Details

Autor(en) / Beteiligte

• Gritti, Fabrice
• Guiochon, Georges

Titel

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

Ist Teil von

• Journal of Chromatography A, 2014-01, Vol.1327, p.49-56

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2014

Quelle

Access via ScienceDirect (Elsevier)

Beschreibungen/Notizen

• •A simple protocol is proposed for the measurement of the true column and system performances.•This protocol is based on the plot of the apparent efficiency versus the reciprocal of (1+k′)2.•The protocol was validated for narrow-bore columns packed with sub-3μm core–shell particles.•The protocol was applied to new narrow-bore columns packed with sub-2μm core–shell particles.•The intrinsic minimum reduced plate height of these prototype columns is 1.50±0.05. 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 R2 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.