Higher molecular weight biomolecules are nonvolatile compounds that may decompose before ionizing or vaporizing during mass analysis with conventional electron impact ionization methods. Accordingly, electrospray ionization (ESI) is the favored method for vaporizing and ionizing biomolecules as it circumvents rapid fragmentation and enables the recording of mass signals for the entire biomolecule.
ESI utilizes electrical energy to transfer ions from the liquid phase of the sample into the gaseous phase. The analyte biomolecule is premixed with an ionic liquid—a nonmolecular compound serving as the ionization source. The solution is sprayed through a high-voltage capillary to generate a fine aerosol. The voltage difference applied between the capillary and the entrance of the analysis chamber causes the movement of charged droplets toward the analysis chamber. The ionic liquid from the aerosol evaporates during this movement, leaving behind a variably protonated or ionized analyte molecule. These charged species are detected in the mass spectrum at an m/z value equivalent to the sum of the analyte molecule's molecular mass and the ions' molecular weight from the ionic liquid associated with the analyte molecule.
The position of mass signals varies depending on the type of cations present in the ionic liquid. For example, the presence of sodium ions in the ionic liquid leads to an (M+23) peak in the mass spectra, while the presence of a proton results in an (M+1) peak.
Biomolecules such as proteins and nucleic acids are low volatility compounds with high molecular weights. Conventional electron-impact ionization mass spectrometry faces challenges with such molecules, including severe molecular fragmentation.
Electrospray ionization is an alternative "soft" ionization method that generates nonradical molecular ions for the mass spectrometry of such biomolecules.
In this method, the sample is mixed with a polar solvent such as methanol, methanol-water, or acetonitrile with or without added salts.
The mixture is sprayed via a high voltage capillary into a vacuum chamber. The rapid evaporation of solvent from the fine spray of charged droplets ultimately leads to the charged gaseous sample molecule.
This charged sample molecule is detected in the mass spectrum as the sum of the sample molecular mass and the masses of the associated ions.
For example, if sodium ions are present in the ionic solution, the spectrum typically features an M+23 peak.
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