Aspects of the “Design Space” in high pressure liquid chromatography method development

The present paper describes a multifactorial optimization of 4 critical HPLC method parameters, i.e. gradient time (tG), temperature (T), pH and ternary composition (B1:B2) based on 36 experiments. The effect of these experimental variables on critical resolution and selectivity was carried out in such a way as to systematically vary all four factors simultaneously. The basic element is a gradient time–temperature (tG–T) plane, which is repeated at three different pH's of the eluent A and at three different ternary compositions of eluent B between methanol and acetonitrile. The so-defined volume enables the investigation of the critical resolution for a part of the Design Space of a given sample. Further improvement of the analysis time, with conservation of the previously optimized selectivity, was possible by reducing the gradient time and increasing the flow rate. Multidimensional robust regions were successfully defined and graphically depicted.