Adsorption phenomena often occur between the packing media (stationary phase) of a reversed-phase silica column and the sample:
Ionic Adsorption between residual silanol groups and basic substances;
Coordination Adsorption between metal impurities on the silica surface and metal-chelating substances;
Accumulation of highly hydrophobic substances within the column.
The above phenomena can lead to decreased column efficiency, peak tailing, and poor reproducibility.
1. Removal of Hydrophobic Substances
Use organic solvents (Acetonitrile, Methanol, or a mixture of Acetonitrile and THF) for cleaning.
Procedure: Considering the solubility of the sample components in organic solvents, select an organic solvent with a higher concentration than the mobile phase or a solvent with lower polarity for flush cleaning.
Precaution: In this case, it is necessary to watch out for salt precipitation from the mobile phase when transitioning to high organic concentrations.
2. Removal of Basic Substances (Metal-Coordinating Substances)
Use a mixture of an acidic aqueous solution and an organic solvent (e.g., 0.1% Phosphoric acid or 0.1% TFA mixed with Acetonitrile or Methanol) for flush cleaning.
Technical Tips
Cleaning Duration: Generally, flushing the column with 5–10 column volumes is sufficient.
Irreversible Adsorption: If the adsorbed components have extremely strong affinity, column performance may not fully recover even after thorough cleaning.
pH Limits: Always fully consider the pH range of the mobile phase and cleaning solvents to avoid damaging the silica matrix or the bonded phase.