ABSTRACT
Reversed-phase columns are widely used in assays based on high-performance liquid chromatography (HPLC). When such assays are repeated over time, it is often necessary to replace the column. In such cases, the selectivity of columns from different production batches may prove sufficiently variable to result in a failed separation. It is possible to compensate for differences in column selectivity by making small changes (adjustments) in separation conditions. The present paper describes an efficient procedure for choosing adjusted conditions and discusses its general applicability.
Subject(s)
Chromatography, High Pressure Liquid/instrumentation , Sensitivity and SpecificityABSTRACT
Computer-facilitated method development has been extended for the simultaneous optimization of any two variables in separations by HPLC and other chromatographic procedures (gas chromatography, supercritical fluid chromatography, capillary electrophoresis, etc.). The application of this approach to HPLC method development is illustrated by the reversed-phase separation of a nine-component mixture of organic acids. Two of four variables (temperature, solvent strength (%B), pH and buffer concentration) were separately optimized in terms of selectivity, and the results are compared in terms of which variables and other conditions are most effective in providing maximum resolution for samples that contain ionizable compounds.
