Volume 35, Issue 16 p. 2018-2025
Research Article

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

Naijun Wu

Corresponding Author

Naijun Wu

Merck & Co., Inc., Rahway, NJ, USA

Correspondence: Dr. Naijun Wu, 86 Morris Avenue, Celgene Corporation, Summit, NJ 07901, USA

E-mail:[email protected]

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Ashley C. Bradley

Ashley C. Bradley

Chemistry Department, North Carolina A&T State University, Greensboro, NC, USA

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Christopher J. Welch

Christopher J. Welch

Merck & Co., Inc, Separation & Purification, Center of Excellence, Rahway, NJ, USA

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Li Zhang

Li Zhang

Merck & Co., Inc, Separation & Purification, Center of Excellence, Rahway, NJ, USA

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First published: 04 July 2012
Citations: 40

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.