Preventing dead volume in chromatography

The dead volume of a chromatography column corresponds to the volume of mobile phase that is required to fill all pores and spaces in a column packing. In practice, however, this dead volume plays a smaller role than the dead volume of an entire HPLC system.

Minimising dead volume

In order to obtain a satisfactory peak resolution even with short retention times, particular attention must be paid to minimise dead volume. Dead volume is the volume that flows through the system from the injection to the detector cell without interacting with the column. Dead volume should be determined before a measurement if there are substances that elute very early or if the separation results of the substances are not satisfactory.

Determination of dead volume

To determine dead volume, connect the injection valve directly to the detector cell. Inject a defined volume of a UV-active substance such as acetone. Dead volume can be determined from the peak of this substance via the retention volume. Very good systems have values of up to 20 µl dead volume, whereby values up to 50 µl are considered acceptable.

The inner diameter of the capillaries also has an effect on dead volume. With an internal diameter of 0.1 mm, a dead volume of 0.1 µl per cm column is calculated. With an inside diameter of 1 mm, a dead volume of 8 µl per cm column is to be expected.

The injected volume itself has no influence on dead volume. Take note to avoid adding the proportion of solvent in the dead volume to the proportion of solvent in the injected sample as this leads to a dilution effect which negatively influences the selectivity.

To prevent dead volume, it is best to connect the column directly to the injection valve and to the detector cells. Inserts such as in-line filters lead to backmixing. A uniform temperature in the injector and detector also helps to minimise dead volume.