Ionization, chemical

Ionization methods that may be utilized in LC-MS include electron ionization (El), chemical ionization (Cl), fast-atom bombardment (FAB), thermospray (TSP), electrospray (ESI) and atmospheric-pressure chemical ionization (APCI). 

El may be used with the moving-belt and particle-beam interfaces, Cl with the moving-belt, particle-beam and direct-liquid-introduction interfaces, and FAB with the continuous-flow FAB interface. A brief description of these ionization methods will be provided here but for further details the book by Ashcroft [8] is recommended. 

ESI and APCI effect ionization from solution and in these cases it is not possible to separate a description of the processes involved in the ionization of an analyte from a description of the interface. These ionization techniques will therefore be described in detail in Chapter 4. 

Interpretation of an El spectrum involves a consideration of the chemical significance of the ions observed in the mass spectrum and then using this information to derive an unequivocal structure. For a detailed consideration of the interpretation of El mass spectra, the text by McLafferty and Turecek [7] is recommended. 

One of the major limitations of El is that the excess energy imparted to the analyte molecule during electron bombardment may bring about such rapid fragmentation that the molecular ion is not observed in the mass spectrum. Under 

Chemical ionization (Cl) is vastly employed in complement to electron ionization, either for the reasons evoked above (accessing the molecular weight of an analyte). 

FIGURE 3.7 Mass spectra of venlafaxine (MW = 277) in electron ionization and in isobutane positive chemical ionization. 

A Cl source is considered a soft ionization source it results in less fragmentation of analyte molecules and a simpler mass spectrum than that resulting from EL Most importantly, the molecular ion is much more abundant using Cl, allowing the determination of the MW. Since proton adduct 

Collisions between the ionic species CH5 or C2H5 and a sample molecule M cause ionization of the sample molecule by proton transfer from the ionized reagent gas species to form MH+ or by hydride (H ) transfer from the sample molecule to form (M - H)+, also written as (M - 1)+  

Hydride transfer from M occurs mainly when the analyte molecule is a saturated hydrocarbon. In addition, the ionized reagent gas can react with M to form, for example, an (M + C2H5)+ ion with m/z = (M + 29). The presence of such an adduct ion of mass 29 Da above a candidate molecular ion in a methane Cl mass spectrum is a good confirmation of the identity of the molecular ion. 

Many commercial sources are designed to switch from El to Cl rapidly to take advantage of the complementary information obtained from each technique. The main advantage of Cl is that fragmentation of the sample molecule is greatly reduced and significant peaks at m/z = (M + 1) or (M - 1) are seen, permitting the identification of the MW of the analyte. 

It is possible to introduce a sample directly into the Cl source on a tungsten or rhenium wire. A drop of sample in solution is applied to the wire, the solvent is allowed to evaporate, and the sample inserted into the Cl source. The sample molecules are desorbed by passing a current through the wire, causing it to heat. The analyte molecules then ionize by interaction with the reagent gas ions as has been described. This technique is called desorption Cl and is used for nonvolatile compounds. 

In general, there are four main pathways leading to the ionization of analyte molecules in Cl [12,13]  

The ionization pathway depends on the type of intermediate ions generated from the reagent gas as well as the properties of the analyte molecules. Therefore, the choice of reagent gas is particularly important. Considering the ionization pathway of proton transfer, intermediates should be able to donate protons while the analyte (A) should exhibit a tendency to accept a proton. The latter property is referred to as proton affinity (PA). 

The proton affinities of reagent gases vary a lot. For example, the proton affinity of methane during formation of protonated species (CH5+) was found to be 131.6 kcal moP [13,16]. In order to cause ionization, the proton affinity of the analyte must be higher than that of 

Reagent gases commonly used in Cl include methane, isobutane, and ammonia. When using methane as the reagent gas, three types of reactions (listed below) occur simultaneously during the ionization process [3, 12, 13]  

In the case of saturated hydrocarbons (RH), ions are formed via hydride abstraction  

Where the sample will not vaporize, even through a short path length, without decomposing one must use one of the methods which produce ions from the condensed phase. The collective term desorption ionization techniques has been used (75) for these methods and biological applications up to mid-1980 are reviewed in ref. (4). 

