On-tubing fluorescence measurements of the band broadening of contemporary injectors in ultra-high performance liquid chromatography

We report on a detailed study of the injection contribution to band broadening in contemporary UHPLC-instruments, using either flow-through needle or fixed loop injection (full loop). Using on-tubing fluorescence measurements at the outlet of the injector valve, very localized and undisturbed measurements were obtained. Varying both the flow rate and the injected volume allowed to split the injection variance (σV2,inj) in a volumetric component (related to the amount injected) and a hydrodynamic component (related to the flow rate). For the flow-through needle injector and for the small injection volumes (<2 μL) typically used in UHPLC, it was found that the volumetric contribution (i.e. the part of σV2,inj, that increases with increasing injection volume) is given by a value of σV2,inj,vol = 0.8 to 1·Vinj2 rather than by the value of 0.125 to 0.2·Vinj2 that is normally assumed in literature. For the hydrodynamic contribution to σV2,inj, (i..e, the part which remains present even for very small injection volumes), a clear increase in dispersion with flow rate is found, reaching a plateau around 0.8ml/min of 0.6 μL² or 1.2 μL² for the 75 μm and 120 μm needle seat capillaries respectively. The difference between both shows the clear advantage of using a low dispersion 75 μm injection needle seat capillary. For a loop-type injector operated in a full-loop mode, the increase in peak variance with the injection volume is much less pronounced, leading to a total injector variance given by σV2,inj = 0.34 μL² + 0.12·Vinj2 over the entire range of investigated injection volumes of 1.1 μL up to 4.5 μL when using 120 μm or narrower ID loops. This expression was nearly completely independent of the flow rate. For larger ID sample loops, a clear increase of peak variance with flow rate at fixed injection volume was observed (σV2,inj increases with 20% for a 170 μm ID loop and with 70% for a 220 μm ID loop from 0.3 to 1 ml/min).