Effect of extra‐column volume on practical chromatographic parameters of sub‐2‐μm particle‐packed columns in ultra‐high pressure liquid chromatography

Effects of extra‐column volume on apparent separation parameters were studied in ultra‐high pressure liquid chromatography with columns and inlet connection tubings of various internal diameters (id) using 50‐mm long columns packed with 1.8‐μm particles under isocratic conditions. The results showed that apparent retention factors were on average 5, 11, 18, and 41% lower than those corrected with extra‐column volumes for 4.6‐, 3.0‐, 2.1‐, and 1.0‐mm id columns, respectively, when the extra‐column volume (11.3 μL) was kept constant. Also, apparent pressures were 31, 16, 12, and 10% higher than those corrected with pressures from extra‐column volumes for 4.6‐, 3.0‐, 2.1‐, and 1.0‐mm id columns at the respective optimum flow rate for a typical ultra‐high pressure liquid chromatography system. The loss in apparent efficiency increased dramatically from 4.6‐ to 3.0‐ to 2.1‐ to 1.0‐mm id columns, less significantly as retention factors increased. The column efficiency was significantly improved as the inlet tubing id was decreased for a given column. The results suggest that maximum ratio of extra‐column volume to column void volume should be approximately 1:10 for column porosity more than 0.6 and a retention factor more than 5, where 80% or higher of theoretically predicted efficiency could be achieved.