[Readers Insight] What Happens If Formic Acid Was Replaced by Acetic Acid in HPLC Analysis?

The purpose of adding an acid is to provide a stable supply of hydrogen ions. The pKa of formic acid is 3.75, whereas the pKa of acetic acid is 4.76. Since the formula for calculating pKa is logarithmic, a difference of one unit means that the acid dissociation constant (Ka) of formic acid is roughly 10 times stronger than that of acetic acid.

For weakly acidic target analytes, we need sufficient acidity to firmly suppress them back into their neutral molecular state, allowing them to be properly retained on the C18 column. If formic acid is replaced with acetic acid, the pH of the mobile phase will increase. Seeing such a mild surrounding environment, the target analytes immediately deprotonate and ionize, running wild inside the column like a runaway horse. The result can be a drastic shift in retention times and, to some extent, exacerbated peak tailing.

Suppose we are using a photodiode array detector (PDA), particularly for detecting compounds whose maximum absorption wavelengths are at low wavelengths (e.g. around 210 nm). Swapping the acid at this point is equivalent to putting sunglasses on the detector. The UV cutoff wavelength of formic acid is very low (around 210 nm), whereas the cutoff wavelength for acetic acid is as high as roughly 230 nm, higher than that of formic acid.

As a result, when switched from formic acid to acetic acid, the detector acts as if it is wearing a pair of astigmatism sunglasses—it is not entirely blind, but it cannot see as clearly as before. Furthermore. the baseline might drift like a kite with a broken string, the noise can become dense and serrated, and system equilibrium becomes difficult to stabilize. Finally, trace impurity peaks that were originally faintly visible can be completely obscured by the high background absorbance of the acetic acid.

To compensate for the loss of "visibility", the target analyte has to be "clearer"—meaning it needs a higher response; however, this not only reduces sensitivity but also compromises quantitative accuracy.

In an electrospray ionization (ESI) source, the positive ion mode typically relies heavily on a continuous stream of hydrogen protons released by formic acid to help the target analytes form [M+H]⁺ ions. If replaced by acetic acid, which has a relatively weaker ionization capability, the efficiency of providing hydrogen ions will be significantly compromised.

While the impact on the response of acidic compounds might be minor, the response values for weakly basic compounds could suffer a catastrophic drop.

On the other hand, acetic acid is not entirely useless for compounds analyzed in negative ion mode. In the analysis of specific highly polar substances (e.g. certain glycosides), the acetate ion can combine with the target analyte to form a stable [M+CH₃COO]^- adduct. In these cases, the response value can sometimes far outperform that of formic acid.