The correct use and maintenance of chromatographic columns are crucial, as even a slight mistake can reduce column efficiency, shorten service life, or even damage the column. During chromatographic manipulation, it is important to pay attention to the following questions in order to maintain the chromatographic column.

① Avoid sudden changes in pressure and temperature, as well as any mechanical vibrations. Sudden changes in temperature or dropping the chromatographic column from a high position can affect the filling condition inside the column; The sudden increase or decrease of column pressure can also stimulate the packing inside the column, so the flow rate should be adjusted slowly, and the rotation of the valve should not be too slow during valve injection (as mentioned earlier).

② The composition of the solvent should be gradually changed, especially in reverse phase chromatography, and it should not be directly changed from organic solvent to all water, and vice versa.

③ Generally speaking, chromatographic columns cannot be backwashed. Only when the producer indicates that the column can be backwashed, can impurities remaining in the column head be backwashed. Otherwise, recoil will quickly reduce column efficiency.

④ Choose the appropriate active phase (especially pH) to avoid damaging the stationary phase. Sometimes a pre column can be connected in front of the sampler. When the analysis column is bonded silica gel, the pre column is silica gel, which can make the active phase 'saturated' with silica gel before entering the analysis column, avoiding the dissolution of the silica gel matrix in the analysis column.

⑤ Avoid directly injecting samples with complex matrices, especially biological samples, into the column. It is necessary to preprocess the samples or connect a protective column between the injector and the chromatographic column. Protective columns are generally short columns filled with similar fixed phases. The protective column can and should be replaced frequently.

⑥ Regularly rinse the chromatography column with strong solvents to remove impurities stored inside the column. When cleaning, the displacement of active phases in the flow path system should gradually transition with miscible solvents, and the volume of each active phase should be about 20 times the volume of the column, which requires 50-75ml for conventional analysis.

Here are some cleaning solvents and sequences for chromatography columns for reference: the silica gel column is washed with n-hexane (or heptane), dichloromethane, and methanol in sequence, and then washed in reverse order. All solvents must be strictly dehydrated. Methanol can wash away residual strong polar impurities, while hexane reactivates the surface of silica gel. Wash the reverse phase column with water, methanol, acetonitrile, and chloromethane (or chloroform) in sequence, and then rinse in reverse order. If the active phase used for the next analysis does not contain buffer solution, the step of rinsing with water can be omitted. Chloromethane can wash away residual non-polar impurities, and repeated injection of 100-200 μ l tetrahydrofuran several times during methanol (acetonitrile) washing can help remove strongly hydrophobic impurities. A mixed solution of tetrahydrofuran with acetonitrile or methanol can remove lipids. Sometimes dimethyl sulfoxide is injected several times. In addition, gradient elution with acetonitrile, acetone, and trifluoroacetic acid (0.1%) can remove protein contamination.

The cation exchange column can be washed with dilute acid buffer solution, and the anion exchange column can be washed with dilute alkali buffer solution to remove salts with strong exchange performance. Then, rinse with water, methanol, dichloromethane (except for organic matter adsorbed on the surface of the stationary phase), methanol, and water in sequence.

⑦ When storing chromatography columns, the column should be filled with acetonitrile or methanol, and the column joints should be tightened to prevent solvent evaporation and drying. Do not leave the buffer solution in the column overnight or for a longer period of time.

⑧ During the use of the chromatographic column, if the pressure increases, one possibility is that the sintered filter is clogged. In this case, the filter should be replaced or removed for cleaning; Another possibility is that large molecules enter the column and contaminate the column head; If the column efficiency decreases or the chromatographic peak deforms, the column head may collapse and the dead volume may increase.

When the latter two situations occur, carefully unscrew the column joint and use clean small steel to remove the column head packing to a height of 1-2mm (pay attention to removing the contaminated packing completely), and then level the packing inside the column. Then fill the chromatography column with a stationary phase moistened with an appropriate solvent (the same as inside the column), flatten it, and tighten the column joints. After this treatment, the column efficiency is improved, but it is difficult to restore it to the level of a new column.