Concluding Remarks. Extreme changes in selectivity and reversal of elution order of phenolic isomeric compounds were obtained after changing either the pH of the mobile phase, the temperature of the system, or the type of organic eluent employed. Changes in the analyte ionization state were observed upon increasing the acetonitrile composition as well as the temperature. Method development for ionizable analytes requires a judicious choice of the mobile-phase conditions and system parameters in order to perform the analysis of the compounds in their desired ionization state. . | METHOD DEVELOPMENT APPROACHES 405 Case Study 2 Concluding Remarks. Extreme changes in selectivity and reversal of elution order of phenolic isomeric compounds were obtained after changing either the pH of the mobile phase the temperature of the system or the type of organic eluent employed. Changes in the analyte ionization state were observed upon increasing the acetonitrile composition as well as the temperature. Method development for ionizable analytes requires a judicious choice of the mobile-phase conditions and system parameters in order to perform the analysis of the compounds in their desired ionization state. Choosing the optimal parameters in the chromatographer s toolbox allows for the development of rugged and reproducible methods. Case Study 3 Method Development for a Diprotic Basic Compound A case study is presented for the method development of a diprotic base compound. The first step in method development is to look at the chemical structure of the analyte and to determine if there are any ionizable sites on the molecule. If there are ionizable sites then their respective pKa values should be determined. The pKa values may have been already determined by the preformulation group and close communication with that group would avoid duplication of work. However commercially available programs such as ACD Labs Advanced Chemistry Development Toronto Canada are also available to allow for in-silico prediction of the analyte pKa. Also using selected fragments of the molecule can also be helpful for pKa determination of the desired molecule because the pKa values for each of these fragments of the molecule may be readily available from the literature. This is only an estimate at best but can guide the chromatographer down the right path for initial mobilephase pH selection. In the following case study for this pharmaceutical compound M the method development scenario and rationale for each iteration in the method development process is .
