Mobile Phase Equilibrium of the HPLC Columns
1. Thoroughly equilibrate the HPLC columns with at least 20 column volumes of mobile phase prior to sample testing. The mobile phase must use a chromatographic grade of solvent. Buffers such as those using an aqueous phase should be formulated daily to keep fresh to avoid bacterial production.
2. The mobile phase should be filtered with a microporous filter membrane before use to eliminate damage to the chromatographic system and HPLC column of the particles in the mobile phase. The buffer should be re-filtered after mixing with other mobile phases to avoid a change in solubility resulting in a new precipitate. Pure water should not be used as a mobile phase to flush the C18 HPLC columns to avoid column performance damage (add 5% organic solvent to the LC column, and at the same time achieve buffer salt cleaning. Also make the LC column more Easy to balance).
3. The mobile phase needs to be degassed and used to avoid the abnormal operation of the pump and detector caused by air bubbles. If the mobile phase used in the test differs significantly from the mobile phase used in the HPLC columns, it should be balanced using an over-distributed form. Avoid excessive column pressure increase due to sudden changes in the mobile phase or damage to the LC columns and instrumentation system due to crystallization of the mobile phase buffer salt. Normal phase liquid chromatography columns require longer equilibration times than reversed phase liquid chromatography columns.