Evaluation of The Peak Capacity of Various RP-Columns for Small Molecule Compounds in Gradient Elution

Peak capacity is the commonly used measure of separation efficiency in gradient elution. This study focuses on the effect of column characteristics (particle size and column length) and operating parameters (gradient time and flow rate) on the peak capacity for small molecule compounds in gradient elution. The goal of this study is to develop a practical strategy to maximize the separation efficiency (i.e., peak capacity) under different constraints (analysis time or pressure limit). Using both experimental data and theoretical modeling, the current study reveals that the peak capacity increases with both gradient time and column length in a non-linear fashion. Marginal peak capacity is proposed to characterize the non-linear increase of peak capacity over the gradient time and column length. This study also attempts to understand the maximum peak capacity achievable under certain pressure limits using Neue’s peak capacity model. The results of this study provide a better understanding of the UPLC technology, and can also help to develop practical strategies to maximize the separation efficiency in gradient elution to meet the separation needs.