Why would a new C18, C8, C4 (or other reversed-phase) column show longer retention than an older, used column of the same part number when running an isocratic method or a gradient method? - WKB57068

ENVIRONMENT

• C18 columns
• Column comparison

ANSWER

New C18 columns tend to have more C18 ligand and endcap groups, than do older, used C18 columns.

Biomolecules might undergo more non-specific adsorption on new columns which might lead to slightly longer retention times.

ADDITIONAL INFORMATION

There are many possible reasons why a new C18 column (compared with an old C18 column of the same part number) exhibits longer retention. 

A common cause is that old, used columns have lost C18 ligand as a result of hydrolysis. 

As a column loses ligand, the surface becomes less and less hydrophobic. Installation of a new C18 column of the same part number may show significantly longer retention in some cases (especially if the used column is several years old).

In addition, old columns may have also lost endcap groups through hydrolysis.  When a column loses endcap groups, a higher number of silanol groups are exposed. This results in a more polar particle surface and increases the amount of negative surface charge on the older particles. This can have a dramatic effect on retention and selectivity.

If you are analyzing biomolecules:  Repeated injections of a biomolecule such as bovine serum albumin might help to passivate the column and minimize non-specific adsorption of your analytes on the new column.