Improved Sensitivity For Trifluoroacetic Acid Gradients On The Alliance™ iS HPLC Systems

By Kimberly Martin, Lise Gauthier, Paula Hong

This research examines the performance of the USP Tryptophan Monograph’s method for organic impurities on two HPLC systems—the Alliance™ iS Bio HPLC and Alliance iS HPLC. The method involves reversed-phase bioseparations with ion-pairing reagents like trifluoroacetic acid (TFA) and utilizes a water/acetonitrile gradient with a 220 nm UV wavelength. However, TFA’s strong retention on the stationary phase causes baseline ripples at low UV wavelengths, impacting sensitivity and reproducibility.

This application note evaluates the systems under default configurations, including stroke volumes, using both a standard mixer and an optional diffusion bonded mixer. While the diffusion bonded mixer minimally affects dwell volume, it is designed to enhance mixing efficiency. This comparison aims to assess the impact of system settings and mixer type on analytical performance.

Results demonstrate that the diffusion bonded mixer improves baseline stability and sensitivity, providing more reliable quantitation. The findings highlight how instrument configurations, particularly mixing efficiency, can significantly influence method performance. Such optimizations are crucial for achieving precise and reproducible results in methods that involve challenging conditions, like those specified in the USP monograph.