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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 (σ) 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 σ, that increases with increasing injection volume) is given by a value of σ = 0.8 to 1·V rather than by the value of 0.125 to 0.2·V that is normally assumed in literature. For the hydrodynamic contribution to σ (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 σ = 0.34 μL² + 0.12·V 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 (σ 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).

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a b s t r a c tLinear gradient programs are very frequently used in reversed phase liquid chromatography to enhance the selectivity compared to isocratic separations. Multi-linear gradient programs on the other hand are only scarcely used, despite their intrinsically larger separation power. Because the gradient-conformity of the latest generation of instruments has greatly improved, a renewed interest in more complex multisegment gradient liquid chromatography can be expected in the future, raising the need for better performing gradient design algorithms. We explored the possibilities of a new type of multi-segment gradient optimization algorithm, the so-called "one-segment-per-group-of-components" optimization strategy. In this gradient design strategy, the slope is adjusted after the elution of each individual component of the sample, letting the retention properties of the different analytes auto-guide the course of the gradient profile. Applying this method experimentally to four randomly selected test samples, the separation time could on average be reduced with about 40% compared to the best single linear gradient. Moreover, the newly proposed approach performed equally well or better than the multi-segment optimization mode of a commercial software package. Carrying out an extensive in silico study, the experimentally observed advantage could also be generalized over a statistically significant amount of different 10 and 20 component samples. In addition, the newly proposed gradient optimization approach enables much faster searches than the traditional multi-step gradient design methods.

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