title: Use of individual retention modeling for gradient optimization in hydrophilic interaction chromatography: separation of nucleobases and nucleosides.
abstract: In this study, the separation of twelve nucleobases and nucleosides was optimized via chromatogram simulation (i.e., prediction of individual retention times and estimation of the peak widths) with the use of an empirical (reversed-phase) non-linear model proposed by Neue and Kuss. Retention time prediction errors of less than 2% were observed for all compounds on different stationary phases. As a single HILIC column could not resolve all peaks, the modeling was extended to coupled-column systems (with different stationary phase chemistries) to increase the separation efficiency and selectivity. The analytical expressions for the gradient retention factor on a coupled column system were derived and accurate retention time predictions were obtained (<2% prediction errors in general). The optimized gradient (predicted by the optimization software) included coupling of an amide and an pentahydroxy functionalized silica stationary phases with a gradient profile from 95 to 85%ACN in 6 min and resulted in almost baseline separation of the twelve nucleobases and nucleosides in less than 7 min. The final separation was obtained in less than 4h of instrument time (including equilibration times) and was fully obtained via computer-based optimization. As such, this study provides an example of a case where individual retention modeling can be used as a way to optimize the gradient conditions in the HILIC mode using a non-linear model such as the Neue and Kuss model.
authors.affiliation: Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium. Electronic address: eva.tyteca@vub.ac.be.
authors.forename: Eva
authors.initials: E
authors.lastname: Tyteca
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 20, Boulevard d'Yvoy, 1211 Geneva 4, Switzerland.
authors.forename: Davy
authors.initials: D
authors.lastname: Guillarme
authors.affiliation: Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Gert
authors.initials: G
authors.lastname: Desmet
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D009705:Nucleosides / Q000302:isolation & purification*; D009711:Nucleotides / Q000302:isolation & purification*; D012822:Silicon Dioxide
chemical_list: D009705:Nucleosides; D009711:Nucleotides; D012822:Silicon Dioxide
keywords: Computer-assisted method development; HILIC; Individual retention modeling; Nucleobases; Nucleosides