How many empty column volumes should I use to re-equilibrate a reverse-phase column between each injection? - WKB49967

ENVIRONMENT

• Reverse-phase columns
• Re-equilibration

ANSWER

5 to 10 column volumes

ADDITIONAL INFORMATION

For example: One empty column volume (pi * r² * L) for a 4.6x150mm column equals 2.5 mL.

It is recommended to re-equilibrate with between 5 and 10 column volumes (12.5 to 25 mL) for most reverse-phase columns.

First, check for reproducible retention times if re-equilibrating with 10 column volumes. If this looks reproducible, then, if desired, you can try re-equilibrating with only 9 column volumes (22.5 mL). If this looks good, you can try only 8 column volumes, and so on.

Brand new reverse-phase columns will usually require more re-equilibration than older, used columns. This is because older columns may have lost some bonded endcap groups and/or ligand. As a result, older columns may have a more polar particle surface. Because the older column particle surface would be more polar, it can be re-equilibrated with less volume; the particle surfaces do not want the residual organic solvents to remain at the particle surface. Instead, the more polar particle surfaces of older columns prefer a higher water content, which is why they can be re-equilibrated with less volume.  

A brand new column will have full coverage of both endcap groups and ligand. As a result, the particle surface will be more nonpolar; the particle surfaces prefer the higher organic mobile phase. When re-equilibration begins, the new column's particle surface does not want to "let go" of the higher organic content as quickly as a used column. This is why it may take up to 10 full column volumes to re-equilibrate a new reverse-phase column.

Be cautious of validating a method to re-equilibrate a reverse-phase column for too small of a volume. To get the best reproducible retention times after re-equilibration, it is recommended to not only verify how much volume is required on an older (used) reverse-phase column but also to compare that to a new column.