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The separation of high-molecular compounds under isocratic conditions is very difficult, if possible at all, and thus gradient elution is needed. The theory of gradient elution for small molecules is well established; however, its applications to reversed-phase gradient separations of biopolymers are not straightforward because of specific problems, such as slow diffusion, limited accessibility of the stationary phase for larger molecules, or possible sample conformation changes during the elution. The first step of our study was the determination of the experimental data, and then these data were used to predict gradient retention times. High performance liquid chromatography was used to investigate the reversed-phase chromatographic behaviour of four proteins. The influence of experimental parameters was examined using a water/organic solvent/trifluoroacetic acid system. Chromatographic results from four Zorbax stationary phases supports were comparable. © 2015, Estonian Academy Publishers. All rights reserved.


Currently several different approaches are used for speed-up and cost reduction for new method development in reversed-phase high-performance liquid chromatography. During this research, application of a solvatic retention model of reversed-phase high-performance liquid chromatography was studied to predict the retention of phenylisothiocyanate derivatives of 25 natural amino acids, working with different stationary phases. The gradient elution mode was used, with methanol and acetonitrile as the aqueous mobile phases. Retention factors were calculated from the molecular parameters of the structures of the analytes and stationary and mobile phase properties. Such step-by-step methods, which include the first-guess prediction of initial conditions from structural formulae and fine tuning parameters of the retention model using data from successive runs, can save time and consequently will reduce the cost of method development and optimization. © 2016, Estonian Academy Publishers. All rights reserved.


Vorslova S.,University of Latvia | Golushko J.,Institute of Chromatography | Galushko S.,Institute of Chromatography | Viksna A.,University of Latvia
Chromatographia | Year: 2014

Application of the solvatic retention model of reversed-phase liquid chromatography was studied to predict retention of phenylisothiocyanate derivatives of amino acids from structural formulae and stationary and mobile phase properties. The gradient elution mode with methanol and acetonitrile aqueous mobile phases was used. It was shown that practically acceptable prediction or retention time values can be achieved after the first approximation step when experimental data of one run are used. The zero approximation level predictions—from structural formulae, column and mobile phase properties can be used as a “first guess” method from which further optimization can begin. © 2014 Springer-Verlag Berlin Heidelberg


Vorslova S.,University of Latvia | Golushko J.,Institute of Chromatography | Galushko S.,Institute of Chromatography | Viksna A.,University of Latvia
Chromatographia | Year: 2014

Application of the solvatic retention model of reversed-phase liquid chromatography was studied to predict retention of phenylisothiocyanate derivatives of amino acids from structural formulae and stationary and mobile phase properties. The gradient elution mode with methanol and acetonitrile aqueous mobile phases was used. It was shown that practically acceptable prediction or retention time values can be achieved after the first approximation step when experimental data of one run are used. The zero approximation level predictions—from structural formulae, column and mobile phase properties can be used as a “first guess” method from which further optimization can begin. © Springer-Verlag Berlin Heidelberg 2014.

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