Journal of Chromatography A, 1324 (2014) 155–163 Contents lists available at ScienceDirect Journal of Chromatography A jou rn al hom epage: www.elsevier.com/locate/chroma Very high pressure liquid chromatography using fully porous particles: Quantitative analysis of fast gradient separations without post-run times Joseph J. Stankovich, Fabrice Gritti, Paul G. Stevenson, Lois Ann Beaver, Georges Guiochon ∗ Department of Chemistry, University of Tennessee, Knoxville, TN, USA a r t i c l e i n f o Article history: Received 1 September 2013 Received in revised form 15 November 2013 Accepted 18 November 2013 Available online 22 November 2013 Keywords: Very high pressure liquid chromatography (VHPLC) Constant pressure gradient elution Constant flow rate gradient elution Thermal equilibrium a b s t r a c t Using a column packed with fully porous particles, four methods for controlling the flow rates at which gradient elution runs are conducted in very high pressure liquid chromatography (VHPLC) were tested to determine whether reproducible thermal conditions could be achieved, such that subsequent analyses would proceed at nearly the same initial temperature. In VHPLC high flow rates are achieved, produc- ing fast analyses but requiring high inlet pressures. The combination of high flow rates and high inlet pressures generates local heat, leading to temperature changes in the column. Usually in this case a post-run time is input into the analytical method to allow the return of the column temperature to its initial state. An alternative strategy involves operating the column without a post-run equilibration period and maintaining constant temperature variations for subsequent analysis after conducting one or a few separations to bring the column to a reproducible starting temperature. A liquid chromatography instrument equipped with a pressure controller was used to perform constant pressure and constant flow rate VHPLC separations. Six replicate gradient separations of a nine component mixture consisting of acetophenone, propiophenone, butyrophenone, valerophenone, hexanophenone, heptanophenone, octanophenone, benzophenone, and acetanilide dissolved in water/acetonitrile (65:35, v/v) were per- formed under various experimental conditions: constant flow rate, two sets of constant pressure, and constant pressure operation with a programmed flow rate. The relative standard deviations of the response factors for all the analytes are lower than 5% across the methods. Programming the flow rate to maintain a fairly constant pressure instead of using instrument controlled constant pressure improves the reproducibility of the retention times by a factor of 5, when plotting the chromatograms in time. © 2013 Elsevier B.V. All rights reserved. 1. Introduction The need for rapid gradient separations is a driving force in the development of HPLC techniques. Continued progress and the need to improve the results obtained in very high pressure liquid chro- matography (VHPLC) are generating new obstacles. Rapid analyses are made with short, efficient columns that have lower variance contributions than their longer predecessors. These contributions are small compared to the variance contributions of the tubing and connections of modern instruments, which became a nuisance for analysts working with short columns and which are now the leading cause of band spreading in VHPLC and HPLC (micro-high performance liquid chromatography) [1]. ∗ Corresponding author. Tel.: +1 8659740733; fax: +1 8659742667. E-mail addresses: guiochon@utk.edu, guiochon@ion.chem.utk.edu (G. Guiochon). To achieve adequate efficiency, VHPLC columns are short and are packed with very fine particles. They must be operated with high back pressures. The high pressures required to run these columns at a flow rate close to optimum generate heat due to the friction of the mobile phase percolating through the tightly packed bed. This heat leads to expansion of the mobile phase and slowly diffuses to the column environment. Under isocratic conditions, a steady state is achieved and analyses are reproducible. In contrast, no steady state can take place under gradient conditions since the mobile phase composition changes, affecting flow rate and pressure. This leads to reproducibility issues if there is no sufficiently long post-run equilibration time between subsequent injections. Conventional gradient separations are run at constant flow rate, the amount of heat generated by friction decreasing during the gradient run. As a result the inlet pressure and the column temperature decrease during constant flow rate gradients. When the gradient separation is started again, the inlet pressure and the column temperature increase, so the initial conditions are different [2–4]. 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.11.036