Ionization Methods

The mass analysis step in MS requires the interaction of charged ions with magnetic or electrical fields, and therefore a means must be found either to create ions from neutral molecules, or to extract ions from a sample solution and transfer them to (or isolate them in) the gas phase. The ionization source in the mass spectrometer accomplishes this task. In mass spectrometers that interface to various methods for separation, particularly column chromatographic methods, only three ionization methods are used for the majority of applications, and will form the focus of the discussion here. EI dates back to the first developments of MS, and is the basis for the extensive mass spectral libraries available. CI was developed in the mid-1960s and is a powerful adjunct to EI. It is especially useful for the determination of molecular masses of compounds that fragment extensively under EI conditions. ESI is more recent in origin, and produces a different type of mass spectrum. Matrix-assisted laser desorption ionization (MALDI) is an even newer ionization method with special applications to high mass biomolecules. As separation methods are developed for separation of truly high mass biomolecules, and mixtures of such compounds, MALDI may become as common an ionization method as the others described in this section. Note that both EI and CI deal with sample molecules in the gas phase, while ESI brings charged species directly out of a liquid solution, and MALDI generates sample-related ions from a mixture of the solid sample and an energy-absorbing matrix.

Electron ionization EI was the first ionization method developed for MS, and it remains the most widely used. The term 'electron impact' is still also used, and the acronym EI covers both terms. Importantly, the most extensive mass spectral libraries assembled are those of EI mass spectra recorded under a 'standard' set of conditions (70 eV electron energy). The gas that flows into an EI source (helium from a gas chromatograph, for example) is confined so that the gas-phase sample molecules interact with the electrons emitted from a metal filament. A high conductance of un-ionized, neutral sample molecules out of the source must also be maintained to minimize cross-peak contamination. EI sources are maintained at a temperature of about 200°C to prevent condensation of sample molecules on the source walls.

The ionization process is the direct result of the interaction of an energetic electron with the sample molecule. The electrons are emitted from a filament through which 3-4 A of current is passed to heat the filament to about 2000°C. Electrons are accelerated into the source by maintenance of the electron filament at a potential more negative than that of the source itself; a potential difference of 70 V (therefore 70 eV) is standard. Then electrons travel across the source to the trap where they are collected and the current is amplified. The trap is used as part of the feedback loop to maintain a constant emission of electrons from the filament. This current is usually about 100 |xA (about 6.25 x 1014 electrons per second). Only a small fraction of the electrons passing through the ion source participates in ionization of sample molecules, and only about 1% of the sample molecules are ionized in EI. The EI process can be written for the gas-phase sample molecule M:

M(gas) + efilament P M+ + efilament + emolecule

The molecular ion M4 (the dot denotes an unpaired odd electron) may subsequently dissociate, since EI imparts more energy to the gas molecule M than is required for ionization alone. The excess energy can cause the dissociation of the molecular ion M 4, or it can be retained in the ion as excess internal energy. Since an electron is far too light to transfer kinetic energy to the sample molecule in a collisional process, the process of EI involves only electronic excitation of M. The molecular ion M4 retains the original structure of the molecule M, at least for a short time after its formation. If dissociations of the molecular ion are prompt, therefore, we can assume that the dissociations represent those of the original molecule and not a structurally reorganized isomer. Some molecular ions formed will be stable enough to pass through the mass spectrometer and reach the detector. Their measured m/z ratio is a direct indication of the molecular mass of the sample molecule itself. For those molecular ions that dissociate, the fragment ions that form are produced from a structure that is a direct analogue of the molecular structure. Clues to the original structure can thus be obtained by piecing together or rationalizing the processes that lead to fragmentation.

Chemical ionization In EI, if too much energy is deposited into the M4 ion during the ionization process, or if the molecule is especially prone to dissociate, fragment ions may be seen in the mass spectrum, but the M 4 may be reduced to such a low intensity that it is indistinguishable from the back ground signal level. Without the molecular ion, the determination of molecular mass is difficult. CI was developed to overcome this difficulty and provide molecular ions for such compounds. CI involves a collision and reaction between an ion and a gas-phase sample molecule. The ion is called the reagent ion and the molecule is the gas-phase neutral sample molecule. There is no common and standard set of operating conditions for measurement of CI mass spectra such as exists with the EI source. As a result, there is not a large CI spectral library, and interpretation of CI mass spectra depends more heavily on the skill and experience of the user.

