How to choose a chromatographic column

In modern high performance liquid chromatography, the separation effect depends largely on the choice of chromatographic packing. However, the choice of chromatographic packing is very wide. To make a suitable choice, we must have a certain understanding and understanding of this.

1. Normal phase chromatography

The stationary phase for normal phase chromatography is usually silica gel (Silica), and other bonded phase fillers with polar functional groups such as amine groups (NH2, APS) and cyano groups (CN, CPS).

Due to the strong polarity of silicon hydroxyl (SiOH) or other groups on the surface of silica gel, the order of separation is based on the polarity of the components in the sample, that is, the components with strong and weak polarities are washed out of the chromatogram column. The mobile phase used in normal phase chromatography is relatively less polar than the stationary phase, such as: hexane (Hexane), chloroform (Chloroform), dichloromethane (Methylene Chloride), etc.

2. Reverse phase chromatography

Reverse-phase chromatography packing is often based on silica gel, with a relatively weak polar functional group bonded to the surface. The mobile phase used in reversed-phase chromatography is highly polar, usually water, buffer and methanol, nitrile and other mixtures. The order in which the samples flow out of the column is that the more polar components are washed out first, and the less polar components will have a stronger retention on the column. Commonly used reverse phase packings are C18 (ODS), C8 (MOS), C4 (B), C6H5 (Phenyl), etc.

Second, the polymer filler

Most of the polymer seasonings are polystyrene-divinylbenzene or polymethyl propionate, etc. The main advantage is that they can be used at PH values ​​of 1-14. Compared with silica gel-based C18 fillers, such fillers are more hydrophobic; macroporous polymer fillers are very effective for the separation of proteins and other samples. The disadvantage of the current polymer packing is that the column efficiency is lower than that of the silica matrix packing.

3. Other inorganic fillers

Other HPLC inorganic packing chromatography columns have also been commercialized. Due to its special nature, it is generally limited to special uses. Such as graphitized carbon is also used as a reverse phase chromatography packing. The separation of this packing is different from the silica-based alkyl-bonded phase. The surface of the graphitized carbon is the basis for retention, and no other surface modification is required. The column packing is generally better than the alkyl-bonded silica or porous polymer filler. The retention capacity is stronger, graphitized carbon can be used to separate certain geometric conductors, and because the HPLC mobile phase will not be dissolved, this type of column can be used at any pH and temperature. Alumina can also be used for HPLC. Alumina particles have strong rigidity and can be made into a stable chromatographic column bed. Its advantage is that it can be used in mobile phases with a pH of up to 12. However, due to the strong interaction between alumina and basic compounds, the scope of application is limited, so it is not widely used. The new zirconia filler can also be used for HPLC. Commercially available polymer-coated porous zirconia microspheres Chromatography column, the application PH range is 1 ～ 14, and the temperature can reach 100 ℃. Because zirconia fillers have only been researched for several years, and the experimental difficulties they face, their important uses and advantages are still in progress.

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