M M e M M H M M H. Ion Sources
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1 Ion Sources Overview of Various Ion Sources After introducing samples into a mass spectrometer, the next important step is the conversion of neutral molecules or compounds to gas phase ions. The ions could be molecular or monatomic in the case of organic or inorganic mass spectrometry respectively. Examples of molecular and monatomic ions are shown below: Cu 63 e 63 Cu Atomic mass 63 Exact mass Figure 1: General representation of ion formation in ions sources. Ions are needed because their speed and direction of travel can be manipulated by using electric and/or magnetic fields. This manipulation is crucial to the separation and detection of the chemical species. Ions can be formed in a variety of ways. A few examples are presented below (In these examples, M represents a neutral molecule while the radical or ionic forms of M are indicated with the dot or charge sign respectively (M, M + and M - ).): 1. ejection of electrons from neutral molecules e M M e 2. electron capture M e M 3. protonation H M M H 4. Deprotonation H M M H
2 5. addition of other cations Na M M Na 6. addition of anions CH3COO M M CH3COO Devices that are used for the conversion of neutral molecules to molecular ions are called ion sources. Many types of Ion sources have been developed. Some examples are presented in the list below: Table 1. Common ion sources and their acronyms Ion Source Electron ionization source Chemical Ionization source Matrix Assisted Laser Desorption ionization source Electrospray Ionization sources Atmospheric Pressure Chemical Ionization source Acronym EI CI MALDI ESI APCI Electron ionization Source (EI) Electron source Repeller e - Ion Extraction Lens To MS Trap Sample in Figure 2: principle of operation of electron ionization ion source In EI, the sample to be analyzed is vaporized into the ionization chamber by heating. Pressure in the chamber is typically about torr. The neutral molecules (represented by the black circles) are bombarded by energetic electrons ( ) produced by the electron source which is made from
3 Relative Abundance tungsten or rhenium filament. Typically, an electron is removed from the neutral molecule to form a radical ion. M e M 2e m/z Figure 3: The EI mass spectrum of decanol (CH 3 (CH 2 ) 9 OH). Notice the absence of a signal at m/z 158 which is the molecular ion (M + ). The base (or tallest peak) is at m/z 42. This is the most stable ion. This indicates that the initially formed molecular ion fragmented significantly. For ions to form, the energy of electrons from the filament of the EI must be higher than the ionization energy of the analyte molecule. Ionization energies of organic molecules are about 10eV while that of the bombarding electrons is typically 70eV. As a result, the ion that is formed has a lot of excess energy which causes it to fragment further. The signals observed in the mass spectrum above (Figure 3) indicate the fragmentation pattern of the molecule. This pattern is often referred to as the fingerprint of the molecule. The expected molecular ion at m/z 158 is absent because of its relative instability. It fragmented into other ions whose signals are seen. The most stable fragment is the one at m/z 42 and this is called the base peak. Due to the extensive fragmentation that occurs in EI it is classified as a hard ionization source. Advantages and disadvantages of EI are presented in Table 2. Table 2. Advantages and disadvantages of electron ionization sources. Advantages Results in reproducible, instrument transferable, extensive fragmentation Information from the mass spectrum produced with EI can be used to elucidate the structure of a molecule. This is done by comparing the spectrum to several spectra in a Disadvantages Analytes must be in gas phase prior to introduction into the ion source This makes it unsuitable for thermally unstable and non-volatile compounds The non-volatility problem can be overcome by derivatization of the analyte to a volatile form (e.g.
4 large database containing spectra of known compounds.. Easily interfaced with a gas chromatograph RCOOH can be derivatized to RCOOR ) Some compounds fragment so extensively that the molecular ion peak is absent from their mass spectrum. This makes interpretation more challenging. Chemical Ionization Chemical ionization utilizes the same apparatus as EI but it is operated at higher pressure (~ Pa in the ion box). Also, the electron energy is increased to ev so that the electron beam can penetrate further into the ion box. Chemical ionization is an indirect process. The first step involves ionization of a reagent gas (such as methane, CH 4 ) with the energetic electrons from the filament CH e CH 2 e ( CH other ions) In the second step, reagents ions are formed CH CH CH CH CH CH C H H In the third step, the analyte molecule is ionized by reacting with the reagent ions through one or more of the following reactions: 5 4 M CH M H CH (Proton transfer) 5 5 M CH M CH (Adduct formation) M C H M C H (Adduct formation) M C H M H C H (Hydride Abstraction) Besides methane other reagents that are utilized include isobutyl chloride, water, ethanol, acetone, and methylamine.