The CI source is a variation of the standard EI source, with modifications required to achieve a higher source pressure (about 1 torr) while keeping the mass analyser pressure within acceptable limits. Methane is a common CI reagent gas, and the reagent ion CH5# will transfer a proton to the gas-phase sample molecule to form (M + H) +. The protonated molecule is relatively stable, and can usually be observed in the mass spectrum of a compound for which the molecular ion M4 formed by EI cannot be distinguished. The source filament is still heated to a high temperature so that electron emission occurs, but in CI the electron energy is usually 250-500 eV; the higher energy allows the electrons to penetrate through the high pressure in the source. The gas pressure caused by sample molecules is still about 10m5-10mfi torr, just as it was in the EI source. As the pressure of methane is 1 torr, in an equal source volume, the electron emitted from the filament is much more likely to encounter a methane molecule. When it does, an EI process occurs, namely:

CH4 4 efilament P CH4 + efilament 4 emethane

The CH/' ion does not travel far before it encounters a neutral gas molecule, and at 1 torr of methane and 10 m6 torr of sample, the molecule it encounters will most likely be a methane molecule. The next reaction creates CH# and CH3. Several other reactions occur, and the final distribution of ions depends explicitly on the source temperature and pressure. The primary reactant ion for methane reagent gas is usually CH54 , and this ion acts as a strong gas-phase acid that protonates anything more basic than methane. The sample molecules are sufficiently basic to accept a proton to form the protonated molecule. The proto-nated molecule then fragments in accordance with the amount of internal energy it contains. In most cases not all of the protonated molecules fragment, and since there is an observable signal for the protonated molecule in the mass spectrum, the molecular mass of the sample compound can be established. The fragmentation processes in CI are different from those observed in the EI mass spectrum, since the (M + H) + ion is an even-electron rather than an odd-electron species. The pattern of fragment ions is still interpreted to support a structure for the sample molecule. Other reagent gases form ions that transfer protons to the neutral sample molecules, forming an ion of the same mass but with a different amount of internal energy. The protonated molecule therefore fragments to a different extent. The degree of fragmentation of the sample molecule can be 'tuned' by choice of the reagent gas, and this experiment can be useful in interpretation of CI mass spectra.

Electrospray ionization In GC, helium carrier gas that enters the source along with the vapours of the sample does not disrupt the EI or CI process. However, both LC and CE separate species in a solvent, and the sample enters the ionization source along with a continuous flow of solvent (aqueous or organic) that generates a tremendous amount of solvent vapour. If EI or CI are to be used, the bulk of the solvent must be removed without loss of the sample, as the great excess of solvent vapour will certainly affect the ionization process. Various means have been devised to accomplish this task, but the efficiency is low and the process cumbersome and subject to many complicating factors. ESI allows ions to be created directly from the sample solution, and conveniently at atmospheric pressure. In ESI, the mechanical need for solvent removal is greatly reduced, albeit at the cost of allowing only a small continuous flow of solution to enter the mass spectrometer.

In ESI the sample solution is passed from the LC column or CE column through a connection junction into a short length of stainless steel capillary. A high positive or negative electrical potential, typically 3-5 kV, is applied to this capillary. There is clearly a need for electrical isolation in the LC connection, and potential management in CE. As the solution is forced to flow through the capillary tip, the solution is nebulized into a spray of very small droplets. This spray is formed at atmospheric pressure. The mass spectrometer operates at a vacuum of 10~5-10~6 torr. The pressure must therefore be reduced before the droplets (and the sample species that they contain) enter the mass spectrometer. The spray of droplets is usually directed through a skimmer that provides a differential pressure aperture, and also acts as a momentum separator. As the droplets move through this region, neutral solvent molecules evaporate rapidly and the droplets become progressively smaller. As droplets leave the charged capillary needle, most of them retain an excess of positive or negative electrical charge, corresponding to the potential applied to the capillary. This excess charge resides on the surface of the droplets. As the droplets get smaller, the electrical surface charge density increases until the natural repulsion between like charges causes ions as well as neutral molecules to be expelled from the droplets. This field-induced evaporation also forces the droplets to become progressively smaller. Note that ions themselves cannot evaporate from the droplet. However, if the charge density is high, a Coulomb-force-induced 'explosion' can expel them from the droplet. As solvent molecules evaporate from the droplets they diffuse in all directions, while the higher momentum, charged droplets are directed towards the first skimmer, and then (usually) through a second concentric skimmer that lowers the pressure even further, by a combination of momentum separation and steering potentials applied to the skimmers. In some ESI sources a drying gas (nitrogen) flows along and past the end of the capillary and skimmer to assist with evaporation of the solvent from the droplets. The end result of the electrospray and progressive desolvation process is a stream of ions that have been extracted directly from the solution in which they were originally found.

