Attention to the use of liquid chromatography column

The chromatographic column is composed of column tube, pressure cap, ferrule (sealing ring), sieve plate (filter), joint, screw, etc. The column tube is mostly made of stainless steel. When the pressure is not higher than 70 bar, thick wall glass or quartz tube can also be used. The inner wall of the tube requires high finish. In order to improve the column efficiency and reduce the tube wall effect, the inner wall of stainless steel column is usually polished. There are also fused silicon or glass lined ones for thin strings. A sieve plate with pore size of 0.2-20 μ m (5-10 μ m) is installed in the column joint at both ends of the chromatographic column, depending on the particle size of the packing, so as to prevent the packing from leaking out.
In the daily separation and analysis work, the correct use and maintenance of chromatographic column is very important. Whether the chromatographic column is used properly or not will directly affect the life of the chromatographic column. A little carelessness will reduce the column efficiency, shorten the service life and even damage it. In the process of chromatographic operation, attention should be paid to the following problems in order to maintain the chromatographic column.
(1) During the loading, unloading and replacement of columns, the action should be light, and the joint should be properly tightened. Strong mechanical vibration must be prevented to avoid voids in the column bed.
(2) If it is used for routine analysis, the sample types are limited, but the analysis times are large, it is advisable to configure a special column for each type of routine analysis, which helps to prolong the life of the column.
(3) Avoid sharp changes in pressure and temperature and any mechanical vibration. The sudden change of temperature or the falling of chromatographic column will affect the filling condition of the column; the sudden increase or decrease of column pressure will also affect the packing in the column, so it should be carried out slowly when adjusting the flow rate, and the rotation of the valve should not be too slow when the valve is injected.
(4) The composition of solvents should be changed gradually, especially in reversed phase chromatography. It should not be changed from organic solvents to water, and vice versa.
(5) If the column temperature control device is used, it should be noted that the temperature can be raised only after the mobile phase is introduced.
(6) Generally speaking, the chromatographic column can not be recoiled. Only when the producer indicates that the column can be recoiled, the impurities left in the column head can be removed by backflushing. Otherwise, the backwash will be light, which will reduce the column efficiency, and if it is heavy, the packing will be lost and the column will be damaged.
(7) Appropriate mobile phase should be selected to avoid the destruction of stationary phase. Sometimes a protective column can be connected in front of the injector, and the packing of the protective column should be consistent with the chromatographic column, so that the mobile phase can be "saturated" by silica gel before entering the analysis column, so as to avoid the dissolution of silica gel matrix in the analysis column.
(8) To avoid injecting complex matrix samples, especially biological samples directly into the column, it is necessary to pretreat the samples or connect a protective column between the injector and the chromatographic column. The protective column is usually a short column filled with similar stationary phase. Protective posts can and should be replaced frequently.
(9) Wash the chromatographic column with strong solvent to remove the impurities retained in the column. In the cleaning process, the displacement of mobile phase in the convection system should be gradually transited with miscible solvent, and the volume of each mobile phase should be about 20 times of the column volume.
(10) The column should be filled with acetonitrile or methanol to prevent bacterial growth. The column joint should be tightened to prevent solvent evaporation and drying. It is forbidden to leave the buffer solution in the column overnight or for a longer time.
(11) During the use of chromatographic column, if the pressure increases, one may be that the sintered filter is blocked, so the filter should be replaced or taken out for cleaning; the other may be that macromolecules enter the column to pollute the column head; if the column efficiency is reduced or the chromatographic peak is deformed, the column head may collapse and the dead volume will increase.
(12) After the separation and analysis work is completed, it is not necessary to stop the machine immediately, but to flush the chromatographic analysis system in time to remove impurities in the chromatographic column.
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