5 Relative Abundance m/z Figure 4: The CI mass spectrum of decanol (CH 3 (CH 2 ) 9 OH). The peak at m/z 141 is the base peak and it is due to loss of OH - [M-OH] +. The peak at m/z 157 is the molecular ion peak due to hydride abstraction [M-H] +. As noted in the mass spectrum representation in Figure 4, chemical ionization produces less fragmentation of the molecular ion. As such, the mass spectrum Is simpler and cleaner compared to that obtained with EI. Additionally, the molecular ion is observed (at [M+H] +, [M-H] +, and/or [M + adduct] + ). However, CI does not produce sufficient fragmentation to facilitate structural information. Additionally, like EI, it is not suitable for thermally unstable compounds. Electrospray Ionization Source (ESI) With ESI, ions are generated under atmospheric pressure. Hence this approach is classified as atmospheric pressure ionization (API) technique. The ESI device is comprised of a needle (~ 0.2mm outer diameter, ~0.1mm inner diameter) that is encased in a sleeve through which a sheath gas flows. This gas (typically N 2 ) is used to nebulize the sample solution that is pumped through the needle. Sample flow rate is typically about L /min. In operation a high voltage (2-6kV relative to the ion extraction cone) is applied to the needle. Ions are typically formed in solution prior to desorption into the gas phase. To facilitate this, sample solutions are made in polar solvents such as aqueous methanol or aqueous acetonitrile. The solvent is typically modified with volatile weak acids such as formic, acetic acids, or trifluoroacetic acid, or salts of weak bases such as ammonium acetates. This facilitates abundance of charged species ( such as H + ) which are necessary for ion formation. Upon nebulization, the droplets are surrounded with charged species such as H +. As the droplets evaporate, the charge density increases. At a certain size, the charge density becomes high enough that the droplet explodes due to columbic repulsion between the charges. Charges are then transferred to the analytes (see Figure 5). The ions are typically adducts of H, ([M+H] + ), adducts of salt ions such as M+Na +, adducts of counter ions of the modifier e.g. M+NH 4 + and M+CH 3 COO -. These ions don t undergo significant fragmentations. Therefore, the mass spectrum obtained with ESI is generally clean and simple. Protonated or deprotonated molecular ions are typically observed.
6 ESI needle Sheath or nebulizing gas Analyte solution Analyte ion Charged aerosol Figure 5: Schematic diagram of an electrospray ionization device and ion formation process. Molecules such as proteins that have multiple sites through which they can be protonated or deprotonated can be multiply charged. The significance of this is that a large molecule can be observed at a lower m/z. One advantage of ESI over EI or CI is that it is readily interfaced with liquid-based separation techniques such as HPLC and capillary electrophoresis. Matrix Assisted Laser Desorption Ionization (MALDI) The MALDI process is split into two steps: sample preparation and ion formation. During sample preparation, the analyte to be transformed into ions is mixed with solution of a substance that is referred to as the matrix. The solvent is evaporated and the solid residue that is produced is placed under vacuum in the mass spectrometer, where it is irradiated with pulses of photons from a laser source (Figure 6). Radiation from Laser hv Desorption Desolvation and protonation Matrix Neutral Analyte
7 Figure 6: Schematic diagram of a Matrix assisted laser desorption ionization (MALDI) device and ion formation process. Energy from the photons causes a rapid heating of the crystals. This results in a localized ablation and sublimation of the matrix crystals. Intact analytes are entrained in the plume that is formed. Ionization can occur during this process via any of the following: proton transfer, ion molecule reactions, desorption of preformed ions. MALDI is commonly used for studying non-volatile and thermally labile compounds such as proteins, oligonucleotides, synthetic polymers and large inorganic compounds. Commonly used matrices ar cyano-4-hydrocinnamic acid for peptides; 3,5-dimethyoxy-4-hydroxycinnamic acid for proteins and polymers; 2,5-dihydroxybenzoic acid for peptides, proteins, lipids, and nucleic acids. 3 hydroxypicolinic acid is used for nucleic acid and DNA analysis. Laser sources that have been used include: nitrogen (337nm), Nd:YAG (355 and 266nm) Er:YAG (2.94 m) and CO 2 (10.6 m). Some advantages of this method include: simple sample preparation and tolerance to contamination from salts, buffers and detergents commonly used in sample preparation for biochemical analysis. Atmospheric Pressure Chemical Ionization (APCI) Atmospheric pressure chemical ionization (APCI) as the name indicates belongs to the group of ion sources that are operated under ambient atmospheric pressures. The principle of operation of APCI is similar to that of chemical ionization (CI) with the major difference that CI is operated under vacuum. APCI is comprised of a heated nebulizing probe, a corona discharge electrode (typically a needle) and a system of ion extraction lenses to transmit ions into a mass analyzer (see figure 1). Figure 1: Schematic diagram of a typical atmospheric pressure chemical ionization (APCI) source
8 In operation, the analyte solution (typically delivered with a syringe or Liquid chromatograph) is directed through a capillary tube (made of stainless steel or fused silica with ~120m internal diameter) at about 0.2-2ml/min into the APCI probe. A nebulizing gas at about 80psi is used to convert the solution into fine droplets. A heated ( o C) coaxial sheath gas flow helps to evaporate and convert the droplets into a stream of gaseous analyte and solvent molecules which are directed to the ionization region. The ionization region contains a discharge electrode that is held at a high potential (2 to 3 kv) with respect to the ion extraction lens. Assuming nitrogen is utilized as the nebulizer and sheath gas, ions such as the N 2 +., N 4 +, O 2 +. and NO + are formed in the corona discharge, These then interact with solvent molecules (e.g. water vapor from the atmosphere or sample) in a cascade of reactions that culminates in the production of secondary ions (e.g. water or solvent cluster ions). Reaction between the solvent ions and analyte molecules leads to the formation of analyte ion. For example: H O H O M M nh O 3 2 n 2 Application This ion sources is typically used in LC-MS for less polar (e.g. PAH, PCB, Fatty acids, Phthalates, triglycerides), thermally stable compounds. When compared to electrospray (ESI), APCI is more tolerant to contaminants that cause ion suppression. This makes it more useful for analysis of biological samples that contain high salt concentrations.
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