Reduction of the extra-column band dispersion by a slow transport and splitting of a sample band in isocratic reversed-phase liquid chromatography.

Abstract Sample introduction method was studied to reduce the extra-column effect in reversed-phase HPLC. Slow transport of a sample band (SToSB) in the pre-column space followed by the introduction of the band into the column at a near-optimum flow rate resulted in larger plate counts for a 1.0 mmID, 5 cm long column as much as 1.4-1.6 times for solutes with a retention factor (k) of 0.5-1.8 compared to a conventional elution method. Further reduction of the extra-column effect was possible by orthogonally splitting the sample band (SplSB) by flow switching during its slow transport followed by the introduction of the leading part of the band into the column. In this case, increased plate counts of up to 2-3 times for solutes with k of 0.5-1.8 were observed for a 1.0 mmID, 5 cm column. The sample introduction method, SToSB in the injector and the pre-column tube of a few μL, was found to reduce the extra-column band variance by 0.4-0.5 μL2 for an UHPLC system with the extra-column volume (Vextra) of ca. 4.6 μl and the system variance (σextra2) of 1.1 μL2 at flow rate of 100 μL/min, while SToSB and subsequent SplSB were found to be more effective, reducing σextra2 by about 0.8 μL2. With an UHPLC instrument with Vextra of about 10 μL and σextra2 of ca. 3.6 μL2 at flow rate of 300 μL/min, 1.4-2.1 times as many plate counts were observed with SToSB and SplSB compared to the normal elution method for early-eluting solutes with k=0.25-1.7 for a column, 2.1 mmID, 5 cm long. With this UHPLC instrument, SToSB and/or SplSB resulted in the reduction of σextra2 by 1.2-2.2 μL2.