Because mass spectrometry (MS) measurements are based on mass-to-charge ratio (m/z), ionization is essential. Ionization causes sample components to become either positively or negatively charged. Following ionization, the ions are transferred into the mass analyzer where they are filtered based on their mass-to-charge ratios (m/z) and detected.
Figure 1. During ionization, the sample components become charged, forming cations and/or anions. The ions are then separated by the magnetic or electric fields in the mass analyzer.
Different ionization techniques have been developed to optimally ionize molecules of different characteristics such as polarity, volatility, thermal lability, stability, and size. Experimental goals also influence the ionization method chosen.
Electron impact (EI)
EI is an ionization method used for samples amenable to gas-phase analysis due to their thermally stable and relatively low molecular weight. For this reason, samples are usually introduced to the ion source for EI after gas chromatography (GC) separation or from a solids probe. An EI source uses a filament set to about 70eV to create a stream of high-energy electrons that interact with the gas phase sample molecules. Ionization occurs when the collision removes an electron from the sample molecule, creating predominantly singly charged positive ions. Because EI is a high-energy process, it cleaves covalent bonds, producing repeatable fragmentation that can be used to identify compounds using mass spectral libraries.
Chemical ionization (CI)
Complementary to EI,CI is used to ionize molecules that would fragment excessively by EI, or to ionize molecules without fragmentation to produce a molecular ion that can be used to determine the molecular weights of sample components. In CI, a reagent gas such as methane, isobutene, or ammonia is introduced to the ion source, where it is ionized by the filament. The ionized gas interacts with the sample, which is subsequently ionized by reactions with reagent gas ions, creating singly charged sample components. Because reagent gas is introduced at high concentration relative to the sample, most of the ionization of the sample occurs by CI rather than EI. Depending on the sample molecule and the reagent gas, ionization reactions include proton transfer, proton abstraction, and adduct formation. Compared to EI, CI is a soft ionization technique because the reagent gas reactions substantially reduce the energy absorbed by sample molecules, producing substantially less fragmentation or predominantly molecular ions.
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ESI is a soft ionization technique used to ionize non-volatile or thermally labile samples amenable to analysis in the liquid phase such as those separated by liquid or ionic chromatography (LC or IC). The ESI source applies a high voltage to the liquid sample stream, generating a fine mist of charged droplets. An opposing flow of heated, neutral gas causes the charged droplets to evaporate, leaving the charges on the sample molecules in a process termed desolvation. Positive and negative ions in a range of charge states are created, including multiply charged ions that enable mass analysis of large molecules such as peptides, proteins, and oligonucleotides.
Figure 2. In ESI, the sample is aerosolized to a fine mist of charged droplets.
Atmospheric pressure chemical ionization (APCI): APCI is a soft ionization technique that is a variation of ESI and relies on an electrified (the corona discharge) needle to ionize nitrogen gas in the source. Once ionized, this nitrogen reacts with the solvent through gas-phase reactions. The charged solvent, in turn, either protonates or deprotonates the analyte depending on its proton affinity and gas-phase acidity. The end result is an ionized sample that carries a +1 or -1 charge. APCI is best used with samples that are small and non-polar, such as steroids and some lipids.
Figure 3. APCI generates a charged solvent, which then charges the sample.
Atmospheric pressure photoionization (APPI): A variation of ESI, APPI, uses high-intensity ultraviolet photons to ionize liquid-phase sample components in a solvent such as toluene or hexane. APPI generates molecular ions for molecules that have an ionization potential below the photon energy of the light emitted by the source. Molecules such as steroids, basic drug entities, and pesticides have ionization potentials below the threshold, and thus generate protonated molecules during APPI. APPI reduces fragmentation because only a small amount of energy is deposited in the molecule. The nitrogen sheath gas and the typical solvents used for LC-MS are not ionized because their ionization potentials are greater than the photon energy. The result is selective ionization of analytes versus background. APPI is useful for samples that cannot be analyzed with ESI or APCI.
Inductively coupled plasma ionization (ICP): ICP ionization coupled with MS is known for its ability to detect metals and certain non-metals in samples at trace levels. The plasma is generated by inductively heating a gas (usually argon) to over 10,000 oK using an electromagnetic coil. At these temperatures, a significant portion of chemical elements are ionized, where atoms lose their most loosely bound electron to form a singly charged ion. Liquid samples are typically introduced as a nebulized spray, while solid samples are introduced following laser ablation.
Glow discharge (GD) ionization: Like ICP, GD is used for elemental analysis. GD relies on the creation of a plasma gas that ionizes the sample. The plasma is generated by passing an electric current through two electrodes in an environment containing a low-pressure gas such as argon. Once the so-called striking voltage is exceeded, the gas is ionized and becomes plasma. This charged gas produces a colored glow. In elemental analysis, the sample itself acts as the cathode and is hit by gas ions. With GD ionization, solid samples can be introduced without the need for a matrix or solvent. This simplifies the process of elemental analysis and negates the need for matrix or solvent-containing spectral libraries.
Figure 4. Schematic diagram of a glow discharge ion source.
Matrix assisted laser desorption and ionization (MALDI): Used as a “soft” ionization technique to minimize fragmentation of large molecules, MALDI involves mixing a sample with a matrix and applying that mixture to a metal plate. The plated mixture is irradiated by a pulsed laser, resulting in sample ablation and desorption. During this process, the analyte molecules become charged by protonation or de-protonation. The choice of matrix is important because it absorbs the energy of the laser and transmits it to the sample for vaporization and ionization, while preventing direct sample irradiation and destruction.
Why are there multiple ionization techniques?
As shown in Figure 5, multiple ionization methods are needed to address the range of samples and sample components requiring analysis. Depending on sample polarity, size, physical makeup, volatility, thermal lability, and other factors, certain ionization techniques work more optimally toward experimental goals than others.
Figure 5. Due to the broad range of samples that are analyzed, various ionization technologies are chosen.