HPLC vs UHPLC - a comparison of peak asymmetry and plan to step forward using Quality by Design (QbD) related to analytical methods

The purpose of this study is to demonstrate, with experiments and with computer simulations based on a firm chromatographic theory, that the wide spread perception of that the United States Pharmacopeia tailing factor must be lower than 2 (Tf < 2) is questionable when using the latest generation of LC equipment. It is shown that highly efficient LC separations like those obtained with sub-2 i­m porous and 2.7 i­m superficially porous particles (UHPLC) produce significantly higher Tf-values than the corresponding separation based on 3 i­m porous particles (HPLC) when the same amount of sample is injected. Still UHPLC separations provide a better resolution to adjacent peaks. Expressions have been derived that describe how the Tf-value changes with particle size or number of theoretical plates. Expressions have also been derived that can be used to scale the injection volume based on particle size or number of theoretical plates to maintain the Tf-value when translating a HPLC separation to the corresponding UHPLC separation. This aspect has been ignored in previous publications. Finally, data obtained from columns with different age/condition indicate that Tf-values should be complemented by a peak width measure to provide a more objective quality measure. A plan to take a further step for using Quality by Design (QbD) related to analytical methods will also be presented [Ref 1]. Continuous improvement of an original HPLC method to an UHPLC method will be the used as a case study. The capability of the two methods will be deeply studied using mechanistic comparisons. Method criteria that must be fulfilled for the two chromatographic methods will be stated. [1] 2012 PDA Europe Workshop Quality by Design The Role of Analytical Science in Implementing QbD − Technical and Regulatory Aspects, Liverpool UK 6-7 March. This is an industrial – academic cooperation in the Fundamental Separation S