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CHROMtalks - Fundamentals and Principles of Extra Column Band Broadening in UHPLC: A Tutorial

With the introduction of sub-2 µm (superficially) porous particles around 15 years ago, significant improvement in separation speed and resolution became possible. These small particles allowed the use of shorter columns and higher velocities and were often packed in smaller ID columns to avoid thermal effects (viscous heating), reduce solvent consumption, and improve compatibility with MS (lower flow rates). These high-performance small-volume columns, however, are very sensitive to dispersion occurring in the rest of the fluidic path of the instrument such as in the injector, mobile-phase preheater, connection tubing, and the detector cell or interface, such as with a mass spectrometer. It was therefore not possible to use these narrow-bore column on “standard” HPLC instrumentation, because they required the use of so-called ultrahigh-pressure (UHPLC) instruments that not only have a higher maximum operating pressure, but also have reduced extra column dispersion. After introducing the importance and consequences of extra column dispersion, this tutorial will highlight the different contributions to extra column broadening occurring in modern UHPLC instrumentation. Methodologies and the pro- and cons to estimate the amplitude of extra column dispersion will be critically reviewed and some easy hands-on tests to reduce and optimize your system configuration will be provided.