Note When a positive ion results from Cl, the term may be used without qualification nonetheless positive-ion chemical ionization (PICI) is frequently found in the literature. When negative ions are formed, the term negative-ion chemical ionization (NICI) should be used. [8] 

The FI mass spectrum shows the fragmentation pattern. The nature (m/z value) and frequency (intensity %) of the fragmentation can be read directly from the line spectrum. The loss of neutral particles Is shown by the difference between the molecular ion and the fragments formed from it. 

Which line in the El spectrum is the molecular ion Only a few stable molecules give dominant M ions, for example, aromatics and their derivatives, such as PCBs and dioxins. The molecular ion is frequently only present with a low intensity. With the small quantities of analytes applied, as is the case with GC-MS, the molecular information can be identified only with difficulty among the noise (matrix), or it fragments completely and does not appear in the spectrum (Howe etal., 1981). 

How can both El and Cl spectra be completed Obviously, Tolclofos fragments completely in El by loss of a Cl atom to m/z 265 as (M — 35). With Cl, this fragmentation does not occur. The attachment of a proton retains the complete molecule with all components of the elemental formula by formation of the quasimolecular ion (M + H) . 

The importance of El spectra for identification and structure confirmation is due to the fragmentation pattern. Searches through libraries of spectra are typically based on El spectra. With the introduction of the Cl capabilities for internal ionization ion trap systems, a commercial Cl library of spectra with more than 300 pesticides was introduced only at that time by Finnigan. 

The term chemical ionization, unlike El, covers all soft ionization techniques which involve an exothermic chemical reaction in the gas phase mediated by a reagent gas and its reagent ions. Stable positive or negative ions are formed as products. Unlike the molecular ions of El ionization, the quasimolecular ions of Cl are not radicals (Harrison, 1992). 

Number of ions produced as a function of the electron energy. A wide maximum appears around 70 eV. 

This equation shows that the sample pressure is directly correlated with the resulting ionic current. This allows such a source to be used in quantitative measurements. 

A modification implies desorbing the sample from a heated rhenium filament near the electronic beam. This method is called desorption electron ionization (DEI). 

Under conventional electron ionization conditions, the formation of negative ions is inefficient compared with the formation of positive ions. 

Electron ionization leads to fragmentation of the molecular ion, which sometimes prevents its detection. Chemical ionization (Cl) is a technique that produces ions with little excess energy. Thus this technique presents the advantage of yielding a spectrum with less fragmentation in which the molecular species is easily recognized. Consequently, chemical ionization is complementary to electron ionization. 

The energy content of the various secondary ions (from, respectively, methane, isobutane, and ammonia) decrease in the order CH5+ f-C4H9+ NH4+. Thus, 

Chemical ionization mass spectrometry is not useful for peak matching (either manually or by computer) nor is it particularly useful for structure elucidation its main use is for the detection of molecular ions and hence molecular weights. 

---

A third method for generating ions in mass spectrometers that has been used extensively in physical chemistry is chemical ionization (Cl) [2]. Chemical ionization can involve the transfer of an electron (charge transfer), proton (or otlier positively charged ion) or hydride anion (or other anion). 

Harrison A G 1992 Chemical Ionization Mass Spectrometry (Boca Raton, FL Chemloal Rubber Company)... 

Brodbelt J, Liou C-C and Donovan T 1991 Selective adduct formation by dimethyl ether chemical ionization is a quadrupole ion trap mass spectrometer and a conventional ion source Ana/. Chem. 63 1205-9... 

Molecular Identification. In the identification of a compound, the most important information is the molecular weight. The mass spectrometer is able to provide this information, often to four decimal places. One assumes that no ions heavier than the molecular ion form when using electron-impact ionization. The chemical ionization spectrum will often show a cluster around the nominal molecular weight. 

Decomposition (fragmentation) of a proportion of the molecular ions (M +) to form fragment ions (A B+, etc.) occurs mostly in the ion source, and the assembly of ions (M +, A+, etc.) is injected into the mass analyzer. For chemical ionization (Cl), the Initial ionization step is the same as in El, but the subsequent steps are different (Figure 1.1). For Cl, the gas pressure in the ion source is typically increased to 10 mbar (and sometimes even up to atmospheric pressure) by injecting a reagent gas (R in Figure 1.1). 

Much of the energy deposited in a sample by a laser pulse or beam ablates as neutral material and not ions. Ordinarily, the neutral substances are simply pumped away, and the ions are analyzed by the mass spectrometer. To increase the number of ions formed, there is often a second ion source to produce ions from the neutral materials, thereby enhancing the total ion yield. This secondary or additional mode of ionization can be effected by electrons (electron ionization, El), reagent gases (chemical ionization. Cl), a plasma torch, or even a second laser pulse. The additional ionization is often organized as a pulse (electrons, reagent gas, or laser) that follows very shortly after the... 

