Equilibration Regeneration and Maintenance of HPLC Column

For a general reversed-phase HPLC column, it is washed and put into pure methanol (acetonitrile) or about 80% methanol (acetonitrile) water, and then both ends of the column are plugged tightly with plugs to avoid evaporation of the preservation solvent. There should be no special protection.

The maintenance of the liquid chromatographic column is divided into the balance of the chromatographic column, the regeneration of the chromatographic column, and finally the maintenance of the HPLC chromatographic column.

HPLC Column equilibration

The reversed-phase column is stored in acetonitrile/water after being tested by the factory. The new column should be flushed with 10-20 column volumes of methanol or acetonitrile. Please make sure that the mobile phase you use for analyzing the sample is miscible with acetonitrile/water. Spend enough time every day to equilibrate the column with the mobile phase, you will get the greatest “compensation” in dealing with the problem, and the life of your column will become longer!

High Purity HPLC Columns

Operation steps: Slowly increase the flow rate at the beginning of equilibrium, and equilibrate the HPLC chromatographic column with the mobile phase until a stable baseline is obtained (if the flow rate of buffer salt or ion pair reagent is low, it will take longer to equilibrate).

If the mobile phase used contains buffer salts, you should pay attention to the “transition” with pure water, that is, you must rinse with pure water for more than 30 minutes before starting the analysis every day, and then balance with the buffer salt mobile phase; after the analysis, you must rinse with pure water 30 After removing the buffer salt for more than minutes, rinse the column with methanol for 30 minutes to protect the column.

HPLC Column regeneration

Chromatographic columns that are used for a long time tend to decrease their efficiency (the number of theoretical plates on the column decreases). The chromatographic column can be regenerated, and a cheap pump should be used to regenerate the column in a qualified laboratory.

When regenerating the NH2-modified chromatographic column, since NH2 may exist in the form of ammonium ion, it should be washed with 0.1M ammonia water after washing with water, and then use water washed until the alkali solution completely flows out.

When cleaning the contaminated column, 0.05M dilute sulfuric acid can be used. If the simple organic solvent/water treatment cannot completely remove the impurities adsorbed on the silica gel surface, it is very effective to add 0.05M dilute sulfuric acid after washing.

HPLC Column maintenance

Use a pre-column to protect the analytical column, because silica gel has a certain solubility in the polar mobile phase/ionic mobile phase. Please try not to exceed the pH range of 2-7.5 for most reversed-phase columns. Avoid The composition and polarity of the mobile phase changing drastically.

Before use, it is necessary to degas and filter the mobile phase. If a polar or ionic buffer solution is used as the mobile phase, the column should be rinsed after the experiment and stored in a methanol or acetonitrile Medium. Chloride solvents are corrosive to a certain extent, so please pay attention to them when using them, and do not store such solvents in the column and connecting pipe for a long time to avoid corrosion.