Gradient elution systematic development

Steps in method development (1) determine the goal of the analysis, (2) select a method of sample preparation, (3) choose a detector, and (4) use a systematic procedure to select solvent for isocratic or gradient elution. Aqueous acetonitrile, methanol, and tetrahydrofuran are customary solvents for reversed-phase separations. A separation can be optimized by varying several solvents or by using one solvent and temperature as the principal variables. If further resolution is required, flow rate can be decreased and you can use a longer column with smaller particle size. Criteria for a successful separation are 0.5 < < 20, resolution >2.0, operating... 

Systematic studies on the selection of the best mobile phases to assure the best micropreparative separation of analyzed taxoids, especially of 10-DAB III, as well as its less polar derivatives baccatin III, paclitaxel, and cephalomannine obtained from the extracts of fresh and dried needles and stems of Taxus baccata L. by Glowniak and Mroczek have been undertaken [2]. The TLC investigation on silica gel included solvent systems with one and two polar modifiers, multicomponent mobile phases, as well as some multiple development techniques and gradient elution. As polar modifiers, methanol, acetone, dioxane, ethyl acetate and ethylmethylketone, as well as their mixtures, have been reinvestigated, but dichloromethane, chloroform, benzene, toluene, heptane, and their mixtures were used as solvents. 

Successful separations of many complex mixtures by HPLC gradient elution have demonstrated the utility of this technique (1-5). In contrast to HPLC, gradient development in TLC has been applied relatively rarely, owing to the rather complex devices required for the generation of reproducible gradients and lack of a simple theory of gradient development. Niederwieser and Honegger (6,7) have systematized many experimental results and outlined some theoretical problems. 

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