Effect of column dimension on observed column efficiency in very high pressure liquid chromatography

The effect of extra-column volume on observed linear velocity was investigated for columns of various internal diameters in very high pressure liquid chromatography. The results showed that the observed linear velocities were approximately 4.5, 9.5, 16.8, and 39.5% lower than the linear velocities corrected for the extra-column volume contribution for 4.6, 3.0, 2.1, and 1.0 mm internal diameter columns, respectively. An empirical relationship between extra-column band broadening and extra-column volume was obtained using 50 cm long tubings of various internal diameters. The peak variance from the extra-column volume is near linearly proportional to the square of the extra-column volume for tubings with 0.0635–0.178 mm (0.025–0.07 in.) i.d. using a 50/50 acetonitrile/water mobile phase at flow rates greater than 0.3 mL/min. The effect of column internal diameter and column length on observed efficiency was studied using 50 mm columns with four different column internal diameters and 2.1 mm i.d columns with three different lengths. The results showed that the observed column efficiencies for 3.0, 2.1, and 1.0 mm internal diameter columns were 18, 33, and 73% lower than that for a 4.6 mm internal diameter column for benzophenone (k = 5.5), respectively. An approximate 20% decrease in theoretical plate number was observed for propiophenone (k = 3.3) using a 50 mm × 2.1 mm column packed with 1.7 μm particles compared to a 150 mm × 2.1 mm column packed with 5.0 μm particles, while the former column provided 9 fold faster separation. It is the column to extra column volume ratio instead of absolute extra-column volume that determines the degree of extra-column band-broadening in VHPLC.