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.
Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600, USA.
Fabrice
F
Gritti
Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600, USA. Electronic address: guiochon@utk.edu.
Georges
G
Guiochon
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation*; D010316:Particle Size; D016062:Porosity
Column efficiency; Extra-column band broadening; Homologous compounds; Intrinsic efficiency; Sub-2μm core–shell particles; vHPLC systems