YMC Europe has considered the use of trifluoroacetic acid (TFA) in high-performance liquid chromatography (HPLC) applications.
TFA is a common additive (typically at 0.1 per cent) to mobile phases for the reverse-phased HPLC separations of peptides and proteins.
The addition of TFA provides a lower pH, improved peak shape and, as a result, improved resolution.
In addition, TFA is able to form ion pairs with these analytes, which increases retention especially for the polar substances, while selectivity can be influenced by varying the TFA concentration.
This influence of concentration on selectivity is important in regards to the robustness of a method.
The TFA must be added in a highly reproducible manner.
This may seem trivial, but there are several parameters to be considered.
First, the density of TFA is 1.53mg/ml, so confusion between 0.1 per cent weight per volume or 0.1 per cent volume per volume may lead to severe reproducibility problems.
Second, due to the viscosity and the vapour pressure differing from those of water (the substance displacement pipettes are calibrated with) false dosing easily is obtained.
For this reason the use of positive displacement pipettes or weighing of the TFA is recommended.
Third, when using TFA as a mobile phase additive, HPLC or spectrophotometric grade quality should be used.
As TFA is not very stable it should be stored in a well-sealed bottle in the refrigerator or freezer to slow down degradation and minimise additional peaks in the chromatogram.
Because the acid is hygroscopic, the contact with air should be minimised by flushing with an inert gas when taking aliquots from the stock solution.
For optimum sensitivity such as when small peaks near the detection limit have to be recorded, glass ampoules of TFA are available.
While there are considerably more expensive, they avoid contamination of the data and overcome many of the variability issues described above, which makes their extra cost more reasonable.
