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 used “as is” with electrochemical detection. Residues from the production process need to be washed out to avoid contamination and high background current. Washing advise for reversed phase columns is 24 hours with high organic modifier (ca 75%) at a low flow rate. Before switching to/from mobile phase, wash with 10% organic in water to avoid precipitation of salts.

Other type of columns need to be treated with care as well. Ion exchange columns can be washed for some time with the recommended solvent by the manufacturer. Never apply organic solvents to enzyme loaded columns as it will make the enzymes inactive.

As a rule of thumb, the background current should not change much with or without the column installed. It is therefore advisable to measure the current also without having the column installed. For that purpose, connect the tubing with a connector instead of the column and measure the I-cell, the uncompensated current, for a while.

In all cases, when replacing a column take enough time (volume) to fill the HPLC incl column with the mobile phase before putting the cell in the flow path. Never switch on the cell if it is still filled with only water or some organic solvent. The cell needs ions for proper operation, or damage to the cell may occur.

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. In practice, it is most convenient to replace a column at the end of a working day so stabilization can occur overnight.