Liquid Chromatography Blog

Buffer Preparation

In this technical article you will learn how effective trifluoroacetic acid (TFA) in mobile-phase preparation is and how a buffer can be a better choice if a specific pH is required.

TFA is widely used as a mobile phase additive in the HPLC separation of biological molecules, such as proteins and peptides, because it acts as an ion-pairing reagent and equilibrates quickly so that it can be used with gradient elution. The standard recipe is to put 0.1% TFA in water for the A-solvent and 0.1% TFA in acetonitrile (ACN) for the B-solvent. The volatility of TFA also has made it a favourite additive for LC-MS applications. But formic acid is favoured for LC-MS in most cases now, because it was found that TFA can suppress ionization of analyte molecules. These popular uses of TFA may lull us into thinking that TFA can be used just like any other acid in mobile-phase preparation. But be cautious! When the mobile phase pH is adjusted by titrating with an acid, the amount of acid required to obtain a certain pH will depend on the pKa of the acid and the pH of the water. If pH is the only concern, it is likely that small variations in the amount of added acid will not be very important, but if the acid is contributing other characteristics to the chromatographic system, the variation may be quite important.

By reading the full article you will learn how to get consistent batch-to-batch retention-time reproducibility using TFA and be provided with alternative solutions such as the use of a buffer.

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