Separation performance in chromatography has been extensively studied since the dawn of the technique. Although the basic principles of band broadening and the resulting separation performance in isocratic elution are in general well known and understood, this is much less the case for gradient separations. In this tutorial, first the basic principles, concepts and parameters that determine separation performance, peak width and variance and analysis time in isocratic separations are reviewed. This is subsequently used to discuss the parameters that affect peak width in gradient elution, together with the concepts of plate count and plate height in this elution mode. In addition, the effect of peak compression in gradient elution is elaborated. Finally, the effect of extra-column dispersion on separation performance in gradient elution is discussed, and an overview of how these contributions can be experimentally evaluated is given.
Keywords: Extra-column band broadening; Gradient elution; Peak compression; Plate height; Separation performance.
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