Efficiency of short, small-diameter columns for reversed-phase liquid chromatography under practical operating conditions

Prototype small-size (1.0 mm I.D., 5 cm long) columns for reversed-phase HPLC were evaluated in relation to instrument requirements. The performance of three types of columns, monolithic silica and particulate silica (2 μm, totally porous and 2.6 μm, core-shell particles) was studied in the presence of considerable or minimal extra-column effects, while the detector contribution to band broadening was minimized by employing a small size UV-detector cell (6- or 90 nL). A micro-LC instrument having small system volume (<1 μL) provided extra-column band variance of only 0.01-0.02 μL2. The three columns generated about 8500 theoretical plates for solutes with retention factor, k > 1-3 (depending on the column), in acetonitrile/water mobile phase (65/35 = vol/vol) at 0.05 mL/min, with the instrument specified above. The column efficiency was lower by up to 30% than that observed with a 2.1 mm I.D. commercial column. The small-size columns also provided 8000-8500 theoretical plates for well retained solutes with a commercial ultrahigh-pressure liquid chromatography (UHPLC) instrument when extra-column contributions were minimized. While a significant extra-column effect was observed for early eluting solutes (k < 2-4, depending on column) with methanol/water (20/80 = vol/vol) as weak-wash solvent, the use of methanol/water = 50/50 as wash solvent affected the column efficiency for most analytes. The results suggest that the band compression effect by the weak-wash solvent associated with partial-loop injection may provide a practical means to reducing the extra-column effect for small-size columns, while the use of an instrument with minimum extra-column effect is highly desirable.