Chemical ionization and atmospheric-pressure ionization are covered in Chapters 1 and 9, respectively.) The corona discharge is relatively gentle in that, at atmospheric pressure, it leads to more sample molecules being ionized without causing much fragmentation. 

One of the first successful techniques for selectively removing solvent from a solution without losing the dissolved solute was to add the solution dropwise to a moving continuous belt. The drops of solution on the belt were heated sufficiently to evaporate the solvent, and the residual solute on the belt was carried into a normal El (electron ionization) or Cl (chemical ionization) ion source, where it was heated more strongly so that it in turn volatilized and could be ionized. However, the moving-belt system had some mechanical problems and could be temperamental. The more recent, less-mechanical inlets such as electrospray have displaced it. The electrospray inlet should be compared with the atmospheric-pressure chemical ionization (APCI) inlet, which is described in Chapter 9. 

Since ions and neutral molecules are formed close together in an API source, many ion/molecule collisions occur as in Cl, and so the ion evaporation process also has impressed upon it the characteristics of Cl. Therefore, API is usually thought to involve a mix of ion evaporation and chemical ionization. 

Another way of improving ion yield is to include a repeller electrode (Figure 11.1). This electrode slows lighter ions more than heavier ones, which catch up and collide, causing enhanced chemical ionization. 

Most of the ions produced by either thermospray or plasmaspray (with or without the repeller electrode) tend to be very similar to those formed by straightforward chemical ionization with lots of protonated or cationated positive ions or negative ions lacking a hydrogen (see Chapter l).This is because, in the first part of the inlet, the ions continually collide with neutral molecules in the early part of their transit. During these collisions, the ions lose excess internal energy. 

This is entirely analogous to the problem with simple chemical ionization, and the solution to it is similar. To give the quasi-molecular ions the extra energy needed for them to fragment, they can be passed through a collision gas and the resulting spectra analyzed for metastable ions or by MS/MS methods (see Chapters 20 through 23). 

The term nebulizer is used generally as a description for any spraying device, such as the hair spray mentioned above. It is normally applied to any means of forming an aerosol spray in which a volume of liquid is broken into a mist of vapor and small droplets and possibly even solid matter. There is a variety of nebulizer designs for transporting a solution of analyte in droplet form to a plasma torch in ICP/MS and to the inlet/ionization sources used in electrospray and mass spectrometry (ES/MS) and atmospheric-pressure chemical ionization and mass spectrometry (APCI/MS). 

A further important property of the two instruments concerns the nature of any ion sources used with them. Magnetic-sector instruments work best with a continuous ion beam produced with an electron ionization or chemical ionization source. Sources that produce pulses of ions, such as with laser desorption or radioactive (Californium) sources, are not compatible with the need for a continuous beam. However, these pulsed sources are ideal for the TOF analyzer because, in such a system, ions of all m/z values must begin their flight to the ion detector at the same instant in... 

The LC/TOF instmment was designed specifically for use with the effluent flowing from LC columns, but it can be used also with static solutions. The initial problem with either of these inlets revolves around how to remove the solvent without affecting the substrate (solute) dissolved in it. Without this step, upon ionization, the large excess of ionized solvent molecules would make it difficult if not impossible to observe ions due only to the substrate. Combined inlet/ionization systems are ideal for this purpose. For example, dynamic fast-atom bombardment (FAB), plas-maspray, thermospray, atmospheric-pressure chemical ionization (APCI), and electrospray (ES)... 

Some mild methods of ionization (e.g., chemical ionization. Cl fast-atom bombardment, FAB electrospray, ES) provide molecular or quasi-molecular ions with so little excess of energy that little or no fragmentation takes place. Thus, there are few, if any, normal fragment ions, and metastable ions are virtually nonexistent. Although these mild ionization techniques are ideal for yielding molecular mass information, they are almost useless for providing details of molecular structure, a decided disadvantage. 

The study of metastable ions concerns substances that have been ionized by electrons and have undergone fragmentation. The stable molecular ions that are formed by soft ionization methods (chemical ionization. Cl field ionization, FI) need a boost of extra energy to make them fragment, but in such cases other methods of investigation than linked scanning are generally used. 

