In selecting the ionisation technique, two criteria are considered essential. First, minimal ionisation fragmentation of pyrolysis products should occur with principally molecular ions being formed. Secondly, the ionisation process must be stable enough to provide a high degree of long-term reproducibility. While no ionisation technique is ideal in both respects, the advantages and disadvantages of the three major "soft" ionisation techniques, i.e. low voltage EI, CI and FI, will be briefly discussed.

Low voltage EI has been the principal technique used throughout the Curie-point Py-MS studies reported so far. Trial studies using compounds typical of pyrolysates indicate that electron energies between 13 and 15 eV appear to be a good compromise between minimum yield of fragment ions and maximum yield of molecular ions (ref. 46). Once the electron energy has been chosen, every effort should be made to keep this energy constant to within 0.1 eV. In reproducibility studies, a variation in electron energy from 13.9 to 14.1 eV resulted in a 3.4% and 4.0% mean relative deviation in the intensities of the 40 most intense peaks in the spectra of albumin and glycogen, respectively (refs. 100, 101). In spite of this sensitivity to minor changes in operating conditions, low voltage EI has the unique advantage of being able to ionise molecular beams efficiently. This allows for a very open design of the ion source and minimises memory effects. In contrast, both CI and FI involve considerable interaction of pyrolysis products with surfaces during the ionization procedure (FI only for polar products). Finally, low voltage EI is readily obtainable on most mass spectrometers whereas the CI and FI techniques may require elaborate equipment modification.

The CI technique is extremely efficient in generating (quasi-)molecular ions thereby allowing the detection of these ions even when the sample is present in very low concentrations (ref. 111). An additional feature of CI is the degree of selectivity as to which particular chemical moiety can be ionised, depending on the selection of the reagent ion (ref. 112). As an example of how this could be used in Py-MS studies, one might conceivably enhance the contribution of nitrogen-containing pyrolysis products, e.g. derived from proteins in the sample, by using a reagent ion with a high proton affinity, such as However, several inherent features of CI

ion source design potentially limit its usefulness in Py-MS studies. First, the prolonged residence time in the tight ion source may cause drastic changes in the chemical composition of the pyrolysate. Secondly, unless the sample is pyrolysed slowly and the products are diluted with reagent gas, pyrolysis compounds formed in high concentrations (e.g. h^O, NH^) could modify the ionisation process by substituting for the original reagent gas (ref. 54). Thus the ionisation process may then be determined by sample-dependent factors. Although CI studies of multicomponent hydrocarbon mixtures have been reported (ref. 113), so far no long-term reproducibility studies on multicomponent mixtures containing highly polar compounds appear to have been carried out. Until such data become available the value of CI in quantitative Py-MS studies ("fingerprinting") cannot be established with certainty. As discussed on page 14, however, CI is potentially a very valuable ancillary technique for qualitative studies of the pyrolysate, using selective reagent gases.

In FI the ionisation process is dependent to some extent on the condition of the emitter surface, especially with polar compounds. This is shown by the frequent occurrence of protonated molecular ions, indicating the role of surface ionisation processes in FI of these compounds (ref. 114). Changes in the condition of the emitter surface may thus change the character and distribution of the ions produced from multicomponent mixtures containing relatively polar compounds. This problem does not exist with mixtures of apolar substances. In fact, FI has been shown to be superior to low voltage EI in the quantitative analysis of hydrocarbon mixtures (ref. 115). Except for some selected geopolymers and synthetic polymers, however, most complex organic materials will yield pyrolysates containing varying amounts of polar components. Therefore, FI is unlikely to become a routine ionisation technique in fingerprinting of biomaterials by Py-MS. However, FI can be a very valuable tool for determining the elemental composition of intact molecular ions in pyrolysates especially in combination with high-resolution mass spectrometry (refs. 53, 71, 116).

Although low-voltage EI may have the drawbacks of residual fragmentation and poor sensitivity in comparison with CI or FI, the inherent advantages of simplicity and ability to operate in a beam mode, thus reducing contamination, have made it the method of choice in most Curie-point Py-MS studies reported so far. Further, it is worth mentioning that the problem of residual fragmentation during low voltage EI is to some extent compensated for by the fact that pyrolysis products represent an unusual collection of relatively stable compounds which often produce stable molecular ions during low-voltage EI (ref. 100). Among the more common biomaterials, lipids are the only class of compounds which consistently produce low voltage EI Py-MS patterns strongly dominated by fragment ions.

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