How to prepare and install a new column?

Applies to

Chromatography, reversed phase columns for ECD

Answer

When installing a new column, a few things have to be kept in mind. New columns from a supplier are usually not suitable to be immediately connected to an electrochemical detector. Residues from the column production process need to be washed out to avoid contamination and high background current. The general advice is to always check the recommended solvent in the column care document from the manufacturer, and to make sure to redirect the column effluent to waste, bypassing the cell. During the process, have no cell connected behind the column.

Additional washing advice for reversed phase columns:

• wash for 1 hour with an intermediate level of organic modifier (ca 20%), followed by at least 16 hours of mobile phase at the application flow rate. This long time of rinsing is particularly important when the mobile phase contains ion pairing agent.
• Never apply organic solvents to enzyme loaded columns (IMER’s), as it will make the enzymes inactive.
• In all cases, make sure to rinse the HPLC and the new column with enough mobile phase before connecting the cell in the flow path. The cell needs ions for proper operation, or damage to the cell may occur (never switch on the cell if there is only water and/or organic solvent running through). In practice, it is most convenient to replace a column before the end of a working day, so stabilization can occur overnight. After filling the HPLC system and flow cell completely with mobile phase, continue with the stabilization process by switching on the cell and monitoring the baseline.
• As a rule of thumb, the background current should not change much with or without the column installed. Measure the current also without column installed and compare. For that purpose, connect the tubing with a connector instead of the column and measure the I-cell, the uncompensated current, for a while.