title: A practical approach for predicting retention time shifts due to pressure and temperature gradients in ultra-high-pressure liquid chromatography.
abstract: Large pressure gradients are generated in ultra-high-pressure liquid chromatography (UHPLC) using sub-2μm particles causing significant temperature gradients over the column due to viscous heating. These pressure and temperature gradients affect retention and ultimately result in important selectivity shifts. In this study, we developed an approach for predicting the retention time shifts due to these gradients. The approach is presented as a step-by-step procedure and it is based on empirical linear relationships describing how retention varies as a function of temperature and pressure and how the average column temperature increases with the flow rate. It requires only four experiments on standard equipment, is based on straightforward calculations, and is therefore easy to use in method development. The approach was rigorously validated against experimental data obtained with a quality control method for the active pharmaceutical ingredient omeprazole. The accuracy of retention time predictions was very good with relative errors always less than 1% and in many cases around 0.5% (n=32). Selectivity shifts observed between omeprazole and the related impurities when changing the flow rate could also be accurately predicted resulting in good estimates of the resolution between critical peak pairs. The approximations which the presented approach are based on were all justified. The retention factor as a function of pressure and temperature was studied in an experimental design while the temperature distribution in the column was obtained by solving the fundamental heat and mass balance equations for the different experimental conditions. We strongly believe that this approach is sufficiently accurate and experimentally feasible for this separation to be a valuable tool when developing a UHPLC method. After further validation with other separation systems, it could become a useful approach in UHPLC method development, especially in the pharmaceutical industry where demands are high for robustness and regulatory oversight.
authors.affiliation: Department of Engineering and Chemical Science, Karlstad University, SE-651 88 Karlstad, Sweden.
authors.forename: Dennis
authors.initials: D
authors.lastname: Åsberg
authors.affiliation: Department of Chemical and Process Engineering, Rzeszów University of Technology, PL-359 59 Rzeszów, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Chutkowski
authors.affiliation: Department of Chemical and Process Engineering, Rzeszów University of Technology, PL-359 59 Rzeszów, Poland.
authors.forename: Marek
authors.initials: M
authors.lastname: Leśko
authors.affiliation: Department of Engineering and Chemical Science, Karlstad University, SE-651 88 Karlstad, Sweden.
authors.forename: Jörgen
authors.initials: J
authors.lastname: Samuelsson
authors.affiliation: Department of Chemical and Process Engineering, Rzeszów University of Technology, PL-359 59 Rzeszów, Poland. Electronic address: kkaczmarski@prz.edu.pl.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Kaczmarski
authors.affiliation: Department of Engineering and Chemical Science, Karlstad University, SE-651 88 Karlstad, Sweden. Electronic address: torgny.fornstedt@kau.se.
authors.forename: Torgny
authors.initials: T
authors.lastname: Fornstedt
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D008962:Models, Theoretical; D009853:Omeprazole / Q000737:chemistry / Q000302:isolation & purification; D011312:Pressure; D013696:Temperature
chemical_list: D009853:Omeprazole
keywords: Gradient effects; Method development; Pressure effects; Retention time; Temperature; UHPLC