Basic knowledge of liquid chromatography column

Before using, the performance of the column was tested, and the results were saved as a reference for evaluating the performance of the column in the future. However, it should be noted that the performance of the column may be different due to the difference of sample, mobile phase, column temperature and other conditions; in addition, the column performance test is conducted according to the conditions in the ex factory report of chromatographic column (the conditions used in the factory test are the best conditions), and only in this way can the measured results be comparable.
Classification of liquid chromatographic columns
Liquid chromatography column can be divided into many kinds: normal column, reverse phase column, ion exchange column, there are many manufacturers of liquid chromatography column, and different types of liquid chromatography column will be different. Each manufacturer has its own name. Some are called liquid chromatography column, some are called chromatographic column, some are called carbooctadecylsilane chemical build-up column.
HPLC columns can be divided into normal chromatographic column, reverse column, gel column, Hilic column, ion chromatography column, hydrophobic column, affinity chromatography, ultra performance liquid chromatography and chiral chromatographic column.
There are also special chromatographic columns for liquid chromatography, such as ephedra, honey, ribavirin, mannitol, lactose, heparin, amino acid, motherwort and chondroitin sulfate. Special column for cephalosporin polymer. A special column for antibody analysis.
mobile phase
Liquid chromatography column is the separation of sample components by mass exchange between column packing and mobile phase. Therefore, the mobile phase is required to have the following characteristics:
a. The flow relative to the sample has a certain solubility to ensure that the sample components will not precipitate in the column (or remain in the column for a long time).
b. The mobile phase is inert and does not react with the sample (except for special cases).
c. The viscosity of the mobile phase should be as small as possible, so as to get a good separation effect when using a long analysis column; at the same time, reduce the pressure drop of the column and prolong the service life of the liquid pump (the viscosity of the mobile phase can be reduced by increasing the temperature).
d. The physicochemical properties of the mobile phase should be compatible with the detector used. If using a UV detector, use a solvent with low UV absorption.
e. The boiling point of the mobile phase should not be too low, otherwise it is easy to produce bubbles, leading to the experiment can not be carried out.
f. After the mobile phase is prepared, degassing must be carried out. Removing the trace gas dissolved in the mobile phase is not only beneficial to the detection, but also can prevent the interaction between the trace oxygen in the mobile phase and the sample.
HPLC columns 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 reversed-phase chromatography. The separation of this kind of packing is different from that of alkyl bonded phase in silica gel matrix. The surface of graphitized carbon is the basis of retention and no other surface modification is needed. The retention capacity of the column packing is generally stronger than that of alkyl bonded silica gel or porous polymer filler. Graphitized carbon can be used for the separation of some geometric isomers and can be used at any pH and temperature since it will not be dissolved in the HPLC mobile phase.
alumina
It can also be used in HPLC. Alumina particles have strong rigidity and can be made into stable column bed. Its advantage is that it can be used in 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 Matrix chromatographic packing can also be used for HPLC. The commercial porous zirconia microsphere chromatographic column with only polymer coating can be used. The pH range is 1-14 and the temperature can reach 100 ℃. Due to the fact that the research of zirconia fillers has only started in recent years, and the experimental difficulties, its important use and advantages are still in progress.
Of course, the same as the principle of liquid chromatography column, the length and inner diameter of the column also have an important influence on the separation effect
Selection of flow rate of mobile phase
Liquid chromatography column is a function of linear flow rate of mobile phase in the column. Different column efficiency can be obtained by using different flow rate. For a specific chromatographic column, it is necessary to pursue good column efficiency and use good flow rate. For a 4.6 mm inner diameter column, the flow rate is generally 1 ml / min. for a 4.0 mm inner diameter column, the flow rate is 0.8 ml / min
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