Precautions for The Use of HPLC Columns

In order to extend the life of the column, we need to pay attention to the following points when using the HPLC column.

1. 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 HPLC columns; a sudden increase or decrease in column pressure will also impede the packing in the column, so it should be slowed when adjusting the flow rate when the valve is injected. The rotation of the valve cannot be too slow (as described above).

2. 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 water, and vice versa.

3. Generally speaking, the HPLC columns 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.

4. Choose to use a suitable mobile phase (especially pH) 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.

5. Avoid injecting samples with complex matrices, especially biological samples, directly into the HPLC columns. 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.

6. Rinse the column with a strong solvent often to remove impurities that remain 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 to 75 ml for routine analysis.