Ionization is the process of charging a sample prior to its analysis by the mass analyzer of the mass spectrometer so that it becomes negatively or positively 'labeled.' The analysis is then performed by measuring both the mass (m) and charge (z) of the sample, also known as its m/z ratio. Once the sample is charged, it can undergo separation, deflection and manipulation by the mass spectrometer.
The process of ionization typically involves placement of the sample into a chamber of the mass spectrometer, where an electron source is located. A heater may vaporize the sample, after which the electron beam charges the atoms and molecules of the sample by removing or adding electrons. This results in the sample becoming cationic or anionic, or positively or negatively charged, respectively. Afterwards, an electric field accelerates these charged particles towards an arced magnetic region. Here, neutral fragments are lost while the charged analytes are deflected.
Figure 1. During ionization, the sample is charged and forms cations and anions; these ions are separated by electromagnetic fields in the mass spectrometer.
As the charged beam passes through the magnet, its particles are deflected at different angles based on their individual masses. Essentially, as the charged particle beam passes through the magnetic field, it undergoes separation based on the m/z ratio of its particles. The separated particles arrive at different locations on a detector within the mass spectrometer, and each location is translated into a molecular ion peak on the spectrograph.
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Biological and chemical samples consist mostly of neutral molecules and cannot be easily “sifted” apart based on the minute differences of their masses alone. A second differential is introduced, and this is typically ionization (i.e., charge).
Samples are charged during many different scientific analyses, including gel electrophoresis. Once charged, the samples become positive (cationic) or negative (anionic) and can be deflected by electrical and magnetic fields.
Which ionization techniques are used?
Different ionization techniques have been developed over the years to best charge sample atoms and molecules based on their inherent polarity, stability and size. The most common ionization processes are described below.
Chemical ionization (CI): CI is often called a “soft” ionization technique in that it does not fragment the molecule, leaving it intact for detection and analysis. The ionization occurs when an additional gas, such as methane, isobutene or ammonia, is added to the ion source, which consists of an EI filament. The protonated gas collides with the target analyte, protonating the sample ions to a +1 charge. Like EI, CI is used in GC-MS analysis. Historically, CI was often used ahead of EI because it could generate the “parent” molecular ions prior to their fragmentation by EI.
Electron impact (EI): The oldest and most common method of ionization is EI, wherein high energy electrons (~70eV) collide with a gas phase sample to form cationic radicals. In EI, molecular fragmentation occurs through bond cleavage and requires only 3-10 eV. This ionization technique is used in GC-MS analysis because it is robust and reliable, producing peaks that can be compared to known spectral libraries.
Electrospray ionization (ESI): ESI is another popular soft ionization technique for LC-MS and LC-MS/MS, producing a fine mist of droplets from a liquid sample. Because the liquid sample is subjected to a “voltage offset” of 2000-3000 V as it exits the sprayer, it can gain a high electrical charge on the surface of the exiting droplets. As they evaporate, those droplets transfer their charges to the analyte, creating both positive and negative ions in a wide range of charged states. Due to its high charge capacity, ESI performs well with larger and polar molecules such as peptides and oligonucleotides.
Figure 2. In ESI, the sample is aerosolized and becomes a fine mist of ions.
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 light to ionize liquid sample compounds that are mixed with a solvent such as toluene or hexane. It is often used for non-polar samples (e.g., anthracenes) that cannot be analyzed with ESI or APCI.
Inductively coupled plasma ionization (ICP): Commonly used for the elemental analysis of unknown compounds, ICP involves fragmenting a sample into its atomic components. The plasma itself is generated by inductively heating a (usually argon) gas with an electromagnetic coil, which results in the gas becoming almost entirely positively charged and very hot (in excess of 10,000K). This plasma is maintained in a torch that consists of three tubes. The liquid sample is typically introduced as a nebulized spray into the second central tube, while solid samples are introduced following laser ablation.
Glow discharge (GD) ionization: Like ICP, GD is used for elemental analysis and relies on the creation of a plasma gas that ionizes the introduced 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 a plasma. This charged gas also 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): Also considered a “soft” ionization technique akin to CI, MALDI involves mixing a sample with a matrix and applying that mixture to a metal plate. The mixture is then irradiated by a pulsed laser, resulting in sample ablation and desorption. During this process, the analyte molecules become positively charged and can be analyzed by the mass spectrometer.
Why are there multiple ionization techniques?
As shown below, the reason why multiple ionization methods exist is because one ionization technique “size” does not fit all samples. Depending on sample polarity, size, physical makeup and other factors, some ionization techniques are more ideal than others. Some ionization source technologies are better suited to LC-MS samples, while others fit better with GC-MS, IC-MS, or GD-MS.
Figure 5. Due to different sample characteristics, various ionization technologies are used.