J. Sep. Sci. 2013, 36, 2709–2717 2709 Joseph J. Stankovich Fabrice Gritti Paul G. Stevenson Georges Guiochon Department of Chemistry, University of Tennessee, Knoxville, TN, USA Received February 15, 2013 Revised April 8, 2013 Accepted April 8, 2013 Research Article The impact of column connection on band broadening in very high pressure liquid chromatography A series of experiments was conducted to evaluate the degree of band broadening in very high pressure LC due to column connections. Different column manufacturers use slightly different designs for their column fittings. If the same column connections are repeatedly used to attach columns of different origins, different void volumes form between capillary tubes and column inlets. An Agilent Ultra Low Dispersion Kit (tubing id 75 m) was installed on an Agilent Infinity 1290 ultra HPLC and used to connect successively an Agilent, a Phenomenex, and a Waters column. A series of uracil (unretained) samples were injected and eluted at a wide range of flow rates with a water/acetonitrile mixture as eluent. In order to determine the variance contribution from column connections as accurately as possible a nonretained probe compound was selected because the variance contribution from the column is the smallest for analytes, which have very low k values. Yet, this effect still has an impact on the resolution for moderately retained compounds (k > 2) for narrow-bore columns packed with fine particles, since variance contributions are additive for linear chro- matographic systems. Each injection was replicated five times under the same experimental conditions. Then NanoViper column connections (tubing id 75 m) were used and the same injections were made. This system was designed to minimize connection void volumes for any column. Band variances were calculated as the second central moment of elution peaks and used to assess the degree of band broadening due to the column connections. Band broadening may increase from 3.8 to 53.9% when conventional metal ferrules were used to join columns to connection sites. The results show that the variance contribution from improper connections can generate as much as 60.5% of the total variance observed. This demonstrates that column connections can play a larger role than the column packing with respect to band dispersion. Keywords: Band broadening / Column connections / Very high pressure liquid chromatography / Void volumes DOI 10.1002/jssc.201300175 1 Introduction Our goal was to measure the degree of band broadening that could occur when void volumes take place in the UPLC (ultra HPLC) column connections made with conventional metal ferrules. We used NanoViper column connections, which are advertised as having a “zero void volume” due to their seamless interface design that is easily adapted to different columns. The importance of having very low (near zero) void volumes at connection sites in HPLC and UPLC has long been recognized by instrument manufacturers, as demon- strated by the current influx of “zero void volume” connec- tions and unions. However, there has not yet been an attempt Correspondence: Dr. Georges Guiochon, University of Tennessee, 1420 Circle Drive, 414 Buehler Hall, Knoxville, TN 37996, USA E-mail: guiochon@utk.edu Fax: +1-865-974-2667 Abbreviations: PEEK, polyether ether ketone; UPLC, ultra HPLC; U.S.P., United States Pharmacopeia at assessing the extent of band broadening experienced when there are gaps in column connections. Voids or gaps in column connections create stagnant zones not flushed by the eluent stream. When an analyte band passes by these zones, a portion of the analyte molecules enters the stagnant zone, their velocities are slowed down or stopped. In these zones, the migration of molecules is gov- erned by diffusion, which is not a fast mechanism but is controlled by the concentration gradient. Solute molecules enter when the band concentration increases and becomes high; when they leave, the axial concentration gradient along the column decreases, concentrations become low and their movement out of the void areas slows down, causing the band broadening, tailing, and peak asymmetry often observed. When short narrow-bore columns, generally preferred for providing short analysis times [1], are used, these void vol- umes contribute relatively more to band broadening than Colour online: See the article online to view the Figs. 1–6 in colour. C  2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com