One of the most important reasons to use formic acid in LC-MS is that formic acid is a commonly used component in mobile phases of RP-HPLC that gives protons for analysis in LC/MS.
The LC-MS (Liquid chromatography-mass spectrometry) separates analytes within a sample solution and gives mass that can assist to provide structural identification of the compound. This separation technique depends on the disparate retention of analytes on a stationary phase.
Liquid chromatography/Mass Spectroscopy (LC/MS) is a method that combines high-performance liquid chromatography (HPLC), a powerful analytical separation technique, with mass spectroscopy, a powerful analytical analysis, and detection tool. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the popular atmospheric pressure ionization (API) in LC/MS processes; they both are the soft ionization technique. Most chromatographic separations are compatible with both of these techniques. Most volatile mobile phase solvents and volatile buffers are also compatible with the API process.
The solvents are normally selected based on the solubility and compatibility of a compound with different ionization techniques used in LC/MS. To improve the peak shape of molecules, many analysts use the low concentration of formic acid in the solvent system. It has been used widely as modifiers in the mobile phase. This is due to the relatively low ability of formic acid to suppress the ion detection at the time of light MS analysis using electrospray ionization (ESI) of samples.
To improve the separation of analytes in liquid chromatography, if the mobile phase pH needs to be decreased in that case acetic and formic acid are the most appropriate. Formic acid is stronger as compared to acetic acid; consequently, it must be added less to reach the required pH. The suppression of acid is on the ion in negative mode ion analysis; weak acidic compounds cannot be formed.
The reasons for using an aqueous formic acid in LC-MS.
- Formic acid is the most common component of reversed-phase mobile phases when liquid chromatography-mass spectrometry methods (LC-MS) are employed because it provides protons for the LC-MS analysis in positive ionization mode by generating [M+H]+ ions.
- The lower molecular weights generate less interference: The molecular weight of formic acid is smaller than that of acetic acid and trifluoroacetate. These acids are observed as deprotonated components and deprotonated dimers in negative ion analysis.
- It allows residual silanol to be kept in an undissociated form in low-pH mobile phases, which inhibits the adsorption (tailing) of basic analytes.
- Basic molecules are often analyzed using added-acid mobile phases, while pharmaceuticals and other basic molecules are frequently analyzed using LC-MS analysis.
- Formic acid solvents are usually considered to have lower contamination levels compared to the acetic acid mobile phases when it comes to preventing contamination of prepared mobile phases.
- Unlike acetic acid, formic acid has a less unpleasant odor, however, this is not a scientific problem. Because of its odor, acetic acid is frequently substituted with formic acid.
- Formic acid is used as a buffer component in liquid chromatography, which is one of its most important applications. It is used when the pH of aqueous mobile phases must be adjusted to complete the separation of target solute with acid-basic characteristics.
The useful solvents and buffers in the mobile phase system of LC-MS are water, acetonitrile, methanol, ethanol, propanol, isopropanol, acetic acid, formic acid, ammonium hydroxide, ammonium formate (10 mm or less), ammonium acetate (10 mm or less), and nonvolatile salts (phosphate, borate, citrate), etc.
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