Selection of HPLC column

base material
The matrix of chromatographic column can be divided into silica gel matrix packing, polymer matrix packing and other inorganic fillers.
Silica gel substrate filler:
1. Normal phase chromatography
The stationary phase for normal chromatography is usually silica gel and other functional groups with strong polarity, such as amino group and cyano group.
Because the polarity of silicon hydroxyl group (Si oh) or other groups on the surface of silica gel is strong, the separation order is based on the polarity of each component in the sample, that is, the component with weak polarity is first washed the excellent column. The polarity of mobile phase used in normal chromatography is lower than that of stationary phase, such as n-ethane, chloroform, dichloromethane, etc.
2. Reversed phase chromatography
The packing of reversed phase chromatography is usually based on silica gel, and its surface is bonded with relatively weak polar functional groups. The mobile phase used in RP-HPLC has strong polarity, which is usually the mixture of water, buffer, methanol, acetonitrile, etc. The order of sample flow out of the column is that the combination with stronger polarity is washed out first, while the component with weak polarity will have stronger retention on the column.
The commonly used reversed phase fillers are C18, C8, C4, phenyl, etc.
Polymer substrate filler
Most of the polymer substrates are polystyrene divinylbenzene or poly (methyl propionate), which can be used in pH range of 1-14.
Compared with the C18 packing, this kind of packing has stronger hydrophobicity; the macroporous polymer packing is very effective for the separation of protein and other samples.
The disadvantage of polymer packing is that the column efficiency is lower than that of silica gel.
Other inorganic fillers
Chromatographic columns with other inorganic fillers have also been commercialized. Due to its special properties, it is generally limited to special uses. For example, graphitized carbon is gradually becoming a packing material for RP-HPLC. The separation of this kind of packing is different from the alkyl bonded phase of silica gel matrix. The surface of graphitized carbon is the basis of retention and no other surface modification is needed. Generally, the retention capacity of the column packing is stronger than that of alkyl bonded silica gel or porous polymer packing. Graphitized carbon can be used to separate some geometric isomers. Because it will not be dissolved in the HPLC mobile phase, this kind of column can be used in the separation of some geometric isomers Use at temperature.
Alumina can also be used in HPLC. Alumina particles have strong rigidity and can be used in the mobile phase with pH up to 12. However, due to the strong interaction between alumina and alkaline compounds, its application scope is limited, so it can not be widely used.
The new zirconia packing can also be used in HPLC. The commercial porous zirconia microspheres column with only polymer coating can be used. The pH range is 1 ~ 14 and the temperature can reach 1000 ℃. Because the research of zirconia fillers was only started a few years ago and the previous experiments were difficult, its important uses and advantages are still under study.
PH range of column
The advantages of the reversed phase column are that the stationary phase is stable and widely used, and a variety of solvents can be used. However, the pH range of the mobile phase should be paid attention to when using silica gel as the filler. Generally, the pH range of C18 column is 2 ~ 8. When the pH value of mobile phase is less than 2, the bonding phase will be hydrolyzed; when the pH value is greater than 7, the silica gel is easy to dissolve; the stationary phase often uses buffer solution to degrade. Once this happens, the column entrance will collapse. The chromatographic columns of different brands with the same packing are not the same. If the pH of the mobile phase is high or buffer solution is often used, it is recommended to choose a column with a wide pH range. For example, waters XBridge series chromatographic column, pH range of 1-12, has a good separation ability for basic compounds.
End base sealing of packing
Sealing the residual silicon hydroxyl groups of fillers with sealing technology can improve the adsorption or tailing of polar compounds; the increase of carbon content is conducive to the separation of difficult retention compounds; the stability of fillers is good, and the retention time reproducibility of components is good. If the sample to be analyzed is acidic or alkaline compound, it is better to use the column with the packing end capped.
How to choose filler size
At present, the commercial chromatographic materials have particle sizes ranging from 1 μ m to more than 30 μ M. at present, 3, 5 and 10 μ m fillers are mainly used for analysis and separation. The particle size of fillers mainly affects two parameters of packed column, namely column efficiency and back pressure. The smaller the particle size is, the higher the column efficiency is; when the packing is less than 3 μ m, the separation can be improved by increasing the column efficiency, but it is not the only factor. If the choice of stationary phase is correct, but the resolution is not enough, it is very useful to choose smaller size packing. The number of columns filled with 3 μ m packing is nearly 30% higher than that of 5 μ m packing under the same conditions; however, the back pressure of 3 μ m chromatography is twice that of 5 μ M. At the same time, the improvement of column efficiency means that shorter column can be selected under the same conditions to shorten the analysis time. In addition, low viscosity solvent can be used as mobile phase or increase the use temperature of chromatographic column, such as using acetonitrile instead of methanol to reduce the pressure of chromatographic column.
For more related technologies, please consult MS technologies technicians