Soft Ionisation Techniques

Tandem mass spectrometry or ms/ms was first introduced in the 1970s and gained rapid acceptance in the analytical community. The technique has been used for stmcture elucidation of unknowns (26) and has the abiUty to provide sensitive and selective analysis of complex mixtures with minimal sample clean-up (27). Developments in the mid-1980s advancing the popularity of ms/ms included the availabiUty of powerhil data systems capable of controlling the ms/ms experiment and the viabiUty of soft ionisation techniques which essentially yield only molecular ion species. 

Table 6.10 reports the main areas of application of the various ionisation methods and the principal ions detected. A breakdown of MS techniques applied to various types of analytes is as follows thermally stable, low-MW Cl, El thermally instable, low-MW APCI (FLA, LC-MS), ESI and high-MW DCI, FD, FAB, LD, ESI (FLA, LC-MS, CZE-MS). Soft ionisation techniques such as FL, FAB and LD are useful for the detection of non-volatile, sometimes oligomeric, polymer additives. Recent developments in ionisation techniques have allowed the analysis of polar, ionic, and high-MW compounds, previously not amenable to mass-spectrometric analysis. Figure 6.4 shows the applicability of various atmospheric pressure ionisation techniques in terms of molar mass and polarity. 

The DCI probe is particularly attractive for samples that are susceptible to thermal decomposition, although it can equally well be used as a general means of introducing samples into the ionisation source, i.e. as an alternative to the direct insertion probe. The types of sample which benefit most from DCI probing are higher-molecular-weight, less-volatile compounds, organometallics, and any thermally sensitive compounds [40,67]. DCI is considered to be a soft ionisation technique. 

Generally FAB produces protonated, MH+, or depro-tonated, (M — H) , quasi-molecular ions with a little excess energy which will sometimes produce fragment ions of low intensity. FAB is therefore a mild to soft ionisation technique which produces primarily molecular weight information and some structural information. Positive and negative ionisation mass spectra are produced with equal facility. FAB was originally used with magnetic sector mass spectrometers, but lately mainly with quadrupole mass spectrometers (Table 6.10). 

Applications Early MS work on the analysis of polymer additives has focused on the use of El, Cl, and GC-MS. The major drawback to these methods is that they are limited to thermally stable and relatively volatile compounds and therefore are not suitable for many high-MW polymer additives. This problem has largely been overcome by the development of soft ionisation techniques, such as FAB, FD, LD, etc. and secondary-ion mass spectrometry. These techniques all have shown their potential in the analysis of additives from solvent extract and/or from bulk polymeric material. Although FAB has a reputation of being the most often used soft ionisation method, Johlman el al. [83] have shown that LD is superior to FAB in the analysis of polymer additives, mainly because polymer additives fragment extensively under FAB conditions. 

Principles and Characteristics The pioneering technique of field ionisation (FI) was the first soft ionisation technique, introduced in 1954 [105]. For FI analysis of a reasonably volatile sample, the compound under investigation is volatilised by heat close to the emitter, so that its vapour can condense on to an emitter needle. Hence,... 

Thermospray (TSP) is another soft ionisation technique which produces predominantly MH+ or (M — H) ions, together with some fragmentation. TSP is best suited to the analysis of organic compounds of low molecular mass (<1000 Da) that exhibit some polarity. Polymer additive molecules fall in this wide category. 

The process of ion formation is extremely soft usually no fragmentation occurs. Mass spectra in ESI+ mode are dominated by pseudo-molecular ions (M+, [M + H] + or [M + Na]+) and cluster ions formed by the addition of one or more solvent molecules. For samples with molecular masses up to ca. 1000 Da (as in case of polymer additives), the ions produced from ESI are similar to those formed by other soft ionisation techniques, namely a protonated molecular ion (MH+) in the positive ionisation mode for basic compounds such... 

MS/MS has especially found analytical applications in combination with soft ionisation techniques, where, without MS/MS, only molecular weight information on... 

