Reduction of the extra-column band dispersion by a slow transport of a sample band from the injector to the column in isocratic reversed-phase liquid chromatography

Abstract

Extra-column band dispersion during the transport of a sample band from the injector to the column can be reduced by a flow rate program starting with a low flow rate until the sample band has approached to, or just entered into the column, followed by an increased flow rate suitable for the solute separation in the column. Such a sample introduction method increased the plate counts of a 50 mm long column, 1.0 or 2.1 mmID, especially for early-eluting solutes by up to several times compared to a conventional elution method, when a 0.254 mmID, 15.2 cm connection tubing was used. Increase in plate counts of up to 50–70% was possible for solutes with retention factors smaller than 1.0 for the columns connected with a 0.13 mmID, 15 cm tube. The method also seems to reduce the contribution of the void space at the column inlet to the band dispersion. The elution method including a slow transport of the sample band in the pre-column space of 10 μL or less may require a little longer separation time than normal elution, but it was shown to be effective for increasing the observed efficiency of a small column for solutes with small retention factors.