Electron ionization occurs in a gas phase reaction described by the expression:
M + e– –> M+. + 2e–
where M is the atom of molecule being ionized, e– is the electron, and M+. is the resulting ion. In an EI source, electrons are produced through thermionic emission by heating a wire filament that has electric current running through it (10, 11). The electrons are accelerated through the ionization space towards an anode; in the ionization space, they interact with analyte molecules in the gas phase, causing them to ionize to a radical ion, and frequently causing numerous cleavage reactions that give rise to fragment ions, which can convey structural information about the analyte.
Chemical ionization. Ions are produced through collision of analyte molecules with ions of a reagent gas in the ion source (12, 13). Reagent gases include: methane, ammonia, and isobutane. Reagent gas is present in large excess compared to analyte molecules in the ion source, electrons that enter the source preferentially ionize reagent gas molecules. Subsequent collisions with other reagent gas molecules create an ionized plasma, and analyte ions are formed by reactions with the plasma (Scheme 1).
Negative ion electron capture ionization can occur by two mechanisms (14). The first is Resonance Electron Capture (REC; Scheme 2). Under chemical ionization (CI) conditions both positive and negative ions can be formed. High-energy electrons produced by EI reactions with a suitable moderating gas (e.g., methane) are collisionally cooled, and the resulting thermal- or near-thermal-energy electrons attach to certain analyte molecules by the process illustrated in Scheme 2. The captured electron occupies the lowest vacant molecular orbital of M and thus forms a short-lived radical anion in an excited state. These collisions have resonant character, and M-. formation follows second-order kinetics with the rate constant k1. The excess internal energy is often large enough to cause auto-detachment by first-order kinetics with a rate constant k2, but in the presence of a moderating gas B (e.g., methane), the excited molecular radical anion may lose its excess internal energy by collision and thereby yield a stabilized thermal-energy molecular radical anion by second order kinetics with rate constant k3. For those compounds for which the reverse reaction is minor, resonance electron capture is a highly efficient ionization process, and this accounts for the high sensitivity of NIEC-MS in many analytical applications.
Dissociative Electron Capture. In addition to stable molecular radical anions, fragment ions can form by dissociative electron capture (Scheme 3). This generally yields negative ions with a distribution of internal energies governed primarily by the ability of the leaving group AB to absorb excess internal energy, which increases with the size of AB (14).
Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique that permits MS analyses of large, involatile molecules that include biopolymers, such as proteins, peptides, carbohydrates, and oligonucleotides (15-18). Ionization is induced by UV laser (usually a nitrogen laser) irradiation of a mixture of analyte and matrix molecules on a solid surface/plate. Matrix molecules absorb UV light and are ionized (Figure 2). Analyte molecules [M] are then ionized by interaction with matrix ions to produce even electron-ions such as [M+H]+, [M+Na]+, or [M-H]–. MALDI generally produces singly-charged ions, but multiply charged ions ([M+nH]n+) can be observed, depending on the matrix, laser intensity, and other factors.
The matrix is thought to protect the target analyte molecules from photodestruction and to facilitate ionization and vaporization/desorption. Among commonly used matrices are 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), α-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix) and 2,5-dihydroxybenzoic acid (DHB). In general, matrix molecules have a relatively low molecular weight to facilitate vaporization, but are large enough and have a sufficiently low vapor pressure not to evaporate during sample preparation or after application. Matrix molecules are also generally acidic and serve as a proton source in producing [M + H]+ ions; contain UV chromophores that permit absorption of energy from the laser; and have polar substituents to facilitate dissolution in solvent mixtures of water and a miscible organic solvent such as ethanol or acetonitrile. When matrix solution is mixed with analyte, the organic solvent and water facilitate dissolution of hydrophobic and hydrophilic molecules, respectively. After the solution is spotted onto a MALDI plate, the solvents evaporate to leave matrix crystals withembedded analyte molecules (15, 16). In some cases, liquid ionic matrices have been used for MALDI analyses of certain classes of analyte molecules, such as phospholipids (19).
Electrospray ionization (ESI) achieves delivery of ions from liquid solutions into the gas phase for MS analyses, including ions from large, non-volatile molecules such as proteins and other biopolymers (20, 21). A solution containing ionic analyte molecules dissolved in a volatile liquid is pumped through a charged capillary (Figure 3), from which it is emitted as an aerosol of small droplets about 10 μm across. The aerosol is thought to be produced by a process involving formation of a “Taylor cone” and a jet from the cone’s tip. An uncharged carrier gas such as nitrogen facilitates nebulization of the liquid and evaporation of the neutral solvent in the droplets. As the solvent evaporates, the analyte molecules are forced closer together, repel each other, and cause the droplets to break into ever smaller daughter droplets. This process is called Coulombic fission and is driven by repulsive forces between charged molecules. The process iterates until single, de-solvated analyte ions are formed. The exact mechanism of the final events leading to formation of lone ions is still debated, and two alternative proposals are illustrated in Figure 4 (21). By whatever mechanism they are produced, lone ions are formed and are drawn into the mass analyzer region of the mass spectrometer.
These ions may be quasi-molecular and result from adding or removing a proton ([M +H]+ or [M-H]–] or adding another cation, such as sodium ([M+Na]+) or lithium ([M+Li]+) ion. Multiply-charged ions, e.g. [M+nH]n+], are often observed (20). For large macromolecules, such as proteins, there can be many charge states that occur with different relative abundances. Proteins tend to be variably protonated at arginine, lysine, and histidine residues, and the multiple charging reduces the m/z ratio to a value that falls within the range accessible to quadrupole and ion trap mass analyzers even in the case of very large molecules. Ions observed in electrospray are even-electron ion species. Electrons (alone) are not added or removed during the ionization process. This stands in contrast to electron ionization, which produces odd electron radical cations, or negative ion electron capture, which produces odd electron radical anions.