Accurate measurements of the true column efficiency and of the instrument band broadening contributions in the presence of a chromatographic column.

A rapid and simple validated experimental protocol is proposed for the accurate determination of the true intrinsic column efficiency and for that of the variance of the extra-column volume of the instrument used, the latter being obtained without requiring the removal of the chromatographic column from the HPLC system. This protocol was applied to 2.1mm×100mm columns packed with sub-3 (2.7μm Halo Peptide ES-C18) and sub-2μm (1.6μm prototype) core-shell particles. It was validated by observing the linear behavior of the plot of the apparent column plate height versus the reciprocal of (1+k')(2) for at least three homologous compounds, with a linear regression coefficient R(2) larger than 0.999. Irrespective of the contribution of the several, different instruments used to the total band broadening, the same column HETP value was obtained within 5%. This new protocol outperform the classical one in which the chromatographic column is replaced with a zero dead volume (ZDV) union connector to measure the extra-column volume variance, which is subtracted from the variance measured with the column to measure the intrinsic HETP. This protocol fails because it significantly underestimates the system volume variance.