Effect of the pressure on pre-column sample dispersion theory, experiments, and practical consequences

• The effect of pressure on dispersion along 0.5/1.0 mm i.d. × 50 cm long open tubes was measured.
• The time variance increases linearly by 70% with increasing pressure from 10 bar to 1 kbar.
• Intrinsic HETP values cannot be accurately measured with the ZDV union connector method.
• The apparent column HETP decreases with increasing flow rate due to pre-column dispersion.
• The pre-column volume should be minimized in very high pressure liquid chromatography.

The effect of the pressure on the dispersion of a low molecular weight compound along 0.508 and 1.016 mm i.d. × 50 cm long open circular tubes was investigated theoretically and experimentally. The theoretical predictions were based on the early models of dispersion derived by Aris and Taylor (1953) and on the approximate model of Alizadeh for the time moments (1980). Experimentally, the system pressure was increased at constant flow rate (0.15 mL/min) from less than 20 to nearly 1000 bar by using a series of capillary tubes (25 μm i.d. PEEKSIL tubes) of increasing flow resistances placed upstream the detection cell of a commercial very high pressure liquid chromatograph (vHPLC) but downstream the 50 cm long tube. Theoretical and experimental results agree that the peak variance increases linearly with increasing pressure in the tube volume. The relative increase of the peak variance is 7% above that measured at low pressure (<20 bar) for each 100 bar increment in the tube volume. This result confirms that accurate measurements of the column efficiency corrected for extra-column contribution cannot be made by replacing the column with a zero dead volume union connector, because the pressures applied in the pre-column volume are significantly different in these two cases. This work shows also that increasing the pressure in the pre-column volume by increasing the flow rate affects the apparent column efficiency that is measured, independently of the direct effect of the flow rate. For a 2.1 × 50 mm column packed with 1.3 μm core–shell particles run with a classic Acquity system, the associated relative decreases of the column efficiency are expected to be −30%, −20%, and −5% for retention factors of 1, 3, and 10, respectively. The column HETP is no longer independent of its length.