Cleaning Solvent and Sequence of the HPLC Column

You often need to flush the HPLC column with a strong solvent 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, and the volume of each mobile phase should be about 20 times the volume of the column.

The cleaning solvents and sequences of HPLC columns are listed below. For reference:

The silica gel column is washed sequentially with n-hexane (or heptane), dichloromethane, and methanol, and then washed sequentially in reverse order. All solvents must be strictly dehydrated. Methanol can wash away residual strong polar impurities, and hexane will reactivate the surface of the silica gel.

The reversed phase column is washed successively with water, methanol, acetonitrile, methyl chloride (or chloroform), and then washed sequentially in reverse order. The mobile phase used is free of the buffer, so the last step of rinsing with water can be omitted. Such as reversed phase C4 HPLC column

A mixture of tetrahydrofuran and acetonitrile or methanol can remove lipids. Sometimes dimethyl sulfoxide is injected several times. In addition, acetonitrile, acetone, and trifluoroacetate are used. Gradient elution of acetic acid (0.1%) can remove protein contamination.

The cation exchange column can be washed with dilute acid buffer, and the anion exchange column can be washed with dilute alkali buffer to remove the exchangeable salt, then remove with water, methanol, and dichloromethane. The organic matter adsorbed on the surface of the stationary phase), methanol, and water are washed successively. Such as SCX HPLC Column