Mobile Phase Additives for Peptide Characterization

The characterization of peptides and proteins can be challenging due to issues such as adsorption, varying hydrophobicities, and specificity to name just a few.

When analyzing peptides by Liquid Chromatography (LC) and Mass Spectrometry (MS), it’s beneficial to introduce mobile phase additives in order to facilitate ionization and enhance the analyte signal.

Additionally, these additives (i.e., strong acids or buffers) are commonly used in LC to improve analyte retentivity and peak shape. But for MS separations, high purity mobile phase additives are necessary to reduce background contribution and ensure quality data.

The most common additives are trifluoroacetic acid (TFA) for LC analysis and formic acid for MS analysis. In addition to enhancing the analyte signal, mobile phase additives such as TFA, are also used to inhibit unwanted secondary interactions in LC applications. However, there is another forgotten solution to consider as well: Difluoroacetic acid (DFA).

Each of the available options offer benefits for certain techniques, but where can the additive selection have a negative impact on the application?

Trifluoroacetic Acid (TFA)

TFA is generally the mobile phase additive of choice in instances where UV detection is being used due to its great solubility and resolving power as a strong acid. On the contrary, TFA provides lackluster results when using MS detection because the deprotonated TFA forms ion pairs with the peptides or analytes of interest, decreasing the analyte abundance delivered to the detector. This phenomenon is referred to as ion suppression. Additionally, TFA is famous for contaminating MS systems and being nearly impossible to remove. Great for UV detection, not great for MS detection.

Formic Acid (FA)

Formic Acid is the mobile phase additive most commonly used in MS separations. It excels for reverse phase LC-MS techniques, however, provides some injustices to UV detection. For instance, even when high purity FA is used, UV baselines are typically higher than TFA separations and the peak shape and symmetry diminishes. Not great for UV detection, great for MS detection.

Difluoroacetic Acid (DFA)

Difluoroacetic acid (DFA) is an ion-pairing agent that achieves a balance between the optical peak shape and MS response desired in peptide LC-UV/MS-based workflows. As a weaker acid than TFA, DFA will not suppress ions or contaminate the MS system. But as a stronger acid than FA, DFA will resolve peaks and improve symmetry of UV detection. Great for UV detection, great for MS detection.

Conclusion

When it comes to peptide analysis, mobile phase additives can significantly improve performance. However, it is important to note which technique (LC, MS, or both) you are using and to make the appropriate selection based on that technique and the application requirements.

For scientists focused solely on LC analysis, TFA may be the right choice for you. For those working solely on MS, FA may be the way to go. For scientists working across both techniques, a single mobile phase additive, such as DFA, may be the right choice for you.