Applications With the current use of soft ionisation techniques in LC-MS, i.e. ESI and APCI, the application of MS/MS is almost obligatory for confirmatory purposes. However, an alternative mass-spectrometric strategy may be based on the use of oaToF-MS, which enables accurate mass determination at 5 ppm. This allows calculation of the elemental composition of an unknown analyte. In combination with retention time data, UV spectra and the isotope pattern in the mass spectrum, this should permit straightforward identification of unknown analytes. Hogenboom et al. [132] used such an approach for identification and confirmation of analytes by means of on-line SPE-LC-ESI-oaToFMS. Off-line SPE-LC-APCI-MS has been used to determine fluorescence whitening agents (FWAs) in surface waters of a Catalan industrialised area [138]. Similarly, Alonso et al. [139] used off-line SPE-LC-DAD-ISP-MS for the analysis of industrial textile waters. SPE functions here mainly as a preconcentration device. 

ESI and APCI are soft ionisation techniques which usually result in quasi-molecular ions such as [M + H]+ with little or no fragmentation molecular weight information can easily be obtained. However, experimental conditions can also be chosen in such a way that a sufficiently characteristic pattern is obtained, allowing verification [540]. ESI is amenable to thermally labile and nonvolatile molecules. Both ESI and APCI are much more sensitive than PB and very well suited for quantitative analysis, but less so for unknown samples. The choice among the two is usually determined by the application. Recently, nanoscale LC-ESI-MS has been developed [541]. The nano-electrospray ion source offers the highest sensitivity available for LC-MS (atto-to femtomole range) and can also be used as an off-line ion source. 

Soft ionisation modes, such as API, which leave the (pseudo)molecular ion intact without much fragmentation, offer more sensitivity, and are ideal for quantitative work at low levels (e.g. breakdown products). With the use of soft ionisation techniques in LC-MS, tandem MS... 

In specific cases, when long chain compounds such as esters and TAGs have survived and have not yet been hydrolysed or oxidised, it may be useful to carry out soft ionisation techniques in order to fully characterise the structure of these biomarkers by direct infusion into an electrospray source after adapted purification treatments. 

Several years later, the next step in the application of MS-MS for mixture analysis was developed by Hunt et al. [3-5] who described a master scheme for the direct analysis of organic compounds in environmental samples using soft chemical ionisation (Cl) to perform product, parent and neutral loss MS-MS experiments for identification [6,7]. The breakthrough in LC-MS was the development of soft ionisation techniques, e.g. desorption ionisation (continuous flow-fast atom bombardment (CF-FAB), secondary ion mass spectrometry (SIMS) or laser desorption (LD)), and nebulisation ionisation techniques such as thermospray ionisation (TSI), and atmospheric pressure ionisation (API) techniques such as atmospheric pressure chemical ionisation (APCI), and electrospray ionisation (ESI). 

One of the most serious drawbacks that has been observed in the ionisation processes with soft ionisation techniques is the very soft generation of ions. This process, which predominately leads to molecular ions or adduct ions but no fragments for identification, however, was also used to improve and speed-up MS analysis. 

MS-MS is a general analytical method for the analysis of unknown compounds. This analytical technique must be applied if soft ionisation techniques are performed and fragment information for identification is needed. With the different types of data obtained by MS-MS application, e.g. product ion scan, parent ion scan, neutral loss scan,... 

Mixture analysis (FIA-MS-MS) with a preceding screening step using flow FIA-MS [5,6,8,9,17] in combination with soft ionisation techniques... 

ESI is a very soft ionisation technique, meaning that very little excess energy is imparted into the molecule during the ionisation process and therefore producing little fragmentation. The prerequisite is that the analyte molecule M has to be basic enough to be protonated... 

API techniques provide efficient ionisation for a variety of molecules including polar, labile and high mass molecules. They are soft ionisation techniques useful... 

The mobile phase enters the instrument directly so that the flow rates can only be ca 10 pl/min. The mobile contains 1% of an involatile matrix, e.g. glycerol. The sample flows out onto the centre of a porous disc and the solvent, apart from the involatile matrix, evaporates. The sample in the matrix is struck by fast atoms (Xe or Cs) from a FAB gun and the high energy of the atoms generates ions from the sample. Soft ionisation technique produces limited fragmentation. Sensitive to 10 g level for lipophilic compounds, mass range up to 2000 amu or more... 

The pseudomolecular ion MH + (which is not a radical ion) is usually more stable than the M + ion produced by electron ionisation. Hence, fewer fragment ions are observed in chemical ionisation and so it is called a soft ionisation technique. 

