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2.1.2 Atmospheric pressure chemical ionization mass spectrometry (APCI/MS) The first reported use of chemical ionization at atmospheric pressure coupled to mass spectrometry is the work by Shahin.9,10 He used a discharge chamber with a platinum wire as anode to study ion-molecule reactions. The group of Horning developed an atmospheric pressure ionization source, in which a radioactive Ni foil is used to initiate gasphase ionization.11 Later, when they coupled liquid chromatography (LC) to mass spectrometry, the use of a corona discharge needle was introduced.12-15 APCI is an ionization technique for mass spectrometry (MS) that uses gas-phase ion-molecule reactions at atmospheric pressure. The method is analogous to chemical ionization (CI), which is normally used in gas chromatography/mass spectrometry (GC/MS). The APCI technique is mainly applied to polar and ionic compounds with moderate molecular weight and generally gives monocharged ions. It is usually coupled with liquid chromatography, especially high-performance liquid chromatography (HPLC), and has become a popular ionization source for mass spectrometry in these years.16,17 The schematic diagram of an APCI source is shown in Fig. 2-3.18,19 The analyte in solution from a direct inlet probe or a liquid chromatography eluate is introduced directly into a pneumatic nebulizer where it is converted into a thin fog by a high-speed nitrogen beam.
- 13 Theoretical Background ————————————————————————————————— then transported by the gas flow through a heated quartz tube called a desolvation/vaporization chamber. The heat transferred to the spray droplets allows vaporization of the mobile phase and of the sample in the gas flow. The temperature of this chamber is controlled, which makes the vaporization conditions independent of the flow and from the nature of the mobile phase. After desolvation, the solvent and analyte molecules are carried along a corona discharge electrode where ionization occurs.
Figure 2-3. Schematic diagram of an APCI source18
The ionization processes in APCI are equivalent to the processes that take place in CI but all of these occur under atmospheric pressure. In the positive ion mode, either proton transfer or addition of reactant gas ion can occur to produce the ions of molecular species, depending on the relative proton affinities of the reactant ions and the gaseous analyte molecules. In the negative mode, the ions of the molecular species are produced either by proton abstraction or adduct formation. Typically, the corona discharge is formed by electron ionization primary ions such as N•+ or O2•+. Then, these ions collide with vaporized solvent molecules to form secondary —————————————————————————————————
- 14 Theoretical Background ————————————————————————————————— reactant gas ions. As shown below, it is a typical proton transfer process.
Here, M is the analyte, and H3O+ plays the role of the reactant gas ion.
Because the ionization of the substrate occurs at atmospheric pressure and thus with a high collision frequency, it is very efficient. Furthermore, the moderate desolvation and vaporization of the droplets considerably reduce the thermal decomposition of the analyte. The result is that the ionization step yields predominantly ions of molecular species with few fragments. In comparison to other ionization techniques working under vacuum, APCI can be regarded as a ‘soft’ ionization technique.
2.1.3 Thin-layer chromatography (TLC) Thin-layer chromatography is a type of chromatography in which the stationary phase is a thin layer of an adsorbent, e.g. silica gel, coated on a rectangular plate and the mobile phase is typically a solvent mixture. For analytical purposes, samples are applied in the form of spots at a point near one edge of a plate. In preparative work, the samples are streaked, often by means of a special device (applicator, streaker) on a line parallel to that edge. The chromatograms are developed in a closed vessel (chamber) by allowing the edge of the plate to dip into the mobile phase, which then advances past the sample to a paralleled line (solvent front) near the opposite edge. The detection is carried out after the TLC plate is dried in order to remove the residues of the solvents from the sorbent.
Since most substances resolved by TLC are colorless, visualization step is used to find the position of the analyte, such as staining techniques, —————————————————————————————————
- 15 Theoretical Background ————————————————————————————————— ultraviolet (UV) absorption and fluorescence extinction. Automated and more convenient detection is based on UV/visible (VIS) absorption or fluorescence imaging allowing the quantification of the analytes.
Normally, TLC detection methods are nondestructive and permit further analysis of the separated substances.20-22 The attractive features of TLC include parallel sample processing for high sample throughput; accessibility of the sample for postchromatographic evaluation free of time constraints; detection in the presence of the stationary phase independent of mobile phase properties;
and the stationary phase is normally used only once. As an economic and handy chromatographic separation technique, TLC has been extensively used for many years not only because of the modest demands on instrumentation, but also due to its sensitivity, general applicability, and its flexibility.23-25 It is generally agreed that TLC is most effective for the low-cost analysis of samples requiring minimal sample clean-up, and it is also preferred for the analysis of substances with poor detection characteristics requiring post-chromatographic treatment for detection.
Since all sample components are located in the chromatogram, TLC is the most suitable technique for surveying sample properties.
