Accurate measurement of dispersion data through short and narrow tubes used in very high-pressure liquid chromatography.

An original method is proposed for the accurate and reproducible measurement of the time-based dispersion properties of short L< 50cm and narrow rc< 50μm tubes at mobile phase flow rates typically used in very high-pressure liquid chromatography (vHPLC). Such tubes are used to minimize sample dispersion in vHPLC; however, their dispersion characteristics cannot be accurately measured at such flow rates due to system dispersion contribution of vHPLC injector and detector. It is shown that using longer and wider tubes (>10μL) enables a reliable measurement of the dispersion data. We confirmed that the dimensionless plot of the reduced dispersion coefficient versus the reduced linear velocity (Peclet number) depends on the aspect ratio, L/rc, of the tube, and unexpectedly also on the diffusion coefficient of the analyte. This dimensionless plot could be easily obtained for a large volume tube, which has the same aspect ratio as that of the short and narrow tube, and for the same diffusion coefficient. The dispersion data for the small volume tube are then directly extrapolated from this plot. For instance, it is found that the maximum volume variances of 75μm×30.5cm and 100μm×30.5cm prototype finger-tightened connecting tubes are 0.10 and 0.30μL(2), respectively, with an accuracy of a few percent and a precision smaller than seven percent.