The separation of vitamin A acetate isomers is essential for quality assurance of e.g. nutrition supplements, cosmetics, and pharmaceutical ingredients. High performance liquid chromatography (HPLC) is currently the most suitable analytical method for tackling this challenging separation task. However, the existing methods based on normal phase chromatography (NPC) are poorly reproducible due to the typical disadvantages of NPC, such as long equilibration times and fluctuation in retention factors. A new reversed phase method developed in our labs allows the separation of the isomers applying a chiral stationary phase (CSP). This phase consists of an immobilized polysaccharide which can be used in every chromatographic mode. However, they are not typically used in reversed phase mode. Through the screening of various stationary phases with different polysaccharide based chiral selectors, the choice of the ideal stationary phase could be confirmed, allowing to draw conclusions about the retention mechanism. The CSP Chiralpak IG-3 was found to be the most suitable among the examined. Regarding the separation mechanism, the spatial helical structure of the polysaccharide derivatives was confirmed to be of particular significance. In addition to the stationary phase, the mobile phase was tested for optimization regarding composition, gradient parameters as well as temperature using chromatographic method optimization software for the sake of method robustness.
Amylose , Diterpenes , Polysaccharides , Retinyl Esters , Amylose/chemistry , Stereoisomerism , Polysaccharides/chemistry , Chromatography, High Pressure Liquid/methods
In striving for the best possible separation, the selectivity of stationary phases as an optimization parameter is often underestimated although there are many ways to influence this powerful tool. This review serves to provide an insight into the various ways of adapting the selectivity of a separation in liquid chromatography. Approaches via temperature and flow rate tuning are discussed as a basis followed by focusing on the stationary phase as the superior optimization parameter. Highly selective stationary phases hereby provide an advantage for groups of similar analytes. For more complex mixtures, separations can be improved using mixed-mode technologies where different retention mechanisms are combined. Serial coupling, mixed-bed columns, and stationary phase optimized selectivity liquid chromatography provide solutions to various degrees. Finally, the advantages of stationary phase tuning over adaption of mobile phase and/or temperature are presented in terms of optimum application range.
