科研之友
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摘要
On-column solute focusing is a simple and powerful method to decrease the influence of precolumn band spreading and increase the allowable volume injected increasing sensitivity. It relies on creating conditions so that the retention factor, k', is transiently increased during the injection process. Both solvent composition and temperature control can be used to effect solute focusing. In the case of temperature, the release of the transiently delayed solute band requires increasing the temperature rapidly and with a minimum of radial thermal gradients. Thus, the focus of attention in temperature-based efforts to carry out on-column focusing has been on capillary columns. As a result, the benefits of this simple and reliable approach, temperature-assisted solute focusing or TASF, are not available to those using larger diameter columns, in particular the highly successful 2.1 mm inside diameter columns. Based on considerations of thermal entrance length at the volume flow rates used with 2.1 mm inside diameter columns, TASF would not be effective with such columns. However, we determined that the thermal entrance length for a 1.0 mm inside diameter precolumn is sufficiently short, about 2 mm, that it could work as a pre-column before a 2.1 mm inside diameter analytical column. Finite element calculations demonstrate that a 1.0 x 20 mm precolumn packed with 5 mu m reversed phase particles is effective at a flow rate of 250 mu L/min, suitable for the 2.1 mm inside diameter column. Eight 1-cm(2) Peltierdevices are used to heat (and cool) the precolumn. The computed axial temperature profile shows that the center of the column heats more rapidly than the ends. Based on the changes in back pressure, the full temperature transient from 5 degrees C (focus) to 80 degrees C (release) takes about 10 s. Experimental van Deemter curves indicate that the reduced velocity in the precolumn at 250 mu L/min flow rate is about 50. Nonetheless, about 1000 theoretical plates are generated. When operating as a precolumn, clear advantages are seen for solutes across a range of modest k' values (2.2-23.4 at the separation conditions at 65 degrees C) using TASF alone (5 degrees C) with 50 mu L injection volumes of methyl through n-butyl parabens, and with 100 mu L injections that also include solvent-based focusing (90:10 aqueous/acetonitrile sample, 80:20 mobile phase).
- 出版日期2017-11-10
