No.9 Regenerating Columns

In reversed phase HPLC, column deterioration causes poor peak shapes or shortened retention time. The column deterioration results from packing materials' chemical alteration such as loss of bonded phase like C18 or dissolution of silica-gel as the base material. Consequently columns in such condition are difficult to restore and reuse.

Meanwhile, 100% aqueous mobile phase in an ODS column sometimes entails steep reduction in retention of compounds as in the figure below. Many may think the reduction of this retention time is due to the column deterioration. However this is not the case. Rather, the cause is considered to be the decrease of apparent hydrophobicity of packing material due to polarity difference between the water of mobile phase and the surfaces of packing material bonded with C18 functional groups and become difficult to solvate. Coping with this and restoring the initial retention time is easily achieved by flushing the column with 10 times its volume of mobile phase containing 50% organic solvent. This situation is believed to result from the decrease of the repulsion between the eluant and the C18 functional groups. If the retention time reduction occurs when using 100% aqueous mobile phase, try to flush with organic solvent/ water mixture to regenerate the column.