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NMR and Mass Spectroscopy of Carboxylic Acids

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In ¹H NMR spectroscopy, acidic protons (–COOH) of carboxylic acids are highly deshielded and absorb far downfield, at around 9–12 ppm. The chemical shift value depends on the concentration and solvent used.
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Propionic Acid Outperforms Formic and Acetic Acid in MS Sensitivity for High-Flow Reversed-Phase LC-MS Bottom-Up

Mykyta R Starovoit¹, Siddharth Jadeja¹, Rudolf Kupčík²

¹Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203/8, Hradec Králové 500 03, Czech Republic.

Analytical Chemistry

|April 3, 2026

Summary

This summary is machine-generated.

Propionic acid enhances liquid chromatography-mass spectrometry (LC-MS) peptide identification by improving electrospray ionization (ESI) efficiency. This overlooked mobile phase additive offers a robust alternative to formic acid, boosting proteome depth without compromising chromatography.

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Area of Science:

Proteomics
Analytical Chemistry
Mass Spectrometry

Background:

Formic acid is the standard acidic additive in reversed-phase LC-MS bottom-up proteomics.
It provides a balance between chromatography and electrospray ionization (ESI) efficiency.
Improving ESI efficiency without impacting chromatography is an ongoing challenge.

Purpose of the Study:

To evaluate propionic acid as an alternative mobile phase acidifier in LC-MS.
To assess its impact on ESI efficiency and chromatographic performance.
To determine its suitability as a replacement for formic acid.

Main Methods:

Comparative analysis of mobile phases containing propionic acid, formic acid, and acetic acid.
Evaluation across analytical- and microflow LC-MS setups.
Assessment of peptide identifications, chromatographic performance, and mobile phase properties.

Main Results:

Propionic acid significantly enhanced ESI efficiency, increasing peptide identifications by 39% over formic acid.
It showed a 12% increase compared to acetic acid.
Chromatographic performance was largely unaffected, with only minor changes in peptide retention.
Results were consistent across different laboratories, LC-MS configurations, and sample types.

Conclusions:

Propionic acid is a highly effective mobile phase additive for LC-MS proteomics.
It offers superior ESI efficiency and peptide identification rates compared to formic acid and acetic acid.
Propionic acid represents a stable, instrument-compatible, and drop-in alternative for optimizing LC-MS workflows.
Its use can lead to increased MS sensitivity and greater proteome depth.