Cleaning of HPLC Reversed Phase Column
When the HPLC Reversed-Phase column is contaminated, its chromatographic behavior will be different from the uncontaminated ones. There would be back pressure problems happened. Therefore, the contaminated reversed-phase columns must be cleaned.
Common Cleaning Method
Generally, the contamination is caused by the accumulation of strongly retained materials during the repeated sampling process. Basically, their chromatographic behavior could be restored with simple procedures to remove the contaminants. After multiple operations, the column could be rinsed with 90-100% solvent B (the stronger solvent in the dual solvent reversed-phase system) to remove contaminants. If a buffer system is employed, do not directly switch to a strong solvent.
Suddenly switching to a strong organic solvent may precipitate the buffer in the HPLC flow system, which can cause larger problems, such as blockage of the column, pump Leakage, or injection valve shaft failure. In general, the solvent used is increased based on the strength of the solvent, and often the last solvent is very hydrophobic (such as ethyl acetate), which can be used to dissolve the non-polar substances, such as lipids and oils.
Protein Residue Cleaning Method
If a biological material on a reversed-phase column, such as plasma or serum accumulates, the operator must use a different cleaning procedure. In most cases, pure organic solvents such as acetonitrile or methanol could not dissolve the polypeptide and protein, which means it could not effectively clean the column. However, a mixture of an organic solvent with a buffer, an acid, an ion-pairing agent or the like can be used to effectively dissolve the proteins.