If sample ions are already present in the solution then it is clear that these ions can be sampled directly. Solvents used in LC and CE also have appreciable ion concentrations, especially as buffers and ionic modifiers are often present in the solutions. In most cases, there is a substantial free proton population. Protons will not evaporate from the droplet as it becomes smaller; the 'pH' rises inexorably as the droplet becomes smaller. During the last stages of solvent evaporation, the protons will be forced to associate with the most basic molecules remaining in the droplet. This is not necessarily an acid-base equilibrium situation, because the dynamics of desolva-tion and sampling play a large role. However, the situation can be considered as one in which a free proton (a strong acid) protonates the sample molecule, which is forced to act as a proton acceptor. Other Lewis acids (cations) present in the droplet act similarly. There is a transition from the lower concentrations of ionic species present in the bulk solution to the near 100% ionic population present in a nano-droplet. The 'pH' drops to such a low value that multiple protonation is common.

The unique nanodroplet environment from which ions are drawn in ESI provides a route to highly protonated, multiply charged ions. It is the formation of multiply charged ions that makes ESI valuable for examination of sample molecules of high molecular masses. In EI and CI, most ions are formed with a single positive or negative charge. The x-axis of the mass spectrum is the m/z ratio, and z is one. Therefore the mass on the 'm/z' axis is directly indicative of the sample molecular mass. In ESI, values of z greater than one are commonplace. ESI-derived positive ions are found as [M + nH]n +, where n ranges from 2 to 30, and is sometimes as high as 100. Several factors contribute to the propensity of ESI to create multiply charged ions. The first is the strongly acidic environment of the nanodroplet. The second is the fact that higher molecular mass molecules are, quite naturally, large molecules, and larger molecules can accommodate a greater number of protons. For a protein, for example, a basic amino acid residue will be the site associated with the proton. Basic amino acid residues will be far enough apart in a typical protein that the protons add independently, and there is minimal Coulombic repulsion between the charged sites. The higher order structure of the protein will therefore determine what sites are accessible for protonation, and this characteristic is the basis for some of the most intriguing and revealing ESI experiments.

Suppose that M, the molecular mass of the sample molecule, is 10 000 Da. In ESI, the (M # 20H)20+ ion may be formed. This ion has a mass-to-change ratio of (M + 20H)20 +, and therefore m/z = 10020/20 = 501. This mass is well within the range of the mass spectrometer, and can be determined accurately. Usually several different forms of the multiply charged ions are found, namely (M + nH)n + , with a distribution of intensities. Each successive molecular ion contains one more proton and therefore one more charge. The series is easy to identify, and the value of n need only be determined for any one ion for the entire ion series to fall into place. The value of n can be determined from the spacing of isotope peaks in the molecular ion isotopic envelope, and the derivation has now been fully automated. The mass spectrum that contains the array of multiply charged ions is plotted in terms of the m/z values of those ions. But M is the same for each of those ions, and each ion is a slightly different pointer to that value of M. Having determined the masses of each of the multiply charged ions, the series of equations can be solved to determine the value of M. The data can now be presented as a transformed spectrum with one molecular ion, M. If there is more than one sample molecule M, the m/z spectrum can appear extraordinarily complex, but the transformed mass spectrum clearly shows the presence of multiple components (although the relative intensities may not accurately reflect the solution concentrations of the sample molecules).

Matrix-assisted laser desorption ionization The title of this section reveals MALDI as the most 'matrix-

dependent' of the ionization methods discussed in this overview. This should not be surprising, as the ionization usually occurs from a solid-phase mixture of sample molecules in a large excess of energy-absorbing matrix molecules. Therefore, MALDI would appear to be the most ill-suited of the techniques discussed for interfacing to column chromatographic methods. However, effluents from LC separations have been deposited onto collection surfaces, and then the trail in space (along the x-dimension, for example) analysed by MALDI to provide the corresponding trace of sample elution in time. MALDI has also been applied to planar separation methods such as thin-layer chromatography (TLC) and gel electrophoresis. As interface technology improves, a miniaturized solid surface may be used to intercept effluents from columns. This may be a particularly attractive interface since MALDI, in conjunction with a time-of-flight mass analyser, has been shown to be a very capable ionization method for the production of simple mass spectra of very high mass biomolecules. Column chromatography will increasingly be used to perform separations of mixtures of such molecules, although not necessarily in the forms described previously in this section, and MALDI may be used increasingly in such applications.

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