Soft ionization

The ionization processes described in the following sections are classified as soft ionization methods. The intention is to reduce the amount of energy added to the analyte to the point where the only ions formed are directly related to the molecular mass and the energy is insufficient to cause the formation of fragment ions. To accomplish this aim, the amount of energy transferred to the analyte is reduced to the minimum required to ionize organic compounds, 10 eV, much less than the 70 eV used in EL Cl is less efficient but more structurally selective than EL 

The predominant process of ion formation in the soft ionization methods is through the association of the analyte with an ionizing agent. Typically a proton is attached to the analyte, yielding [M + H]+ ions, but there are other species that can be adducted onto the analyte to form alternative ions in the molecular ion region. 

A protonated molecrdar ion wordd have two charges the proton and the molecular ion. 

Electrons from the filament react with a reagent gas (methane, isobutane, or ammonia) generating protonated reagent species that, in turn, transfer a proton onto, or form an adduct with, the analyte. 

The efficiency of Cl is enhanced by raising the energy of the electron beam to 200 eV thereby increasing the formation of the reagent species. Also, the vacuum system must include differential pumping (Section 2.5) to remove the excess reagent gas so that it does not affect the pressure in the mass analyzer. 

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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. 

Multidimensional or hyphenated instmments employ two or more analytical instmmental techniques, either sequentially, or in parallel. Hence, one can have multidimensional separations, eg, hplc/gc, identifications, ms/ms, or separations/identifications, such as gc/ms (see CHROMATOGRAPHY Mass spectrometry). The purpose of interfacing two or more analytical instmments is to increase the analytical information while reducing data acquisition time. For example, in tandem-mass spectrometry (ms/ms) (17,18), the first mass spectrometer appHes soft ionization to separate the mixture of choice into molecular ions the second mass spectrometer obtains the mass spectmm of each ion. 

The analysis of penicillins by mass spectrometry (qv) has developed with the advent of novel techniques such as fast atom bombardment. The use of soft ionization techniques has enabled the analysis of thermally labile nonvolatile compounds. These techniques have proven extremely valuable in providing abundant molecular weight information from underivatized penicillins, both as free acids and as metal salts (15). 

Alternative ( soft ) ionization techniques are not usually required for aromatic isothiazoles because of the stability of the molecular ions under electron impact. This is not the case for the fully saturated ring systems, which fragment readily. The sultam (25) has no significant molecular ion under electron impact conditions, but using field desorption techniques the M + lY ion. is the base peak (73X3861) and enables the molecular weight to be confirmed. 

Low ionizing potentials or soft ionization methods are necessary to observe the parent ions in the mass spectra of many S-N compounds because of their facile thermal decomposition. Mass spectrometry has been used to investigate the thermal breakdown of S4N4 in connection with the formation of the polymer (SN). On the basis of the appearance potentials of various S Ny fragments, two important steps were identified ... 

Unfortunately, not every compound shows a molecular ion in its mass spectrum. Although M+ is usually easy to identify if it s abundant, some compounds, such as 2,2-dimelhylpropane, fragment so easily that no molecular ion is observed (Figure 12.3). In such cases, alternative "soft" ionization methods that do not use electron bombardment can prevent or minimize fragmentation. 

Most biochemical analyses by MS use either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALD1), typically linked to a time-of-flight (TOF) mass analyzer. Both ESI and MALDl are "soft" ionization methods that produce charged molecules with little fragmentation, even with biological samples of very high molecular weight. 

Chapter 12, Structure Determination Mass Spectrometiy and Infrared Spectroscopy—A new Section 12.4 discusses mass spectrometry of biological molecules, focusing on time-of-flight instruments and soft ionization methods such as MAI.DI. 

Electrospray ionization mass spectrometry (ESI-MS) is an analytical method for mass determination of ionized molecules. It is a commonly used method for soft ionization of peptides and proteins in quadmpole, ion-trap, or time-of-flight mass spectrometers. The ionization is performed by application of a high voltage to a stream of liquid emitted from a capillaty. The highly charged droplets are shrunk and the resulting peptide or protein ions are sampled and separated by the mass spectrometer. 

Fortunately, this backgronnd is often less of a problem than might be anticipated from the above. The majority of ionization techniques employed in LC-MS are soft ionization techniqnes which provide primarily molecular ions that occur at relatively high values of mass-to-charge ratio (m/z), rather than fragment ions which occur at relatively low m/z values. In the majority of cases, the molecular weight of the analyte is higher than those of the solvent impurities and the effect of these may therefore be minimized. 

Chemical ionization (Cl) is a technique that has been developed specifically to enhance the production of molecular species, i.e. to reduce the fragmentation associated with ionization. A number of such techniques exist and these are known collectively as soft ionization techniques . 