2.2 TLC-MS The major inherent drawback to the usual detection techniques of TLC is the inability to identify the analytes and have a low specificity with partially overlapping substances. Recently, mass spectrometry has become the method of choice for identification of compounds in conjunction with TLC techniques, which was first realized by Kaiser.26 In his work, a small H2-O2 flame was used to desorb samples directly from a silica plate, and then desorbed materials were simultaneously swept into a mass spectrometer by Helium.
- 16 Theoretical Background ————————————————————————————————— In the last few decades, a series of attempts have been made to couple TLC and MS.23,24,26,27 There are two approaches to remove the analyte molecules from the TLC plate. One possibility is to extract the analytes with a solvent either directly from the TLC plate or after removing a part of the stationary phase that contains the separated analyte. Typically, the extraction step is then followed by gas chromatography /mass spectrometry (GC/MS) analysis. Unfortunately, this extraction method can destroy the chromatographic integrity and it is also quite time consuming.
The other basic technique is the desorption of analytes with energy, such as secondary ion mass spectrometry (SIMS), fast atom bombardment (FAB) or laser desorption/ionization and some related techniques. These techniques need fewer experimental steps, however, due to an intrinsic coupling of desorption and ionization of the analytes they do have high requirements for the instrumentation.
2.2.1 Electron impact (EI) and Chemical ionization (CI) EI is the classical ionization technique in mass spectrometry, in which a gaseous sample is bombarded by electrons usually generated from a tungsten filament.28,29 Because the pressure inside the ion source is kept low, the technique induces extensive fragmentation. Ion-molecule reactions do not occur, e.g. a [M+H]+ signal due to proton transfer is not observed. The application of EI is restricted to the thermally stable samples with low molecular masses.18 Because EI is a popular ionization method for GC/MS, the coupling is generally performed by using a GC mass spectrometer system. There are two methods to introduce analytes into the EI ion source after removing the stationary phase containing the analyte. In the first method, the analyte is extracted with a solvent, and then the solvent is injected as a liquid phase. This method is technically undemanding and efficient, but involves extraction and concentration —————————————————————————————————
- 17 Theoretical Background ————————————————————————————————— steps, which is usually time-consuming.30,31 In another method, the TLC piece is put on a heating probe. The analyte absorbed in the stationary phase is thermally evaporated and injected in its gaseous phase. The method has less experimental steps but is restrictedly for polar or involatile compounds on silica gel plates because of the strong absorption of such analytes to silica.32 One solution for the problem is the use of less absorbing stationary phase, such as polyamide TLC plates.33-35 The basic principle of CI is similar to that of APCI, but ion-molecule reactions happen in much lower pressure (about 10-3 Pa).36 Compared with EI, it yields less fragmentation and often gives information about the molecular weight of the analyty.18 CI is not widely used in combination with TLC, but very often used in GC/MS. A prominent work about TLCCI/MS was performed by Ramaley et al.37,38 A remarkable advance in this work was that they developed a device for plate scanning, in which a chamber containing a TLC plate was put on a stepping motor to provide motion. The analyte on the TLC plate was desorbed with a quartz-halogen projection lamp or a CO2 laser. The desorbed molecules were swept by a CI regent gas to the ion source of the mass spectrometer after passing through a heated glass tube.
- 18 Theoretical Background ————————————————————————————————— Compared with EI, these ionization techniques are ‘soft’ and produce only few fragments. They are more appropriate for polar or thermally unstable substances than EI.18 The use of TLC in combination with these ionization techniques was realized for a whole range of analytes, including drugs and their metabolites, pesticides, natural products, synthetic chemicals and dyes.26 Because these ionization techniques can be applied on solid surfaces directly, the approach of extraction with solvent is not necessary. The normal procedure of the combinations includes the removal of the stationary phase with substrate; condensation with a solvent, such as methanol, for analytes with small amount or in a diffuse spot, which is applied around the sample spot; application of matrix (for FABMS and LSIMS); and subsequent mass spectroscopic analysis.43-45 Suzuki et al.
developed an improved condensation technique, in which the area including the desired spot was cut, and a trapezoidal shape was scribed, out of which the stationary phase was scratched. A small amount of methanol was deposited on the lower base of the trapezoid, which causes the migration of the analyte to the upper base by penetration of methanol.
Finally, the analyte was concentrated in a line of 0.5-1 mm.46 Nakagawa et al. designed a simple device allowing a stripe of the TLC plate to be source.47 scanned by a LSIMS It supplies the information of chromatography, but has just a few modifications of the ion source at the same time.
2.2.3 Electrospray ionization (ESI) ESI is another popular ionization method for mass spectrometry, suitable for large bio-molecules or synthetic polymers. Like APCI, it also works under atmospheric pressure and enables LC/MS coupling. An ESI source consists of a very fine needle and a series of skimmers. A high potential is —————————————————————————————————