Performance optimizing injection sequence for minimizing injection band broadening contributions in high efficiency liquid chromatographic separations.

The observed performance when using high efficiency, low volume (U)HPLC columns has and continues to be limited by the extra column dispersion introduced by the chromatographic system. Even with the latest UHPLC instruments the injection and detection systems are still major contributors to peak broadening, especially when using 50 mm × 2.1mm columns. A previously described injection technique now termed Performance Optimizing Injection Sequence (POISe) was shown to reduce or eliminate the impact of the injection system on the observed chromatographic performance. The POISe technique involves injecting a defined volume of weak solvent along with the sample in order to increase retention factors during sample loading. In the present study, a newly developed equation describing the phenomena involved during sample introduction is presented and shows that analyte bands are compressed at the head of the column in proportion to their retention factor independent of the elution mode (i.e. gradient or isocratic). This phenomenon is termed isocratic focusing and is shown to be most effective with analytes having retention factors in the range of 0.4-3. Additionally, it is shown that gradient compression plays a minor role in band compression when using this technique with analytes having retention factors of 1 or higher. The POISe technique is further investigated experimentally to determine its optimum configuration. It is also demonstrated to be effective with different HPLC and UHPLC instrument platforms and different high efficiency columns, such as those packed sub-2 μm and core-shell particles.