Preventing Dead Volume in Chromatography

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

Minimising dead volume

To achieve satisfactory peak resolution even with short retention times, special attention must be paid to minimising dead volume. Dead volume is the volume that flows through the system from injection to detector cell without interacting with the column. Dead volume should be determined prior to a measurement if there are compounds that elute very early or if the separation results of the compounds are not satisfactory.

Determination of dead volume

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

The inner diameter of the capillaries also affects the dead volume. With an inner diameter of 0.1 mm, a dead volume of 0.1 µl per cm column is calculated. With an inner diameter of 1 mm, a dead volume of 8 µl per cm column can be expected.

The injected volume itself does not affect the dead volume. Note that the percentage of solvent in the dead volume should not be added to the percentage of solvent in the injected sample, as this will cause a dilution effect which will negatively affect selectivity.

The best way to avoid dead volume is to connect the column directly to the injection valve and detector cells. Inserts such as in-line filters cause backmixing. A uniform temperature in the injector and detector also helps to minimise dead volume.