In the daily separation and analysis work, the use and maintenance of the liquid chromatography column is very important. The proper use of the liquid chromatography column directly affects the life of the column. A slight carelessness will reduce the efficiency of the column, shorten the service life and even damage.
Correct use of liquid chromatography columns:
(1) When loading and unloading or replacing the column, the action should be light and the joint should be tightened properly. Strong mechanical vibration must be prevented to avoid voids in the column bed.
(2) If the instrument is used for routine analysis, the sample type is limited, but the number of analyses is large, it may be useful to configure a special column for each type of routine analysis, which helps to extend the life of the column.
(3) Avoid sudden changes in pressure and temperature and any mechanical shock. Sudden changes in temperature or dropping the column from a height will affect the filling condition in the column; a sudden increase or decrease in column pressure will also impede the packing in the column, so it should be slow when adjusting the flow rate, when the valve is injected. The rotation of the valve cannot be too slow.
(4) The composition of the solvent should be gradually changed, especially in reversed-phase chromatography, and should not be changed directly from organic solvent to all, and vice versa.
(5) If the column temperature control device is used, it should be noted that the temperature can be raised after the mobile phase is passed.
(6) Generally speaking, the column cannot be backflushed. Only when the producer indicates that the column can be backflushed, the impurities remaining in the column head can be backflushed. Otherwise, the recoil will quickly reduce the efficiency of the column.
(7) Choose to use a suitable mobile phase to avoid damage to the stationary phase. Sometimes a pre-column can be connected in front of the injector. When the analytical column is bonded to silica gel, the pre-column is silica gel, so that the mobile phase can be “saturated†by the silica gel before entering the analytical column to avoid dissolution of the silica matrix in the analytical column. .
(8) Avoid injecting samples with complex matrices, especially biological samples, directly into the column. Pretreatment of the sample is required or a guard column is connected between the injector and the column. The guard column is typically a short column filled with a similar stationary phase. The guard column can and should be replaced frequently.
(9) The column is often washed with a strong solvent to remove impurities remaining in the column. When cleaning, the displacement of the mobile phase in the flow path system should be gradually transitioned with a solvent that is miscible. The volume of each mobile phase should be about 20 times the volume of the column, that is, 50-75 mL is required for routine analysis.
(10) When storing the column, the column should be filled with acetonitrile or methanol, and the column joint should be tightened to prevent the solvent from evaporating and drying. It is absolutely forbidden to leave the buffer solution in the column for an overnight or longer period of time.
(11) During the use of the column, if the pressure rises, one may be that the sintered filter is blocked. At this time, the filter should be replaced or taken out for cleaning; the other possibility is that the macromolecule enters the column and makes the column head. Contaminated; if the efficiency of the column is reduced or the peak of the chromatogram is deformed, the stigma may collapse and the dead volume increases.
(12) After the separation and analysis work is completed, it should not be stopped immediately. It is necessary to rinse the chromatographic analysis system in time, generally 0.5h or more, to remove impurities in the column.
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