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The recent advances in chromatography have brought us columns that can produce such sharp and narrow peaks that the extra-column volumes of the current generation of instruments inevitably causes an efficiency loss. This also puts a serious strain on the ability to measure the "column-only" efficiency of these columns, especially when using weakly retained components, which are most sensitive to the hydrodynamic quality of the packing. In column performance studies, the effect of extra-column band broadening (ECBB) is traditionally eliminated using the variance subtraction method, owing its popularity to the fact that it is based on a simple subtraction. However, by focusing only on its variance, information describing the shape and the asymmetry of the peak is discarded. Since the variance subtraction method can only be correctly applied when the variance is calculated via the 2nd order moment integral, the observed “column-only” variance and its associated plate height may gravely overestimate the band broadening compared to the parameters that are more relevant for the practical reality such as the plate heights based on the half height width or on the 4-5- or even 6- width. The peak deconvolution method on the other hand can filter out the extra-column contributions while preserving all the shape features of the peak. The present study provides evidence for the fact that the peak deconvolution method can be applied to the current generation of high speed and high efficiency columns and instruments and indeed allows to eliminate all ECBB while retaining all the peak asymmetry information of the “column-only” dispersion. This has been shown by testing the same column on 3 different systems, with different extra-column volumes and detector, and showing that, after deconvolution, the resulting "column-only" peaks remain (slightly) asymmetric but coincide perfectly, at least within the variability on the flow and injection reproducibility of the instrument.

The advances in particle and column manufacturing (nowadays offering sub-2µm fully porous and core-shell particle columns capable of withstanding inlet pressures of 1000 bar and more) that have been made in the past decade have opened the road towards unprecedented separation efficiencies and speeds. Using these columns in combination with the high-temperature and ultra-high pressure modes in which modern day HPLC instruments can be operated, one frequently runs into a situation wherein the quality of the columns and separation methods has improved to such an extent that the quality of the instrument (and its contribution to the band broadening) has become the limiting factor. In the present contribution, we will review the most important factors affecting the instrument contribution (tubing length and diameter, injection volume, detector size) on the observed column performance and illustrate these effects using a fully optimized state-of-the-art instrument in combination with the most efficient columns that are available today. Emphasis will also be put on a correct determination of the column and system efficiency, discussing the tedious problem of the use of the method of moments to determine the variance of the chromatographic peaks. In addition, an overall view on the optimal choice of particle and column diameter (cf. the trade-off between 4.6 mm columns and 2.1 mm columns) as a function of the required separation efficiency will be given by presenting an extension of the kinetic plot method which allows to account for the influence of the fixed extra-column volumes that exist in a given HPLC or UHPLC instrument.

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Plate Height and Kinetic Plot Analysis of the Performance of New LC Columns

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