Other Useful Ionization Techniques

For organic compounds that are sufficiently volatile, introduction by vaporization or by gas chromatography 

Less volatile but thermally stable compounds can be thermally vaporized in the direct inlet probe (DIP) situated close to the ionizing molecular beam. This DIP is standard equipment on most instruments. An electron-impact spectrum results. 

For compounds that are not thermally stable enough for the direct inlet probe, field desorption (FD) is the next resort. 

In recent years, several procedures have been developed for handling high molecular weight, water-soluble biomolecules. Several of these procedures are here briefly described. [See the Chapman (1993) and Watson (1985) references for a thorough discussion of these techniques. The Harrison (1992) reference presents a thorough treatment of chemical ionizations.] 

1 Chemical Ionization (Cl) The vaporized sample is introduced into the mass spectrometer with an excess of a reagent gas (commonly methane) at a pressure of about 1 torr. The excess carrier gas is ionized by electron impact to the primary ions CH4,+ and CH3+. These react with the excess methane to give secondary ions. 

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One of the advantages of FAB over other soft ionization techniques is that it uses glycerol (or some other suitable liquid phase) which sets up a condition where the surface of the droplet is constantly replenished with sample molecules. A second advantage of the liquid matrix is that it allows solution chemistry to take place virtually at the site of analysis. 

Among the other soft ionization techniques is laser microprobe mass spectrometry (LAMMA) in which a laser pulse is used to vaporize a small amount of sample, as discussed in a 1982 review (108). Of interest to us is the application to the study of some cobalamins (109). (M + H) and (M - H) ions were observed in the positive and negative ion modes, respectively. However, there were few other high-mass fragments that could be used to impart structural information. 

The limit of detection depends considerably on the abundance of the ionic species that is measured. The more abundant the measured ionic species is with respect to all of the ions derived from the analysed molecule, the higher is the limit of detection, as shown in Figure 6.12. The goal is thus to produce a signal that is as intense as possible. Several methods allow this goal to be reached, such as the modification of the ionization conditions, reversal into the negative mode, the use of other softer ionization techniques or the derivative of the sample, in order to increase the number of ions produced in the source or to reduce their fragmentation. 

By modifying the laser wavelength (and intensity) or using a second laser, a photoionization process may be included (LPI) to generate ions from the analyte that will be further separated and detected in the mass spectrometer. When the ion formation from the plume of the first laser is low, some other secondary ionization techniques such as Cl can also be used. 

While ESI and MALDI are the most commonly used ionization techniques for the MS analysis of biological samples, many other ionization mechanisms do exist and some of them were already implemented on microfluidic devices. APCI and various laser desorption ionization sources were pursued. A miniaturized APCI nebulizer chip, fabricated from silicon and Pyrex glass wafers, was designed to accommodate sample inlet capillaries, a stopper, a vaporizer channel, and a nozzle. The nebulizer chip was used to interface capillary LC to MS, but could be integrated within CE separation chips as well. 

APCI is a relatively soft ionization technique. In fact, only few fragment ions are normally recorded. Nevertheless, analyte decomposition may occur due to heating. It can be used in the analysis of polar and low polar analytes with molecular weights up to 1500 Da [29] as long as the proton affinity of the analytes is higher than that of the solvents. On the other hand, ionization techniques such as ESI and matrix-assisted laser desorption/ionization (MALDI) are mainly used in the analyses of polar, less volatile, and thermally labile analytes, for example, large biomolecules. These two ionization techniques are discussed in the following (Sections 2.4 and 2.6). 

Advances in mass spectrometry have made it a tool of exceptional power for analysis of large biomolecules. Electrospray ionization, MALDI, and other soft ionization techniques for nonvolatile compounds and macromolecules make possible analyses of proteins, nucleic acids, and other biologically relevant compounds with molecular weights up to and in excess of 100,000 daltons. Electrospray ionization with quadrupole mass analysis is now routine for biomolecule analysis as is analysis using MALDI-TOF instruments. Extremely high resolution can be achieved using Fourier transform-ion cyclotron resonance (FT ICR, or FTMS). We shall discuss ESI and MALDI applications of mass spectrometry to protein sequencing and analysis in Sections 24.5E, 24.13B, and 24.14. 

On the other hand, there are some ionization techniques that are very useful, particularly at very high mass, but produce ions only in pulses. For these sources, the ion extraction field can be left on continuously. Two prominent examples are Californium radionuclide and laser desorption ionization. In the former, nuclear disintegration occurs within a very short time frame to give a... 

The use of mass spectrometry for the analysis of peptides, proteins, and enzymes has been summarized. This chapter should be read in conjunction with others, including Chapter 45, An Introduction to Biotechnology, and Chapters 1 through 5, which describe specific ionization techniques in detail. 

In other articles in this section, a method of analysis is described called Secondary Ion Mass Spectrometry (SIMS), in which material is sputtered from a surface using an ion beam and the minor components that are ejected as positive or negative ions are analyzed by a mass spectrometer. Over the past few years, methods that post-ion-ize the major neutral components ejected from surfaces under ion-beam or laser bombardment have been introduced because of the improved quantitative aspects obtainable by analyzing the major ejected channel. These techniques include SALI, Sputter-Initiated Resonance Ionization Spectroscopy (SIRIS), and Sputtered Neutral Mass Spectrometry (SNMS) or electron-gas post-ionization. Post-ionization techniques for surface analysis have received widespread interest because of their increased sensitivity, compared to more traditional surface analysis techniques, such as X-Ray Photoelectron Spectroscopy (XPS) and Auger Electron Spectroscopy (AES), and their more reliable quantitation, compared to SIMS. 

For many years, electron ionization, then more usually known as electron impact, was the only ionization method used in analytical mass spectrometry and the spectra encountered showed exclusively the positively charged species produced during this process. Electron ionization also produces negatively charged ions although these are not usually of interest as they have almost no structural significance. Other ionization techniques, such as Cl, FAB, thermospray, electrospray and APCI, however, can be made to yield negative ions which are of analytical utility. 

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. 

The ability to produce ions using electrospray ionization is more reliant on the solution chemistry of the analyte than the other ionization techniques described and this feature may be used by the analyst to advantage. It may also confuse the unwary ... 

An electrospray spectrum is unusual in that while it provides only ions from molecular species, it consists (usually) of ions at a number of m/z values, i.e. it appears more like an El than a Cl spectrum, with the number of these ions increasing with the molecular weight of the analyte. The appearance of the spectrum is much more dependent upon the environment from which it has been produced than those produced using other ionization techniques. The spectra produced from a single analyte under different experimental conditions may therefore vary considerably in appearance. 

In contrast to ions generated by other ionization techniques, these ions do not occur at integer m jz values and this non-integer part of the mass is of crucial importance when making a precise determination of the molecular weight of the analyte involved - this being one of the major uses of electrospray ionization. 

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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