As described above, the mobile phase carrying mixture components along a gas chromatographic column is a gas, usually nitrogen or helium. This gas flows at or near atmospheric pressure at a rate generally about 0,5 to 3.0 ml/min and evenmally flows out of the end of the capillary column into the ion source of the mass spectrometer. The ion sources in GC/MS systems normally operate at about 10 mbar for electron ionization to about 10 mbar for chemical ionization. This large pressure... 

As each mixture component elutes and appears in the ion source, it is normally ionized either by an electron beam (see Chapter 3, Electron Ionization ) or by a reagent gas (see Chapter I, Chemical Ionization ), and the resulting ions are analyzed by the mass spectrometer to give a mass spectmm (Figure 36.4). 

Liquids that are sufficiently volatile to be treated as gases (as in GC) are usually not very polar and have little or no hydrogen bonding between molecules. As molecular mass increases and as polar and hydrogen-bonding forces increase, it becomes increasingly difficult to treat a sample as a liquid with inlet systems such as El and chemical ionization (Cl), which require the sample to be in vapor form. Therefore, there is a transition from volatile to nonvolatile liquids, and different inlet systems may be needed. At this point, LC begins to become important for sample preparation and connection to a mass spectrometer. 

For solids, there is now a very wide range of inlet and ionization opportunities, so most types of solids can be examined, either neat or in solution. However, the inlet/ionization methods are often not simply interchangeable, even if they use the same mass analyzer. Thus a direct-insertion probe will normally be used with El or Cl (and desorption chemical ionization, DCl) methods of ionization. An LC is used with ES or APCI for solutions, and nebulizers can be used with plasma torches for other solutions. MALDI or laser ablation are used for direct analysis of solids. 

El = electron ionization Cl = chemical ionization ES = electrospray APCI = atmospheric-pressure chemical ionization MALDI = matrix-assisted laser desorption ionization PT = plasma torch (isotope ratios) TI = thermal (surface) ionization (isotope ratios). 

There are methods for vaporizing solids of low volatility by placing them on a thin wire, which is then raised to a high temperature within a fraction of a second (direct chemical ionization, DCI). This rapid heating allows some vaporization without decomposition, but with the development of later ionization methods, it is now rarely used. 

Electrospray Ionization (ES) and Atmospheric Pressure Chemical Ionization (APCI)... 

Because this chemical reaction occurs between the and M species, the original methane (CH4) is called a reagent gas, the CH5+ species are reagent gas ions, and the process is known as chemical ionization (Cl). 

Chemical ionization produces quasi-molecular or protonated molecular ions that do not fragment as readily as the molecular ions formed by electron ionization. Therefore, Cl spectra are normally simpler than El spectra in that they contain abundant quasi-molecular ions and few fragment ions. It is advantageous to run both Cl and El spectra on the same compound to obtain complementary information. 

Some of the target molecules gain so much excess internal energy in a short space of time that they lose an electron and become ions. These are the molecular cation-radicals found in mass spectrometry by the direct absorption of radiation. However, these initial ions may react with accompanying neutral molecules, as in chemical ionization, to produce protonated molecules. 

The above direct process does not produce a high yield of ions, but it does form many molecules in the vapor phase. The yield of ions can be greatly increased by applying a second ionization method (e.g., electarn ionization) to the vaporized molecules. Therefore, laser desorption is often used in conjunction with a second ionization step, such as electron ionization, chemical ionization, or even a second laser ionization pulse. 

Thus, either the emitted light or the ions formed can be used to examine samples. For example, the mass spectrometric ionization technique of atmospheric-pressure chemical ionization (APCI) utilizes a corona discharge to enhance the number of ions formed. Carbon arc discharges have been used to generate ions of otherwise analytically intractable inorganic substances, with the ions being examined by mass spectrometry. 

The beam of substrate molecules then passes straight into the ion source (electron ionization, El, or chemical ionization. Cl) for ionization before entry into the mass analyzer. 

Samples containing mixtures of peptides can be analyzed directly by electrospray. Alternatively, the peptides can be separated and analyzed by LC/MS coupling techniques such as electrospray or atmospheric pressure chemical ionization (APCI). 

The ion guides are frequently used to transmit ions from an atmospheric-pressure inlet/source system (electrospray ionization, atmospheric-pressure chemical ionization) into the vacuum region of an m/z analyzer. 

---