded to the surface. The mobile phase used in reversed phase chromatography is more polar, usually a mixture of water, buffer and methanol, acetonitrile, and the like. The order in which the sample flows out of the column is that the more polar component is first washed out, while the less polar component is more retained on the column.
Commonly used reversed phase fillers are: C18 (ODS), C8 (MOS), C4 (Butyl), C6H5 (Phenyl) and the like.
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