title: Effect of extra-column volume on practical chromatographic parameters of sub-2-μm particle-packed columns in ultra-high pressure liquid chromatography.
abstract: 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.
authors: Merck & Co, Inc, Rahway, NJ, USA. nawu@celgene.com
authors: Naijun
authors: N
authors: Wu
authors: Ashley C
authors: AC
authors: Bradley
authors: Christopher J
authors: CJ
authors: Welch
authors: Li
authors: L
authors: Zhang
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods; D008962:Models, Theoretical