Solvent mixing is critical to obtaining optimal LC separations. Currently, there are two commonly available pump mixing designs for reversed phase gradient HPLC separations - high pressure (typically binary) and low pressure (typically quaternary) systems. Both high- and low-pressure systems are subject to noise and mobile phase composition fluctuations. Mixers of various volumes and designs are utilized to minimize these issues by reducing baseline noise and oscillations in mobile phase composition.

The use of certain mobile phase modifiers is known to impact chromatographic baselines. Trifluoroacetic acid (TFA) is a common ion-pairing reagent that is used in combination with acetonitrile for many gradient reversed phase applications. TFA absorbs strongly at wavelengths below 250 nm. Additionally, TFA is slightly retained on reversed phase columns resulting in fluctuations in TFA concentration as the acetonitrile gradient passes through the column. In combination, these factors result in significant baseline disturbances (ripples) when TFA-acetonitrile gradients are used at low wavelengths. These baseline ripples can impact the sensitivity and retention time precision of the method.

Most modern HPLC systems include a standard mixer in the pump design. Additionally, different mixers may be available to improve mixing performance for specific applications. In this study, mixers of varying volumes and design were evaluated. The study utilized the USP method for organic impurities in tryptophan (procedure 1) which uses a shallow TFA-acetonitrile gradient at 220 nm. Mixing performance was determined through comparison of retention time precision and signal to noise values obtained with each mixer.