The ionization techniques most widely used for LC-MS, however, are termed soft ionization in that they produce primarily molecular species with little fragmentation. It is unlikely that the molecular weight alone will allow a structural assignment to be made and it is therefore desirable to be able to generate structural information from such techniques. There are two ways in which this may be done, one of which, the so-called cone-voltage or in-source fragmentation, is associated specifically with the ionization techniques of electiospray and APCl and is discussed later in Section 4.7.4. The other, termed mass spectrometry-mass spectrometry (MS-MS) or tandem mass spectrometry, is applicable to all forms of ionization, provided that appropriate hardware is available, and is described here. 

In this chapter, the main aspects of mass spectrometry that are necessary for the application of LC-MS have been described. In particular, the use of selected-ion monitoring (SIM) for the development of sensitive and specific assays, and the use of MS-MS for generating structural information from species generated by soft ionization techniques, have been highlighted. Some important aspects of both qualitative and quantitative data analysis have been described and the power of using mass profiles to enhance selectivity and sensitivity has been demonstrated. 

Electrospray is a soft-ionization method prodncing intact molecular species and structural information is not usually available. Electrospray sources are capable of producing structural information from cone-voltage fragmentation but these spectra are not always easily interpretable. Experimentally, the best solution is to use a mass spectrometer capable of MS-MS operation but this has not inconsequential financial implications. 

APCl is a soft ionization technique which usually enables the molecular weight of the analyte under study to be determined. 

Atmospheric-pressure chemical ionization (APCI) and electrospray ionization are both soft ionization techniques which give rise, almost exclusively, to the production of molecular species. Structural information. 

This is a task for which electrospray ionization is well suited although, as discussed earlier in Section 4.7, this is a soft ionization technique that yields almost exclusively molecular ions with little fragmentation and consequently, in the case of biopolymers, little sequence information directly. 

MS-MS is a term that covers a number of techniques in which two stages of mass spectrometry are used to investigate the relationship between ions found in a mass spectrum. In particular, the product-ion scan is used to derive structural information from a molecular ion generated by a soft ionization technique such as electrospray and, as such, is an alternative to CVF. The advantage of the product-ion scan over CVF is that it allows a specific ion to be selected and its fragmentation to be studied in isolation, while CVF bring about the fragmentation of all species in the ion source and this may hinder interpretation of the data obtained. 

The specificity of the ions/decompositions must be considered. Both electrospray ionization and APCI are soft ionization techniques and the resulting mass... 

The fact that APCl and electrospray are soft ionization techniques is often advantageous because the molecular ion alone, in conjunction with HPLC separation, often provides adequate selectivity and sensitivity to allow an analytical method to be developed. Again, method development is important, particularly when more than one analyte is to be determined, when the effect of experimental parameters, such as pH, flow rate, etc., is not likely to be the same for each. Electrospray, in particular, is susceptible to matrix effects and the method of standard additions is often required to provide adequate accuracy and precision. 

Soft ionization technique An ionization technique that produces molecular species with few, if any, fragment ions. 

The success of the soft ionization techniques (DCI, FAB, and LSIMS) presents several possibilities for detection of brevetoxins in complex matrices. Positive-ion DCI was used for the analysis of PbTx-3 metabolites generated in vitro by isolated rat hepatocytes (see below). Unmetabolized parent was conclusively identified and metabolites were tentatively identified, pending confirmation by alternate methods (see below). 

In line with the policy of Advances to provide periodic coverage of major developments in physical methodology for the study of carbohydrates, A. Dell (London) here surveys the use of fast-atom-bombardment mass spectrometry in application to carbohydrates. This technique has achieved rapid prominence as the soft ionization technique of choice for structural investigation of complex carbohydrate sequences in biological samples. The author s extensive personal involvement in this field makes her chapter a critical, state-of-the-art overview for the specialist, as well as a valuable primer for the reader unfamiliar with this technique. 

ESI-MS has emerged as a powerful technique for the characterization of biomolecules, and is the most versatile ionization technique in existence today. This highly sensitive and soft ionization technique allows mass spectrometric analysis of thermolabile, non-volatile, and polar compounds and produces intact ions from large and complex species in solution. In addition, it has the ability to introduce liquid samples to a mass detector with minimum manipulation. Volatile acids (such as formic acid and acetic acid) are often added to the mobile phase as well to protonate anthocyanins. A chromatogram with only the base peak for every mass spectrum provides more readily interpretable data because of fewer interference peaks. Cleaner mass spectra are achieved if anthocyanins are isolated from other phenolics by the use of C18 solid phase purification. - ... 

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