Flow rate dependent extra-column variance from injection in capillary liquid chromatography

• The extra-column variance in capillary LC due to the injection valve was characterized.
• The extra-column variance was found to vary from 34 to 23 nL.
• The variance was due to both injection volume and flow rate.
• Column efficiency was compromised by ∼130% due to injector dead volume.

Efficiency and resolution in capillary liquid chromatography (LC) can be significantly affected by extra-column band broadening, especially for isocratic separations. This is particularly a concern in evaluating column bed structure using non-retained test compounds. The band broadening due to an injector supplied with a commercially available capillary LC system was characterized from experimental measurements. The extra-column variance from the injection valve was found to have an extra-column contribution independent of the injection volume, showing an exponential dependence on flow rate. The overall extra-column variance from the injection valve was found to vary from 34 to 23 nL. A new mathematical model was derived that explains this exponential contribution of extra-column variance on chromatographic performance. The chromatographic efficiency was compromised by ∼130% for a non-retained analyte because of injection valve dead volume. The measured chromatographic efficiency was greatly improved when a new nano-flow pumping system with integrated injection valve was used.