![[Readers Insight] Why is Formic Acid Added to the Mobile Phase in Mass Spectrometry?](http://www.welch-us.com/cdn/shop/articles/formic-acid-1080_285b53f7-6c39-44e1-98eb-566f7e73016d_750x.jpg?v=1765503777)
[Readers Insight] Why is Formic Acid Added to the Mobile Phase in Mass Spectrometry?
Author: Chromatography Mound
Introduction
In methods that involve mass spectrometry, it is common to see formic acid added to the mobile phase. Readers may have wondered: what specific role formic acid, as a critical component, plays in MS? It seems like it is required in many assays, but can other acids substitute?
In this article, we will explore this question from the perspective of chemical reaction equilibrium.
Formic Acid as a Proton Donor in MS
If you are familiar with mass spectrometry, you certainly know that formic acid is a good proton donor. Since formic acid is a weak acid, a 0.1% aqueous solution of it possesses moderate acidity, meaning it will not damage the column.
Moreover, formic acid is volatile, evaporating easily under high-temperature conditions, and thus it does not leave residues in the ion source, preventing contamination to the mass spectrometer as well.
How Do Formic Acid Function in Ionization Processes?
According to the Lewis acid-base theory, substances containing lone pair electrons (for example, compounds containing nitrogen (N) or oxygen (O) atoms) can donate those lone pairs into the empty orbital of hydrogen ions (H⁺), initiating an acid-base reaction. A typical example is NH₃ combining with H⁺ to form NH₄⁺.
A similar reaction occurs during the ionization process in mass spectrometry, as seen in the diagram below.
In this example, the amino nitrogen atom of Carbofuran in the ion source combines with the H⁺ provided by formic acid, forming a precursor ion carrying a single positive charge. This reaction is reversible, as the degree of ionization can never reach 100%, and the position of the equilibrium depends on reactant concentrations.
To enhance the signal of the precursor ion and shift the reaction equilibrium to the right, it is necessary to provide additional H⁺ to the system by formic acid, thereby increase the concentration of the protonated species.
Why not Simply Increase Analyte Concentration?
Although increasing the analyte concentration could also shift the equilibrium toward more protonated ions, doing so is impractical: excessive analyte concentrations can contaminate the mass spectrometer, leading to carryover residues and false positives in subsequent assays. For low-abundance samples, raising sample mass or enrichment factors greatly complicates the experiment.
Practical Guidelines on When to Add Formic Acid
Now that we understand the role of formic acid, we can draw two tentative conclusions:
- If the target compound contains N or O atoms, we should consider adding formic acid to the mobile phase to facilitate the precursor ion response, although this is not guaranteed to be effective in 100% of cases.
- For target compounds where the precursor ion form is [M-H]⁻, rather than [M+H]⁺, adding formic acid may inhibit the reaction from shifting to the right. This is because the formation of the [M-H]⁻ precursor ion requires deprotonation (loss of H), not protonation (addition of H).
Can Other Acids Substitute for Formic Acid?
As written above, formic acid is a volatile weak acid, with moderate acidity in aqueous solution and no residue in ion source. A similar acid that also meets these criteria is acetic acid, but the dissociation constant (pKa) of acetic acid is 4.74, where the pKa of formic acid is 3.77. Therefore, formic acid is stronger in acidity than acetic acid, supplying a higher quantity of H⁺ with equal molar amounts.
For this reason, formic acid is generally more suitable for promoting protonation in mass-spectrometric analyses.