Improved Sensitivity for Trifluoroacetic Acid Gradients on the Alliance™ iS HPLC Systems

Given the nature of biomolecules, such as amino acids and peptides, many HPLC reversed phase bio-separations often require ion-pairing reagents, such as Trifluoroacetic acid (TFA), in combination with low wavelengths (<250 nm) for analysis. However, due to the strong retention of TFA on the stationary phase and continual displacement with acetonitrile/water gradients, baseline ripples are often observed at low UV wavelengths. For methods that require the combination of conditions, baseline ripples can impact sensitivity and reproducible quantitation. One such method, the USP Monograph for Tryptophan, Organic Impurities (Procedure 1) specifies these conditions including 0.1% TFA in water/acetonitrile gradient and 220 nm wavelength for analysis.

In this work, we will test the monograph on the Alliance™ iS Bio HPLC System and Alliance iS HPLC System and evaluate the performance of the method with systems in the default configurations, including the stroke volumes. Both systems will be tested with the standard mixer and an optional diffusion bonded mixer. The latter optional mixer has a minimal impact on dwell volume but is designed to improve mixing efficiency. Results will demonstrate the improved sensitivity observed with the diffusion bonded mixer, as well as the impact of instrument settings.

Benefits
- Diffusion bonded mixer reduces baseline fluctuations for TFA gradients on the Alliance iS HPLC System
- Improved sensitivity achieved for challenging TFA gradients at low wavelengths
- Reduced baseline noise with diffusion bonded mixer allows for more stable baseline and improved peak area precision for USP Monograph of Tryptophan