Abstract |
This work focuses on one of the most important analytical techniques in chemistry: electrospray ionization under atmospheric pressure conditions. The complexity of the highly factorial nature of the technique makes its study interesting. The work focuses on studying the effect of nebulization conditions on the final formation of ions. The process involves converting a solution into charged droplets, which then evaporate, leaving behind ions that are analyzed by high-resolution mass spectrometry. The ionization mechanism varies depending on whether small or large molecules are being ionized. The study was conducted using a Q Exactive Plus Hybrid Quadrupole mass spectrometer with an Orbitrap detector, and the sample was introduced directly into the nebulizer using a pump and syringe. This technique allows for immediate and fast analysis, and the high resolution of the instrument enables the observation of small differences in spectra. During electrospraying, the sodium trifluoroacetate solution forms complex clusters, which are detected and cover the entire mass range, resulting in distinct peaks. Due to this characteristic, it is used as a calibration solution for the instrument. These complexes form singly charged, doubly charged, positive, and negative ions. Specifically, the following ions were detected: (CF3COONa)nNa+ (n=1-43), (CF3COONa)nCF3COO- (n=1-26), [(CF3COONa)nNa2] 2+ (n=27-87) and [(CF3COONa)n(CF3COO) 2] 2- (n=15-43). In the first part of the work, the effect of ionization conditions on the distribution of the produced ions is examined. Various instrument conditions were investigated, including s-lens, sheath gas, fluid flow, potential, capillary temperature, different STFA solvents, and different concentrations. The results do not show a significant change in the distribution of STFA clusters. It is found that certain changes optimize the signal for singly charged ions, while others favor doubly charged ions. Additionally, partial discrimination between negative and positive ions is observed. The S-lens agent works adjuvantly and selectively by assisting higher m/z ions. Towards the end of the study, the formation of formic acid clusters with alkali metals discovered. The presence of formic acid originated from the waste connected to the nebulization chamber. This observation led to experiments investigating the effect of vapors on the droplets produced during electrospraying. By delivering formic acid vapors to the aqueous potassium chloride solution droplets, the formation of potassium formate clusters was observed. The final part of the work focuses on the reactions of droplets produced from different protein solutions with formic acid or ammonium hydroxide vapors. Five different proteins were used (lysozyme, α-lactalbumin, cytochrome-c, myoglobin, and β-casein). The addition of vapors resulted in differentiated ionization and structural states of the proteins, which were reflected in the mass spectra. The ionization mechanisms varied depending on the protein's disordered or naturally folded structure. Signal intensity and charge distribution provided clues that correlated with specific states of the protein and ionization mechanisms. This technique provides direct indications of non-covalently attached substituents, structural stability under acidic conditions, and intermediate states formed at low pH. Protein samples were studied in water, deuterated water, ammonium acetate buffer, and dithiothreitol solution. Of interest were the hydrogen/deuterium exchange monitoring experiments in favor of the different ionization mechanisms. Ammonium hydroxide vapors were used to deprotonate the proteins, thereby shifting the distribution to smaller charges. Overall, the work presents a comprehensive investigation of electrospray ionization under atmospheric pressure conditions, highlighting the impact of nebulization conditions on ion formation.
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