This desorption ionisation technique leads to weak fragmentation. The analyte is incorporated into a solid organic matrix (such as hydroxybenzoic acid) and the mixture is placed on a sample holder that is irradiated with UV laser pulses (e.g. N2 laser, A = 337 nm, pulse width = 5 ns). The laser energy is absorbed by the matrix and transferred to the analyte, which becomes desorbed and ionised (Fig. 16.18c). Although MALDI is considered to be a soft ionisation technique, a substantial amount of energy is involved. Because the technique involves pulsed ionisation, it is well suited for time-of-flight mass analysis of biomolecules. The analysis of small molecules (M < 500 Da) is limited because the matrix decomposes upon absorption of the laser radiation. However, solid supports such as silicone can be used as the matrix to overcome this disadvantage. 

One of the main advantages of these soft ionisation techniques is that they lead to the formation of multiply charged, pseudomolecular ions (z can be greater than 30). Hence the mass range of the spectrometer can be extended to over 105 Da (to include proteins, polysaccharides and other polymers) (Fig. 16.20). These ionisation devices are often coupled to the mass spectrometer through a heated capillary transmitting the ions. 

The most important piece of information which may be obtained from a mass spectrum is the molecular weight. However, certain classes of compounds do not show molecular ions and in other cases it is not always possible to identify unequivocally the molecular ion. Therefore a family of so called soft ionisation techniques has been developed. These generate a molecular ion or quasi-molecular ion and fragmentation is kept to a minimum. The most commonly used technique is chemical ionisation (Cl)... 

Ofrier soft ionisation techniques include field desorption, field ionisation, atmospheric pressure ionisation, and fast atom bombardment and each increases the rmolecular weight but because of the production of cluster ions such as (M + Na), (M + 2Na), 2M, etc., they are not used in the investigation of unknowns. 

Fragmentation is characteristic for a given compound providing a uniquely identifiable mass spectrum Soft ionisation technique can form pseudomolecular ions with little fragmentation Very little fragmentation, predominantly singly charged species formed low sample volumes required... 

Lawson et al. (2000) examined the migration of constituents from solvent-free adhesives used to bond 12 pm PET film to 45 pm LDPE. The technique of MALDl-MS, a soft ionisation technique capable of looking at sample mixtures over a mass range of 150-500,000 Da without prior separation, was employed. The adhesives studied were based on a solution of mixed isomers of MDl in polymeric MDI with either polyether or polyester-based polyols. Pouch testing of cured laminates with distilled water was undertaken (two hours at 70 °C) with the LDPE surface in contact with the water. 

It is preferable to have a mass spectrum which shows the molecular ion of the compound and for this reason soft ionisation techniques such as chemical ionization (Cl), fast atom bombardment (FAB) and electrospray are now widely used instead of or as well as electron impact (El) ionization. 

Recent advances in mass spectrometry have produced a number of soft ionisation techniques such as fast atom bombardment (FAB) or electrospray ionisation. The major advantage of these techniques is that they are less likely to break the sample into small fragments and are more likely to produce a molecular ion. This is particularly important in the analysis of macromolecules such as proteins and nucleic acids. 

Particularly with overtly reactive labelling reagents such as halocarbonyl compounds, it is desirable to have confirmation that the labelling is active site directed. Only one molecule of the reagent should be incorporated per active site inactivated (these days this readily established by mass spectrometry with soft ionisation techniques such as electrospray) and the kinetics of inactivation in the presence of excess inactivator should be first order (if A is activity, then At = oexp(—kobsO- particular, quantitative protection by a reversible... 

Another way to alleviate the problem of lack of molecular weight information resides in the use of so-called "soft ionisation techniques". These techniques have been developed to provide the mass spectrometrist with means to obtain mass spectra that almost invariably contained molecular weight information. In addition, in many instances, the limit of detection is enhanced as a result of the lesser number of ion species being produced for a same quantity of ionised material (less ion species for the same total ion count). However, these techniques suffer from reproducibility difficulties and, generally, offer less structural information. These factors might account for the fact that libraries for such mass spectra were never tabulated. 

We will introduce only two soft ionisation techniques. Both techniques selected here, chemical ionisation and fast atom bombardment, make use of very different phenomena. Our choice is not intended to be exhaustive, nor to be representative of all processes involved, it is merely used to demonstrate two main variations brought forth to obtain novel, innovative ways of ionising various substances, and yet yield informative mass spectra. 

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