Thermospray ionization , HPLC

If a buffer is present in the HPLC mobile phase, and this is essential for true thermospray ionization, it should ideally be volatile and this may necessitate modifying existing HPLC methodology. 

Yinon et al. (228) used an HPLC interfaced with a triple-quadrupole mass spectrometer by means of a particle beam for the identification of several azo dyes. Characterization of the dyes was achieved by observing typical fragment ions formed by cleavage of the N-C and C-N bond on either side of the azo linkage and/or cleavage of the N=N double bond with the transfer of two hydrogen atoms to form an amine. Sensitivity was observed to be two to three orders of magnitude worse than with thermospray ionization. 

In the late 1980s, thermospray ionization (TSI) techniques offered what would be the precursor to a universal and reliable LC/MS interface for compounds of pharmaceutical interest. Conventional HPLC flow rates (l-2mL/min) were accommodated by this interface, using volatile buffers that contain ammonium acetate. New applications were realized, and higher standards of analytical performance were established for pharmaceutical analysis (Voyksner et al., 1985 Beattie and Blake, 1989 Oxford and Lant, 1989 Malcolm et al., 1990 Bowers et al., 1991). 

Thermospray ionization 8,000 Can be interfaced with standard-bore HPLC can be used with NP- or RP-HPLC simple to operate suitable for large, polar, and involatile molecules as well as ions Decomposition of thermolabile compounds only volatile solvents and volatile buffers can be used limited structural information... 

Figure 8. Ion chromatogram (top) of a lysozyme derived tryptic peptide fraction obtained using thermospray discharge ionization HPLC-MS. The resulting mass spectrum of one of the peptides (val-phe-gly-arg) is shown (bottom).

Figure 9. Hass spectrum of thiamine dihydrochloride obtained using thermospray discharge ionization HPLC-MS.

The solvents were HPLC grade acetonitrile, EM OMNISOLV solvent, (EM Science, N.J.) and distilled, deionized water using a MILLI-Q water Purification System (Millipore Corp., MA). The ammonium acetate used to prepare the 0.5M solution which is added postcolumn to aid the thermospray ionization was Baker Analyzed Reagent (J. T. Baker, NJ). 

Thermospray ionization First to interface with HPLC, quasimoleular ions, +ve or—ve... 

The emergence of thermospray ionization heralded a first ideal interface for a wide range of molecules [30,31]. With the introduction of this interface, LC/MS was accepted as a routine analytical technique. A major beneficiary of this interface was the pharmaceutical industry, which used this system to characterize drugs and metabolites. The construction and basic principle of thermospray ion source was discussed in Section 2.14 briefly, it consists of a heated probe, a desolvation chamber, and an ion extraction skimmer. When passed through a resistively heated capillary, the HPLC effluent, emerges as a mist of fine droplets into a heated desolvation chamber. Ionization of the solute molecules occurs by direct evaporation of the preformed ions or solvent-mediated chemical ionization. Thus, unlike the interfaces discussed above, the thermospray system acts as an ion source as well as an interface. Thermospray is ideally suited to coupling with conventional wide-bore columns. It is, however, confined primarily to reversed-phase HPLC separations, and it is less compatible with nonvolatile... 

Normal phase and to a lesser extent reversed and chiral phase HPLC have been combined online with thermospray ionization (TSI)-MS, ESI-MS, or APCI-MS for the purpose of identification and... 

The basic function of the mass spectrometer is to measure the mass-to-charge ratios of analyte ions, and the various designs of mass spectrometers have been described in detail in the literature. The HPLC-MS system has four main components consisting of a sample inlet, an ion source, a mass analyzer, and finally an ion detector. The sample introduction system vaporizes the HPLC column effluent. The ion source produces ions from the neutral analyte molecules in the vapor phase. Several designs of ion sources have been used over the past years including electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), thermospray ionization (TSP), continuous flow fast atom bombardment (FAB), and atmospheric pressure photoionization (APPI). The inductively coupled plasma (ICP) is a hard ionization source and is used specifically for the detection of metals and metals in adducts or in organometallic compounds. Generally, ICP-MS is used for elemental speciation analysis with HPLC, which has been described elsewhere in... 

The thermospray ionization interface was the first device to provide a viable means of coupling HPLC to MS for quantitative analysis, particularly for relatively involatile analytes. In its first form (Blakley 1978), HPLC eluant... 

The advent of atmospheric-pressure ionization (API) provided a method of ionizing labile and nonvolatile substances so that they could be examined by mass spectrometry. API has become strongly linked to HPLC as a basis for ionizing the eluant on its way into the mass spectrometer, although it is also used as a stand-alone inlet for introduction of samples. API is important in thermospray, plasmaspray, and electrospray ionization (see Chapters 8 and 11). 

The nebulization and evaporation processes used for the particle-beam interface have closely similar parallels with atmospheric-pressure ionization (API), thermospray (TS), plasmaspray (PS), and electrospray (ES) combined inlet/ionization systems (see Chapters 8, 9, and 11). In all of these systems, a stream of liquid, usually but not necessarily from an HPLC column, is first nebulized... 

The addition of a discharge electrode and a filament to the thermospray source widens the range of compounds that may be studied and HPLC solvents that may be accommodated. Optimum ionization conditions for a particular compouud ueed to be determiued empirically and it is essential that switching between the possible ionization modes may be accomplished easily and quickly. 

Atmospheric-pressure chemical ionization (APCI) is another of the techniques in which the stream of liquid emerging from an HPLC column is dispersed into small droplets, in this case by the combination of heat and a nebulizing gas, as shown in Figure 4.21. As such, APCI shares many common features with ESI and thermospray which have been discussed previously. The differences between the techniques are the methods used for droplet generation and the mechanism of subsequent ion formation. These differences affect the analytical capabilities, in particular the range of polarity of analyte which may be ionized and the liquid flow rates that may be accommodated. 

After the development of larger and more efficient vacuum pumps, more user-friendly LC/MS interfaces of thermospray,4 5 and atmosphere pressure ionization,6 7 LC/MS earned its place in bio-analytical laboratories. The resulting device was a powerful instrument that required significantly more capital investment than HPLC/UV, GC, or GC/MS. 

Much data on the structure of flavonoids in crude or semipurified plant extracts have been obtained by HPLC coupled with MS, in order to obtain information on sugar and acyl moieties not revealed by ultraviolet spectrum, without the need to isolate and hydrolyze the compounds. In the last decade, soft ionization MS techniques have been used in this respect, e.g., thermospray (TSP) and atmospheric pressure ionization (API). However, the most used methods for the determination of phenols in crude plant extracts were the coupling of liquid chromatography (LC) and MS with API techniques such as electrospray ionization (ESI) MS and atmospheric pressure chemical ionization (APCI) MS. ESI and APCI are soft ionization techniques that generate mainly protonated molecules for relatively small metabolites such as flavonoids. 

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